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High-Performance Intensiometric Direct- and Inverse-Response Genetically Encoded Biosensors for Citrate

  • Yufeng Zhao
    Yufeng Zhao
    Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada
    More by Yufeng Zhao
    Orcidhttp://orcid.org/0000-0002-1788-7969
  • , 
  • Yi Shen
    Yi Shen
    Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada
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  • , 
  • Yurong Wen*
    Yurong Wen
    Department of Talent Highland, The First Affiliated Hospital, Xi’an Jiaotong University, Xi’an, Shaanxi 710061, China
    *Email: [email protected]. (Y.W.; regarding x-ray crystallography)
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  • , and 
  • Robert E. Campbell*
    Robert E. Campbell
    Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada
    Department of Chemistry, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan
    *Email: [email protected]. (R.E.C.)
    More by Robert E. Campbell
    Orcidhttp://orcid.org/0000-0003-0604-092X
Cite this: ACS Cent. Sci. 2020, 6, 8, 1441–1450
Publication Date (Web):July 9, 2020
https://doi.org/10.1021/acscentsci.0c00518

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Abstract

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Motivated by the growing recognition of citrate as a central metabolite in a variety of biological processes associated with healthy and diseased cellular states, we have developed a series of high-performance genetically encoded citrate biosensors suitable for imaging of citrate concentrations in mammalian cells. The design of these biosensors was guided by structural studies of the citrate-responsive sensor histidine kinase and took advantage of the same conformational changes proposed to propagate from the binding domain to the catalytic domain. Following extensive engineering based on a combination of structure guided mutagenesis and directed evolution, we produced an inverse-response biosensor (ΔF/Fmin ≈ 18) designated Citroff1 and a direct-response biosensor (ΔF/Fmin ≈ 9) designated Citron1. We report the X-ray crystal structure of Citron1 and demonstrate the utility of both biosensors for qualitative and quantitative imaging of steady-state and pharmacologically perturbed citrate concentrations in live cells.

Synopsis

Development of high-performance genetically encoded biosensors for citrate, based on engineered fluorescent proteins, enables visualization and quantification of intracellular citrate concentrations.

Introduction

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Citrate is primarily known as the first major intermediate in the tricarboxylic acid (TCA) cycle, but in recent years, there has been a growing appreciation of the wider range of roles for citrate in biological processes including inflammation, cancer, and insulin secretion, among others. (1) Citrate is produced in the mitochondria by the enzyme citrate synthase (CS) and is metabolized into isocitrate by the enzyme aconitase 2 (ACO2) in the next step of the TCA cycle. Citrate can be transported from the mitochondria to the cytosol by the citrate carrier (CiC; SLC25A1) or transported into the cell from the bloodstream by a plasma-membrane-specific variant (pmCiC; SLC13A5) of the mitochondrial CiC. Once in the cytosol, citrate can be cleaved by ATP citrate lyase (ACLY) into acetyl-CoA and oxaloacetate. Acetyl-CoA is the substrate for various biochemical processes including fatty acid synthesis and histone acetylation for the epigenetic modifications of gene expression, (2) and oxaloacetate plays a key role in various biochemical processes including amino acid biosynthesis. As some of citrate’s many roles are associated with the mitochondrial pool, and others are associated with the cytoplasmic pool, methods that can enable the accurate determination of the concentration in both subcellular compartments are of high importance.
Much remains to be learned about the roles of citrate, as published reports support apparently contradictory roles for this key metabolite in cancer cells. Huang et al. have noted that citrate can have both antitumor and tumor-promoting effects, depending on its local concentration. (2) For example, despite the decreased reliance on the TCA cycle in tumor cells (the Warburg effect), (3) some cell types appear to have an increased cytosolic pool of citrate that may serve as a source of acetyl-CoA to be used for increased fatty acid biosynthesis. (4) Conversely, other evidence suggests that a decrease in cytosolic citrate is associated with tumor aggressiveness, possibly due to increased resistance to apoptosis as a consequence of decreased acetyl-CoA-dependent protein acetylation. (5) As these examples demonstrate, citrate is central to the dysregulated metabolism of cancer cells, and enzymes that produce (i.e., CS), transport (i.e., CiC), and metabolize (i.e., ACLY and ACO2) citrate have been proposed as potential targets for new therapeutic approaches to cancer. (2,4) Further investigation of the role of citrate in tumor development will require new methods that could be used for sensitive detection of citrate in subcellular compartments of cells in in vivo tumor models.
In addition to its roles in cancer cells, citrate functions as a regulatory metabolite in endocrine cells such as beta cells (6) and immune cells such as dendritic cells and macrophages. (7) Previous studies suggest that citrate (and isocitrate) exported by CiC to the cytosol can function as a signaling molecule to regulate downstream insulin secretion in beta cells. (8,9) Furthermore, activated dendritic cells and macrophages undergo a metabolic reprogramming that leads to the accumulation of citrate, which, in turn, regulates the immune cell functions through carbohydrate and fatty acid metabolism and citrate-derived acetylation of histones. (10) Techniques that enable spatially and temporally resolved imaging of citrate concentrations would facilitate the understanding of its central functions in these metabolism-based signaling processes.
A variety of synthetic fluorescent indicators for citrate have been reported, including ones based on indicator displacement assays, (11−13) turn-on fluorescence complexation, (14) competitive binding of metal ions such as Eu3+ and Pb2+ (refs (15) and (16)), and aggregation-induced emission. (17) These indicators have proven useful for the quantitative analysis of citrate in urine, but none of these designs are practical for measurement of citrate in the mitochondria and cytosol of living cells. Challenges with their use in such applications include delivery into cells, competition from other endogenous small molecules, potential toxicity, and the lack of control over subcellular location.
For in vitro or in vivo application in tissue, an attractive alternative to synthetic indicators are fully genetically encoded proteinaceous biosensors. (18) Genetically encoded biosensors are engineered proteins composed of a fusion between a fluorescent protein (FP), such as Aequorea victoria green FP (GFP), and a sensing domain that undergoes a conformational change upon binding its cognate ligand of interest or in response to some other biological parameter of interest. This conformational change in the sensing domain is propagated to the FP fluorophore environment, leading to a change in fluorescence intensity. First demonstrated in 1999 by Tsien and co-workers for the construction of Ca2+ and Zn2+ biosensors, (19) this general approach has now been expanded to a multitude of other ligands of interest. (18) Advantages of such biosensors include their ability to be delivered relatively noninvasively in the form of their DNA genes, high specificity conferred by the molecular recognition properties of the sensing domain, low toxicity due to being protein-based, and the ability to be genetically targeted to subcellular compartments.
A key challenge in developing a genetically encoded biosensor is first identifying a protein that both specifically binds to a target ligand of interest and undergoes substantial conformational change upon binding. A protein that satisfies both of these criteria are sensor histidine kinases (SHK), which are one of the two proteins in a bacterial two-component regulatory system (with the other protein being the response regulator). (20) A typical SHK is a homodimer protein with an N-terminal periplasmic sensory domain that has both its N-terminus (TM1) and C-terminus (TM2) linked to transmembrane α-helices (Figure 1a). Linked to TM2 are several intracellular domains, including the C-terminal histidine kinase (HK) catalytic domain. (21) Necessarily, binding of a cognate ligand to the periplasmic sensory domain leads to a conformational change that propagates across the membrane and modulates the activity of the C-terminal HK domain. (21−23) Various models have been proposed for these membrane-spanning structural changes including the “piston” model, in which the two helices are vertically displaced relative to each other, the “scissor blade” model, in which the helices undergo a diagonal scissor-like displacement, and the “rotating helix” model, in which one helix is proposed to rotate relative to the other. (21) Regardless of the precise mechanism, it is evident that ligand binding to the periplasmic domain must lead to substantial conformational changes in the transmembrane portion of the HKS.

Figure 1

Figure 1. Rationale for the design of a single-FP-based citrate biosensor. (a) Schematic representation of Klebsiella pneumoniae SHK CitA, which is composed of a periplasmic citrate-binding domain (CitAP; light blue, unbound; magenta, bound), connected to transmembrane helices at both its N- (transmembrane helix 1, TM1) and C-termini (transmembrane helix 2, TM2). TM2 is, in turn, connected to an intracellular HK catalytic domain. (26) (b) The structures of citrate-free CitAP (left; light blue; PDB ID 2V9A), (25) citrate-bound CitAP (right; magenta; PDB ID 2J80), (25) and a superposition of the citrate-free and -bound structures (middle). (c) We hypothesized that the piston-type conformational motion at the CitAP termini could be communicated to GFP to allosterically control the chromophore environment and its fluorescent brightness. In this way, the CitAP domain could serve as the basis of construction of a genetically encoded citrate biosensor. (d) To realize this biosensor design, we inserted CitAP into GFP by replacing the CaM-RS20 domain of ncpGCaMP6s (27) with CitAP.

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One relatively well characterized SHK is the citrate-responsive CitA protein from Klebsiella pneumoniae. (22,24,25) X-ray crystal structures are available for the Per-Arnt-Sim (PAS)-fold citrate-binding periplasmic domain (CitAP) both with (PDB IDs 1P0Z (24) and 2J80) (25) and without (PDB ID 2V9A) (25) citrate (Figure 1b). As described by Sevanna et al., (25) the major loop of CitAP is disordered in the citrate-free structure but “wraps over” the citrate-binding site in the bound state. In addition, there is substantial movement of a minor loop that “folds in” on the citrate-binding site in the bound state. This “folding in” of the minor loop is associated with a compaction of the central β-sheet that pulls the C-terminus away from the transmembrane domain and causes a piston-like “pull” on TM2. This model is also supported by NMR studies of a truncated and membrane embedded form of CitA. (22)
Honda and Kirimura previously reported a genetically encoded biosensor, designated CF98, based on insertion of circularly permuted (cp) GFP into the CitAP domain of CitA. (28) To develop CF98, Honda and Kirimura made a series of nine prototype constructs, in which circularly permuted cpGFP was systematically inserted at positions within the minor loop of CitAP (Figures 1b and S1ab). The most promising construct (CF98) was based on insertion of cpGFP between residues 98 and 99 of CitAP (all numbering as in PDB ID 2J80) (25) and exhibited an intensiometric fluorescence increase (ΔF/F, calculated as (Fmax – Fmin)/ Fmin) of approximately 0.7. CF98 was used to detect intracellular citrate concentration changes in a suspension of E. coli treated with citrate. To the best of our knowledge, there have been no reports of using this biosensor to visualize citrate concentrations in eukaryotic cells. In other work, Ewald et al. reported a series of genetically encoded Förster resonance energy transfer (FRET)-based biosensors, composed of a cyan FP and yellow FP fused to the termini of CitAP, that exhibit up to a 55% increase in emission ratio upon binding citrate. (29) These biosensors were originally used to monitor citrate in E. coli after starvation and were later used to monitor citrate oscillations in glucose-treated islet β-cells. (30)
With the growing recognition of the central importance of citrate in healthy and diseased cells (1) and the need for new tools to probe the concentration of citrate in tissues, we have developed a new generation of high-performance intensiometric direct-response (Citron) and inverse-response (Citroff) genetically encoded biosensors for citrate. Further motivation came from the growing recognition that, with appropriate optimization and directed evolution, high-performance single-FP-based biosensors can be engineered with ΔF/F > 10 (refs (31−33)) or even >100 (refs (34) and (35)). Such large fluorescence changes, combined with high fluorescent brightness and ligand affinity tuned to the relevant physiological concentration, are critical if such biosensors are to be robust and effective tools for tissue-based imaging experiments.

Results

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Development of Citrate Biosensors Citron1 and Citroff1

To construct a genetically encoded biosensor for citrate, we replaced the calmodulin (CaM)-RS20 domain of ncpGCaMP6s (27) (from pcDNA-ncpGCaMP6s, Addgene plasmid #113674) with residues 4–133 of the CitAP domain of Klebsiella pneumoniae CitA (Figures 1c,d and S1cd). From this point on, residues are numbered according to the full length biosensor constructs, as shown in Figure S2. The initial variant with linker sequences copied from ncpGCaMP6s (linker 1 (L1) = L145–P146 and linker 2 (L2) = L277–E278–N279) were nonfluorescent. To rescue the fluorescence, we randomized each of the two linkers individually and performed colony-based screening. Screening of a library (∼1000 colonies) in which both residues of L1 were randomized led to the identification of ∼50 fluorescent colonies with relatively high brightness. These colonies were picked and cultured overnight at 37 °C in 4 mL of LB media. The bacteria were lysed, the protein extract was dispensed into wells of a 384-well plate, and a microplate reader was used to evaluate fluorescence changes upon citrate binding. This screening led to a variant, designated Citron0.1 (L1 = L145M-P146 V), with a ΔF/F, calculated as (Fmax – Fmin)/Fmin ≈ 0.2 direct response (that is, an increase in fluorescence upon citrate binding), and a second variant, designated Citroff0.1 (L1 = L145W–P146Q), with ΔF/F ≈ 1.9 inverse-response (that is, a decrease in fluorescence upon citrate binding). A similar randomization and screening of L2 was then performed for both variants, leading to the identification of Citron0.2 (ΔF/F ≈ 0.6; L2 = L277S–E278N–N279L) and Citroff0.2 (ΔF/F ≈ 4.5; L2 = L277P–E278N–N279F).
Following linker optimization, the two variants were further optimized in parallel by directed evolution, which involved screening of randomly mutated libraries generated by an error-prone polymerase chain reaction of the entire biosensor gene. After each round, the most promising variants were used as the template for the next round of library construction and screening. Following 10 rounds of directed evolution, the best direct-response biosensor (with 14 mutations in GFP and 8 mutations in CitAP; Figure S2) had ΔF/F ≈ 9 in vitro (Figure 2a and Table S1) and was named as Citron1. For the inverse-response biosensor, three rounds of directed evolution led to a variant (with three mutations in GFP and one mutation in CitAP; Figure S2) that exhibited ΔF/F ≈ 18 in vitro (Figure 2b and Table S1) and was designated Citroff1. Further screening did not produce any variants with improved performance. The dynamic range of both biosensors is substantially higher than the ΔF/F ≈ 1.1 that we measured for purified CF98 under identical experimental conditions (Figure S3a). The molecular brightnesses of the bright states of Citron1 (+ citrate) and Citroff1 (− citrate), calculated as the product of extinction coefficient and quantum yield, are 21 and 25, respectively (Table S1). For reference, EGFP has a brightness of 37.5 (ref (36)), and the Ca2+-bound state of GCaMP6s has a brightness of 41.8 (ref (37)).

Figure 2

Figure 2. In vitro characterization of new citrate biosensors and crystal structure of Citron1. (a,b) Normalized excitation and emission spectra of purified Citron1 (a) and Citroff1 (b) in the presence and absence of 20 mM citrate. (c,d) In vitro citrate titration curves of purified Citron1 (c) and Citroff1 variants (d). Error bars represent standard deviation of triplicates. (e) Overall representation of the Citron1 structure with the position of all mutations indicated. The CitAP domain with citrate is colored in magenta, and the cpGFP domain is colored in green. The chromophore, citrate, and the Cα of Met145 (blue) and Asn278 (magenta) are represented as spheres. (f) Zoom-in view of the hydrogen bonding interaction between Asn278 and the chromophore. Additional residues in the vicinity of Asn278 of Citron1 are shown and labeled with magenta text, with the identity of the corresponding residue of Citroff1 labeled with black text.

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In Vitro Characterization and Affinity Tuning

Determination of the citrate-binding affinities of the two biosensors revealed that the Kd of the direct-response biosensor, Citron1, is 1.1 mM (Figure 2c), which is a substantially lower affinity than that of CitAP itself (Kd = 5.5 μM at pH 7). (38) This decreased affinity is attributed to mutations in the CitAP domain that were accumulated during directed evolution (Figure S2). Specifically, mutation E223 V is immediately adjacent to the citrate-binding pocket (equivalent to E80 V as numbered in PDB ID 2J80) (25) and is likely to directly affect citrate binding (Figure S4). However, as the intracellular citrate concentration in mammalian cells ranges from hundreds of micromolar to several millimolar, Citron1 should be particularly responsive to physiologically relevant concentration changes. We determined that CF98 demonstrates an even lower binding affinity (Kd ≈ 9 mM), which is likely to make it poorly responsive to physiologically relevant changes in citrate concentration (Figure S3b).
In contrast to Citron1, Citroff1 retains the high binding affinity of CitAP (Figure 2d). In an effort to fine-tune the affinity of Citroff1 into a physiologically relevant concentration range, we used site-directed mutagenesis to introduce mutations (Y199F, T201A, S227A, S244A, S248A, and S267A) that were expected to directly or indirectly disrupt interactions between the protein and citrate, based on the crystal structure of CitAP (PDB ID 2J80). (25) Mutations that have previously been reported to lower the binding affinity (R192A, K220A, K235A, and R250A) were also assessed. (39) Single-mutations S244A and R250A resulted in biosensors with substantially lower affinity (Kd = 35 and 153 μM, respectively) while maintaining a large dynamic range (Figure 2d and Table S2). A combination of S244A and Y199F further increased the Kd to 965 μM but with a slightly lowered ΔF/F of ∼11 (Figure 2d and Table S2). Other single or pairwise combinations of mutations did not yield variants that had both appropriate affinities and large fluorescence responses (Figure S5).

pH Dependence and Nonbinding Control Biosensors

To determine the pH dependence of the response of Citron1 and Citroff1, the fluorescence was measured in the presence and absence of citrate at various pH values (Figure S6a,b). Citron1 exhibits a 60% increase in fluorescence intensity for the citrate-bound state and a 150% increase in fluorescence intensity for the citrate-free state, when going from a pH of 6.0 to 8.0. For Citroff1, the values are 730 and 350%, respectively. In terms of specificity, the two citrate biosensors showed a negligible response to several potentially interfering metabolites at high concentration (20 mM) (Figure S6c,d). This excellent specificity is similar to that of the previously reported citrate biosensors that employed CitAP as the sensing domain. (28,29) In a previous study, a bound sodium ion (Na+) was observed in the crystal structure of CitAP, which might potentially affect the citrate-induced conformational change. (24) The dependence of Citron1 and Citroff1 on Na+ was examined, and no obvious Na+-dependent fluorescence change was found (Figure S7).
While Citron1 is only modestly pH-sensitive through the physiologically relevant pH range, the possibility of artifactual fluorescence changes due to changes in pH is a persistent concern for both Citron1 and Citroff1 when performing cell imaging with genetically encoded biosensors. Accordingly, we created citrate-insensitive (but pH-sensitive) control constructs by introducing two key mutations (R209A and H212A, equivalent to CitAP R66A and H69A, respectively; Figures S2 and S8a) to disable citrate binding. These variants, named as CitronRH and CitroffRH, showed no response to citrate (Figure S8b,c), but pH dependence that was similar to Citron1 and Citroff1 in the citrate-free states (Figure S8d,e). These variants are appropriate controls for evaluating the possible contribution of pH changes and other physiological changes to fluorescence responses observed during cell imaging experiments.

Crystal Structure of Citron1

In an effort to obtain molecular insight into the structure and mechanism of these citrate biosensors, we crystallized Citron1 in the citrate-bound state. The Citron1 crystal structure was determined to 2.99 Å resolution using molecular replacement (Figure 2e,f and Table S3). The overall structure is represented in Figure 2e, with all mutations labeled. Within the biosensor structure, the CitAP domain and its citrate-binding pocket are essentially identical to previously determined CitAP structures. (24,25)
Inspection of the molecular interactions between the GFP chromophore and its immediate environment reveals a possible mechanism for the citrate-dependent fluorescence modulation. Specifically, we observe a hydrogen bond between the GFP chromophore phenolate group and the side chain of Asn278 in linker 2 (Figure 2f). This interaction is likely to stabilize the fluorescent phenolate form (deprotonated form), resulting in bright fluorescence in the citrate-bound state. Presumably, in the citrate-free state, Asn278 is positioned in a different conformation such that this hydrogen bond interaction is not present, and the dimly fluorescent phenol form (protonated form) of the chromophore predominates. A very similar mechanism has been previously suggested for the red Ca2+ biosensor K-GECO1 (ref (40)). In the Ca2+-bound state of K-GECO1, Asn32 is positioned similarly to Asn278 of Citron1 and is similarly engaged in a hydrogen bond with the chromophore. In the case of Citroff1, we speculate that Asn278 stabilizes the phenolate form of the chromophore through a hydrogen bond interaction in the citrate-free state but not in the citrate-bound state.

Quantification of Citrate Concentrations in HeLa Cells

To evaluate the utility of Citron1 and Citroff1 for visualization of intracellular citrate concentration changes, we expressed each construct in HeLa cells (ATCC CCL-2). The genes were expressed under a CMV promoter with either no targeting (that is, cytoplasmic and nuclear localization), or as a fusion to two copies of a mitochondrial-targeting sequence (41) (that is, mitochondrial localization). As shown in Figure 3a, the biosensors exhibited strong fluorescence intensity in both the cytoplasm and mitochondria of HeLa cells. Artificial changes in the intracellular citrate concentration were achieved by permeabilization of the cell membrane with digitonin followed by citrate addition to the imaging buffer. Substantial fluorescence changes were observed for both Citron1 and Citroff1, and the changes were similar for both the cytoplasmic and mitochondria-localized biosensors (Figures 3b and S9a). No substantial fluorescence changes were observed when cells expressing the control variants, CitronRH and CitroffRH, were subjected to the same treatments (Figure S10a,b). These results demonstrate that Citron1 and Citroff1 remain responsive to citrate concentration changes when expressed in live cells.

Figure 3

Figure 3. Expression of citrate biosensors in HeLa cell and permeabilization tests. (a) Representative fluorescence images of HeLa cells expressing Citron1 in the cytosol (left panel) and mitochondria (right panel). (b) Fluorescence intensity of Citron1 in the cytosol in response to treatment with digitonin and citrate (n = 78 cells). The analogous chart for mitochondrial Citron1 is provided as Figure S9a. (c,d) In situ titration of Citron1 (c) and Citroff1 (d) in the cytosol. Digitonin was added as indicated, and the concentration of citrate in the external buffer is indicated in millimolar (n = 128 for Citron1 and n = 26 for Citroff1). (e) In situ titration curve of Citron1 in the cytosol averaged from 128 cells in panel (c). At the highest concentrations (14.6 and 24.6 mM), the biosensor may not have fully equilibrated with the added citrate (see panel (c)), and the in situ Kd may therefore be underestimated. The analogous chart for Citroff1 is provided as Figure S9b. Error bars represent s.e.m. for panels (b–e). (f) Quantification of citrate concentration in the cytosol and mitochondria with or without 5.5 mM glucose in the buffer. Each dot is quantified using the average signal from tens of cells in a single experiment. Center box and whiskers represent the average and s.e.m. of the four data points, respectively.

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To perform an in situ titration to quantify the citrate concentration in the cytosol, cell membranes were permeabilized with digitonin, and various concentrations (0.1 to ∼20 mM) of citrate were added to the imaging buffer (Figure 3c,d). After each titration step, we allowed at least 250 s for the biosensors to reach the maximal signal change before the next step. The average response from cells expressing Citron1 or Citroff1 vs concentration of citrate was plotted to provide an in situ calibration curve (Figures 3e and S9b). To quantify the concentration of citrate in cells, we acquired a fluorescence image of individual intact cells, then acquired a second image following treatment with digitonin to fully deplete intracellular citrate, and then acquired a third image after adding a saturating concentration of citrate (20 mM). The initial fluorescence intensity of intact cells (first image) was normalized according to the intensities at 0 mM (second image) and 20 mM citrate (third image), and the initial concentration was determined using the previously acquired calibration curve (Figures 3e and S9b). Using this approach, the average citrate concentrations in the cytosol and mitochondria of HeLa cells conditioned in HEPES buffered HBSS solution with 5.5 mM glucose were determined to be 0.15 ± 0.07 and 0.44 ± 0.13 mM, respectively, using Citron1 (four replicates each). In the absence of glucose in the same buffer, citrate concentration remained essentially constant both in the cytosol and mitochondria (Figure 3f). Quantification using Citroff1 gave concentrations (0.12 ± 0.01 mM in the cytosol and 0.45 ± 0.19 mM in the mitochondria) consistent with the Citron1 measurements. The experimentally determined cytosolic citrate concentration is comparable to that previously reported for Hep G2 cells (168.0 ± 51.0 μM) (42) and human blood plasma (100–150 μM). (43)

Physiological and Pharmacological Alteration of Subcellular Citrate Concentration

Next, we explored the use of Citron1, Citroff1, and the affinity variants for imaging of changes in citrate concentration induced by changes in culture media composition or by pharmacological treatments. Both Citron1 and Citroff1 biosensors demonstrated a larger Kd in cultured cells than that in vitro (5.7 mM in situ (Figure 3e) vs 1.1 mM in vitro for Citron1 and 130 μM in situ (Figure S9b) vs 5 μM in vitro for Citroff1). Among the inverse-response biosensors, Citroff1 has an optimal in situ Kd for imaging citrate dynamics in the cytosol, where the citrate concentration is determined to be 0.12–0.15 mM. Citroff1 S244A, with a slightly lower affinity than Citroff1, is presumably suitable for imaging citrate in the mitochondria where the citrate concentration was ∼0.45 mM. To investigate glucose-induced changes in intracellular citrate concentration, Citron1- and Citroff1-expressing HeLa cells were kept in a glucose-free buffer for 0.5–1 h. Upon glucose addition, both Citron1 and Citroff1, targeted to either the cytosol or mitochondria, exhibited fluorescence intensity changes consistent with an increased citrate concentration (that is, increased fluorescence intensity for Citron1 (Figure 4a) and decreased signal for Citroff1 (Figure S11b)). The cytosolic citrate concentration substantially increased upon glucose treatment as revealed using both Citron1 and Citroff1. For mitochondrial citrate, treatment with glucose induced an initial transient decrease followed by a slower increase as reported by the mitochondria-localized Citron1. Consistent results were obtained with mitochondrially targeted Citroff1 S244A (with Kd = 35 μM vs Kd = 5 μM for Citroff1), though the initial transient decrease was not as pronounced (Figure S11d). Similar experiments using the control variants, CitronRH and CitroffRH, confirmed that the observed signal changes were attributable to changes in citrate concentration rather than other potentially confounding factors such as pH changes (Figure S11).

Figure 4

Figure 4. Use of Citron1 for imaging of citrate concentration changes induced by changes in glucose or pharmacologically in HeLa cells. (a) Glucose-induced citrate concentration changes detected with cytosolic (orange trace, n = 32) and mitochondrial (green trace, n = 28) Citron1. (b) BMS-303141-induced citrate concentration changes detected by cytosolic (n = 22) and mitochondrial (n = 24) Citron1. (c) UK-5099 (in DMSO)-induced citrate concentration changes detected by Citron1 (blue trace, n = 36) expressed in mitochondria. Control experiments include Citron1 + DMSO (orange trace, n = 23), CitronRH + UK-5099 (gray trace, n = 38), and CitronRH + DMSO (green trace, n = 23). The arrow indicates the addition of UK-5099 or DMSO solutions. Error bars in a–c represent s.e.m. (d–f) Dual color imaging of citrate and ATP concentration changes in mitochondria using Citron1 and MalionR. (44) (d,e) Representative fluorescence images of cells coexpressing Citron1 (d) and MalionR (e). (f) Representative glucose-induced citrate and ATP concentration changes reported by Citron1 (green traces) and MalionR (red traces).

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The observed increases in cytoplasmic and mitochondrial citrate concentrations following glucose treatment are consistent with increased glucose availability fueling the TCA cycle, resulting in an increase in TCA intermediates including citrate. Notably, citrate concentration changes occurred approximately 50 s earlier in the mitochondria compared to the cytosol (Figure 4a), consistent with the expected production of citrate by citrate synthase in mitochondria. (10)
We next used Citron1 and Citroff1 to investigate changes in citrate concentration associated with inhibition of ACLY by the inhibitor BMS 303141 (ref (45)). Using cytosolic Citron1 and Citroff1, we observed a transient slight increase of citrate concentration, followed by a sustained decrease in concentration (Figure S12a,b). The initial transient increase in mitochondrial citrate concentration was most pronounced when using Citroff1 S244A (Figure S12d) but less apparent with Citron1 (Figures 4b and S12c). These differences in biosensor response are most likely due to differences in Kd for Citron1 vs Citroff1 vs Citroff1 S244A (1100, 5, and 35 μM, respectively). Mechanistically, the transient citrate accumulation is to be expected upon ACLY inhibition, due to cessation of citrate to acetyl-CoA conversion. The subsequent sustained decrease in mitochondrial citrate concentration may be due to the suspended production of oxaloacetate catalyzed by ACLY and downstream malate catalyzed by malate dehydrogenase (46) in the cytosol. Since the mitochondrial CiC imports malate to the mitochondria in exchange for citrate export, (47,48) the reduced concentration of cytosolic malate could potentially suppress citrate export and therefore cause a decrease in the concentration of citrate in the cytosol. Parallel experiments with the control variants CitronRH and CitroffRH confirmed that the observed changes in fluorescence were induced by citrate concentration changes (Figure S12). Taken together, our results demonstrate that imaging of citrate concentration dynamics with Citron1 and Citroff1 provides a sensitive method for the detection of ACLY activity that could potentially be used in cell-based screens for ACLY inhibitors. (4)
To investigate changes in citrate concentration associated with pharmacological manipulation of its upstream metabolic regulator, mitochondrial pyruvate carrier (MPC), we applied MPC inhibitor UK-5099 to HeLa cells expressing the mitochondria-localized citrate biosensors. (49) We hypothesized that inhibition of MPC would block pyruvate uptake into mitochondria and lead to decreased flux of the TCA cycle and a lower citrate concentration. As shown in Figure 4c (blue trace), treatment with UK-5099 (1 mM stock in 10% DMSO; diluted 1:40 to give a final concentration of 25 μM) resulted in a transient 20% increase of Citron1 fluorescence, followed by a sustained decrease. Cells expressing the CitronRH control construct in mitochondria exhibited a smaller and slower transient increase in fluorescence and no sustained decrease (Figure 4c, gray trace). In parallel control experiments, we found that DMSO alone elicited even larger transient fluorescence increases for both Citron1 (Figure 4c, orange trace) and CitronRH in mitochondria (Figure 4c, green trace), possibly due to a pH increase related to DMSO-dependent modulation of mitochondrial membrane potential and structural integrity. (50,51) This result suggests that the initial increase observed for Citron1 is likely an experimental artifact. Nevertheless, the sustained decrease in Citron1 fluorescence upon treatment of UK-5099 (and not in any of the control experiments) is consistent with UK-5099-dependent inhibition of pyruvate uptake into mitochondria. This result suggests that Citron1 could be potentially used for indirect monitoring of pyruvate uptake and TCA cycle activity in mitochondria. This example also illustrates the importance of nonbinding control constructs to decouple true from artifactual fluorescence changes.

Concurrent Imaging of Citrate and ATP Dynamics in HeLa Cells

To explore the possibility of using the new citrate biosensors for multicolor and multiparameter metabolite imaging, we coexpressed green fluorescent Citron1 (Figure 4d) with the red fluorescent ATP biosensor, MalionR (Figure 4e), (44) both targeted to the mitochondria. Upon treatment of starved (glucose-free) HeLa cells with 20 mM glucose, we observed a change in MalionR fluorescence that was consistent with an increase in ATP concentration, as has been previously reported. (52) Similar to our previous results (Figure S11c), Citron1 again reported an initial small dip and then a large increase in mitochondrial citrate (Figure 4f). Overall, these results support the notion of tight coupling between ATP and citrate concentrations in mitochondria. (53)

Imaging Citrate in INS-1 Beta Cells

Citrate export from the mitochondria plays a critical role in regulation of insulin secretion from pancreatic beta cells. (8) To investigate the utility of the citrate biosensors in beta cells, we expressed Citron1 in INS-1 rat insulinoma cells (Figure 5a) and acquired in situ calibration curves (Figure 5b,c). Using the in situ calibration curves, we determined the concentration of citrate in the cytosol and mitochondria of INS-1 cells, conditioned in Krebs-Ringer buffer, to be 2.93 ± 0.09 and 2.81 ± 0.24 mM, respectively (Figure 5d). These concentration values are substantially higher than those of HeLa cells (0.15 ± 0.07 and 0.44 ± 0.13 mM). The higher concentrations of citrate are consistent with a higher flux through the TCA cycle, as further supported by the higher oxygen consumption rates (OCRs) of INS-1 cells compared to that of HeLa cells. (52) Addition of 20 mM glucose increased both cytosolic and mitochondrial citrate concentration to ∼25 mM (Figure 5d–f). This result is in agreement with a previous study, in which it was demonstrated that stimulatory glucose can result in an approximately an 8-fold increase in the concentration of citrate. (54)

Figure 5

Figure 5. Characterization of Citron1 in INS-1 cells. (a) Representative fluorescence images of INS-1 cells expressing Citron1 in the cytosol (left panel) and mitochondria (right panel). (b,c) In situ titration curve of Citron1 in the cytosol (b, n = 62) and mitochondria (c, n = 34). (d) Citrate concentration in the cytosol (gray) and mitochondria (green) in Krebs-Ringer buffer with or without 20 mM glucose/2 mM BTC treatment. Each quantification result is averaged from triplicates. (e,f) Glucose- and BTC-induced citrate changes in the cytosol (e, n = 49) and mitochondria (f, n = 14). (g,h) BTC-induced citrate changes in the cytosol (g, n = 7) and mitochondria (h, n = 10) in the absence of glucose. The results of quantification in (e–h) are summarized in (d). Error bars in (b–h) marks s.e.m.

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An inhibitor of mitochondrial CiC, 1,2,3-benzenetricarboxylate (BTC), suppresses glucose-stimulated insulin secretion by blocking the mitochondrial export of citrate. (8) We found that the citrate concentration in INS-1 cells treated with 2 mM BTC after glucose addition exhibited a pronounced decrease from ∼25 to ∼10 mM, followed by an increase to ∼18 mM (Figure 5d–f). Cells treated with 2 mM BTC in the absence of glucose decreased in citrate concentration from ∼3 to less than 2 mM (Figure 5d,g,h). The citrate concentration in the cytosol and mitochondria are found to be nearly identical and changed in parallel during our experiments. Imaging with the nonbinding variant CitronRH confirmed that the fluorescence changes are indeed attributable to changes in citrate concentration (Figure S13).

Discussion

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We have engineered two new high-performance citrate biosensors, Citron1 and Citroff1, through the use of semirational design and directed protein evolution. The initial prototype for both of these new biosensors was constructed by effectively inserting the CitA sensing domain into GFP at the uniquely permissive site in the vicinity of residues 146–147. Insertions at this site are generally well tolerated, and such hybrid proteins (that is, GFP with a conformationally response domain inserted at this site) can generally be optimized to produce high-performance constructs such as Ca2+ and K+ biosensors. (27,55) In such biosensors, a conformational change in the sensing domain (for example, as induced by binding to an analyte of interest), is communicated to the GFP chromophore environment in such a way that the fluorescence intensity is reversible modulated.
To develop biosensors with effective coupling between the sensing domain and the GFP chromophore environment, we first attempted to optimize the two linkers that connect the CitA domain to GFP. This effort resulted in two distinct variants: one with a direct fluorescence response and one with an inverse fluorescence response, to citrate binding. Further optimization by random mutagenesis and screening ultimately led to the Citron1 and Citroff1 biosensors, respectively. Variants of Citroff1 with Kd values ranging from 5 μM to ∼1 mM were engineered by introducing rationally designed mutations in the binding pocket or at previously reported positions. (39)
As a series, Citron1 and Citroff1 (and its affinity variants) exhibit substantially larger dynamic responses and much more sensitivity in the physiologically relevant range of citrate concentrations than the previously reported single-FP-based citrate biosensors CF98. Furthermore, the availability of citrate-insensitive but pH-sensitive variants provides researchers with an ideal experimental control to confirm that signal changes from the biosensor are attributable to citrate rather than other physiological changes such as in pH. Together, these advantages mean that the advent of Citron1 and Citroff1 represent a major advance for the detection of citrate concentration dynamics in live mammalian cells using fluorescence imaging. Notably, the CitAP domain of the thermophilic bacterium Geobacillus thermoleovorans CitA SHK has recently been shown to be amenable to “binding pocket grafting” to convert its binding specificity to that of close homologues including l-malate, phthalate, and ethylmalonate. (56) Potentially, similar binding pocket grafting could be used to create a series of new biosensors based on the Citron1 and Citroff1 templates.
We have demonstrated the utility of these new citrate biosensors for the quantitative determination of citrate steady-state concentrations in both the mitochondria and cytosol of HeLa and INS-1 cells. In addition, we have used these biosensors to follow dynamic changes in citrate concentration in cells induced by changes in glucose concentration in the media or by pharmacological treatment. These imaging results have generally reaffirmed what was already known, or expected, regarding citrate concentrations in these two cell types. For example, INS-1 cells were found to have substantially higher concentrations of citrate than HeLa cells, consistent with the higher metabolic rates of INS-1 cells. (52) However, these citrate indicators also enable intracellular citrate concentrations to be probed in ways that would be otherwise impractical or impossible. As one example, these indicators have enabled us to determine that, upon treatment of starved cells with glucose, the increase in cytosolic citrate concentration is delayed ∼50 s relative to the increase in mitochondrial citrate concentration. As another example, these indicators have enabled us to observe transient changes in citrate concentration that would likely escape notice using traditional biochemical approaches that lack high temporal precision. For example, treatment of cells with the ACLY inhibitor BMS 303141 caused a transient increase in citrate concentration followed by a sustained decrease in concentration. In summary, we expect these high-performance citrate biosensors will see widespread adoption for use in tracking the metabolism of various types of cells for biochemical and pharmacological studies conducted either in vitro and in vivo. Both particularly low and particularly high concentrations of citrate have been reported to be key characteristics of normal and diseased cell types and to play a role in a variety of fundamental processes. For example, citrate concentration in prostate cancer cells is substantially lower than in noncancerous cells, (57) and astrocytes produce and release large amounts of citrate into cerebrospinal fluid. (58) In the extracellular milieu, citrate might function as a chelator of free Ca2+ and Zn2+, altering the excitable state of neurons. (58) To provide insight into this hypothesis, the green citrate biosensors reported here could potentially be used simultaneously for multiparameter, multicolor imaging with red fluorescent Ca2+ and Zn2+ biosensors (32,40) in order to correlate changes in citrate concentration with changes in the concentration of these divalent cations. Finally, it has been found that early onset epilepsy in children is related to loss-of-function mutations in the Na+-coupled citrate transporter (SLC13A5) in neurons, but the mechanism remains unclear. (59−62) For these example biological problems, and many others not mentioned here, imaging citrate concentration changes using citrate biosensors will undoubtedly provide us with a deeper understanding of the underlying mechanism and further highlight citrate’s role as one of life’s most central metabolites.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acscentsci.0c00518.

  • Experimental methods, Tables S1–S3, Figures S1–S14, data availability statement, and supplementary references (PDF)

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Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

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  • Corresponding Authors
    • Yurong Wen - Department of Talent Highland, The First Affiliated Hospital, Xi’an Jiaotong University, Xi’an, Shaanxi 710061, China;  Email: [email protected]
    • Robert E. Campbell - Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada;  Department of Chemistry, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan;  Orcidhttp://orcid.org/0000-0003-0604-092X;  Email: [email protected]
  • Authors
    • Yufeng Zhao - Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada;  Orcidhttp://orcid.org/0000-0002-1788-7969
    • Yi Shen - Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada
  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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The authors thank the University of Alberta Molecular Biology Services Unit for technical support and Dr. Patrick E. McDonald and Dr. Wen-hong Li for helpful comments and providing INS-1 cells. MaLionR was a gift from Tetsuya Kitaguchi (Addgene plasmid # 113908). This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC; RGPIN 2018 04364) and the Canadian Institutes of Health Research (CIHR; FS 154310). Y.W. was supported by the National Natural Science Foundation of China (NO.31870132, NO.81741088). We thank the staff from the BL19U1 beamline for technical support during data collection at the National Center for Protein Sciences Shanghai (NCPSS) at the Shanghai Synchrotron Radiation Facility.

References

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    Scientific reports (2018), 8 (1), 286 ISSN:.
    The selective detection of citrate anions is essential for various biological functions in living systems. A quantitative assessment of citrate is required for the diagnosis of various diseases in the human body; however, it is extremely challenging to develop efficient fluorescence and color-detecting molecular probes for sensing citrate in water. Herein, we report a macrocycle-based dinuclear foldamer (1) assembled with eosin Y (EY) that has been studied for anion binding by fluorescence and colorimetric techniques in water at neutral pH. Results from the fluorescence titrations reveal that the 1·EY ensemble strongly binds citrate anions, showing remarkable selectivity over a wide range of inorganic and carboxylate anions. The addition of citrate anions to the 1·EY adduct led to a large fluorescence enhancement, displaying a detectable color change under both visible and UV light in water up to 2 μmol. The biocompatibility of 1·EY as an intracellular carrier in a biological system was evaluated on primary human foreskin fibroblast (HF) cells, showing an excellent cell viability. The strong binding properties of the ensemble allow it to be used as a highly sensitive, detective probe for biologically relevant citrate anions in various applications.
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  14. 14
    Akdeniz, A.; Caglayan, M. G.; Anzenbacher, P. A tri-serine tri-lactone scaffold for the quantification of citrate in urine. Chem. Commun. 2016, 52, 1827– 1830,  DOI: 10.1039/C5CC08759G
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    14
    A tri-serine tri-lactone scaffold for the quantification of citrate in urine
    Akdeniz, Ali; Caglayan, Mehmet Gokhan; Anzenbacher, Pavel
    Chemical Communications (Cambridge, United Kingdom) (2016), 52 (9), 1827-1830CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
    Tri-serine tri-lactone based C3 symmetry fluorescent sensors were synthesized. Citrate is shown to bind to sensors, while displaying an increase in fluorescence intensity for the sensor with thiourea and a quenching for the sensor with sulfonamide. Information-rich responses of the sensors enable the authors to discriminate structurally similar anions, including mono-, di- and tri-carboxylates with 100% correct classification. A simple two-sensor array enables the detn. of the concn. of citrate in urine without any sample prepn. with high accuracy (error < 2%).
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  15. 15
    Li, C.-Y. Colorimetric and fluorescent chemosensor for citrate based on a rhodamine and Pb2+ complex in aqueous solution. Anal. Chim. Acta 2013, 774, 79– 84,  DOI: 10.1016/j.aca.2013.02.040
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    15
    Colorimetric and fluorescent chemosensor for citrate based on a rhodamine and Pb2+ complex in aqueous solution
    Li, Chun-Yan; Zhou, Yu; Li, Yong-Fei; Kong, Xue-Fei; Zou, Chun-Xiang; Weng, Chao
    Analytica Chimica Acta (2013), 774 (), 79-84CODEN: ACACAM; ISSN:0003-2670. (Elsevier B.V.)
    In this paper we unveil a novel rhodamine compd. based fluorescent chemosensor (1-Pb2+) for colormetric and fluorescent detection of citrate in aq. soln. This is the first fluorescent chemosensor for citrate based on rhodamine compd. The comparison of this method with some other fluorescence methods for citrate indicates that the method can detect citrate in aq. soln. by both color changes and fluorescent changes with long emission wavelength. In the new developed sensing system, 1-Pb2+ is fluorescent due to Pb2+-induced fluorescence enhancement of 1. However, the addn. of citrate may release 1 into the soln. with quenching of fluorescence. The chemosensor can be applied to the quantification of citrate with a linear range covering from 1.0 × 10-7 to 5.0 × 10-5 M and a detection limit of 2.5 × 10-8 M. The expt. results show that the response behavior of 1-Pb2+ towards citrate is pH independent in medium condition (pH 6.0-8.0). Most importantly, the fluorescence changes of the chemosensor are remarkably specific for citrate in the presence of other anions (even those that exist in high concn.), which meet the selective requirements for practical application. Moreover, the response of the chemosensor toward citrate is fast (response time less than 1 min). In addn., the chemosensor has been used for detn. of citrate in urine samples with satisfactory results.
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  16. 16
    Zhuo, S.; Gong, J.; Zhang, P.; Zhu, C. High-throughput and rapid fluorescent visualization sensor of urinary citrate by CdTe quantum dots. Talanta 2015, 141, 21– 25,  DOI: 10.1016/j.talanta.2015.03.054
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    16
    High-throughput and rapid fluorescent visualization sensor of urinary citrate by CdTe quantum dots
    Zhuo, Shujuan; Gong, Jiajia; Zhang, Ping; Zhu, Changqing
    Talanta (2015), 141 (), 21-25CODEN: TLNTA2; ISSN:0039-9140. (Elsevier B.V.)
    In this paper, we have presented a novel CdTe quantum dots (QDs) based fluorescent sensor for visual and turn-on sensing of citrate in human urine samples. The europium ion (Eu3+) can lead to the fluorescence quenching of thioglycollic acid (TGA) modified CdTe QDs due to photoinduced electron transfer accompanied by the change of emission color from yellow to orange. Next, addn. of citrate breaks the preformed assembly because citrate can replace the CdTe QDs, based on the fact that the Eu3+ ion displays higher affinity with citrate than the CdTe QDs. Thus the photoinduced electron transfer is switched off, and the fluorescence emission of CdTe QDs is rapidly (within 5 min) recovered, simultaneously, the orange emission color restores to yellow. Such proposed strategy may conveniently discriminate the patient of renal stone from normal person by naked eyes. In addn. to visualization detection, the fluorescence responses can be used for well quantifying citrate in the range of 0.67-133 μM. So, the present, simple, low-cost and visualized citrate fluorescence sensor has great potential in the applications for earlier screening in clin. detection.
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  17. 17
    Hang, Y.; Wang, J.; Jiang, T.; Lu, N.; Hua, J. Diketopyrrolopyrrole-Based Ratiometric/Turn-on Fluorescent Chemosensors for Citrate Detection in the Near-Infrared Region by an Aggregation-Induced Emission Mechanism. Anal. Chem. 2016, 88, 1696– 1703,  DOI: 10.1021/acs.analchem.5b03715
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    17
    Diketopyrrolopyrrole-Based Ratiometric/Turn-on Fluorescent Chemosensors for Citrate Detection in the Near-Infrared Region by an Aggregation-Induced Emission Mechanism
    Hang, Yandi; Wang, Jian; Jiang, Tao; Lu, Niannian; Hua, Jianli
    Analytical Chemistry (Washington, DC, United States) (2016), 88 (3), 1696-1703CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    This work reports two new diketoprrrolopyrrole-based fluorescent chemosensors (DPP-Py1 and DPP-Py2) using sym. diamides as recognition groups for selective and fast detection of citrate in the near-IR region. To the authors' delight, DPP-Py1 is a ratiometric sensor, whereas DPP-Py2 is a turn-on fluorescent sensor. It is worth noting that DPP-Py1 has higher accuracy and sensitivity with a relatively lower detection limit (1.8 × 10-7 M) and better stability in different pH buffers than DPP-Py2. SEM, dynamic light scattering analyses, 1H NMR titrn., and 2-dimensional-NOESY NMR suggested that the fluorescence increment of the probes DPP-Py1 and DPP-Py2 for citrate could probably originate from aggregation-induced emission (AIE) from the complexation of the pyridinium-based sym. diamides, DPPs, with carboxyl anions of citrate. The authors' work may provide a simpler and faster means for qual. and quant. anal. of citrate through an AIE mechanism.
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  18. 18
    Greenwald, E. C.; Mehta, S.; Zhang, J. Genetically Encoded Fluorescent Biosensors Illuminate the Spatiotemporal Regulation of Signaling Networks. Chem. Rev. 2018, 118, 11707– 11794,  DOI: 10.1021/acs.chemrev.8b00333
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    18
    Genetically Encoded Fluorescent Biosensors Illuminate the Spatiotemporal Regulation of Signaling Networks
    Greenwald, Eric C.; Mehta, Sohum; Zhang, Jin
    Chemical Reviews (Washington, DC, United States) (2018), 118 (24), 11707-11794CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
    A review. Cellular signaling networks are the foundation which dets. the fate and function of cells as they respond to various cues and stimuli. The discovery of fluorescent proteins over 25 years ago enabled the development of a diverse array of genetically encodable fluorescent biosensors that are capable of measuring the spatiotemporal dynamics of signal transduction pathways in live cells. In an effort to encapsulate the breadth over which fluorescent biosensors have expanded, we endeavored to assemble a comprehensive list of published engineered biosensors and discuss many of the mol. designs utilized in their development. Then, we review how the high temporal and spatial resoln. afforded by fluorescent biosensors has aided our understanding of the spatiotemporal regulation of signaling networks at the cellular and subcellular level. Finally, we highlight some emerging areas of research in both biosensor design and application that are on the forefront of biosensor development.
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  19. 19
    Baird, G. S.; Zacharias, D. A.; Tsien, R. Y. Circular permutation and receptor insertion within green fluorescent proteins. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 11241– 11246,  DOI: 10.1073/pnas.96.20.11241
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    19
    Circular permutation and receptor insertion within green fluorescent proteins
    Baird, Geoffrey S.; Zacharias, David A.; Tsien, Roger Y.
    Proceedings of the National Academy of Sciences of the United States of America (1999), 96 (20), 11241-11246CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Many areas of biol. and biotechnol. have been revolutionized by the ability to label proteins genetically by fusion to the Aequorea green fluorescent protein (GFP). In previous fusions, the GFP has been treated as an indivisible entity, usually appended to the amino or carboxyl terminus of the host protein, occasionally inserted within the host sequence. The tightly interwoven, three-dimensional structure and intricate posttranslational self-modification required for chromophore formation would suggest that major rearrangements or insertions within GFP would prevent fluorescence. However, we now show that several rearrangements of GFPs, in which the amino and carboxyl portions are interchanged and rejoined with a short spacer connecting the original termini, still become fluorescent. These circular permutations have altered pKa values and orientations of the chromophore with respect to a fusion partner. Furthermore, certain locations within GFP tolerate insertion of entire proteins, and conformational changes in the insert can have profound effects on the fluorescence. For example, insertions of calmodulin or a zinc finger domain in place of Tyr-145 of a yellow mutant (enhanced yellow fluorescent protein) of GFP result in indicator proteins whose fluorescence can be enhanced severalfold upon metal binding. The calmodulin graft into enhanced yellow fluorescent protein can monitor cytosolic Ca2+ in single mammalian cells. The tolerance of GFPs for circular permutations and insertions shows the folding process is surprisingly robust and offers a new strategy for creating genetically encodable, physiol. indicators.
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  20. 20
    West, A. H.; Stock, A. M. Histidine kinases and response regulator proteins in two-component signaling systems. Trends Biochem. Sci. 2001, 26, 369– 376,  DOI: 10.1016/S0968-0004(01)01852-7
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    20
    Histidine kinases and response regulator proteins in two-component signaling systems
    West, A. H.; Stock, A. M.
    Trends in Biochemical Sciences (2001), 26 (6), 369-376CODEN: TBSCDB; ISSN:0968-0004. (Elsevier Science Ltd.)
    A review with 60 refs. Phosphotransfer-mediated signaling pathways allow cells to sense and respond to environmental stimuli. Autophosphorylating protein histidine kinases provide phosphoryl groups for response regulator proteins which, in turn, function as mol. switches that control diverse effector activities. Structural studies of proteins involved in 2-component signaling systems have revealed a modular architecture with versatile conserved domains that are readily adapted to the specific needs of individual systems.
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  21. 21
    Bhate, M. P.; Molnar, K. S.; Goulian, M.; DeGrado, W. F. Signal transduction in histidine kinases: insights from new structures. Structure 2015, 23, 981– 994,  DOI: 10.1016/j.str.2015.04.002
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    21
    Signal Transduction in Histidine Kinases: Insights from New Structures
    Bhate, Manasi P.; Molnar, Kathleen S.; Goulian, Mark; DeGrado, William F.
    Structure (Oxford, United Kingdom) (2015), 23 (6), 981-994CODEN: STRUE6; ISSN:0969-2126. (Elsevier Ltd.)
    A review. Histidine kinases (HKs) are major players in bacterial signaling. There has been an explosion of new HK crystal structures in the last 5 years. The authors globally analyze the structures of HKs to yield insights into the mechanisms by which signals are transmitted to and across protein structures in this family. The authors interpret known enzymol. data in the context of new structural data to show how asymmetry across the dimer interface is a key feature of signal transduction in HKs, and discuss how different HK domains undergo asym. to sym. transitions during signal transduction and catalysis. A thermodn. framework for signaling that encompasses these various properties is presented, and the consequences of weak thermodn. coupling are discussed. The synthesis of observations from enzymol., structural biol., protein engineering, and thermodn. paves the way for a deeper mol. understanding of HK signal transduction.
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    Salvi, M. Sensory domain contraction in histidine kinase CitA triggers transmembrane signaling in the membrane-bound sensor. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, 3115– 3120,  DOI: 10.1073/pnas.1620286114
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    22
    Sensory domain contraction in histidine kinase CitA triggers transmembrane signaling in the membrane-bound sensor
    Salvi, Michele; Schomburg, Benjamin; Giller, Karin; Graf, Sabrina; Unden, Gottfried; Becker, Stefan; Lange, Adam; Griesinger, Christian
    Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (12), 3115-3120CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Bacteria use membrane-integral sensor histidine kinases (HK) to perceive stimuli and transduce signals from the environment to the cytosol. Information on how the signal is transmitted across the membrane by HKs is still scarce. Here, by combining both liq.- and solid-state NMR, we demonstrated that structural rearrangements in the extracytoplasmic, citrate-sensing Per-Arnt-Sim (PAS) domain of HK CitA were identical for the isolated domain in soln. and in a longer construct contg. the membrane-embedded HK and lacking only the kinase core. We showed that upon citrate binding, the PAS domain contracted, resulting in a shortening of the C-terminal β-strand. We demonstrated that this contraction of the PAS domain, which is well characterized for the isolated domain, is the signal transmitted to the transmembrane (TM) helixes in a CitA construct in liposomes. Putting the extracytoplasmic PAS domain into context of the membrane-embedded CitA construct slows down citrate-binding kinetics by at least a factor of 60, confirming that TM helix motions are linked to the citrate-binding event. These results were confirmation of a hallmark of the HK signal transduction mechanism with at. resoln. on a full-length construct lacking only the kinase core domain.
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  23. 23
    Gushchin, I. Mechanism of transmembrane signaling by sensor histidine kinases. Science 2017, 356, eaah6345,  DOI: 10.1126/science.aah6345
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    Reinelt, S.; Hofmann, E.; Gerharz, T.; Bott, M.; Madden, D. R. The structure of the periplasmic ligand-binding domain of the sensor kinase CitA reveals the first extracellular PAS domain. J. Biol. Chem. 2003, 278, 39189– 39196,  DOI: 10.1074/jbc.M305864200
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    24
    The Structure of the Periplasmic Ligand-binding Domain of the Sensor Kinase CitA Reveals the First Extracellular PAS Domain
    Reinelt, Stefan; Hofmann, Eckhard; Gerharz, Tanja; Bott, Michael; Madden, Dean R.
    Journal of Biological Chemistry (2003), 278 (40), 39189-39196CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    The integral membrane sensor kinase CitA of Klebsiella pneumoniae is part of a two-component signal transduction system that regulates the transport and metab. of citrate in response to its environmental concn. Two-component systems are widely used by bacteria for such adaptive processes, but the stereochem. of periplasmic ligand binding and the mechanism of signal transduction across the membrane remain poorly understood. The crystal structure of the CitAP periplasmic sensor domain in complex with citrate reveals a PAS fold, a versatile ligand-binding structural motif that has not previously been obsd. outside the cytoplasm or implicated in the transduction of conformational signals across the membrane. Citrate is bound in a pocket that is shared among many PAS domains but that shows structural variation according to the nature of the bound ligand. In CitAP, some of the citrate contact residues are located in the final strand of the central β-sheet, which is connected to the C-terminal transmembrane helix. These secondary structure elements thus provide a potential conformational link between the periplasmic ligand binding site and the cytoplasmic signaling domains of the receptor.
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    Sevvana, M. A ligand-induced switch in the periplasmic domain of sensor histidine kinase CitA. J. Mol. Biol. 2008, 377, 512– 523,  DOI: 10.1016/j.jmb.2008.01.024
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    25
    A Ligand-Induced Switch in the Periplasmic Domain of Sensor Histidine Kinase CitA
    Sevvana, Madhumati; Vijayan, Vinesh; Zweckstetter, Markus; Reinelt, Stefan; Madden, Dean R.; Herbst-Irmer, Regine; Sheldrick, George M.; Bott, Michael; Griesinger, Christian; Becker, Stefan
    Journal of Molecular Biology (2008), 377 (2), 512-523CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
    Sensor histidine kinases of two-component signal-transduction systems are essential for bacteria to adapt to variable environmental conditions. However, despite their prevalence, it is not well understood how extracellular signals such as ligand binding regulate the activity of these sensor kinases. CitA is the sensor histidine kinase in Klebsiella pneumoniae that regulates the transport and anaerobic metab. of citrate in response to its extracellular concn. We report here the X-ray structures of the periplasmic sensor domain of CitA in the citrate-free and citrate-bound states. A comparison of the two structures shows that ligand binding causes a considerable contraction of the sensor domain. This contraction may represent the mol. switch that activates transmembrane signaling in the receptor.
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    Stock, A. M.; Robinson, V. L.; Goudreau, P. N. Two-component signal transduction. Annu. Rev. Biochem. 2000, 69, 183– 215,  DOI: 10.1146/annurev.biochem.69.1.183
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    Two-component signal transduction
    Stock, Ann M.; Robinson, Victoria L.; Goudreau, Paul N.
    Annual Review of Biochemistry (2000), 69 (), 183-215CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews Inc.)
    A review with 276 refs. Most prokaryotic signal-transduction systems and a few eukaryotic pathways use phosphotransfer schemes involving two conserved components, a histidine protein kinase and a response regulator protein. The histidine protein kinase, which is regulated by environmental stimuli, autophosphorylates at a histidine residue, creating a high-energy phosphoryl group that is subsequently transferred to an aspartate residue in the response regulator protein. Phosphorylation induces a conformational change in the regulatory domain that results in activation of an assocd. domain that effects the response. The basic scheme is highly adaptable, and numerous variations have provided optimization within specific signaling systems. The domains of two-component proteins are modular and can be integrated into proteins and pathways in a variety of ways, but the core structures and activities are maintained. Thus detailed analyses of a relatively small no. of representative proteins provide a foundation for understanding this large family of signaling proteins.
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    Qian, Y.; Rancic, V.; Wu, J.; Ballanyi, K.; Campbell, R. E. A Bioluminescent Ca2+ Indicator Based on a Topological Variant of GCaMP6s. ChemBioChem 2019, 20, 516– 520,  DOI: 10.1002/cbic.201800255
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    27
    A Bioluminescent Ca2+ Indicator Based on a Topological Variant of GCaMP6s
    Qian, Yong; Rancic, Vladimir; Wu, Jiahui; Ballanyi, Klaus; Campbell, Robert E.
    ChemBioChem (2019), 20 (4), 516-520CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
    There is no expanded citation for this reference.
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    Honda, Y.; Kirimura, K. Generation of circularly permuted fluorescent-protein-based indicators for in vitro and in vivo detection of citrate. PLoS One 2013, 8, e64597,  DOI: 10.1371/journal.pone.0064597
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    28
    Generation of circularly permuted fluorescent-protein-based indicators for in vitro and in vivo detection of citrate
    Honda, Yuki; Kirimura, Kohtaro
    PLoS One (2013), 8 (5), e64597CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    Indicators for citrate, particularly those applicable to its in vivo detection and quantitation, have attracted much interest in both biochem. studies and industrial applications since citrate is a key metabolic intermediate playing important roles in living cells. We generated novel fluorescence indicators for citrate by fusing the circularly permuted fluorescent protein (cpFP) and the periplasmic domain of the bacterial histidine kinase CitA, which can bind to citrate with high specificity. The ratiometric fluorescent signal change was obsd. with one of these cpFP-based indicators, named CF98: upon addn. of citrate, the excitation peak at 504 nm increased proportionally to the decrease in the peak at 413 nm, suitable for build-in quant. estn. of the binding compd. We confirmed that CF98 can be used for detecting citrate in vitro at millimolar levels in the range of 0.1 to 50 mM with high selectivity; even in the presence of other org. acids such as isocitrate and malate, the fluorescence intensity of CF98 remains unaffected. We finally demonstrated the in vivo applicability of CF98 to estn. of the intracellular citrate concn. in Escherichia coli co-expressing the genes encoding CF98 and the citrate carrier CitT. The novel indicator CF98 can be a specific and simple detection tool for citrate in vitro and a non-invasive tool for real-time estn. of intracellular concns. of the compd. in vivo.
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    Ewald, J. C.; Reich, S.; Baumann, S.; Frommer, W. B.; Zamboni, N. Engineering genetically encoded nanosensors for real-time in vivo measurements of citrate concentrations. PLoS One 2011, 6, e28245,  DOI: 10.1371/journal.pone.0028245
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    29
    Engineering genetically encoded nanosensors for real-time in vivo measurements of citrate concentrations
    Ewald, Jennifer C.; Reich, Sabrina; Baumann, Stephan; Frommer, Wolf B.; Zamboni, Nicola
    PLoS One (2011), 6 (12), e28245CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    Citrate is an intermediate in catabolic as well as biosynthetic pathways and is an important regulatory mol. in the control of glycolysis and lipid metab. Mass spectrometric and NMR based metabolomics allow measuring citrate concns., but only with limited spatial and temporal resoln. Methods are so far lacking to monitor citrate levels in real-time in-vivo. Here, we present a series of genetically encoded citrate sensors based on Forster resonance energy transfer (FRET). We screened databases for citrate-binding proteins and tested three candidates in vitro. The citrate binding domain of the Klebsiella pneumoniae histidine sensor kinase CitA, inserted between the FRET pair Venus/CFP, yielded a sensor highly specific for citrate. We optimized the peptide linkers to achieve maximal FRET change upon citrate binding. By modifying residues in the citrate binding pocket, we were able to construct seven sensors with different affinities spanning a concn. range of three orders of magnitude without losing specificity. In a first in vivo application we show that E. coli maintains the capacity to take up glucose or acetate within seconds even after long-term starvation.
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  30. 30
    Gregg, T. Obesity-dependent CDK1 signaling stimulates mitochondrial respiration at complex I in pancreatic β-cells. J. Biol. Chem. 2019, 294, 4656– 4666,  DOI: 10.1074/jbc.RA118.006085
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    30
    Obesity-dependent CDK1 signaling stimulates mitochondrial respiration at complex I in pancreatic β-cells
    Gregg, Trillian; Sdao, Sophia M.; Dhillon, Rashpal S.; Rensvold, Jarred W.; Lewandowski, Sophie L.; Pagliarini, David J.; Denu, John M.; Merrins, Matthew J.
    Journal of Biological Chemistry (2019), 294 (12), 4656-4666CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    β-Cell mitochondria play a central role in coupling glucose metab. with insulin secretion. Here, we identified a metabolic function of cyclin-dependent kinase 1 (CDK1)/cyclin B1, the activation of mitochondrial respiratory complex I, that is active in quiescent adult β-cells and hyperactive in β-cells from obese (ob/ob) mice. In WT islets, respirometry revealed that 65% of complex I flux and 49% of state 3 respiration is sensitive to CDK1 inhibition. Islets from ob/ob mice expressed more cyclin B1 and exhibited a higher sensitivity to CDK1 blockade, which reduced complex I flux by 76% and state 3 respiration by 79%. The ensuing redn. in mitochondrial NADH utilization, measured with two-photon NAD(P)H fluorescence lifetime imaging (FLIM), was matched in the cytosol by a lag in citrate cycling, as shown with a FRET reporter targeted to β-cells. Moreover, time-resolved measurements revealed that in ob/ob islets, where complex I flux dominates respiration, CDK1 inhibition is sufficient to restrict the duty cycle of ATP/ADP and calcium oscillations, the parameter that dynamically encodes β-cell glucose sensing. Direct complex I inhibition with rotenone mimicked the restrictive effects of CDK1 inhibition on mitochondrial respiration, NADH turnover, ATP/ADP, and calcium influx. These findings identify complex I as a crit. mediator of obesity assocd. metabolic remodeling in β-cells and implicate CDK1 as a regulator of complex I that enhances β-cell glucose sensing.
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    Zhao, Y. An expanded palette of genetically encoded Ca2+ indicators. Science 2011, 333, 1888– 1891,  DOI: 10.1126/science.1208592
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    31
    An Expanded Palette of Genetically Encoded Ca2+ Indicators
    Zhao, Yongxin; Araki, Satoko; Wu, Jiahui; Teramoto, Takayuki; Chang, Yu-Fen; Nakano, Masahiro; Abdelfattah, Ahmed S.; Fujiwara, Manabi; Ishihara, Takeshi; Nagai, Takeharu; Campbell, Robert E.
    Science (Washington, DC, United States) (2011), 333 (6051), 1888-1891CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    Engineered fluorescent protein (FP) chimeras that modulate their fluorescence in response to changes in calcium ion (Ca2+) concn. are powerful tools for visualizing intracellular signaling activity. However, despite a decade of availability, the palette of single FP-based Ca2+ indicators has remained limited to a single green hue. The authors have expanded this palette by developing blue, improved green, and red intensiometric indicators, as well as an emission ratiometric indicator with an 11,000% ratio change. This series enables improved single-color Ca2+ imaging in neurons and transgenic Caenorhabditis elegans. In HeLa cells, Ca2+ was imaged in three subcellular compartments, and, in conjunction with a cyan FP-yellow FP-based indicator, Ca2+ and ATP were simultaneously imaged. This palette of indicators paints the way to a colorful new era of Ca2+ imaging.
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    Chen, Z.; Ai, H.-W. Single Fluorescent Protein-Based Indicators for Zinc Ion (Zn2+). Anal. Chem. 2016, 88, 9029– 9036,  DOI: 10.1021/acs.analchem.6b01653
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    Single Fluorescent Protein-Based Indicators for Zinc Ion (Zn2+)
    Chen, Zhijie; Ai, Hui-wang
    Analytical Chemistry (Washington, DC, United States) (2016), 88 (18), 9029-9036CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Genetically encoded fluorescent Zn2+ indicators (GEZIs) are highly attractive research tools for studying Zn2+ homeostasis and signaling in mammalian cells. Most current GEZIs are based on Forster resonance energy transfer (FRET) between a select pair of fluorescent proteins (FPs) fused with Zn2+-binding motifs. One drawback of such FRET-based GEZIs is their broad spectral profile bandwidths, creating challenges when monitoring multiple targets or parameters. To address this issue, we have engineered a group of intensiometric GEZIs based on single teal and red FPs that can be utilized to monitor subcellular Zn2+ diffusion and glucose-induced Zn2+ secretion in pancreatic INS-1E β-cells. These GEZIs offer the simplicity of intensiometric measurements, compatibility in multicolor imaging, large dynamic ranges, and relatively small mol. sizes, making them valuable addns. to the mol. toolbox for imaging Zn2+.
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    Minckley, T. F. Sub-nanomolar sensitive GZnP3 reveals TRPML1-mediated neuronal Zn2+ signals. Nat. Commun. 2019, 10, 4806,  DOI: 10.1038/s41467-019-12761-x
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    Sub-nanomolar sensitive GZnP3 reveals TRPML1-mediated neuronal Zn(2+) signals
    Minckley Taylor F; Zhang Chen; Fudge Dylan H; Dischler Anna M; LeJeune Kate D; Qin Yan; Xu Haoxing
    Nature communications (2019), 10 (1), 4806 ISSN:.
    Although numerous fluorescent Zn(2+) sensors have been reported, it is unclear whether and how Zn(2+) can be released from the intracellular compartments into the cytosol due to a lack of probes that can detect physiological dynamics of cytosolic Zn(2+). Here, we create a genetically encoded sensor, GZnP3, which demonstrates unprecedented sensitivity for Zn(2+) at sub-nanomolar concentrations. Using GZnP3 as well as GZnP3-derived vesicular targeted probes, we provide the first direct evidence that Zn(2+) can be released from endolysosomal vesicles to the cytosol in primary hippocampal neurons through the TRPML1 channel. Such TRPML1-mediated Zn(2+) signals are distinct from Ca(2+) in that they are selectively present in neurons, sustain longer, and are significantly higher in neurites as compared to the soma. Together, our work not only creates highly sensitive probes for investigating sub-nanomolar Zn(2+) dynamics, but also reveals new pools of Zn(2+) signals that can play critical roles in neuronal function.
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    Akerboom, J. Optimization of a GCaMP Calcium Indicator for Neural Activity Imaging. J. Neurosci. 2012, 32, 13819– 13840,  DOI: 10.1523/JNEUROSCI.2601-12.2012
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    Optimization of a GCaMP calcium indicator for neural activity imaging
    Akerboom, Jasper; Chen, Tsai-Wen; Wardill, Trevor J.; Tian, Lin; Marvin, Jonathan S.; Mutlu, Sevinc; Calderon, Nicole Carreras; Esposti, Federico; Borghuis, Bart G.; Sun, Xiaonan Richard; Gordus, Andrew; Orger, Michael B.; Portugues, Ruben; Engert, Florian; Macklin, John J.; Filosa, Alessandro; Aggarwal, Aman; Kerr, Rex A.; Takagi, Ryousuke; Kracun, Sebastian; Shigetomi, Eiji; Khakh, Baljit S.; Baier, Herwig; Lagnado, Leon; Wang, Samuel S.-H.; Bargmann, Cornelia I.; Kimmel, Bruce E.; Jayaraman, Vivek; Svoboda, Karel; Kim, Douglas S.; Schreiter, Eric R.; Looger, Loren L.
    Journal of Neuroscience (2012), 32 (40), 13819-13840CODEN: JNRSDS; ISSN:0270-6474. (Society for Neuroscience)
    Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a no. of model organisms and can reliably detect three or more action potentials in short bursts in several systems in vivo. Through protein structure detn., targeted mutagenesis, high-throughput screening, and a battery of in vitro assays, we have increased the dynamic range of GCaMP3 by severalfold, creating a family of "GCaMP5" sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, and in vivo in Caenorhabditis chemosensory neurons, Drosophila larval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2- to 3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combining in vivo imaging with electrophysiol. we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3. GCaMP5 allows more sensitive detection of neural activity in vivo and may find widespread applications for cellular imaging in general.
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    Dana, H. High-performance calcium sensors for imaging activity in neuronal populations and microcompartments. Nat. Methods 2019, 16, 649– 657,  DOI: 10.1038/s41592-019-0435-6
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    High-performance calcium sensors for imaging activity in neuronal populations and microcompartments
    Dana, Hod; Sun, Yi; Mohar, Boaz; Hulse, Brad K.; Kerlin, Aaron M.; Hasseman, Jeremy P.; Tsegaye, Getahun; Tsang, Arthur; Wong, Allan; Patel, Ronak; Macklin, John J.; Chen, Yang; Konnerth, Arthur; Jayaraman, Vivek; Looger, Loren L.; Schreiter, Eric R.; Svoboda, Karel; Kim, Douglas S.
    Nature Methods (2019), 16 (7), 649-657CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)
    Calcium imaging with genetically encoded calcium indicators (GECIs) is routinely used to measure neural activity in intact nervous systems. GECIs are frequently used in one of two different modes: to track activity in large populations of neuronal cell bodies, or to follow dynamics in subcellular compartments such as axons, dendrites and individual synaptic compartments. Despite major advances, calcium imaging is still limited by the biophys. properties of existing GECIs, including affinity, signal-to-noise ratio, rise and decay kinetics and dynamic range. Using structure-guided mutagenesis and neuron-based screening, we optimized the green fluorescent protein-based GECI GCaMP6 for different modes of in vivo imaging. The resulting jGCaMP7 sensors provide improved detection of individual spikes (jGCaMP7s,f), imaging in neurites and neuropil (jGCaMP7b), and may allow tracking larger populations of neurons using two-photon (jGCaMP7s,f) or wide-field (jGCaMP7c) imaging.
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    Cranfill, P. J. Quantitative assessment of fluorescent proteins. Nat. Methods 2016, 13, 557– 562,  DOI: 10.1038/nmeth.3891
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    Quantitative assessment of fluorescent proteins
    Cranfill, Paula J.; Sell, Brittney R.; Baird, Michelle A.; Allen, John R.; Lavagnino, Zeno; Martijn de Gruiter, H.; Kremers, Gert-Jan; Davidson, Michael W.; Ustione, Alessandro; Piston, David W.
    Nature Methods (2016), 13 (7), 557-562CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
    The advent of fluorescent proteins (FPs) for genetic labeling of mols. and cells has revolutionized fluorescence microscopy. Genetic manipulations have created a vast array of bright and stable FPs spanning blue to red spectral regions. Common to autofluorescent FPs is their tight β-barrel structure, which provides the rigidity and chem. environment needed for effectual fluorescence. Despite the common structure, each FP has unique properties. Thus, there is no single 'best' FP for every circumstance, and each FP has advantages and disadvantages. To guide decisions about which FP is right for a given application, we have quant. characterized the brightness, photostability, pH stability and monomeric properties of more than 40 FPs to enable straightforward and direct comparison between them. We focus on popular and/or top-performing FPs in each spectral region.
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    Chen, T.-W. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 2013, 499, 295– 300,  DOI: 10.1038/nature12354
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    Ultrasensitive fluorescent proteins for imaging neuronal activity
    Chen, Tsai-Wen; Wardill, Trevor J.; Sun, Yi; Pulver, Stefan R.; Renninger, Sabine L.; Baohan, Amy; Schreiter, Eric R.; Kerr, Rex A.; Orger, Michael B.; Jayaraman, Vivek; Looger, Loren L.; Svoboda, Karel; Kim, Douglas S.
    Nature (London, United Kingdom) (2013), 499 (7458), 295-300CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    Fluorescent calcium sensors are widely used to image neural activity. Using structure-based mutagenesis and neuron-based screening, we developed a family of ultrasensitive protein calcium sensors (GCaMP6) that outperformed other sensors in cultured neurons and in zebrafish, flies and mice in vivo. In layer 2/3 pyramidal neurons of the mouse visual cortex, GCaMP6 reliably detected single action potentials in neuronal somata and orientation-tuned synaptic calcium transients in individual dendritic spines. The orientation tuning of structurally persistent spines was largely stable over timescales of weeks. Orientation tuning averaged across spine populations predicted the tuning of their parent cell. Although the somata of GABAergic neurons showed little orientation tuning, their dendrites included highly tuned dendritic segments (5-40-μm long). GCaMP6 sensors thus provide new windows into the organization and dynamics of neural circuits over multiple spatial and temporal scales.
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    Gerharz, T.; Reinelt, S.; Kaspar, S.; Scapozza, L.; Bott, M. Identification of basic amino acid residues important for citrate binding by the periplasmic receptor domain of the sensor kinase CitA. Biochemistry 2003, 42, 5917– 5924,  DOI: 10.1021/bi0340595
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    Identification of Basic Amino Acid Residues Important for Citrate Binding by the Periplasmic Receptor Domain of the Sensor Kinase CitA
    Gerharz, Tanja; Reinelt, Stefan; Kaspar, Sibylle; Scapozza, Leonardo; Bott, Michael
    Biochemistry (2003), 42 (19), 5917-5924CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    The sensor kinase CitA and the response regulator CitB of Klebsiella pneumoniae form the paradigm of a subfamily of bacterial two-component regulatory systems that are capable of sensing tri- or dicarboxylates in the environment and then induce transporters for the uptake of these compds. We recently showed that the sepd. periplasmic domain of CitA, termed CitAP (encompasses residues 45-176 supplemented with an N-terminal methionine residue and a C-terminal hexahistidine tag), is a highly specific citrate receptor with a Kd of 5.5 μM at pH 7. To identify pos. charged residues involved in binding the citrate anion, each of the arginine, lysine, and histidine residues in CitAP was exchanged for alanine, and the resulting 17 muteins were analyzed by isothermal titrn. calorimetry (ITC). In 12 cases, the Kd for citrate was identical to that of wild-type CitAP or slightly changed (3.9-17.2 μM). In one case (R98A), the Kd was 6-fold decreased (0.8 μM), whereas in four cases (R66A, H69A, R107A, and K109A) the Kd was 38- to >300-fold increased (0.2 to >1 mM). The secondary structure of the latter five proteins in their apo-form as deduced from far-UV CD spectra did not differ from the apo-form of wild-type CitAP; however, all of them showed an increased thermostability. Citrate increased the m.p. (Tm) of wild-type CitAP and mutein R98A by 6.2 and 9.5°, resp., but had no effect on the Tm of the four proteins with disturbed binding. Three of the residues important for citrate binding (R66, H69, and R107) are highly conserved in the CitA subfamily of sensor kinases, indicating that they might be involved in ligand binding by many of these sensor kinases.
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    Ewald, J. C.; Reich, S.; Baumann, S.; Frommer, W. B.; Zamboni, N. Engineering genetically encoded nanosensors for real-time in vivo measurements of citrate concentrations. PLoS One 2011, 6, e28245,  DOI: 10.1371/journal.pone.0028245
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    Engineering genetically encoded nanosensors for real-time in vivo measurements of citrate concentrations
    Ewald, Jennifer C.; Reich, Sabrina; Baumann, Stephan; Frommer, Wolf B.; Zamboni, Nicola
    PLoS One (2011), 6 (12), e28245CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    Citrate is an intermediate in catabolic as well as biosynthetic pathways and is an important regulatory mol. in the control of glycolysis and lipid metab. Mass spectrometric and NMR based metabolomics allow measuring citrate concns., but only with limited spatial and temporal resoln. Methods are so far lacking to monitor citrate levels in real-time in-vivo. Here, we present a series of genetically encoded citrate sensors based on Forster resonance energy transfer (FRET). We screened databases for citrate-binding proteins and tested three candidates in vitro. The citrate binding domain of the Klebsiella pneumoniae histidine sensor kinase CitA, inserted between the FRET pair Venus/CFP, yielded a sensor highly specific for citrate. We optimized the peptide linkers to achieve maximal FRET change upon citrate binding. By modifying residues in the citrate binding pocket, we were able to construct seven sensors with different affinities spanning a concn. range of three orders of magnitude without losing specificity. In a first in vivo application we show that E. coli maintains the capacity to take up glucose or acetate within seconds even after long-term starvation.
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    Shen, Y. A genetically encoded Ca2+ indicator based on circularly permutated sea anemone red fluorescent protein eqFP578. BMC Biol. 2018, 16, 9,  DOI: 10.1186/s12915-018-0480-0
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    A genetically encoded Ca2+ indicator based on circularly permutated sea anemone red fluorescent protein eqFP578
    Shen, Yi; Dana, Hod; Abdelfattah, Ahmed S.; Patel, Ronak; Shea, Jamien; Molina, Rosana S.; Rawal, Bijal; Rancic, Vladimir; Chang, Yu-Fen; Wu, Lanshi; Chen, Yingche; Qian, Yong; Wiens, Matthew D.; Hambleton, Nathan; Ballanyi, Klaus; Hughes, Thomas E.; Drobizhev, Mikhail; Kim, Douglas S.; Koyama, Minoru; Schreiter, Eric R.; Campbell, Robert E.
    BMC Biology (2018), 16 (), 9/1-9/16CODEN: BBMIF7; ISSN:1741-7007. (BioMed Central Ltd.)
    Genetically encoded calcium ion (Ca2+) indicators (GECIs) are indispensable tools for measuring Ca2+ dynamics and neuronal activities in vitro and in vivo. Red fluorescent protein (RFP)-based GECIs have inherent advantages relative to green fluorescent protein-based GECIs due to the longer wavelength light used for excitation. Longer wavelength light is assocd. with decreased phototoxicity and deeper penetration through tissue. Red GECI can also enable multicolor visualization with blue- or cyan-excitable fluorophores. Here we report the development, structure, and validation of a new RFP-based GECI, K-GECO1, based on a circularly permutated RFP derived from the sea anemone Entacmaea quadricolor. We have characterized the performance of K-GECO1 in cultured HeLa cells, dissocd. neurons, stem-cell-derived cardiomyocytes, organotypic brain slices, zebrafish spinal cord in vivo, and mouse brain in vivo. K-GECO1 is the archetype of a new lineage of GECIs based on the RFP eqFP578 scaffold. It offers high sensitivity and fast kinetics, similar or better than those of current state-of-the-art indicators, with diminished lysosomal accumulation and minimal blue-light photoactivation. Further refinements of the K-GECO1 lineage could lead to further improved variants with overall performance that exceeds that of the most highly optimized red GECIs.
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    Wu, J. Red fluorescent genetically encoded Ca2+ indicators for use in mitochondria and endoplasmic reticulum. Biochem. J. 2014, 464, 13– 22,  DOI: 10.1042/BJ20140931
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    Red fluorescent genetically encoded Ca2+ indicators for use in mitochondria and endoplasmic reticulum
    Wu, Jiahui; Prole, David L.; Shen, Yi; Lin, Zhihong; Gnanasekaran, Aswini; Liu, Yingjie; Chen, Lidong; Zhou, Hang; Chen, S. R. Wayne; Usachev, Yuriy M.; Taylor, Colin W.; Campbell, Robert E.
    Biochemical Journal (2014), 464 (1), 13-22CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)
    Ca2+ is a key intermediary in a variety of signalling pathways and undergoes dynamic changes in its cytoplasmic concn. due to release from stores within the endoplasmic reticulum (ER) and influx from the extracellular environment. In addn. to regulating cytoplasmic Ca2+ signals, these responses also affect the concn. of Ca2+ within the ER and mitochondria. Single fluorescent protein-based Ca2+ indicators, such as the GCaMP series based on GFP, are powerful tools for imaging changes in the concn. of Ca2+ assocd. with intracellular signalling pathways. Most GCaMP-type indicators have dissocn. consts. (Kd) for Ca2+ in the high nanomolar to low micromolar range and are therefore optimal for measuring cytoplasmic [Ca2+], but poorly suited for use in mitochondria and ER where [Ca2+] can reach concns. of several hundred micromolar. We now report GCaMP-type low-affinity red fluorescent genetically encoded Ca2+ indicators for optical imaging (LAR-GECO), engineered to have Kd values of 24 μM (LAR-GECO1) and 12 μM (LAR-GECO1.2). We demonstrate that these indicators can be used to image mitochondrial and ER Ca2+ dynamics in several cell types. In addn., we perform two-color imaging of intracellular Ca2+ dynamics in cells expressing both cytoplasmic GCaMP and ER-targeted LAR-GECO1. The development of these low-affinity intensiometric red fluorescent Ca2+ indicators enables monitoring of ER and mitochondrial Ca2+ in combination with GFP-based reporters.
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    van de Wier, B. Elevated citrate levels in non-alcoholic fatty liver disease: the potential of citrate to promote radical production. FEBS Lett. 2013, 587, 2461– 2466,  DOI: 10.1016/j.febslet.2013.06.019
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    Elevated citrate levels in non-alcoholic fatty liver disease: The potential of citrate to promote radical production
    van de Wier, Bregje; Balk, Jiska M.; Haenen, Guido R. M. M.; Giamouridis, Dimosthenis; Bakker, Jaap A.; Bast, Bertine C.; den Hartog, Gertjan J. M.; Koek, Ger H.; Bast, Aalt
    FEBS Letters (2013), 587 (15), 2461-2466CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)
    Plasma citrate levels were found to be elevated in non-alc. fatty liver disease (NAFLD) patients. Cellular expts. indicated that increased citrate levels might originate from an excess of fatty acids. The impact of elevated citrate levels on oxidative stress was examd. It was found that citrate stimulated hydrogen peroxide induced intracellular oxidative stress in HepG2 cells. This was related to the promotion of iron mediated hydroxyl radical formation from hydrogen peroxide by citrate. The stimulating effect of citrate on the reactivity of iron promotes oxidative stress, a crucial process in the progression of NAFLD.
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    Costello, L. C.; Franklin, R. B. Plasma Citrate Homeostasis: How It Is Regulated; And Its Physiological and Clinical Implications. An Important, But Neglected, Relationship in Medicine. HSOA J. Hum Endocrinol 2016, 1, 5,  DOI: 10.24966/HE-9640/100005
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    Arai, S. RGB-Color Intensiometric Indicators to Visualize Spatiotemporal Dynamics of ATP in Single. Angew. Chem., Int. Ed. 2018, 57, 10873– 10878,  DOI: 10.1002/anie.201804304
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    RGB-Color Indicators to Visualize Spatiotemporal Dynamics of ATP in Single Cells
    Arai, Satoshi; Kriszt, Rokus; Harada, Kazuki; Looi, Liang-Sheng; Matsuda, Shogo; Wongso, Devina; Suo, Satoshi; Ishiura, Shoichi; Tseng, Yu-Hua; Raghunath, Michael; Ito, Toshiro; Tsuboi, Takashi; Kitaguchi, Tetsuya
    Angewandte Chemie, International Edition (2018), 57 (34), 10873-10878CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    ATP provides energy for the regulation of multiple cellular processes in living organisms. Capturing the spatiotemporal dynamics of ATP in single cells is fundamental to the understanding of the mechanisms underlying cellular energy metab. However, it has remained challenging to visualize the dynamics of ATP in and between distinct intracellular organelles and its interplay with other signaling mols. Using single fluorescent proteins, multicolor ATP indicators were developed, enabling the simultaneous visualization of subcellular ATP dynamics in the cytoplasm and mitochondria of cells derived from mammals, plants, and worms. Furthermore, in combination with addnl. fluorescent indicators, the dynamic interplay of ATP, cAMP, and Ca2+ could be visualized in activated brown adipocyte. This set of indicator tools will facilitate future research into energy metab.
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    Li, J. J. 2-hydroxy-N-arylbenzenesulfonamides as ATP-citrate lyase inhibitors. Bioorg. Med. Chem. Lett. 2007, 17, 3208– 3211,  DOI: 10.1016/j.bmcl.2007.03.017
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    2-Hydroxy-N-arylbenzenesulfonamides as ATP-citrate lyase inhibitors
    Li, James J.; Wang, Haixia; Tino, Joseph A.; Robl, Jeffrey A.; Herpin, Timothy F.; Lawrence, R. Michael; Biller, Scott; Jamil, Haris; Ponticiello, Randy; Chen, Luping; Chu, Ching-hsuen; Flynn, Neil; Cheng, Dong; Zhao, Rulin; Chen, Bangchi; Schnur, Dora; Obermeier, Mary T.; Sasseville, Vito; Padmanabha, Ramesh; Pike, Kristen; Harrity, Thomas
    Bioorganic & Medicinal Chemistry Letters (2007), 17 (11), 3208-3211CODEN: BMCLE8; ISSN:0960-894X. (Elsevier Ltd.)
    A novel series of 2-hydroxy-N-arylbenzenesulfonamides were identified to be ATP-citrate lyase (ACL) inhibitors with compd. 9 displaying potent in vitro activity (IC50 = 0.13 μM). Chronic oral dosing of compd. 9 in high-fat fed mice lowered plasma cholesterol, triglyceride, and glucose, as well as inhibited wt. gain.
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    Malate dehydrogenases - structure and function
    Minarik, P.; Tomaskova, N.; Kollarova, M.; Antalik, M.
    General Physiology and Biophysics (2002), 21 (3), 257-265CODEN: GPBIE2; ISSN:0231-5882. (Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences)
    A review with 33 refs. Malate dehydrogenase (MDH; EC 1.1.1.37) catalyzes the NAD/NADH-dependent interconversion of malate and oxalacetate. This reaction plays a key part in the malate/aspartate shuttle across the mitochondrial membrane, and in the tricarboxylic acid cycle within the mitochondrial matrix. MDHs are homodimeric mols. in most organisms, including all eukaryotes and the most bacterial species. The enzymes share a common catalytic mechanism and their kinetic properties are similar, which demonstrates a high degree of structural similarity. The 3-dimensional structures and elements essential for catalysis are conserved between mitochondrial and cytoplasmic forms of MDH in eukaryotic cells even though these isoenzymes are only marginally related at the level of primary structure.
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    Bisaccia, F.; De Palma, A.; Prezioso, G.; Palmieri, F. Kinetic characterization of the reconstituted tricarboxylate carrier from rat liver mitochondria. Biochim. Biophys. Acta, Bioenerg. 1990, 1019, 250– 256,  DOI: 10.1016/0005-2728(90)90201-E
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    Kinetic characterization of the reconstituted tricarboxylate carrier from rat liver mitochondria
    Bisaccia, F.; De Palma, A.; Prezioso, G.; Palmieri, F.
    Biochimica et Biophysica Acta, Bioenergetics (1990), 1019 (3), 250-6CODEN: BBBEB4; ISSN:0005-2728.
    The tricarboxylate carrier from rat liver mitochondria was purified by chromatog. on hydroxyapatite/celite and reconstituted in phospholipid vesicles by removing the detergent using hydrophobic chromatog. on Amberlite. Optimal transport activity was obtained by using a Triton X-114/phospholipid ratio of 0.8, 6% cardiolipin and 24 passages through a single Amberlite column. In the reconstituted system the incorporated tricarboxylate carrier catalyzed a first-order reaction of citrate/citrate or citrate/malate exchange. The activation energy of the exchange reaction was 70.1 kJ/mol. The rate of the exchange had a pH optimum between 7 and 8. The half-satn. const. was 0.13 mM for citrate and 0.76 mM for malate. All these properties were similar to those described for the tricarboxylate transport system in intact mitochondria. In proteoliposomes the max. exchange rate at 25° reached μmol/min per g protein. This value was independent of the type of substrate present at the external or internal space of the liposomes (citrate or malate).
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    Factors affecting the kinetics and equilibrium of exchange reactions of the citrate-transporting system of rat liver mitochondria
    Robinson, B. H.; Williams, G. R.; Halperin, Mitchell L.; Leznoff, C. C.
    Journal of Biological Chemistry (1971), 246 (17), 5280-6CODEN: JBCHA3; ISSN:0021-9258.
    Using an "inhibitor stop" technique with benzene 1,2,3-tricarboxylic acid as the inhibitor, the initial rates of exchange reactions catalyzed by the citrate-transporting system were measured and the dependence of the rates on temp. and the concn. of citrate (I), L-malate (II), and magnesium were studied. Both the rate and extent of exchange II with intramitochondrial [14C]-I were less than those of I with intramitochondrial [14C]-I. This difference in the rate and extent between the 2 exchanges was thought to be due to the fact that II-I exchange resulted in a disequil. of either charge or pH across the mitochondrial membrane. Malate2- probably exchanges for citrate2- rather than citrate3-, thereby setting up a pH differential which restricts further exchange.
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    Halestrap, A. P. The mechanism of the inhibition of the mitochondrial pyruvate transportater by alpha-cyanocinnamate derivatives. Biochem. J. 1976, 156, 181– 183,  DOI: 10.1042/bj1560181
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    The mechanism of the inhibition of the mitochondrial pyruvate transporter by α-cyanocinnamate derivatives
    Halestrap, Andrew P.
    Biochemical Journal (1976), 156 (1), 181-3CODEN: BIJOAK; ISSN:0264-6021.
    α-Cyano-4-hydroxycinnamate (I) (O.1mM) totally, rapidly, and reversibly inhibited mitochondrial pyruvate transport at 6 and 22°. I reacted reversibly with mercaptoethanol and cysteine to form addn. products, suggesting that I reacts with an essential thiol group on the pyruvate carrier.
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    Levenson, R.; Macara, I. G.; Smith, R. L.; Cantley, L.; Housman, D. Role of mitochondrial membrane potential in the regulation of murine erythroleukemia cell differentiation. Cell 1982, 28, 855– 863,  DOI: 10.1016/0092-8674(82)90064-2
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    Role of mitochondrial membrane potential in the regulation of murine erythroleukemia cell differentiation
    Levenson, Robert; Macara, Ian G.; Smith, Roderick L.; Cantley, Lewis; Housman, David
    Cell (Cambridge, MA, United States) (1982), 28 (4), 855-63CODEN: CELLB5; ISSN:0092-8674.
    The fluorescent membrane probe DiOC6 (3,3'-dihexyloxacarbocyanine iodide) was used to exam. the relation between murine erythroleukemia (MEL) cell mitochondria and the changes in cytoplasmic Ca levels occurring at the initiation of the commitment to differentiate. Fluorescence microscopy reveals the selective assocn. of DiOC6 with MEL cell mitochondria, where an enhanced fluorescence is obsd. Treatment of cells with DMSO or other inducers causes a decrease in mitochondria-assocd. fluorescence levels that occurs with the initiation of commitment. A decrease in DiOC6 fluorescence is caused by agents that reduce mitochondrial membrane potential, but is only slightly affected by agents that alter plasma membrane potential. Amiloride and EGTA, agents that prevent commitment and inhibit Ca uptake, also prevent the decrease in DiOC6 uptake caused by DMSO. The effect of DMSO on MEL cell mitochondria is mimicked by FCCP (carbonyl cyanine p-trifluoromethoxyphenyl hydrazone), a proton ionophore that dissipates mitochondrial membrane potential. FCCP also causes MEL cell mitochondria to release Ca into the cytoplasm. When MEL cells are treated with DMSO plus FCCP, commitment is initiated without the lag period obsd. when cells are treated with DMSO alone. These results are consistent with the hypothesis that mitochondrial transmembrane potential is important in the regulation of cytoplasmic Ca levels at the time of commitment of MEL cells to terminal differentiation.
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    Yuan, C. Dimethyl sulfoxide damages mitochondrial integrity and membrane potential in cultured astrocytes. PLoS One 2014, 9, e107447,  DOI: 10.1371/journal.pone.0107447
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    Dimethyl sulfoxide damages mitochondrial integrity and membrane potential in cultured astrocytes
    Yuan, Chan; Gao, Junying; Guo, Jichao; Bai, Lei; Marshall, Charles; Cai, Zhiyou; Wang, Linmei; Xiao, Ming
    PLoS One (2014), 9 (9), e107447/1-e107447/9, 9 pp.CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    DMSO (DMSO) is a polar org. solvent that is used to dissolve neuroprotective or neurotoxic agents in neuroscience research. However, DMSO itself also has pharmacol. and pathol. effects on the nervous system. Astrocytes play a central role in maintaining brain homeostasis, but the effect and mechanism of DMSO on astrocytes has not been studied. The present study showed that exposure of astrocyte cultures to 1% DMSO for 24 h did not significantly affect cell survival, but decreased cell viability and glial glutamate transporter expression, and caused mitochondrial swelling, membrane potential impairment and reactive oxygen species prodn., and subsequent cytochrome c release and caspase-3 activation. DMSO at concns. of 5% significantly inhibited cell variability and promoted apoptosis of astrocytes, accompanied with more severe mitochondrial damage. These results suggest that mitochondrial impairment is a primary event in DMSO-induced astrocyte toxicity. The potential cytotoxic effects on astrocytes need to be carefully considered during investigating neuroprotective or neurotoxic effects of hydrophobic agents dissolved by DMSO.
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    Depaoli, M. R. Real-Time Imaging of Mitochondrial ATP Dynamics Reveals the Metabolic Setting of Single Cells. Cell Rep. 2018, 25, 501– 512,  DOI: 10.1016/j.celrep.2018.09.027
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    Real-Time Imaging of Mitochondrial ATP Dynamics Reveals the Metabolic Setting of Single Cells
    Depaoli, Maria R.; Karsten, Felix; Madreiter-Sokolowski, Corina T.; Klec, Christiane; Gottschalk, Benjamin; Bischof, Helmut; Eroglu, Emrah; Waldeck-Weiermair, Markus; Simmen, Thomas; Graier, Wolfgang F.; Malli, Roland
    Cell Reports (2018), 25 (2), 501-512.e3CODEN: CREED8; ISSN:2211-1247. (Cell Press)
    Reprogramming of metabolic pathways dets. cell functions and fate. In our work, we have used organelle-targeted ATP biosensors to evaluate cellular metabolic settings with high resoln. in real time. Our data indicate that mitochondria dynamically supply ATP for glucose phosphorylation in a variety of cancer cell types. This hexokinase-dependent process seems to be reversed upon the removal of glucose or other hexose sugars. Our data further verify that mitochondria in cancer cells have increased ATP consumption. Similar subcellular ATP fluxes occurred in young mouse embryonic fibroblasts (MEFs). However, pancreatic beta cells, senescent MEFs, and MEFs lacking mitofusin 2 displayed completely different mitochondrial ATP dynamics, indicative of increased oxidative phosphorylation. Our findings add perspective to the variability of the cellular bioenergetics and demonstrate that live cell imaging of mitochondrial ATP dynamics is a powerful tool to evaluate metabolic flexibility and heterogeneity at a single-cell level.
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    MacDonald, M. J. Citrate oscillates in liver and pancreatic beta cell mitochondria and in INS-1 insulinoma cells. J. Biol. Chem. 2003, 278, 51894– 51900,  DOI: 10.1074/jbc.M309038200
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    Citrate Oscillates in Liver and Pancreatic Beta Cell Mitochondria and in INS-1 Insulinoma Cells
    MacDonald, Michael J.; Fahien, Leonard A.; Buss, Julian D.; Hasan, Noaman M.; Fallon, Michael J.; Kendrick, Mindy A.
    Journal of Biological Chemistry (2003), 278 (51), 51894-51900CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Oscillations in citric acid cycle intermediates have never been previously reported in any type of cell. Here we show that adding pyruvate to isolated mitochondria from liver, pancreatic islets, and INS-1 insulinoma cells or adding glucose to intact INS-1 cells causes sustained oscillations in citrate levels. Other citric acid cycle intermediates measured either did not oscillate or possibly oscillated with a low amplitude. In INS-1 mitochondria citrate oscillations are in phase with NAD(P) oscillations, and in intact INS-1 cells citrate oscillations parallel oscillations in ATP, suggesting that these processes are co-regulated. Oscillations have been extensively studied in the pancreatic beta cell where oscillations in glycolysis, NAD(P)/NAD(P)H and ATP/ADP ratios, plasma membrane elec. activity, calcium levels, and insulin secretion have been well documented. Because the mitochondrion is the major site of ATP synthesis and NADH oxidn. and the only site of citrate synthesis, mitochondria need to be synchronized for these factors to oscillate. In suspensions of mitochondria from various organs, most of the citrate is exported from the mitochondria. In addn., citrate inhibits its own synthesis. We propose that this enables citrate itself to act as one of the cellular messengers that synchronizes mitochondria. Furthermore, because citrate is a potent inhibitor of the glycolytic enzyme phosphofructokinase, the pacemaker of glycolytic oscillations, citrate may act as a metabolic link between mitochondria and glycolysis. Citrate oscillations may coordinate oscillations in mitochondrial energy prodn. and anaplerosis with glycolytic oscillations, which in the beta cell are known to parallel oscillations in insulin secretion.
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    Lorenz, M. A.; El Azzouny, M. A.; Kennedy, R. T.; Burant, C. F. Metabolome response to glucose in the β-cell line INS-1 832/13. J. Biol. Chem. 2013, 288, 10923– 10935,  DOI: 10.1074/jbc.M112.414961
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    Metabolome Response to Glucose in the β-Cell Line INS-1 832/13
    Lorenz, Matthew A.; El Azzouny, Mahmoud A.; Kennedy, Robert T.; Burant, Charles F.
    Journal of Biological Chemistry (2013), 288 (15), 10923-10935CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells is triggered by metab. of the sugar to increase ATP/ADP ratio that blocks the KATP channel leading to membrane depolarization and insulin exocytosis. Other metabolic pathways believed to augment insulin secretion have yet to be fully elucidated. To study metabolic changes during GSIS, liq. chromatog. with mass spectrometry was used to det. levels of 87 metabolites temporally following a change in glucose from 3 to 10 mm glucose and in response to increasing concns. of glucose in the INS-1 832/13 β-cell line. U-[13C]Glucose was used to probe flux in specific metabolic pathways. Results include a rapid increase in ATP/ADP, anaplerotic tricarboxylic acid cycle flux, and increases in the malonyl CoA pathway, support prevailing theories of GSIS. Novel findings include that aspartate used for anaplerosis does not derive from the glucose fuel added to stimulate insulin secretion, glucose flux into glycerol-3-phosphate, and esterification of long chain CoAs resulting in rapid consumption of long chain CoAs and de novo generation of phosphatidic acid and diacylglycerol. Further, novel metabolites with potential roles in GSIS such as 5-aminoimidazole-4-carboxamide ribotide (ZMP), GDP-mannose, and farnesyl pyrophosphate were found to be rapidly altered following glucose exposure.
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    Shen, Y. Genetically encoded fluorescent indicators for imaging intracellular potassium ion concentration. Commun. Biol. 2019, 2, 18,  DOI: 10.1038/s42003-018-0269-2
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    Genetically encoded fluorescent indicators for imaging intracellular potassium ion concentration
    Shen Yi; Miyashita Shin-Ichiro; Dong Min; Shen Yi; Wu Sheng-Yi; Aggarwal Abhi; Qian Yong; Campbell Robert E; Rancic Vladimir; Ballanyi Klaus; Campbell Robert E
    Communications biology (2019), 2 (), 18 ISSN:.
    Potassium ion (K(+)) homeostasis and dynamics play critical roles in biological activities. Here we describe three genetically encoded K(+) indicators. KIRIN1 (potassium (K) ion ratiometric indicator) and KIRIN1-GR are Forster resonance energy transfer (FRET)-based indicators with a bacterial K(+) binding protein (Kbp) inserting between the fluorescent protein FRET pairs mCerulean3/cp173Venus and Clover/mRuby2, respectively. GINKO1 (green indicator of K(+) for optical imaging) is a single fluorescent protein-based K(+) indicator constructed by insertion of Kbp into enhanced green fluorescent protein (EGFP). These indicators are suitable for detecting K(+) at physiologically relevant concentrations in vitro and in cells. KIRIN1 enabled imaging of cytosolic K(+) depletion in live cells and K(+) efflux and reuptake in cultured neurons. GINKO1, in conjunction with red fluorescent Ca(2+) indicator, enable dual-color imaging of K(+) and Ca(2+) dynamics in neurons and glial cells. These results demonstrate that KIRIN1 and GINKO1 are useful tools for imaging intracellular K(+) dynamics.
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    Cormann, K. U.; Baumgart, M.; Bott, M. Structure-Based Design of Versatile Biosensors for Small Molecules Based on the PAS Domain of a Thermophilic Histidine Kinase. ACS Synth. Biol. 2018, 7, 2888– 2897,  DOI: 10.1021/acssynbio.8b00348
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    Structure-Based Design of Versatile Biosensors for Small Molecules Based on the PAS Domain of a Thermophilic Histidine Kinase
    Cormann, Kai U.; Baumgart, Meike; Bott, Michael
    ACS Synthetic Biology (2018), 7 (12), 2888-2897CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
    The development of biosensors for in vitro quantification of small mols. such as metabolites or man-made chems. is still a major challenge. Here we show that engineered variants of the sensory PAS domain of the histidine kinase CitA of the thermophilic bacterium Geobacillus thermoleovorans represent promising alternatives to established biorecognition elements. By combining binding site grafting and rational design we constructed protein variants binding L-malate, ethylmalonate, or the arom. compd. phthalate instead of the native ligand citrate. Due to more favorable entropy contributions, the wild-type protein and its engineered variants exhibited increased (nano- to micromolar) affinities and improved enantioselectivity compared to CitA homologues of mesophilic organisms. Ligand binding was directly converted into an optical signal that was preserved after immobilization of the protein. A fluorescently labeled variant was used to quantify ethylmalonate, an urinary biomarker for ethylmalonic encephalopathy, in synthetic urine, thereby demonstrating the applicability of the sensor in complex samples.
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    Costello, L. C.; Franklin, R. B. Prostatic Fluid Electrolyte Composition for the Screening of Prostate Cancer: A Potential Solution to a Major Problem. Prostate Cancer Prostatic Dis. 2009, 12 (1), 17– 24,  DOI: 10.1038/pcan.2008.19
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    Prostatic fluid electrolyte composition for the screening of prostate cancer: a potential solution to a major problem
    Costello, L. C.; Franklin, R. B.
    Prostate Cancer and Prostatic Diseases (2009), 12 (1), 17-24CODEN: PCPDFW; ISSN:1365-7852. (Nature Publishing Group)
    A review. Early detection is the key to effective treatment of prostate cancer, and to the prevention of deaths due to progression to untreatable advanced stage cancer. Because of mitigating factors, esp. benign prostatic hyperplasia (BPH), that result in a low accuracy (about 60%) of prostate-specific antigen (PSA) testing, there is an urgent need for a more reliable biomarker for the identification of early stage through advanced stage prostate cancer and at-risk' individuals. To address this issue we propose that changes in prostatic fluid compn. could provide accurate and reliable biomarkers for the screening of prostate cancer. Most notable is the consistent and significant decrease in citrate and zinc that is assocd. with the development and progression of prostate cancer. In this review we provide the clin. and physiol. basis and the evidence in support of the utility of prostatic fluid anal. as an effective approach for screening/detection of prostate cancer, esp. early stage and at-risk' subjects. The problem of BPH interference that plagues PSA testing is eliminated in the potential prostatic fluid biomarkers. The potential development of rapid, simple, direct, accurate clin. tests provides addnl. advantageous conditions. Further exploration and development of citrate, zinc and other electrolytes as prostatic fluid biomarkers are urgently needed to address this crit. prostate cancer issue.
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    Westergaard, N.; Waagepetersen, H. S.; Belhage, B.; Schousboe, A. Citrate, a Ubiquitous Key Metabolite with Regulatory Function in the CNS. Neurochem. Res. 2017, 42, 1583– 1588,  DOI: 10.1007/s11064-016-2159-7
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    Citrate, a Ubiquitous Key Metabolite with Regulatory Function in the CNS
    Westergaard, Niels; Waagepetersen, Helle S.; Belhage, Bo; Schousboe, Arne
    Neurochemical Research (2017), 42 (6), 1583-1588CODEN: NEREDZ; ISSN:0364-3190. (Springer)
    Citrate is key constituent of the tricarboxylic acid (TCA) cycle, serves as substrate for fatty acid and sterol biosynthesis, and functions as a key regulator of intermediary energy metab. Ursula Sonnewald had initiated studies using for the first time both proton- and 13C-NMR to investigate metabolic processes in cultured neurons and astrocytes resulting in the important observation that citrate was specifically synthesized in and released from astrocytes in large amts. which is in keeping with the high concn. found in the CSF. The aim of this review is to highlight the possible roles of citrate in physiol. and pathophysiol. processes in the CNS. An interesting feature of citrate is its ability to chelate Ca2+, Mg2+ and Zn2+and thereby playing a pivotal role as an endogenous modulator of glutamate receptors and in particular the NMDA subtypes of these receptors in the CNS. Besides its presence in cerebrospinal fluid (CSF) citrate is also found in high amts. in prostate fluid reaching concns. as high as 180 mM and here Zn2+ seems also to play an important role, which makes prostate cells interesting for comparison of features of citrate and Zn2+ between these cells and cells in the CNS.
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    Thevenon, J. Mutations in SLC13A5 cause autosomal-recessive epileptic encephalopathy with seizure onset in the first days of life. Am. J. Hum. Genet. 2014, 95, 113– 120,  DOI: 10.1016/j.ajhg.2014.06.006
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    Mutations in SLC13A5 Cause Autosomal-Recessive Epileptic Encephalopathy with Seizure Onset in the First Days of Life
    Thevenon, Julien; Milh, Mathieu; Feillet, Francois; St-Onge, Judith; Duffourd, Yannis; Juge, Clara; Roubertie, Agathe; Heron, Delphine; Mignot, Cyril; Raffo, Emmanuel; Isidor, Bertrand; Wahlen, Sandra; Sanlaville, Damien; Villeneuve, Nathalie; Darmency-Stamboul, Veronique; Toutain, Annick; Lefebvre, Mathilde; Chouchane, Mondher; Huet, Frederic; Lafon, Arnaud; de Saint Martin, Anne; Lesca, Gaetan; El Chehadeh, Salima; Thauvin-Robinet, Christel; Masurel-Paulet, Alice; Odent, Sylvie; Villard, Laurent; Philippe, Christophe; Faivre, Laurence; Riviere, Jean-Baptiste
    American Journal of Human Genetics (2014), 95 (1), 113-120CODEN: AJHGAG; ISSN:0002-9297. (Cell Press)
    Epileptic encephalopathy (EE) refers to a clin. and genetically heterogeneous group of severe disorders characterized by seizures, abnormal interictal electro-encephalogram, psychomotor delay, and/or cognitive deterioration. The authors ascertained two multiplex families (including one consanguineous family) consistent with an autosomal-recessive inheritance pattern of EE. All seven affected individuals developed subclin. seizures as early as the first day of life, severe epileptic disease, and profound developmental delay with no facial dysmorphism. Given the similarity in clin. presentation in the two families, the authors hypothesized that the obsd. phenotype was due to mutations in the same gene, and the authors performed exome sequencing in three affected individuals. Anal. of rare variants in genes consistent with an autosomal-recessive mode of inheritance led to identification of mutations in SLC13A5, which encodes the cytoplasmic sodium-dependent citrate carrier, notably expressed in neurons. Disease assocn. was confirmed by cosegregation anal. in addnl. family members. Screening of 68 addnl. unrelated individuals with early-onset epileptic encephalopathy for SLC13A5 mutations led to identification of one addnl. subject with compd. heterozygous mutations of SLC13A5 and a similar clin. presentation as the index subjects. Mutations affected key residues for sodium binding, which is crit. for citrate transport. These findings underline the value of careful clin. characterization for genetic investigations in highly heterogeneous conditions such as EE and further highlight the role of citrate metab. in epilepsy.
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    Hardies, K. Recessive mutations in SLC13A5 result in a loss of citrate transport and cause neonatal epilepsy, developmental delay and teeth hypoplasia. Brain 2015, 138, 3238– 3250,  DOI: 10.1093/brain/awv263
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    59
    Recessive mutations in SLC13A5 result in a loss of citrate transport and cause neonatal epilepsy, developmental delay and teeth hypoplasia
    Hardies Katia; Deconinck Tine; Suls Arvid; de Kovel Carolien G F; Brilstra Eva; Braun Kees P J; van 't Slot Ruben; Koeleman Bobby P C; Weckhuysen Sarah; Asselbergh Bob; Geuens Thomas; Timmerman Vincent; Azmi Abdelkrim; May Patrick; Becker Felicitas; Schubert Julian; Weber Yvonne; Lerche Holger; Barisic Nina; Craiu Dana; Lal Dennis; Thiele Holger; Nurnberg Peter; Balling Rudi; Maudsley Stuart; Helbig Ingo; De Jonghe Peter
    Brain : a journal of neurology (2015), 138 (Pt 11), 3238-50 ISSN:.
    The epileptic encephalopathies are a clinically and aetiologically heterogeneous subgroup of epilepsy syndromes. Most epileptic encephalopathies have a genetic cause and patients are often found to carry a heterozygous de novo mutation in one of the genes associated with the disease entity. Occasionally recessive mutations are identified: a recent publication described a distinct neonatal epileptic encephalopathy (MIM 615905) caused by autosomal recessive mutations in the SLC13A5 gene. Here, we report eight additional patients belonging to four different families with autosomal recessive mutations in SLC13A5. SLC13A5 encodes a high affinity sodium-dependent citrate transporter, which is expressed in the brain. Neurons are considered incapable of de novo synthesis of tricarboxylic acid cycle intermediates; therefore they rely on the uptake of intermediates, such as citrate, to maintain their energy status and neurotransmitter production. The effect of all seven identified mutations (two premature stops and five amino acid substitutions) was studied in vitro, using immunocytochemistry, selective western blot and mass spectrometry. We hereby demonstrate that cells expressing mutant sodium-dependent citrate transporter have a complete loss of citrate uptake due to various cellular loss-of-function mechanisms. In addition, we provide independent proof of the involvement of autosomal recessive SLC13A5 mutations in the development of neonatal epileptic encephalopathies, and highlight teeth hypoplasia as a possible indicator for SLC13A5 screening. All three patients who tried the ketogenic diet responded well to this treatment, and future studies will allow us to ascertain whether this is a recurrent feature in this severe disorder.
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  61. 61
    Klotz, J.; Porter, B. E.; Colas, C.; Schlessinger, A.; Pajor, A. M. Mutations in the Na(+)/citrate cotransporter NaCT (SLC13A5) in pediatric patients with epilepsy and developmental delay. Mol. Med. 2016, 22, 310– 321,  DOI: 10.2119/molmed.2016.00077
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    60
    Mutations in the Na+/citrate cotransporter NaCT (SLC13A5) in pediatric patients with epilepsy and developmental delay
    Klotz, Jenna; Porter, Brenda E.; Colas, Claire; Schlessinger, Avner; Pajor, Ana M.
    Molecular Medicine (Manhasset, NY, United States) (2016), 22 (), 310-321CODEN: MOMEF3; ISSN:1528-3658. (Feinstein Institute for Medical Research)
    Mutations in the SLC13A5 gene that codes for the Na+/citrate cotransporter, NaCT, are assocd. with early onset epilepsy, developmental delay and tooth dysplasia in children. In this study, we identify addnl. SLC13A5 mutations in nine epilepsy patients from six families. To better characterize the syndrome, families with affected children answered questions about the scope of illness and the treatment strategies. Currently, there are no effective treatments, but some antiepileptic drugs targeting the γ-aminobutyric acid system reduce seizure frequency. Acetazolamide, a carbonic anhydrase inhibitor and atypical antiseizure medication, decreases seizures in four patients. In contrast to previous reports, the ketogenic diet and fasting resulted in worsening of symptoms. The effects of the mutations on NaCT transport function and protein expression were examd. by transient transfections of COS-7 cells. There was no transport activity from any of the mutant transporters, although some of the mutant transporter proteins were present on the plasma membrane. The structural model of NaCT suggests that these mutations can affect helix packing or substrate binding. We tested various treatments, including chem. chaperones and low temps., but none improved transport function in the NaCT mutants. Interestingly, coexpression of NaCT and the mutants results in decreased protein expression and activity of the wild-type transporter, indicating functional interaction. In conclusion, this study has identified addnl. SLC13A5 mutations in patients with chronic epilepsy starting in the neonatal period, with the mutations producing inactive Na+/citrate transporters.
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  62. 62
    Bhutia, Y. D.; Kopel, J. J.; Lawrence, J. J.; Neugebauer, V.; Ganapathy, V. Plasma Membrane Na+-Coupled Citrate Transporter (SLC13A5) and Neonatal Epileptic Encephalopathy. Molecules 2017, 22, 378,  DOI: 10.3390/molecules22030378
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    61
    Plasma membrane Na+-coupled citrate transporter (SLC13A5) and neonatal epileptic encephalopathy
    Bhutia, Yangzom D.; Kopel, Jonathan J.; Lawrence, John J.; Neugebauer, Volker; Ganapathy, Vadivel
    Molecules (2017), 22 (3), 378/1-378/15CODEN: MOLEFW; ISSN:1420-3049. (MDPI AG)
    SLC13A5 is a Na+-coupled transporter for citrate that is expressed in the plasma membrane of specific cell types in the liver, testis, and brain. It is an electrogenic transporter with a Na+:citrate3+ stoichiometry of 4:1. In humans, the Michaelis const. for SLC13A5 to transport citrate is ∼600 μM, which is physiol. relevant given that the normal concn. of citrate in plasma is in the range of 150-200 μM. Li+ stimulates the transport function of human SLC13A5 at concns. that are in the therapeutic range in patients on lithium therapy. Human SLC13A5 differs from rodent Slc13a5 in two important aspects: the affinity of the human transporter for citrate is ∼30-fold less than that of the rodent transporter, thus making human SLC13A5 a low-affinity/high-capacity transporter and the rodent Slc13a5 a high-affinity/low-capacity transporter. In the liver, SLC13A5 is expressed exclusively in the sinusoidal membrane of the hepatocytes, where it plays a role in the uptake of circulating citrate from the sinusoidal blood for metabolic use. In the testis, the transporter is expressed only in spermatozoa, which is also only in the mid piece where mitochondria are located; the likely function of the transporter in spermatozoa is to mediate the uptake of citrate present at high levels in the seminal fluid for subsequent metab. in the sperm mitochondria to generate biol. energy, thereby supporting sperm motility. In the brain, the transporter is expressed mostly in neurons. As astrocytes secrete citrate into extracellular medium, the potential function of SLC13A5 in neurons is to mediate the uptake of circulating citrate and astrocyte-released citrate for subsequent metab. Slc13a5-knockout mice have been generated; these mice do not have any overt phenotype but are resistant to exptl. induced metabolic syndrome. Recently however, loss-of-function mutations in human SLC13A5 have been found to cause severe epilepsy and encephalopathy early in life. Interestingly, there is no evidence of epilepsy or encephalopathy in Slc13a5-knockout mice, underlining the significant differences in clin. consequences of the loss of function of this transporter between humans and mice. The markedly different biochem. features of human SLC13A5 and mouse Slc13a5 likely contribute to these differences between humans and mice with regard to the metabolic consequences of the transporter deficiency. The exact mol. mechanisms by which the functional deficiency of the citrate transporter causes epilepsy and impairs neuronal development and function remain to be elucidated, but available literature implicate both dysfunction of GABA (γ-aminobutyrate) signaling and hyperfunction of NMDA (N-methyl-D-aspartate) receptor signaling. Plausible synaptic mechanisms linking loss-of-function mutations in SLC13A5 to epilepsy are discussed.
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  • Abstract

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    Figure 1

    Figure 1. Rationale for the design of a single-FP-based citrate biosensor. (a) Schematic representation of Klebsiella pneumoniae SHK CitA, which is composed of a periplasmic citrate-binding domain (CitAP; light blue, unbound; magenta, bound), connected to transmembrane helices at both its N- (transmembrane helix 1, TM1) and C-termini (transmembrane helix 2, TM2). TM2 is, in turn, connected to an intracellular HK catalytic domain. (26) (b) The structures of citrate-free CitAP (left; light blue; PDB ID 2V9A), (25) citrate-bound CitAP (right; magenta; PDB ID 2J80), (25) and a superposition of the citrate-free and -bound structures (middle). (c) We hypothesized that the piston-type conformational motion at the CitAP termini could be communicated to GFP to allosterically control the chromophore environment and its fluorescent brightness. In this way, the CitAP domain could serve as the basis of construction of a genetically encoded citrate biosensor. (d) To realize this biosensor design, we inserted CitAP into GFP by replacing the CaM-RS20 domain of ncpGCaMP6s (27) with CitAP.

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    Figure 2

    Figure 2. In vitro characterization of new citrate biosensors and crystal structure of Citron1. (a,b) Normalized excitation and emission spectra of purified Citron1 (a) and Citroff1 (b) in the presence and absence of 20 mM citrate. (c,d) In vitro citrate titration curves of purified Citron1 (c) and Citroff1 variants (d). Error bars represent standard deviation of triplicates. (e) Overall representation of the Citron1 structure with the position of all mutations indicated. The CitAP domain with citrate is colored in magenta, and the cpGFP domain is colored in green. The chromophore, citrate, and the Cα of Met145 (blue) and Asn278 (magenta) are represented as spheres. (f) Zoom-in view of the hydrogen bonding interaction between Asn278 and the chromophore. Additional residues in the vicinity of Asn278 of Citron1 are shown and labeled with magenta text, with the identity of the corresponding residue of Citroff1 labeled with black text.

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    Figure 3

    Figure 3. Expression of citrate biosensors in HeLa cell and permeabilization tests. (a) Representative fluorescence images of HeLa cells expressing Citron1 in the cytosol (left panel) and mitochondria (right panel). (b) Fluorescence intensity of Citron1 in the cytosol in response to treatment with digitonin and citrate (n = 78 cells). The analogous chart for mitochondrial Citron1 is provided as Figure S9a. (c,d) In situ titration of Citron1 (c) and Citroff1 (d) in the cytosol. Digitonin was added as indicated, and the concentration of citrate in the external buffer is indicated in millimolar (n = 128 for Citron1 and n = 26 for Citroff1). (e) In situ titration curve of Citron1 in the cytosol averaged from 128 cells in panel (c). At the highest concentrations (14.6 and 24.6 mM), the biosensor may not have fully equilibrated with the added citrate (see panel (c)), and the in situ Kd may therefore be underestimated. The analogous chart for Citroff1 is provided as Figure S9b. Error bars represent s.e.m. for panels (b–e). (f) Quantification of citrate concentration in the cytosol and mitochondria with or without 5.5 mM glucose in the buffer. Each dot is quantified using the average signal from tens of cells in a single experiment. Center box and whiskers represent the average and s.e.m. of the four data points, respectively.

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    Figure 4

    Figure 4. Use of Citron1 for imaging of citrate concentration changes induced by changes in glucose or pharmacologically in HeLa cells. (a) Glucose-induced citrate concentration changes detected with cytosolic (orange trace, n = 32) and mitochondrial (green trace, n = 28) Citron1. (b) BMS-303141-induced citrate concentration changes detected by cytosolic (n = 22) and mitochondrial (n = 24) Citron1. (c) UK-5099 (in DMSO)-induced citrate concentration changes detected by Citron1 (blue trace, n = 36) expressed in mitochondria. Control experiments include Citron1 + DMSO (orange trace, n = 23), CitronRH + UK-5099 (gray trace, n = 38), and CitronRH + DMSO (green trace, n = 23). The arrow indicates the addition of UK-5099 or DMSO solutions. Error bars in a–c represent s.e.m. (d–f) Dual color imaging of citrate and ATP concentration changes in mitochondria using Citron1 and MalionR. (44) (d,e) Representative fluorescence images of cells coexpressing Citron1 (d) and MalionR (e). (f) Representative glucose-induced citrate and ATP concentration changes reported by Citron1 (green traces) and MalionR (red traces).

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    Figure 5

    Figure 5. Characterization of Citron1 in INS-1 cells. (a) Representative fluorescence images of INS-1 cells expressing Citron1 in the cytosol (left panel) and mitochondria (right panel). (b,c) In situ titration curve of Citron1 in the cytosol (b, n = 62) and mitochondria (c, n = 34). (d) Citrate concentration in the cytosol (gray) and mitochondria (green) in Krebs-Ringer buffer with or without 20 mM glucose/2 mM BTC treatment. Each quantification result is averaged from triplicates. (e,f) Glucose- and BTC-induced citrate changes in the cytosol (e, n = 49) and mitochondria (f, n = 14). (g,h) BTC-induced citrate changes in the cytosol (g, n = 7) and mitochondria (h, n = 10) in the absence of glucose. The results of quantification in (e–h) are summarized in (d). Error bars in (b–h) marks s.e.m.

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  • References

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      A review. The Krebs cycle is an amphibolic pathway operating in the mitochondrial matrix of all eukaryotic organisms. In response to proinflammatory stimuli, macrophages and dendritic cells undergo profound metabolic remodelling to support the biosynthetic and bioenergetic requirements of the cell. Recently, it has been discovered that this metabolic shift also involves the rewiring of the Krebs cycle to regulate cellular metabolic flux and the accumulation of Krebs cycle intermediates, notably, citrate, succinate and fumarate. Interestingly, a new role for Krebs cycle intermediates as signaling mols. and immunomodulators that dictate the inflammatory response has begun to emerge. This review will discuss the latest developments in Krebs cycle rewiring and immune cell effector functions, with a particular focus on the regulation of cytokine prodn.
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    8. 8
      Joseph, J. W. The mitochondrial citrate/isocitrate carrier plays a regulatory role in glucose-stimulated insulin secretion. J. Biol. Chem. 2006, 281, 35624– 35632,  DOI: 10.1074/jbc.M602606200
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      8
      The mitochondrial citrate/isocitrate carrier plays a regulatory role in glucose-stimulated insulin secretion
      Joseph, Jamie W.; Jensen, Mette V.; Ilkayeva, Olga; Palmieri, Ferdinando; Alarcon, Cristina; Rhodes, Christopher J.; Newgard, Christopher B.
      Journal of Biological Chemistry (2006), 281 (47), 35624-35632CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Glucose-stimulated insulin secretion (GSIS) is mediated in part by glucose metab.-driven increases in ATP/ADP ratio, but byproducts of mitochondrial glucose metab. also play an important role. Here we investigate the role of the mitochondrial citrate/isocitrate carrier (CIC) in regulation of GSIS. Inhibition of CIC activity in INS-1-derived 832/13 cells or primary rat islets by the substrate analog 1,2,3-benzenetricarboxylate (BTC) resulted in potent inhibition of GSIS, involving both first and second phase secretion. A recombinant adenovirus contg. a CIC-specific siRNA (Ad-siCIC) dose-dependently reduced CIC expression in 832/13 cells and caused parallel inhibitory effects on citrate accumulation in the cytosol. Ad-siCIC treatment did not affect glucose utilization, glucose oxidn., or ATP/ADP ratio but did inhibit glucose incorporation into fatty acids and glucose-induced increases in NADPH/NADP+ ratio relative to cells treated with a control siRNA virus (Ad-siControl). Ad-siCIC also inhibited GSIS in 832/13 cells, whereas overexpression of CIC enhanced GSIS and raised cytosolic citrate levels. In normal rat islets, Ad-siCIC treatment also suppressed CIC mRNA levels and inhibited GSIS. We conclude that export of citrate and/or isocitrate from the mitochondria to the cytosol is an important step in control of GSIS.
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    9. 9
      Ferdaoussi, M. Isocitrate-to-SENP1 signaling amplifies insulin secretion and rescues dysfunctional β cells. J. Clin. Invest. 2015, 125, 3847– 3860,  DOI: 10.1172/JCI82498
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      9
      Isocitrate-to-SENP1 signaling amplifies insulin secretion and rescues dysfunctional β cells
      Ferdaoussi Mourad; Dai Xiaoqing; Jensen Mette V; Wang Runsheng; Peterson Brett S; Huang Chao; Ilkayeva Olga; Smith Nancy; Miller Nathanael; Hajmrle Catherine; Spigelman Aliya F; Wright Robert C; Plummer Gregory; Suzuki Kunimasa; Mackay James P; van de Bunt Martijn; Gloyn Anna L; Ryan Terence E; Norquay Lisa D; Brosnan M Julia; Trimmer Jeff K; Rolph Timothy P; Kibbey Richard G; Manning Fox Jocelyn E; Colmers William F; Shirihai Orian S; Neufer P Darrell; Yeh Edward T H; Newgard Christopher B; MacDonald Patrick E
      The Journal of clinical investigation (2015), 125 (10), 3847-60 ISSN:.
      Insulin secretion from β cells of the pancreatic islets of Langerhans controls metabolic homeostasis and is impaired in individuals with type 2 diabetes (T2D). Increases in blood glucose trigger insulin release by closing ATP-sensitive K+ channels, depolarizing β cells, and opening voltage-dependent Ca2+ channels to elicit insulin exocytosis. However, one or more additional pathway(s) amplify the secretory response, likely at the distal exocytotic site. The mitochondrial export of isocitrate and engagement with cytosolic isocitrate dehydrogenase (ICDc) may be one key pathway, but the mechanism linking this to insulin secretion and its role in T2D have not been defined. Here, we show that the ICDc-dependent generation of NADPH and subsequent glutathione (GSH) reduction contribute to the amplification of insulin exocytosis via sentrin/SUMO-specific protease-1 (SENP1). In human T2D and an in vitro model of human islet dysfunction, the glucose-dependent amplification of exocytosis was impaired and could be rescued by introduction of signaling intermediates from this pathway. Moreover, islet-specific Senp1 deletion in mice caused impaired glucose tolerance by reducing the amplification of insulin exocytosis. Together, our results identify a pathway that links glucose metabolism to the amplification of insulin secretion and demonstrate that restoration of this axis rescues β cell function in T2D.
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    10. 10
      Williams, N. C.; O’Neill, L. A. J. A Role for the Krebs Cycle Intermediate Citrate in Metabolic Reprogramming in Innate Immunity and Inflammation. Front. Immunol. 2018, 9, 141,  DOI: 10.3389/fimmu.2018.00141
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      10
      A role for the krebs cycle intermediate citrate in metabolic reprogramming in innate immunity and inflammation
      Williams, Niamh C.; O'Neill, Luke A. J.
      Frontiers in Immunology (2018), 9 (), 141/1-141/11CODEN: FIRMCW; ISSN:1664-3224. (Frontiers Media S.A.)
      Metab. in immune cells is no longer thought of as merely a process for ATP (ATP) prodn., biosynthesis, and catabolism. The reprogramming of metabolic pathways upon activation is also for the prodn. of metabolites that can act as immune signaling mols. Activated dendritic cells (DCs) and macrophages have an altered Krebs cycle, one consequence of which is the accumulation of both citrate and succinate. Citrate is exported from the mitochondria via the mitochondrial citrate carrier. Cytosolic metab. of citrate to acetyl-CoA (acetyl-CoA) is important for both fatty-acid synthesis and protein acetylation, both of which have been linked to macrophage and DC activation. Citrate-derived itaconate has a direct antibacterial effect and also has been shown to act as an anti-inflammatory agent, inhibiting succinate dehydrogenase. These findings identify citrate as an important metabolite for macrophage and DC effector function.
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    11. 11
      Metzger, A.; Anslyn, E. V. A Chemosensor for Citrate in Beverages. Angew. Chem., Int. Ed. 1998, 37, 649– 652,  DOI: 10.1002/(SICI)1521-3773(19980316)37:5<649::AID-ANIE649>3.0.CO;2-H
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      11
      A chemosensor for citrate in beverages
      Metzger, Axel; Anslyn, Eric V.
      Angewandte Chemie, International Edition (1998), 37 (5), 649-652CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH)
      N,N',N''-tris(4,5-dihydro-1H-imidazol-2-yl)-1,3,5-Benzenetrimethanamine (I) has been shown to serve as a receptor which can bind citrate. A competitive assay has been developed in which citrate can be measured by the change in absorbance of 5-carboxyfluorescein (II), when citrate is added to a soln. of chemosensor I and II, which as a fluorescent probe. This chemosensor system was applied to the detn. of citrate in soft drinks.
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    12. 12
      Ghosh, K.; Ranjan Sarkar, A. Pyridinium-based symmetrical diamides as chemosensors in visual sensing of citrate through indicator displacement assay (IDA) and gel formation. Org. Biomol. Chem. 2011, 9, 6551– 6558,  DOI: 10.1039/c1ob05707c
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      12
      Pyridinium-based symmetrical diamides as chemosensors in visual sensing of citrate through indicator displacement assay (IDA) and gel formation
      Ghosh, Kumaresh; Ranjan Sarkar, Avik
      Organic & Biomolecular Chemistry (2011), 9 (19), 6551-6558CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
      The design and synthesis of pyridinium-based sym. diamides I (R = Pr) and I (R = pyrene) along with their anion binding studies through indicator-diplacement assay' are reported. Both the chemosensors effectively respond in CH3CN-H2O (4:1 vol./vol.) at pH = 6.3 for the selective naked-eye detection of citrate. Addnl., chemosensor I (R = pyrene) (c 6.29 × 10-3 M) forms a stable gel only with citrate in CH3CN, which validates its visual sensing.
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    13. 13
      Rhaman, M. M. Highly selective and sensitive macrocycle-based dinuclear foldamer for fluorometric and colorimetric sensing of citrate in water. Sci. Rep. 2018, 8, 286,  DOI: 10.1038/s41598-017-18322-w
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      13
      Highly selective and sensitive macrocycle-based dinuclear foldamer for fluorometric and colorimetric sensing of citrate in water
      Rhaman Md Mhahabubur; Alamgir Azmain; Hossain Md Alamgir; Hasan Mohammad H; Tandon Ritesh; Xu Lihua; Wong Bryan M; Powell Douglas R
      Scientific reports (2018), 8 (1), 286 ISSN:.
      The selective detection of citrate anions is essential for various biological functions in living systems. A quantitative assessment of citrate is required for the diagnosis of various diseases in the human body; however, it is extremely challenging to develop efficient fluorescence and color-detecting molecular probes for sensing citrate in water. Herein, we report a macrocycle-based dinuclear foldamer (1) assembled with eosin Y (EY) that has been studied for anion binding by fluorescence and colorimetric techniques in water at neutral pH. Results from the fluorescence titrations reveal that the 1·EY ensemble strongly binds citrate anions, showing remarkable selectivity over a wide range of inorganic and carboxylate anions. The addition of citrate anions to the 1·EY adduct led to a large fluorescence enhancement, displaying a detectable color change under both visible and UV light in water up to 2 μmol. The biocompatibility of 1·EY as an intracellular carrier in a biological system was evaluated on primary human foreskin fibroblast (HF) cells, showing an excellent cell viability. The strong binding properties of the ensemble allow it to be used as a highly sensitive, detective probe for biologically relevant citrate anions in various applications.
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    14. 14
      Akdeniz, A.; Caglayan, M. G.; Anzenbacher, P. A tri-serine tri-lactone scaffold for the quantification of citrate in urine. Chem. Commun. 2016, 52, 1827– 1830,  DOI: 10.1039/C5CC08759G
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      14
      A tri-serine tri-lactone scaffold for the quantification of citrate in urine
      Akdeniz, Ali; Caglayan, Mehmet Gokhan; Anzenbacher, Pavel
      Chemical Communications (Cambridge, United Kingdom) (2016), 52 (9), 1827-1830CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      Tri-serine tri-lactone based C3 symmetry fluorescent sensors were synthesized. Citrate is shown to bind to sensors, while displaying an increase in fluorescence intensity for the sensor with thiourea and a quenching for the sensor with sulfonamide. Information-rich responses of the sensors enable the authors to discriminate structurally similar anions, including mono-, di- and tri-carboxylates with 100% correct classification. A simple two-sensor array enables the detn. of the concn. of citrate in urine without any sample prepn. with high accuracy (error < 2%).
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    15. 15
      Li, C.-Y. Colorimetric and fluorescent chemosensor for citrate based on a rhodamine and Pb2+ complex in aqueous solution. Anal. Chim. Acta 2013, 774, 79– 84,  DOI: 10.1016/j.aca.2013.02.040
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      15
      Colorimetric and fluorescent chemosensor for citrate based on a rhodamine and Pb2+ complex in aqueous solution
      Li, Chun-Yan; Zhou, Yu; Li, Yong-Fei; Kong, Xue-Fei; Zou, Chun-Xiang; Weng, Chao
      Analytica Chimica Acta (2013), 774 (), 79-84CODEN: ACACAM; ISSN:0003-2670. (Elsevier B.V.)
      In this paper we unveil a novel rhodamine compd. based fluorescent chemosensor (1-Pb2+) for colormetric and fluorescent detection of citrate in aq. soln. This is the first fluorescent chemosensor for citrate based on rhodamine compd. The comparison of this method with some other fluorescence methods for citrate indicates that the method can detect citrate in aq. soln. by both color changes and fluorescent changes with long emission wavelength. In the new developed sensing system, 1-Pb2+ is fluorescent due to Pb2+-induced fluorescence enhancement of 1. However, the addn. of citrate may release 1 into the soln. with quenching of fluorescence. The chemosensor can be applied to the quantification of citrate with a linear range covering from 1.0 × 10-7 to 5.0 × 10-5 M and a detection limit of 2.5 × 10-8 M. The expt. results show that the response behavior of 1-Pb2+ towards citrate is pH independent in medium condition (pH 6.0-8.0). Most importantly, the fluorescence changes of the chemosensor are remarkably specific for citrate in the presence of other anions (even those that exist in high concn.), which meet the selective requirements for practical application. Moreover, the response of the chemosensor toward citrate is fast (response time less than 1 min). In addn., the chemosensor has been used for detn. of citrate in urine samples with satisfactory results.
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    16. 16
      Zhuo, S.; Gong, J.; Zhang, P.; Zhu, C. High-throughput and rapid fluorescent visualization sensor of urinary citrate by CdTe quantum dots. Talanta 2015, 141, 21– 25,  DOI: 10.1016/j.talanta.2015.03.054
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      16
      High-throughput and rapid fluorescent visualization sensor of urinary citrate by CdTe quantum dots
      Zhuo, Shujuan; Gong, Jiajia; Zhang, Ping; Zhu, Changqing
      Talanta (2015), 141 (), 21-25CODEN: TLNTA2; ISSN:0039-9140. (Elsevier B.V.)
      In this paper, we have presented a novel CdTe quantum dots (QDs) based fluorescent sensor for visual and turn-on sensing of citrate in human urine samples. The europium ion (Eu3+) can lead to the fluorescence quenching of thioglycollic acid (TGA) modified CdTe QDs due to photoinduced electron transfer accompanied by the change of emission color from yellow to orange. Next, addn. of citrate breaks the preformed assembly because citrate can replace the CdTe QDs, based on the fact that the Eu3+ ion displays higher affinity with citrate than the CdTe QDs. Thus the photoinduced electron transfer is switched off, and the fluorescence emission of CdTe QDs is rapidly (within 5 min) recovered, simultaneously, the orange emission color restores to yellow. Such proposed strategy may conveniently discriminate the patient of renal stone from normal person by naked eyes. In addn. to visualization detection, the fluorescence responses can be used for well quantifying citrate in the range of 0.67-133 μM. So, the present, simple, low-cost and visualized citrate fluorescence sensor has great potential in the applications for earlier screening in clin. detection.
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    17. 17
      Hang, Y.; Wang, J.; Jiang, T.; Lu, N.; Hua, J. Diketopyrrolopyrrole-Based Ratiometric/Turn-on Fluorescent Chemosensors for Citrate Detection in the Near-Infrared Region by an Aggregation-Induced Emission Mechanism. Anal. Chem. 2016, 88, 1696– 1703,  DOI: 10.1021/acs.analchem.5b03715
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      17
      Diketopyrrolopyrrole-Based Ratiometric/Turn-on Fluorescent Chemosensors for Citrate Detection in the Near-Infrared Region by an Aggregation-Induced Emission Mechanism
      Hang, Yandi; Wang, Jian; Jiang, Tao; Lu, Niannian; Hua, Jianli
      Analytical Chemistry (Washington, DC, United States) (2016), 88 (3), 1696-1703CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      This work reports two new diketoprrrolopyrrole-based fluorescent chemosensors (DPP-Py1 and DPP-Py2) using sym. diamides as recognition groups for selective and fast detection of citrate in the near-IR region. To the authors' delight, DPP-Py1 is a ratiometric sensor, whereas DPP-Py2 is a turn-on fluorescent sensor. It is worth noting that DPP-Py1 has higher accuracy and sensitivity with a relatively lower detection limit (1.8 × 10-7 M) and better stability in different pH buffers than DPP-Py2. SEM, dynamic light scattering analyses, 1H NMR titrn., and 2-dimensional-NOESY NMR suggested that the fluorescence increment of the probes DPP-Py1 and DPP-Py2 for citrate could probably originate from aggregation-induced emission (AIE) from the complexation of the pyridinium-based sym. diamides, DPPs, with carboxyl anions of citrate. The authors' work may provide a simpler and faster means for qual. and quant. anal. of citrate through an AIE mechanism.
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    18. 18
      Greenwald, E. C.; Mehta, S.; Zhang, J. Genetically Encoded Fluorescent Biosensors Illuminate the Spatiotemporal Regulation of Signaling Networks. Chem. Rev. 2018, 118, 11707– 11794,  DOI: 10.1021/acs.chemrev.8b00333
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      18
      Genetically Encoded Fluorescent Biosensors Illuminate the Spatiotemporal Regulation of Signaling Networks
      Greenwald, Eric C.; Mehta, Sohum; Zhang, Jin
      Chemical Reviews (Washington, DC, United States) (2018), 118 (24), 11707-11794CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      A review. Cellular signaling networks are the foundation which dets. the fate and function of cells as they respond to various cues and stimuli. The discovery of fluorescent proteins over 25 years ago enabled the development of a diverse array of genetically encodable fluorescent biosensors that are capable of measuring the spatiotemporal dynamics of signal transduction pathways in live cells. In an effort to encapsulate the breadth over which fluorescent biosensors have expanded, we endeavored to assemble a comprehensive list of published engineered biosensors and discuss many of the mol. designs utilized in their development. Then, we review how the high temporal and spatial resoln. afforded by fluorescent biosensors has aided our understanding of the spatiotemporal regulation of signaling networks at the cellular and subcellular level. Finally, we highlight some emerging areas of research in both biosensor design and application that are on the forefront of biosensor development.
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      Baird, G. S.; Zacharias, D. A.; Tsien, R. Y. Circular permutation and receptor insertion within green fluorescent proteins. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 11241– 11246,  DOI: 10.1073/pnas.96.20.11241
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      19
      Circular permutation and receptor insertion within green fluorescent proteins
      Baird, Geoffrey S.; Zacharias, David A.; Tsien, Roger Y.
      Proceedings of the National Academy of Sciences of the United States of America (1999), 96 (20), 11241-11246CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Many areas of biol. and biotechnol. have been revolutionized by the ability to label proteins genetically by fusion to the Aequorea green fluorescent protein (GFP). In previous fusions, the GFP has been treated as an indivisible entity, usually appended to the amino or carboxyl terminus of the host protein, occasionally inserted within the host sequence. The tightly interwoven, three-dimensional structure and intricate posttranslational self-modification required for chromophore formation would suggest that major rearrangements or insertions within GFP would prevent fluorescence. However, we now show that several rearrangements of GFPs, in which the amino and carboxyl portions are interchanged and rejoined with a short spacer connecting the original termini, still become fluorescent. These circular permutations have altered pKa values and orientations of the chromophore with respect to a fusion partner. Furthermore, certain locations within GFP tolerate insertion of entire proteins, and conformational changes in the insert can have profound effects on the fluorescence. For example, insertions of calmodulin or a zinc finger domain in place of Tyr-145 of a yellow mutant (enhanced yellow fluorescent protein) of GFP result in indicator proteins whose fluorescence can be enhanced severalfold upon metal binding. The calmodulin graft into enhanced yellow fluorescent protein can monitor cytosolic Ca2+ in single mammalian cells. The tolerance of GFPs for circular permutations and insertions shows the folding process is surprisingly robust and offers a new strategy for creating genetically encodable, physiol. indicators.
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      West, A. H.; Stock, A. M. Histidine kinases and response regulator proteins in two-component signaling systems. Trends Biochem. Sci. 2001, 26, 369– 376,  DOI: 10.1016/S0968-0004(01)01852-7
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      20
      Histidine kinases and response regulator proteins in two-component signaling systems
      West, A. H.; Stock, A. M.
      Trends in Biochemical Sciences (2001), 26 (6), 369-376CODEN: TBSCDB; ISSN:0968-0004. (Elsevier Science Ltd.)
      A review with 60 refs. Phosphotransfer-mediated signaling pathways allow cells to sense and respond to environmental stimuli. Autophosphorylating protein histidine kinases provide phosphoryl groups for response regulator proteins which, in turn, function as mol. switches that control diverse effector activities. Structural studies of proteins involved in 2-component signaling systems have revealed a modular architecture with versatile conserved domains that are readily adapted to the specific needs of individual systems.
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    21. 21
      Bhate, M. P.; Molnar, K. S.; Goulian, M.; DeGrado, W. F. Signal transduction in histidine kinases: insights from new structures. Structure 2015, 23, 981– 994,  DOI: 10.1016/j.str.2015.04.002
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      Signal Transduction in Histidine Kinases: Insights from New Structures
      Bhate, Manasi P.; Molnar, Kathleen S.; Goulian, Mark; DeGrado, William F.
      Structure (Oxford, United Kingdom) (2015), 23 (6), 981-994CODEN: STRUE6; ISSN:0969-2126. (Elsevier Ltd.)
      A review. Histidine kinases (HKs) are major players in bacterial signaling. There has been an explosion of new HK crystal structures in the last 5 years. The authors globally analyze the structures of HKs to yield insights into the mechanisms by which signals are transmitted to and across protein structures in this family. The authors interpret known enzymol. data in the context of new structural data to show how asymmetry across the dimer interface is a key feature of signal transduction in HKs, and discuss how different HK domains undergo asym. to sym. transitions during signal transduction and catalysis. A thermodn. framework for signaling that encompasses these various properties is presented, and the consequences of weak thermodn. coupling are discussed. The synthesis of observations from enzymol., structural biol., protein engineering, and thermodn. paves the way for a deeper mol. understanding of HK signal transduction.
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      Salvi, M. Sensory domain contraction in histidine kinase CitA triggers transmembrane signaling in the membrane-bound sensor. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, 3115– 3120,  DOI: 10.1073/pnas.1620286114
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      22
      Sensory domain contraction in histidine kinase CitA triggers transmembrane signaling in the membrane-bound sensor
      Salvi, Michele; Schomburg, Benjamin; Giller, Karin; Graf, Sabrina; Unden, Gottfried; Becker, Stefan; Lange, Adam; Griesinger, Christian
      Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (12), 3115-3120CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Bacteria use membrane-integral sensor histidine kinases (HK) to perceive stimuli and transduce signals from the environment to the cytosol. Information on how the signal is transmitted across the membrane by HKs is still scarce. Here, by combining both liq.- and solid-state NMR, we demonstrated that structural rearrangements in the extracytoplasmic, citrate-sensing Per-Arnt-Sim (PAS) domain of HK CitA were identical for the isolated domain in soln. and in a longer construct contg. the membrane-embedded HK and lacking only the kinase core. We showed that upon citrate binding, the PAS domain contracted, resulting in a shortening of the C-terminal β-strand. We demonstrated that this contraction of the PAS domain, which is well characterized for the isolated domain, is the signal transmitted to the transmembrane (TM) helixes in a CitA construct in liposomes. Putting the extracytoplasmic PAS domain into context of the membrane-embedded CitA construct slows down citrate-binding kinetics by at least a factor of 60, confirming that TM helix motions are linked to the citrate-binding event. These results were confirmation of a hallmark of the HK signal transduction mechanism with at. resoln. on a full-length construct lacking only the kinase core domain.
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    23. 23
      Gushchin, I. Mechanism of transmembrane signaling by sensor histidine kinases. Science 2017, 356, eaah6345,  DOI: 10.1126/science.aah6345
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      Reinelt, S.; Hofmann, E.; Gerharz, T.; Bott, M.; Madden, D. R. The structure of the periplasmic ligand-binding domain of the sensor kinase CitA reveals the first extracellular PAS domain. J. Biol. Chem. 2003, 278, 39189– 39196,  DOI: 10.1074/jbc.M305864200
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      24
      The Structure of the Periplasmic Ligand-binding Domain of the Sensor Kinase CitA Reveals the First Extracellular PAS Domain
      Reinelt, Stefan; Hofmann, Eckhard; Gerharz, Tanja; Bott, Michael; Madden, Dean R.
      Journal of Biological Chemistry (2003), 278 (40), 39189-39196CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      The integral membrane sensor kinase CitA of Klebsiella pneumoniae is part of a two-component signal transduction system that regulates the transport and metab. of citrate in response to its environmental concn. Two-component systems are widely used by bacteria for such adaptive processes, but the stereochem. of periplasmic ligand binding and the mechanism of signal transduction across the membrane remain poorly understood. The crystal structure of the CitAP periplasmic sensor domain in complex with citrate reveals a PAS fold, a versatile ligand-binding structural motif that has not previously been obsd. outside the cytoplasm or implicated in the transduction of conformational signals across the membrane. Citrate is bound in a pocket that is shared among many PAS domains but that shows structural variation according to the nature of the bound ligand. In CitAP, some of the citrate contact residues are located in the final strand of the central β-sheet, which is connected to the C-terminal transmembrane helix. These secondary structure elements thus provide a potential conformational link between the periplasmic ligand binding site and the cytoplasmic signaling domains of the receptor.
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    25. 25
      Sevvana, M. A ligand-induced switch in the periplasmic domain of sensor histidine kinase CitA. J. Mol. Biol. 2008, 377, 512– 523,  DOI: 10.1016/j.jmb.2008.01.024
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      25
      A Ligand-Induced Switch in the Periplasmic Domain of Sensor Histidine Kinase CitA
      Sevvana, Madhumati; Vijayan, Vinesh; Zweckstetter, Markus; Reinelt, Stefan; Madden, Dean R.; Herbst-Irmer, Regine; Sheldrick, George M.; Bott, Michael; Griesinger, Christian; Becker, Stefan
      Journal of Molecular Biology (2008), 377 (2), 512-523CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
      Sensor histidine kinases of two-component signal-transduction systems are essential for bacteria to adapt to variable environmental conditions. However, despite their prevalence, it is not well understood how extracellular signals such as ligand binding regulate the activity of these sensor kinases. CitA is the sensor histidine kinase in Klebsiella pneumoniae that regulates the transport and anaerobic metab. of citrate in response to its extracellular concn. We report here the X-ray structures of the periplasmic sensor domain of CitA in the citrate-free and citrate-bound states. A comparison of the two structures shows that ligand binding causes a considerable contraction of the sensor domain. This contraction may represent the mol. switch that activates transmembrane signaling in the receptor.
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      Stock, A. M.; Robinson, V. L.; Goudreau, P. N. Two-component signal transduction. Annu. Rev. Biochem. 2000, 69, 183– 215,  DOI: 10.1146/annurev.biochem.69.1.183
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      Two-component signal transduction
      Stock, Ann M.; Robinson, Victoria L.; Goudreau, Paul N.
      Annual Review of Biochemistry (2000), 69 (), 183-215CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews Inc.)
      A review with 276 refs. Most prokaryotic signal-transduction systems and a few eukaryotic pathways use phosphotransfer schemes involving two conserved components, a histidine protein kinase and a response regulator protein. The histidine protein kinase, which is regulated by environmental stimuli, autophosphorylates at a histidine residue, creating a high-energy phosphoryl group that is subsequently transferred to an aspartate residue in the response regulator protein. Phosphorylation induces a conformational change in the regulatory domain that results in activation of an assocd. domain that effects the response. The basic scheme is highly adaptable, and numerous variations have provided optimization within specific signaling systems. The domains of two-component proteins are modular and can be integrated into proteins and pathways in a variety of ways, but the core structures and activities are maintained. Thus detailed analyses of a relatively small no. of representative proteins provide a foundation for understanding this large family of signaling proteins.
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      Qian, Y.; Rancic, V.; Wu, J.; Ballanyi, K.; Campbell, R. E. A Bioluminescent Ca2+ Indicator Based on a Topological Variant of GCaMP6s. ChemBioChem 2019, 20, 516– 520,  DOI: 10.1002/cbic.201800255
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      A Bioluminescent Ca2+ Indicator Based on a Topological Variant of GCaMP6s
      Qian, Yong; Rancic, Vladimir; Wu, Jiahui; Ballanyi, Klaus; Campbell, Robert E.
      ChemBioChem (2019), 20 (4), 516-520CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
      There is no expanded citation for this reference.
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      Honda, Y.; Kirimura, K. Generation of circularly permuted fluorescent-protein-based indicators for in vitro and in vivo detection of citrate. PLoS One 2013, 8, e64597,  DOI: 10.1371/journal.pone.0064597
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      Generation of circularly permuted fluorescent-protein-based indicators for in vitro and in vivo detection of citrate
      Honda, Yuki; Kirimura, Kohtaro
      PLoS One (2013), 8 (5), e64597CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
      Indicators for citrate, particularly those applicable to its in vivo detection and quantitation, have attracted much interest in both biochem. studies and industrial applications since citrate is a key metabolic intermediate playing important roles in living cells. We generated novel fluorescence indicators for citrate by fusing the circularly permuted fluorescent protein (cpFP) and the periplasmic domain of the bacterial histidine kinase CitA, which can bind to citrate with high specificity. The ratiometric fluorescent signal change was obsd. with one of these cpFP-based indicators, named CF98: upon addn. of citrate, the excitation peak at 504 nm increased proportionally to the decrease in the peak at 413 nm, suitable for build-in quant. estn. of the binding compd. We confirmed that CF98 can be used for detecting citrate in vitro at millimolar levels in the range of 0.1 to 50 mM with high selectivity; even in the presence of other org. acids such as isocitrate and malate, the fluorescence intensity of CF98 remains unaffected. We finally demonstrated the in vivo applicability of CF98 to estn. of the intracellular citrate concn. in Escherichia coli co-expressing the genes encoding CF98 and the citrate carrier CitT. The novel indicator CF98 can be a specific and simple detection tool for citrate in vitro and a non-invasive tool for real-time estn. of intracellular concns. of the compd. in vivo.
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    29. 29
      Ewald, J. C.; Reich, S.; Baumann, S.; Frommer, W. B.; Zamboni, N. Engineering genetically encoded nanosensors for real-time in vivo measurements of citrate concentrations. PLoS One 2011, 6, e28245,  DOI: 10.1371/journal.pone.0028245
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      29
      Engineering genetically encoded nanosensors for real-time in vivo measurements of citrate concentrations
      Ewald, Jennifer C.; Reich, Sabrina; Baumann, Stephan; Frommer, Wolf B.; Zamboni, Nicola
      PLoS One (2011), 6 (12), e28245CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
      Citrate is an intermediate in catabolic as well as biosynthetic pathways and is an important regulatory mol. in the control of glycolysis and lipid metab. Mass spectrometric and NMR based metabolomics allow measuring citrate concns., but only with limited spatial and temporal resoln. Methods are so far lacking to monitor citrate levels in real-time in-vivo. Here, we present a series of genetically encoded citrate sensors based on Forster resonance energy transfer (FRET). We screened databases for citrate-binding proteins and tested three candidates in vitro. The citrate binding domain of the Klebsiella pneumoniae histidine sensor kinase CitA, inserted between the FRET pair Venus/CFP, yielded a sensor highly specific for citrate. We optimized the peptide linkers to achieve maximal FRET change upon citrate binding. By modifying residues in the citrate binding pocket, we were able to construct seven sensors with different affinities spanning a concn. range of three orders of magnitude without losing specificity. In a first in vivo application we show that E. coli maintains the capacity to take up glucose or acetate within seconds even after long-term starvation.
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    30. 30
      Gregg, T. Obesity-dependent CDK1 signaling stimulates mitochondrial respiration at complex I in pancreatic β-cells. J. Biol. Chem. 2019, 294, 4656– 4666,  DOI: 10.1074/jbc.RA118.006085
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      Obesity-dependent CDK1 signaling stimulates mitochondrial respiration at complex I in pancreatic β-cells
      Gregg, Trillian; Sdao, Sophia M.; Dhillon, Rashpal S.; Rensvold, Jarred W.; Lewandowski, Sophie L.; Pagliarini, David J.; Denu, John M.; Merrins, Matthew J.
      Journal of Biological Chemistry (2019), 294 (12), 4656-4666CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      β-Cell mitochondria play a central role in coupling glucose metab. with insulin secretion. Here, we identified a metabolic function of cyclin-dependent kinase 1 (CDK1)/cyclin B1, the activation of mitochondrial respiratory complex I, that is active in quiescent adult β-cells and hyperactive in β-cells from obese (ob/ob) mice. In WT islets, respirometry revealed that 65% of complex I flux and 49% of state 3 respiration is sensitive to CDK1 inhibition. Islets from ob/ob mice expressed more cyclin B1 and exhibited a higher sensitivity to CDK1 blockade, which reduced complex I flux by 76% and state 3 respiration by 79%. The ensuing redn. in mitochondrial NADH utilization, measured with two-photon NAD(P)H fluorescence lifetime imaging (FLIM), was matched in the cytosol by a lag in citrate cycling, as shown with a FRET reporter targeted to β-cells. Moreover, time-resolved measurements revealed that in ob/ob islets, where complex I flux dominates respiration, CDK1 inhibition is sufficient to restrict the duty cycle of ATP/ADP and calcium oscillations, the parameter that dynamically encodes β-cell glucose sensing. Direct complex I inhibition with rotenone mimicked the restrictive effects of CDK1 inhibition on mitochondrial respiration, NADH turnover, ATP/ADP, and calcium influx. These findings identify complex I as a crit. mediator of obesity assocd. metabolic remodeling in β-cells and implicate CDK1 as a regulator of complex I that enhances β-cell glucose sensing.
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      Zhao, Y. An expanded palette of genetically encoded Ca2+ indicators. Science 2011, 333, 1888– 1891,  DOI: 10.1126/science.1208592
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      An Expanded Palette of Genetically Encoded Ca2+ Indicators
      Zhao, Yongxin; Araki, Satoko; Wu, Jiahui; Teramoto, Takayuki; Chang, Yu-Fen; Nakano, Masahiro; Abdelfattah, Ahmed S.; Fujiwara, Manabi; Ishihara, Takeshi; Nagai, Takeharu; Campbell, Robert E.
      Science (Washington, DC, United States) (2011), 333 (6051), 1888-1891CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      Engineered fluorescent protein (FP) chimeras that modulate their fluorescence in response to changes in calcium ion (Ca2+) concn. are powerful tools for visualizing intracellular signaling activity. However, despite a decade of availability, the palette of single FP-based Ca2+ indicators has remained limited to a single green hue. The authors have expanded this palette by developing blue, improved green, and red intensiometric indicators, as well as an emission ratiometric indicator with an 11,000% ratio change. This series enables improved single-color Ca2+ imaging in neurons and transgenic Caenorhabditis elegans. In HeLa cells, Ca2+ was imaged in three subcellular compartments, and, in conjunction with a cyan FP-yellow FP-based indicator, Ca2+ and ATP were simultaneously imaged. This palette of indicators paints the way to a colorful new era of Ca2+ imaging.
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      Chen, Z.; Ai, H.-W. Single Fluorescent Protein-Based Indicators for Zinc Ion (Zn2+). Anal. Chem. 2016, 88, 9029– 9036,  DOI: 10.1021/acs.analchem.6b01653
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      Single Fluorescent Protein-Based Indicators for Zinc Ion (Zn2+)
      Chen, Zhijie; Ai, Hui-wang
      Analytical Chemistry (Washington, DC, United States) (2016), 88 (18), 9029-9036CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Genetically encoded fluorescent Zn2+ indicators (GEZIs) are highly attractive research tools for studying Zn2+ homeostasis and signaling in mammalian cells. Most current GEZIs are based on Forster resonance energy transfer (FRET) between a select pair of fluorescent proteins (FPs) fused with Zn2+-binding motifs. One drawback of such FRET-based GEZIs is their broad spectral profile bandwidths, creating challenges when monitoring multiple targets or parameters. To address this issue, we have engineered a group of intensiometric GEZIs based on single teal and red FPs that can be utilized to monitor subcellular Zn2+ diffusion and glucose-induced Zn2+ secretion in pancreatic INS-1E β-cells. These GEZIs offer the simplicity of intensiometric measurements, compatibility in multicolor imaging, large dynamic ranges, and relatively small mol. sizes, making them valuable addns. to the mol. toolbox for imaging Zn2+.
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    33. 33
      Minckley, T. F. Sub-nanomolar sensitive GZnP3 reveals TRPML1-mediated neuronal Zn2+ signals. Nat. Commun. 2019, 10, 4806,  DOI: 10.1038/s41467-019-12761-x
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      Sub-nanomolar sensitive GZnP3 reveals TRPML1-mediated neuronal Zn(2+) signals
      Minckley Taylor F; Zhang Chen; Fudge Dylan H; Dischler Anna M; LeJeune Kate D; Qin Yan; Xu Haoxing
      Nature communications (2019), 10 (1), 4806 ISSN:.
      Although numerous fluorescent Zn(2+) sensors have been reported, it is unclear whether and how Zn(2+) can be released from the intracellular compartments into the cytosol due to a lack of probes that can detect physiological dynamics of cytosolic Zn(2+). Here, we create a genetically encoded sensor, GZnP3, which demonstrates unprecedented sensitivity for Zn(2+) at sub-nanomolar concentrations. Using GZnP3 as well as GZnP3-derived vesicular targeted probes, we provide the first direct evidence that Zn(2+) can be released from endolysosomal vesicles to the cytosol in primary hippocampal neurons through the TRPML1 channel. Such TRPML1-mediated Zn(2+) signals are distinct from Ca(2+) in that they are selectively present in neurons, sustain longer, and are significantly higher in neurites as compared to the soma. Together, our work not only creates highly sensitive probes for investigating sub-nanomolar Zn(2+) dynamics, but also reveals new pools of Zn(2+) signals that can play critical roles in neuronal function.
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    34. 34
      Akerboom, J. Optimization of a GCaMP Calcium Indicator for Neural Activity Imaging. J. Neurosci. 2012, 32, 13819– 13840,  DOI: 10.1523/JNEUROSCI.2601-12.2012
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      Optimization of a GCaMP calcium indicator for neural activity imaging
      Akerboom, Jasper; Chen, Tsai-Wen; Wardill, Trevor J.; Tian, Lin; Marvin, Jonathan S.; Mutlu, Sevinc; Calderon, Nicole Carreras; Esposti, Federico; Borghuis, Bart G.; Sun, Xiaonan Richard; Gordus, Andrew; Orger, Michael B.; Portugues, Ruben; Engert, Florian; Macklin, John J.; Filosa, Alessandro; Aggarwal, Aman; Kerr, Rex A.; Takagi, Ryousuke; Kracun, Sebastian; Shigetomi, Eiji; Khakh, Baljit S.; Baier, Herwig; Lagnado, Leon; Wang, Samuel S.-H.; Bargmann, Cornelia I.; Kimmel, Bruce E.; Jayaraman, Vivek; Svoboda, Karel; Kim, Douglas S.; Schreiter, Eric R.; Looger, Loren L.
      Journal of Neuroscience (2012), 32 (40), 13819-13840CODEN: JNRSDS; ISSN:0270-6474. (Society for Neuroscience)
      Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a no. of model organisms and can reliably detect three or more action potentials in short bursts in several systems in vivo. Through protein structure detn., targeted mutagenesis, high-throughput screening, and a battery of in vitro assays, we have increased the dynamic range of GCaMP3 by severalfold, creating a family of "GCaMP5" sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, and in vivo in Caenorhabditis chemosensory neurons, Drosophila larval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2- to 3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combining in vivo imaging with electrophysiol. we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3. GCaMP5 allows more sensitive detection of neural activity in vivo and may find widespread applications for cellular imaging in general.
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      Dana, H. High-performance calcium sensors for imaging activity in neuronal populations and microcompartments. Nat. Methods 2019, 16, 649– 657,  DOI: 10.1038/s41592-019-0435-6
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      High-performance calcium sensors for imaging activity in neuronal populations and microcompartments
      Dana, Hod; Sun, Yi; Mohar, Boaz; Hulse, Brad K.; Kerlin, Aaron M.; Hasseman, Jeremy P.; Tsegaye, Getahun; Tsang, Arthur; Wong, Allan; Patel, Ronak; Macklin, John J.; Chen, Yang; Konnerth, Arthur; Jayaraman, Vivek; Looger, Loren L.; Schreiter, Eric R.; Svoboda, Karel; Kim, Douglas S.
      Nature Methods (2019), 16 (7), 649-657CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)
      Calcium imaging with genetically encoded calcium indicators (GECIs) is routinely used to measure neural activity in intact nervous systems. GECIs are frequently used in one of two different modes: to track activity in large populations of neuronal cell bodies, or to follow dynamics in subcellular compartments such as axons, dendrites and individual synaptic compartments. Despite major advances, calcium imaging is still limited by the biophys. properties of existing GECIs, including affinity, signal-to-noise ratio, rise and decay kinetics and dynamic range. Using structure-guided mutagenesis and neuron-based screening, we optimized the green fluorescent protein-based GECI GCaMP6 for different modes of in vivo imaging. The resulting jGCaMP7 sensors provide improved detection of individual spikes (jGCaMP7s,f), imaging in neurites and neuropil (jGCaMP7b), and may allow tracking larger populations of neurons using two-photon (jGCaMP7s,f) or wide-field (jGCaMP7c) imaging.
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      Cranfill, P. J. Quantitative assessment of fluorescent proteins. Nat. Methods 2016, 13, 557– 562,  DOI: 10.1038/nmeth.3891
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      Quantitative assessment of fluorescent proteins
      Cranfill, Paula J.; Sell, Brittney R.; Baird, Michelle A.; Allen, John R.; Lavagnino, Zeno; Martijn de Gruiter, H.; Kremers, Gert-Jan; Davidson, Michael W.; Ustione, Alessandro; Piston, David W.
      Nature Methods (2016), 13 (7), 557-562CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
      The advent of fluorescent proteins (FPs) for genetic labeling of mols. and cells has revolutionized fluorescence microscopy. Genetic manipulations have created a vast array of bright and stable FPs spanning blue to red spectral regions. Common to autofluorescent FPs is their tight β-barrel structure, which provides the rigidity and chem. environment needed for effectual fluorescence. Despite the common structure, each FP has unique properties. Thus, there is no single 'best' FP for every circumstance, and each FP has advantages and disadvantages. To guide decisions about which FP is right for a given application, we have quant. characterized the brightness, photostability, pH stability and monomeric properties of more than 40 FPs to enable straightforward and direct comparison between them. We focus on popular and/or top-performing FPs in each spectral region.
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      Chen, T.-W. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 2013, 499, 295– 300,  DOI: 10.1038/nature12354
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      Ultrasensitive fluorescent proteins for imaging neuronal activity
      Chen, Tsai-Wen; Wardill, Trevor J.; Sun, Yi; Pulver, Stefan R.; Renninger, Sabine L.; Baohan, Amy; Schreiter, Eric R.; Kerr, Rex A.; Orger, Michael B.; Jayaraman, Vivek; Looger, Loren L.; Svoboda, Karel; Kim, Douglas S.
      Nature (London, United Kingdom) (2013), 499 (7458), 295-300CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      Fluorescent calcium sensors are widely used to image neural activity. Using structure-based mutagenesis and neuron-based screening, we developed a family of ultrasensitive protein calcium sensors (GCaMP6) that outperformed other sensors in cultured neurons and in zebrafish, flies and mice in vivo. In layer 2/3 pyramidal neurons of the mouse visual cortex, GCaMP6 reliably detected single action potentials in neuronal somata and orientation-tuned synaptic calcium transients in individual dendritic spines. The orientation tuning of structurally persistent spines was largely stable over timescales of weeks. Orientation tuning averaged across spine populations predicted the tuning of their parent cell. Although the somata of GABAergic neurons showed little orientation tuning, their dendrites included highly tuned dendritic segments (5-40-μm long). GCaMP6 sensors thus provide new windows into the organization and dynamics of neural circuits over multiple spatial and temporal scales.
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      Gerharz, T.; Reinelt, S.; Kaspar, S.; Scapozza, L.; Bott, M. Identification of basic amino acid residues important for citrate binding by the periplasmic receptor domain of the sensor kinase CitA. Biochemistry 2003, 42, 5917– 5924,  DOI: 10.1021/bi0340595
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      38
      Identification of Basic Amino Acid Residues Important for Citrate Binding by the Periplasmic Receptor Domain of the Sensor Kinase CitA
      Gerharz, Tanja; Reinelt, Stefan; Kaspar, Sibylle; Scapozza, Leonardo; Bott, Michael
      Biochemistry (2003), 42 (19), 5917-5924CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      The sensor kinase CitA and the response regulator CitB of Klebsiella pneumoniae form the paradigm of a subfamily of bacterial two-component regulatory systems that are capable of sensing tri- or dicarboxylates in the environment and then induce transporters for the uptake of these compds. We recently showed that the sepd. periplasmic domain of CitA, termed CitAP (encompasses residues 45-176 supplemented with an N-terminal methionine residue and a C-terminal hexahistidine tag), is a highly specific citrate receptor with a Kd of 5.5 μM at pH 7. To identify pos. charged residues involved in binding the citrate anion, each of the arginine, lysine, and histidine residues in CitAP was exchanged for alanine, and the resulting 17 muteins were analyzed by isothermal titrn. calorimetry (ITC). In 12 cases, the Kd for citrate was identical to that of wild-type CitAP or slightly changed (3.9-17.2 μM). In one case (R98A), the Kd was 6-fold decreased (0.8 μM), whereas in four cases (R66A, H69A, R107A, and K109A) the Kd was 38- to >300-fold increased (0.2 to >1 mM). The secondary structure of the latter five proteins in their apo-form as deduced from far-UV CD spectra did not differ from the apo-form of wild-type CitAP; however, all of them showed an increased thermostability. Citrate increased the m.p. (Tm) of wild-type CitAP and mutein R98A by 6.2 and 9.5°, resp., but had no effect on the Tm of the four proteins with disturbed binding. Three of the residues important for citrate binding (R66, H69, and R107) are highly conserved in the CitA subfamily of sensor kinases, indicating that they might be involved in ligand binding by many of these sensor kinases.
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    39. 39
      Ewald, J. C.; Reich, S.; Baumann, S.; Frommer, W. B.; Zamboni, N. Engineering genetically encoded nanosensors for real-time in vivo measurements of citrate concentrations. PLoS One 2011, 6, e28245,  DOI: 10.1371/journal.pone.0028245
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      39
      Engineering genetically encoded nanosensors for real-time in vivo measurements of citrate concentrations
      Ewald, Jennifer C.; Reich, Sabrina; Baumann, Stephan; Frommer, Wolf B.; Zamboni, Nicola
      PLoS One (2011), 6 (12), e28245CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
      Citrate is an intermediate in catabolic as well as biosynthetic pathways and is an important regulatory mol. in the control of glycolysis and lipid metab. Mass spectrometric and NMR based metabolomics allow measuring citrate concns., but only with limited spatial and temporal resoln. Methods are so far lacking to monitor citrate levels in real-time in-vivo. Here, we present a series of genetically encoded citrate sensors based on Forster resonance energy transfer (FRET). We screened databases for citrate-binding proteins and tested three candidates in vitro. The citrate binding domain of the Klebsiella pneumoniae histidine sensor kinase CitA, inserted between the FRET pair Venus/CFP, yielded a sensor highly specific for citrate. We optimized the peptide linkers to achieve maximal FRET change upon citrate binding. By modifying residues in the citrate binding pocket, we were able to construct seven sensors with different affinities spanning a concn. range of three orders of magnitude without losing specificity. In a first in vivo application we show that E. coli maintains the capacity to take up glucose or acetate within seconds even after long-term starvation.
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      Shen, Y. A genetically encoded Ca2+ indicator based on circularly permutated sea anemone red fluorescent protein eqFP578. BMC Biol. 2018, 16, 9,  DOI: 10.1186/s12915-018-0480-0
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      A genetically encoded Ca2+ indicator based on circularly permutated sea anemone red fluorescent protein eqFP578
      Shen, Yi; Dana, Hod; Abdelfattah, Ahmed S.; Patel, Ronak; Shea, Jamien; Molina, Rosana S.; Rawal, Bijal; Rancic, Vladimir; Chang, Yu-Fen; Wu, Lanshi; Chen, Yingche; Qian, Yong; Wiens, Matthew D.; Hambleton, Nathan; Ballanyi, Klaus; Hughes, Thomas E.; Drobizhev, Mikhail; Kim, Douglas S.; Koyama, Minoru; Schreiter, Eric R.; Campbell, Robert E.
      BMC Biology (2018), 16 (), 9/1-9/16CODEN: BBMIF7; ISSN:1741-7007. (BioMed Central Ltd.)
      Genetically encoded calcium ion (Ca2+) indicators (GECIs) are indispensable tools for measuring Ca2+ dynamics and neuronal activities in vitro and in vivo. Red fluorescent protein (RFP)-based GECIs have inherent advantages relative to green fluorescent protein-based GECIs due to the longer wavelength light used for excitation. Longer wavelength light is assocd. with decreased phototoxicity and deeper penetration through tissue. Red GECI can also enable multicolor visualization with blue- or cyan-excitable fluorophores. Here we report the development, structure, and validation of a new RFP-based GECI, K-GECO1, based on a circularly permutated RFP derived from the sea anemone Entacmaea quadricolor. We have characterized the performance of K-GECO1 in cultured HeLa cells, dissocd. neurons, stem-cell-derived cardiomyocytes, organotypic brain slices, zebrafish spinal cord in vivo, and mouse brain in vivo. K-GECO1 is the archetype of a new lineage of GECIs based on the RFP eqFP578 scaffold. It offers high sensitivity and fast kinetics, similar or better than those of current state-of-the-art indicators, with diminished lysosomal accumulation and minimal blue-light photoactivation. Further refinements of the K-GECO1 lineage could lead to further improved variants with overall performance that exceeds that of the most highly optimized red GECIs.
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      Wu, J. Red fluorescent genetically encoded Ca2+ indicators for use in mitochondria and endoplasmic reticulum. Biochem. J. 2014, 464, 13– 22,  DOI: 10.1042/BJ20140931
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      Red fluorescent genetically encoded Ca2+ indicators for use in mitochondria and endoplasmic reticulum
      Wu, Jiahui; Prole, David L.; Shen, Yi; Lin, Zhihong; Gnanasekaran, Aswini; Liu, Yingjie; Chen, Lidong; Zhou, Hang; Chen, S. R. Wayne; Usachev, Yuriy M.; Taylor, Colin W.; Campbell, Robert E.
      Biochemical Journal (2014), 464 (1), 13-22CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)
      Ca2+ is a key intermediary in a variety of signalling pathways and undergoes dynamic changes in its cytoplasmic concn. due to release from stores within the endoplasmic reticulum (ER) and influx from the extracellular environment. In addn. to regulating cytoplasmic Ca2+ signals, these responses also affect the concn. of Ca2+ within the ER and mitochondria. Single fluorescent protein-based Ca2+ indicators, such as the GCaMP series based on GFP, are powerful tools for imaging changes in the concn. of Ca2+ assocd. with intracellular signalling pathways. Most GCaMP-type indicators have dissocn. consts. (Kd) for Ca2+ in the high nanomolar to low micromolar range and are therefore optimal for measuring cytoplasmic [Ca2+], but poorly suited for use in mitochondria and ER where [Ca2+] can reach concns. of several hundred micromolar. We now report GCaMP-type low-affinity red fluorescent genetically encoded Ca2+ indicators for optical imaging (LAR-GECO), engineered to have Kd values of 24 μM (LAR-GECO1) and 12 μM (LAR-GECO1.2). We demonstrate that these indicators can be used to image mitochondrial and ER Ca2+ dynamics in several cell types. In addn., we perform two-color imaging of intracellular Ca2+ dynamics in cells expressing both cytoplasmic GCaMP and ER-targeted LAR-GECO1. The development of these low-affinity intensiometric red fluorescent Ca2+ indicators enables monitoring of ER and mitochondrial Ca2+ in combination with GFP-based reporters.
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      van de Wier, B. Elevated citrate levels in non-alcoholic fatty liver disease: the potential of citrate to promote radical production. FEBS Lett. 2013, 587, 2461– 2466,  DOI: 10.1016/j.febslet.2013.06.019
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      Elevated citrate levels in non-alcoholic fatty liver disease: The potential of citrate to promote radical production
      van de Wier, Bregje; Balk, Jiska M.; Haenen, Guido R. M. M.; Giamouridis, Dimosthenis; Bakker, Jaap A.; Bast, Bertine C.; den Hartog, Gertjan J. M.; Koek, Ger H.; Bast, Aalt
      FEBS Letters (2013), 587 (15), 2461-2466CODEN: FEBLAL; ISSN:0014-5793. (Elsevier B.V.)
      Plasma citrate levels were found to be elevated in non-alc. fatty liver disease (NAFLD) patients. Cellular expts. indicated that increased citrate levels might originate from an excess of fatty acids. The impact of elevated citrate levels on oxidative stress was examd. It was found that citrate stimulated hydrogen peroxide induced intracellular oxidative stress in HepG2 cells. This was related to the promotion of iron mediated hydroxyl radical formation from hydrogen peroxide by citrate. The stimulating effect of citrate on the reactivity of iron promotes oxidative stress, a crucial process in the progression of NAFLD.
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      Costello, L. C.; Franklin, R. B. Plasma Citrate Homeostasis: How It Is Regulated; And Its Physiological and Clinical Implications. An Important, But Neglected, Relationship in Medicine. HSOA J. Hum Endocrinol 2016, 1, 5,  DOI: 10.24966/HE-9640/100005
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      Arai, S. RGB-Color Intensiometric Indicators to Visualize Spatiotemporal Dynamics of ATP in Single. Angew. Chem., Int. Ed. 2018, 57, 10873– 10878,  DOI: 10.1002/anie.201804304
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      RGB-Color Indicators to Visualize Spatiotemporal Dynamics of ATP in Single Cells
      Arai, Satoshi; Kriszt, Rokus; Harada, Kazuki; Looi, Liang-Sheng; Matsuda, Shogo; Wongso, Devina; Suo, Satoshi; Ishiura, Shoichi; Tseng, Yu-Hua; Raghunath, Michael; Ito, Toshiro; Tsuboi, Takashi; Kitaguchi, Tetsuya
      Angewandte Chemie, International Edition (2018), 57 (34), 10873-10878CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      ATP provides energy for the regulation of multiple cellular processes in living organisms. Capturing the spatiotemporal dynamics of ATP in single cells is fundamental to the understanding of the mechanisms underlying cellular energy metab. However, it has remained challenging to visualize the dynamics of ATP in and between distinct intracellular organelles and its interplay with other signaling mols. Using single fluorescent proteins, multicolor ATP indicators were developed, enabling the simultaneous visualization of subcellular ATP dynamics in the cytoplasm and mitochondria of cells derived from mammals, plants, and worms. Furthermore, in combination with addnl. fluorescent indicators, the dynamic interplay of ATP, cAMP, and Ca2+ could be visualized in activated brown adipocyte. This set of indicator tools will facilitate future research into energy metab.
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      Li, J. J. 2-hydroxy-N-arylbenzenesulfonamides as ATP-citrate lyase inhibitors. Bioorg. Med. Chem. Lett. 2007, 17, 3208– 3211,  DOI: 10.1016/j.bmcl.2007.03.017
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      2-Hydroxy-N-arylbenzenesulfonamides as ATP-citrate lyase inhibitors
      Li, James J.; Wang, Haixia; Tino, Joseph A.; Robl, Jeffrey A.; Herpin, Timothy F.; Lawrence, R. Michael; Biller, Scott; Jamil, Haris; Ponticiello, Randy; Chen, Luping; Chu, Ching-hsuen; Flynn, Neil; Cheng, Dong; Zhao, Rulin; Chen, Bangchi; Schnur, Dora; Obermeier, Mary T.; Sasseville, Vito; Padmanabha, Ramesh; Pike, Kristen; Harrity, Thomas
      Bioorganic & Medicinal Chemistry Letters (2007), 17 (11), 3208-3211CODEN: BMCLE8; ISSN:0960-894X. (Elsevier Ltd.)
      A novel series of 2-hydroxy-N-arylbenzenesulfonamides were identified to be ATP-citrate lyase (ACL) inhibitors with compd. 9 displaying potent in vitro activity (IC50 = 0.13 μM). Chronic oral dosing of compd. 9 in high-fat fed mice lowered plasma cholesterol, triglyceride, and glucose, as well as inhibited wt. gain.
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      Minárik, P.; Tomásková, N.; Kollárová, M.; Antalík, M. Malate dehydrogenases--structure and function. Gen. Physiol. Biophys. 2002, 21, 257– 265
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      General Physiology and Biophysics (2002), 21 (3), 257-265CODEN: GPBIE2; ISSN:0231-5882. (Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences)
      A review with 33 refs. Malate dehydrogenase (MDH; EC 1.1.1.37) catalyzes the NAD/NADH-dependent interconversion of malate and oxalacetate. This reaction plays a key part in the malate/aspartate shuttle across the mitochondrial membrane, and in the tricarboxylic acid cycle within the mitochondrial matrix. MDHs are homodimeric mols. in most organisms, including all eukaryotes and the most bacterial species. The enzymes share a common catalytic mechanism and their kinetic properties are similar, which demonstrates a high degree of structural similarity. The 3-dimensional structures and elements essential for catalysis are conserved between mitochondrial and cytoplasmic forms of MDH in eukaryotic cells even though these isoenzymes are only marginally related at the level of primary structure.
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      Bisaccia, F.; De Palma, A.; Prezioso, G.; Palmieri, F. Kinetic characterization of the reconstituted tricarboxylate carrier from rat liver mitochondria. Biochim. Biophys. Acta, Bioenerg. 1990, 1019, 250– 256,  DOI: 10.1016/0005-2728(90)90201-E
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      Kinetic characterization of the reconstituted tricarboxylate carrier from rat liver mitochondria
      Bisaccia, F.; De Palma, A.; Prezioso, G.; Palmieri, F.
      Biochimica et Biophysica Acta, Bioenergetics (1990), 1019 (3), 250-6CODEN: BBBEB4; ISSN:0005-2728.
      The tricarboxylate carrier from rat liver mitochondria was purified by chromatog. on hydroxyapatite/celite and reconstituted in phospholipid vesicles by removing the detergent using hydrophobic chromatog. on Amberlite. Optimal transport activity was obtained by using a Triton X-114/phospholipid ratio of 0.8, 6% cardiolipin and 24 passages through a single Amberlite column. In the reconstituted system the incorporated tricarboxylate carrier catalyzed a first-order reaction of citrate/citrate or citrate/malate exchange. The activation energy of the exchange reaction was 70.1 kJ/mol. The rate of the exchange had a pH optimum between 7 and 8. The half-satn. const. was 0.13 mM for citrate and 0.76 mM for malate. All these properties were similar to those described for the tricarboxylate transport system in intact mitochondria. In proteoliposomes the max. exchange rate at 25° reached μmol/min per g protein. This value was independent of the type of substrate present at the external or internal space of the liposomes (citrate or malate).
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      Factors affecting the kinetics and equilibrium of exchange reactions of the citrate-transporting system of rat liver mitochondria
      Robinson, B. H.; Williams, G. R.; Halperin, Mitchell L.; Leznoff, C. C.
      Journal of Biological Chemistry (1971), 246 (17), 5280-6CODEN: JBCHA3; ISSN:0021-9258.
      Using an "inhibitor stop" technique with benzene 1,2,3-tricarboxylic acid as the inhibitor, the initial rates of exchange reactions catalyzed by the citrate-transporting system were measured and the dependence of the rates on temp. and the concn. of citrate (I), L-malate (II), and magnesium were studied. Both the rate and extent of exchange II with intramitochondrial [14C]-I were less than those of I with intramitochondrial [14C]-I. This difference in the rate and extent between the 2 exchanges was thought to be due to the fact that II-I exchange resulted in a disequil. of either charge or pH across the mitochondrial membrane. Malate2- probably exchanges for citrate2- rather than citrate3-, thereby setting up a pH differential which restricts further exchange.
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      Halestrap, A. P. The mechanism of the inhibition of the mitochondrial pyruvate transportater by alpha-cyanocinnamate derivatives. Biochem. J. 1976, 156, 181– 183,  DOI: 10.1042/bj1560181
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      Halestrap, Andrew P.
      Biochemical Journal (1976), 156 (1), 181-3CODEN: BIJOAK; ISSN:0264-6021.
      α-Cyano-4-hydroxycinnamate (I) (O.1mM) totally, rapidly, and reversibly inhibited mitochondrial pyruvate transport at 6 and 22°. I reacted reversibly with mercaptoethanol and cysteine to form addn. products, suggesting that I reacts with an essential thiol group on the pyruvate carrier.
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      Levenson, R.; Macara, I. G.; Smith, R. L.; Cantley, L.; Housman, D. Role of mitochondrial membrane potential in the regulation of murine erythroleukemia cell differentiation. Cell 1982, 28, 855– 863,  DOI: 10.1016/0092-8674(82)90064-2
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      Role of mitochondrial membrane potential in the regulation of murine erythroleukemia cell differentiation
      Levenson, Robert; Macara, Ian G.; Smith, Roderick L.; Cantley, Lewis; Housman, David
      Cell (Cambridge, MA, United States) (1982), 28 (4), 855-63CODEN: CELLB5; ISSN:0092-8674.
      The fluorescent membrane probe DiOC6 (3,3'-dihexyloxacarbocyanine iodide) was used to exam. the relation between murine erythroleukemia (MEL) cell mitochondria and the changes in cytoplasmic Ca levels occurring at the initiation of the commitment to differentiate. Fluorescence microscopy reveals the selective assocn. of DiOC6 with MEL cell mitochondria, where an enhanced fluorescence is obsd. Treatment of cells with DMSO or other inducers causes a decrease in mitochondria-assocd. fluorescence levels that occurs with the initiation of commitment. A decrease in DiOC6 fluorescence is caused by agents that reduce mitochondrial membrane potential, but is only slightly affected by agents that alter plasma membrane potential. Amiloride and EGTA, agents that prevent commitment and inhibit Ca uptake, also prevent the decrease in DiOC6 uptake caused by DMSO. The effect of DMSO on MEL cell mitochondria is mimicked by FCCP (carbonyl cyanine p-trifluoromethoxyphenyl hydrazone), a proton ionophore that dissipates mitochondrial membrane potential. FCCP also causes MEL cell mitochondria to release Ca into the cytoplasm. When MEL cells are treated with DMSO plus FCCP, commitment is initiated without the lag period obsd. when cells are treated with DMSO alone. These results are consistent with the hypothesis that mitochondrial transmembrane potential is important in the regulation of cytoplasmic Ca levels at the time of commitment of MEL cells to terminal differentiation.
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      Yuan, C. Dimethyl sulfoxide damages mitochondrial integrity and membrane potential in cultured astrocytes. PLoS One 2014, 9, e107447,  DOI: 10.1371/journal.pone.0107447
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      Dimethyl sulfoxide damages mitochondrial integrity and membrane potential in cultured astrocytes
      Yuan, Chan; Gao, Junying; Guo, Jichao; Bai, Lei; Marshall, Charles; Cai, Zhiyou; Wang, Linmei; Xiao, Ming
      PLoS One (2014), 9 (9), e107447/1-e107447/9, 9 pp.CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
      DMSO (DMSO) is a polar org. solvent that is used to dissolve neuroprotective or neurotoxic agents in neuroscience research. However, DMSO itself also has pharmacol. and pathol. effects on the nervous system. Astrocytes play a central role in maintaining brain homeostasis, but the effect and mechanism of DMSO on astrocytes has not been studied. The present study showed that exposure of astrocyte cultures to 1% DMSO for 24 h did not significantly affect cell survival, but decreased cell viability and glial glutamate transporter expression, and caused mitochondrial swelling, membrane potential impairment and reactive oxygen species prodn., and subsequent cytochrome c release and caspase-3 activation. DMSO at concns. of 5% significantly inhibited cell variability and promoted apoptosis of astrocytes, accompanied with more severe mitochondrial damage. These results suggest that mitochondrial impairment is a primary event in DMSO-induced astrocyte toxicity. The potential cytotoxic effects on astrocytes need to be carefully considered during investigating neuroprotective or neurotoxic effects of hydrophobic agents dissolved by DMSO.
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      Depaoli, M. R. Real-Time Imaging of Mitochondrial ATP Dynamics Reveals the Metabolic Setting of Single Cells. Cell Rep. 2018, 25, 501– 512,  DOI: 10.1016/j.celrep.2018.09.027
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      Real-Time Imaging of Mitochondrial ATP Dynamics Reveals the Metabolic Setting of Single Cells
      Depaoli, Maria R.; Karsten, Felix; Madreiter-Sokolowski, Corina T.; Klec, Christiane; Gottschalk, Benjamin; Bischof, Helmut; Eroglu, Emrah; Waldeck-Weiermair, Markus; Simmen, Thomas; Graier, Wolfgang F.; Malli, Roland
      Cell Reports (2018), 25 (2), 501-512.e3CODEN: CREED8; ISSN:2211-1247. (Cell Press)
      Reprogramming of metabolic pathways dets. cell functions and fate. In our work, we have used organelle-targeted ATP biosensors to evaluate cellular metabolic settings with high resoln. in real time. Our data indicate that mitochondria dynamically supply ATP for glucose phosphorylation in a variety of cancer cell types. This hexokinase-dependent process seems to be reversed upon the removal of glucose or other hexose sugars. Our data further verify that mitochondria in cancer cells have increased ATP consumption. Similar subcellular ATP fluxes occurred in young mouse embryonic fibroblasts (MEFs). However, pancreatic beta cells, senescent MEFs, and MEFs lacking mitofusin 2 displayed completely different mitochondrial ATP dynamics, indicative of increased oxidative phosphorylation. Our findings add perspective to the variability of the cellular bioenergetics and demonstrate that live cell imaging of mitochondrial ATP dynamics is a powerful tool to evaluate metabolic flexibility and heterogeneity at a single-cell level.
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      MacDonald, M. J. Citrate oscillates in liver and pancreatic beta cell mitochondria and in INS-1 insulinoma cells. J. Biol. Chem. 2003, 278, 51894– 51900,  DOI: 10.1074/jbc.M309038200
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      Citrate Oscillates in Liver and Pancreatic Beta Cell Mitochondria and in INS-1 Insulinoma Cells
      MacDonald, Michael J.; Fahien, Leonard A.; Buss, Julian D.; Hasan, Noaman M.; Fallon, Michael J.; Kendrick, Mindy A.
      Journal of Biological Chemistry (2003), 278 (51), 51894-51900CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Oscillations in citric acid cycle intermediates have never been previously reported in any type of cell. Here we show that adding pyruvate to isolated mitochondria from liver, pancreatic islets, and INS-1 insulinoma cells or adding glucose to intact INS-1 cells causes sustained oscillations in citrate levels. Other citric acid cycle intermediates measured either did not oscillate or possibly oscillated with a low amplitude. In INS-1 mitochondria citrate oscillations are in phase with NAD(P) oscillations, and in intact INS-1 cells citrate oscillations parallel oscillations in ATP, suggesting that these processes are co-regulated. Oscillations have been extensively studied in the pancreatic beta cell where oscillations in glycolysis, NAD(P)/NAD(P)H and ATP/ADP ratios, plasma membrane elec. activity, calcium levels, and insulin secretion have been well documented. Because the mitochondrion is the major site of ATP synthesis and NADH oxidn. and the only site of citrate synthesis, mitochondria need to be synchronized for these factors to oscillate. In suspensions of mitochondria from various organs, most of the citrate is exported from the mitochondria. In addn., citrate inhibits its own synthesis. We propose that this enables citrate itself to act as one of the cellular messengers that synchronizes mitochondria. Furthermore, because citrate is a potent inhibitor of the glycolytic enzyme phosphofructokinase, the pacemaker of glycolytic oscillations, citrate may act as a metabolic link between mitochondria and glycolysis. Citrate oscillations may coordinate oscillations in mitochondrial energy prodn. and anaplerosis with glycolytic oscillations, which in the beta cell are known to parallel oscillations in insulin secretion.
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      Lorenz, M. A.; El Azzouny, M. A.; Kennedy, R. T.; Burant, C. F. Metabolome response to glucose in the β-cell line INS-1 832/13. J. Biol. Chem. 2013, 288, 10923– 10935,  DOI: 10.1074/jbc.M112.414961
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      Metabolome Response to Glucose in the β-Cell Line INS-1 832/13
      Lorenz, Matthew A.; El Azzouny, Mahmoud A.; Kennedy, Robert T.; Burant, Charles F.
      Journal of Biological Chemistry (2013), 288 (15), 10923-10935CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells is triggered by metab. of the sugar to increase ATP/ADP ratio that blocks the KATP channel leading to membrane depolarization and insulin exocytosis. Other metabolic pathways believed to augment insulin secretion have yet to be fully elucidated. To study metabolic changes during GSIS, liq. chromatog. with mass spectrometry was used to det. levels of 87 metabolites temporally following a change in glucose from 3 to 10 mm glucose and in response to increasing concns. of glucose in the INS-1 832/13 β-cell line. U-[13C]Glucose was used to probe flux in specific metabolic pathways. Results include a rapid increase in ATP/ADP, anaplerotic tricarboxylic acid cycle flux, and increases in the malonyl CoA pathway, support prevailing theories of GSIS. Novel findings include that aspartate used for anaplerosis does not derive from the glucose fuel added to stimulate insulin secretion, glucose flux into glycerol-3-phosphate, and esterification of long chain CoAs resulting in rapid consumption of long chain CoAs and de novo generation of phosphatidic acid and diacylglycerol. Further, novel metabolites with potential roles in GSIS such as 5-aminoimidazole-4-carboxamide ribotide (ZMP), GDP-mannose, and farnesyl pyrophosphate were found to be rapidly altered following glucose exposure.
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      Shen, Y. Genetically encoded fluorescent indicators for imaging intracellular potassium ion concentration. Commun. Biol. 2019, 2, 18,  DOI: 10.1038/s42003-018-0269-2
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      Genetically encoded fluorescent indicators for imaging intracellular potassium ion concentration
      Shen Yi; Miyashita Shin-Ichiro; Dong Min; Shen Yi; Wu Sheng-Yi; Aggarwal Abhi; Qian Yong; Campbell Robert E; Rancic Vladimir; Ballanyi Klaus; Campbell Robert E
      Communications biology (2019), 2 (), 18 ISSN:.
      Potassium ion (K(+)) homeostasis and dynamics play critical roles in biological activities. Here we describe three genetically encoded K(+) indicators. KIRIN1 (potassium (K) ion ratiometric indicator) and KIRIN1-GR are Forster resonance energy transfer (FRET)-based indicators with a bacterial K(+) binding protein (Kbp) inserting between the fluorescent protein FRET pairs mCerulean3/cp173Venus and Clover/mRuby2, respectively. GINKO1 (green indicator of K(+) for optical imaging) is a single fluorescent protein-based K(+) indicator constructed by insertion of Kbp into enhanced green fluorescent protein (EGFP). These indicators are suitable for detecting K(+) at physiologically relevant concentrations in vitro and in cells. KIRIN1 enabled imaging of cytosolic K(+) depletion in live cells and K(+) efflux and reuptake in cultured neurons. GINKO1, in conjunction with red fluorescent Ca(2+) indicator, enable dual-color imaging of K(+) and Ca(2+) dynamics in neurons and glial cells. These results demonstrate that KIRIN1 and GINKO1 are useful tools for imaging intracellular K(+) dynamics.
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    56. 56
      Cormann, K. U.; Baumgart, M.; Bott, M. Structure-Based Design of Versatile Biosensors for Small Molecules Based on the PAS Domain of a Thermophilic Histidine Kinase. ACS Synth. Biol. 2018, 7, 2888– 2897,  DOI: 10.1021/acssynbio.8b00348
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      56
      Structure-Based Design of Versatile Biosensors for Small Molecules Based on the PAS Domain of a Thermophilic Histidine Kinase
      Cormann, Kai U.; Baumgart, Meike; Bott, Michael
      ACS Synthetic Biology (2018), 7 (12), 2888-2897CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
      The development of biosensors for in vitro quantification of small mols. such as metabolites or man-made chems. is still a major challenge. Here we show that engineered variants of the sensory PAS domain of the histidine kinase CitA of the thermophilic bacterium Geobacillus thermoleovorans represent promising alternatives to established biorecognition elements. By combining binding site grafting and rational design we constructed protein variants binding L-malate, ethylmalonate, or the arom. compd. phthalate instead of the native ligand citrate. Due to more favorable entropy contributions, the wild-type protein and its engineered variants exhibited increased (nano- to micromolar) affinities and improved enantioselectivity compared to CitA homologues of mesophilic organisms. Ligand binding was directly converted into an optical signal that was preserved after immobilization of the protein. A fluorescently labeled variant was used to quantify ethylmalonate, an urinary biomarker for ethylmalonic encephalopathy, in synthetic urine, thereby demonstrating the applicability of the sensor in complex samples.
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    57. 57
      Costello, L. C.; Franklin, R. B. Prostatic Fluid Electrolyte Composition for the Screening of Prostate Cancer: A Potential Solution to a Major Problem. Prostate Cancer Prostatic Dis. 2009, 12 (1), 17– 24,  DOI: 10.1038/pcan.2008.19
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      100
      Prostatic fluid electrolyte composition for the screening of prostate cancer: a potential solution to a major problem
      Costello, L. C.; Franklin, R. B.
      Prostate Cancer and Prostatic Diseases (2009), 12 (1), 17-24CODEN: PCPDFW; ISSN:1365-7852. (Nature Publishing Group)
      A review. Early detection is the key to effective treatment of prostate cancer, and to the prevention of deaths due to progression to untreatable advanced stage cancer. Because of mitigating factors, esp. benign prostatic hyperplasia (BPH), that result in a low accuracy (about 60%) of prostate-specific antigen (PSA) testing, there is an urgent need for a more reliable biomarker for the identification of early stage through advanced stage prostate cancer and at-risk' individuals. To address this issue we propose that changes in prostatic fluid compn. could provide accurate and reliable biomarkers for the screening of prostate cancer. Most notable is the consistent and significant decrease in citrate and zinc that is assocd. with the development and progression of prostate cancer. In this review we provide the clin. and physiol. basis and the evidence in support of the utility of prostatic fluid anal. as an effective approach for screening/detection of prostate cancer, esp. early stage and at-risk' subjects. The problem of BPH interference that plagues PSA testing is eliminated in the potential prostatic fluid biomarkers. The potential development of rapid, simple, direct, accurate clin. tests provides addnl. advantageous conditions. Further exploration and development of citrate, zinc and other electrolytes as prostatic fluid biomarkers are urgently needed to address this crit. prostate cancer issue.
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    58. 58
      Westergaard, N.; Waagepetersen, H. S.; Belhage, B.; Schousboe, A. Citrate, a Ubiquitous Key Metabolite with Regulatory Function in the CNS. Neurochem. Res. 2017, 42, 1583– 1588,  DOI: 10.1007/s11064-016-2159-7
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      57
      Citrate, a Ubiquitous Key Metabolite with Regulatory Function in the CNS
      Westergaard, Niels; Waagepetersen, Helle S.; Belhage, Bo; Schousboe, Arne
      Neurochemical Research (2017), 42 (6), 1583-1588CODEN: NEREDZ; ISSN:0364-3190. (Springer)
      Citrate is key constituent of the tricarboxylic acid (TCA) cycle, serves as substrate for fatty acid and sterol biosynthesis, and functions as a key regulator of intermediary energy metab. Ursula Sonnewald had initiated studies using for the first time both proton- and 13C-NMR to investigate metabolic processes in cultured neurons and astrocytes resulting in the important observation that citrate was specifically synthesized in and released from astrocytes in large amts. which is in keeping with the high concn. found in the CSF. The aim of this review is to highlight the possible roles of citrate in physiol. and pathophysiol. processes in the CNS. An interesting feature of citrate is its ability to chelate Ca2+, Mg2+ and Zn2+and thereby playing a pivotal role as an endogenous modulator of glutamate receptors and in particular the NMDA subtypes of these receptors in the CNS. Besides its presence in cerebrospinal fluid (CSF) citrate is also found in high amts. in prostate fluid reaching concns. as high as 180 mM and here Zn2+ seems also to play an important role, which makes prostate cells interesting for comparison of features of citrate and Zn2+ between these cells and cells in the CNS.
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    59. 59
      Thevenon, J. Mutations in SLC13A5 cause autosomal-recessive epileptic encephalopathy with seizure onset in the first days of life. Am. J. Hum. Genet. 2014, 95, 113– 120,  DOI: 10.1016/j.ajhg.2014.06.006
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      58
      Mutations in SLC13A5 Cause Autosomal-Recessive Epileptic Encephalopathy with Seizure Onset in the First Days of Life
      Thevenon, Julien; Milh, Mathieu; Feillet, Francois; St-Onge, Judith; Duffourd, Yannis; Juge, Clara; Roubertie, Agathe; Heron, Delphine; Mignot, Cyril; Raffo, Emmanuel; Isidor, Bertrand; Wahlen, Sandra; Sanlaville, Damien; Villeneuve, Nathalie; Darmency-Stamboul, Veronique; Toutain, Annick; Lefebvre, Mathilde; Chouchane, Mondher; Huet, Frederic; Lafon, Arnaud; de Saint Martin, Anne; Lesca, Gaetan; El Chehadeh, Salima; Thauvin-Robinet, Christel; Masurel-Paulet, Alice; Odent, Sylvie; Villard, Laurent; Philippe, Christophe; Faivre, Laurence; Riviere, Jean-Baptiste
      American Journal of Human Genetics (2014), 95 (1), 113-120CODEN: AJHGAG; ISSN:0002-9297. (Cell Press)
      Epileptic encephalopathy (EE) refers to a clin. and genetically heterogeneous group of severe disorders characterized by seizures, abnormal interictal electro-encephalogram, psychomotor delay, and/or cognitive deterioration. The authors ascertained two multiplex families (including one consanguineous family) consistent with an autosomal-recessive inheritance pattern of EE. All seven affected individuals developed subclin. seizures as early as the first day of life, severe epileptic disease, and profound developmental delay with no facial dysmorphism. Given the similarity in clin. presentation in the two families, the authors hypothesized that the obsd. phenotype was due to mutations in the same gene, and the authors performed exome sequencing in three affected individuals. Anal. of rare variants in genes consistent with an autosomal-recessive mode of inheritance led to identification of mutations in SLC13A5, which encodes the cytoplasmic sodium-dependent citrate carrier, notably expressed in neurons. Disease assocn. was confirmed by cosegregation anal. in addnl. family members. Screening of 68 addnl. unrelated individuals with early-onset epileptic encephalopathy for SLC13A5 mutations led to identification of one addnl. subject with compd. heterozygous mutations of SLC13A5 and a similar clin. presentation as the index subjects. Mutations affected key residues for sodium binding, which is crit. for citrate transport. These findings underline the value of careful clin. characterization for genetic investigations in highly heterogeneous conditions such as EE and further highlight the role of citrate metab. in epilepsy.
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    60. 60
      Hardies, K. Recessive mutations in SLC13A5 result in a loss of citrate transport and cause neonatal epilepsy, developmental delay and teeth hypoplasia. Brain 2015, 138, 3238– 3250,  DOI: 10.1093/brain/awv263
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      59
      Recessive mutations in SLC13A5 result in a loss of citrate transport and cause neonatal epilepsy, developmental delay and teeth hypoplasia
      Hardies Katia; Deconinck Tine; Suls Arvid; de Kovel Carolien G F; Brilstra Eva; Braun Kees P J; van 't Slot Ruben; Koeleman Bobby P C; Weckhuysen Sarah; Asselbergh Bob; Geuens Thomas; Timmerman Vincent; Azmi Abdelkrim; May Patrick; Becker Felicitas; Schubert Julian; Weber Yvonne; Lerche Holger; Barisic Nina; Craiu Dana; Lal Dennis; Thiele Holger; Nurnberg Peter; Balling Rudi; Maudsley Stuart; Helbig Ingo; De Jonghe Peter
      Brain : a journal of neurology (2015), 138 (Pt 11), 3238-50 ISSN:.
      The epileptic encephalopathies are a clinically and aetiologically heterogeneous subgroup of epilepsy syndromes. Most epileptic encephalopathies have a genetic cause and patients are often found to carry a heterozygous de novo mutation in one of the genes associated with the disease entity. Occasionally recessive mutations are identified: a recent publication described a distinct neonatal epileptic encephalopathy (MIM 615905) caused by autosomal recessive mutations in the SLC13A5 gene. Here, we report eight additional patients belonging to four different families with autosomal recessive mutations in SLC13A5. SLC13A5 encodes a high affinity sodium-dependent citrate transporter, which is expressed in the brain. Neurons are considered incapable of de novo synthesis of tricarboxylic acid cycle intermediates; therefore they rely on the uptake of intermediates, such as citrate, to maintain their energy status and neurotransmitter production. The effect of all seven identified mutations (two premature stops and five amino acid substitutions) was studied in vitro, using immunocytochemistry, selective western blot and mass spectrometry. We hereby demonstrate that cells expressing mutant sodium-dependent citrate transporter have a complete loss of citrate uptake due to various cellular loss-of-function mechanisms. In addition, we provide independent proof of the involvement of autosomal recessive SLC13A5 mutations in the development of neonatal epileptic encephalopathies, and highlight teeth hypoplasia as a possible indicator for SLC13A5 screening. All three patients who tried the ketogenic diet responded well to this treatment, and future studies will allow us to ascertain whether this is a recurrent feature in this severe disorder.
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    61. 61
      Klotz, J.; Porter, B. E.; Colas, C.; Schlessinger, A.; Pajor, A. M. Mutations in the Na(+)/citrate cotransporter NaCT (SLC13A5) in pediatric patients with epilepsy and developmental delay. Mol. Med. 2016, 22, 310– 321,  DOI: 10.2119/molmed.2016.00077
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      60
      Mutations in the Na+/citrate cotransporter NaCT (SLC13A5) in pediatric patients with epilepsy and developmental delay
      Klotz, Jenna; Porter, Brenda E.; Colas, Claire; Schlessinger, Avner; Pajor, Ana M.
      Molecular Medicine (Manhasset, NY, United States) (2016), 22 (), 310-321CODEN: MOMEF3; ISSN:1528-3658. (Feinstein Institute for Medical Research)
      Mutations in the SLC13A5 gene that codes for the Na+/citrate cotransporter, NaCT, are assocd. with early onset epilepsy, developmental delay and tooth dysplasia in children. In this study, we identify addnl. SLC13A5 mutations in nine epilepsy patients from six families. To better characterize the syndrome, families with affected children answered questions about the scope of illness and the treatment strategies. Currently, there are no effective treatments, but some antiepileptic drugs targeting the γ-aminobutyric acid system reduce seizure frequency. Acetazolamide, a carbonic anhydrase inhibitor and atypical antiseizure medication, decreases seizures in four patients. In contrast to previous reports, the ketogenic diet and fasting resulted in worsening of symptoms. The effects of the mutations on NaCT transport function and protein expression were examd. by transient transfections of COS-7 cells. There was no transport activity from any of the mutant transporters, although some of the mutant transporter proteins were present on the plasma membrane. The structural model of NaCT suggests that these mutations can affect helix packing or substrate binding. We tested various treatments, including chem. chaperones and low temps., but none improved transport function in the NaCT mutants. Interestingly, coexpression of NaCT and the mutants results in decreased protein expression and activity of the wild-type transporter, indicating functional interaction. In conclusion, this study has identified addnl. SLC13A5 mutations in patients with chronic epilepsy starting in the neonatal period, with the mutations producing inactive Na+/citrate transporters.
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    62. 62
      Bhutia, Y. D.; Kopel, J. J.; Lawrence, J. J.; Neugebauer, V.; Ganapathy, V. Plasma Membrane Na+-Coupled Citrate Transporter (SLC13A5) and Neonatal Epileptic Encephalopathy. Molecules 2017, 22, 378,  DOI: 10.3390/molecules22030378
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      61
      Plasma membrane Na+-coupled citrate transporter (SLC13A5) and neonatal epileptic encephalopathy
      Bhutia, Yangzom D.; Kopel, Jonathan J.; Lawrence, John J.; Neugebauer, Volker; Ganapathy, Vadivel
      Molecules (2017), 22 (3), 378/1-378/15CODEN: MOLEFW; ISSN:1420-3049. (MDPI AG)
      SLC13A5 is a Na+-coupled transporter for citrate that is expressed in the plasma membrane of specific cell types in the liver, testis, and brain. It is an electrogenic transporter with a Na+:citrate3+ stoichiometry of 4:1. In humans, the Michaelis const. for SLC13A5 to transport citrate is ∼600 μM, which is physiol. relevant given that the normal concn. of citrate in plasma is in the range of 150-200 μM. Li+ stimulates the transport function of human SLC13A5 at concns. that are in the therapeutic range in patients on lithium therapy. Human SLC13A5 differs from rodent Slc13a5 in two important aspects: the affinity of the human transporter for citrate is ∼30-fold less than that of the rodent transporter, thus making human SLC13A5 a low-affinity/high-capacity transporter and the rodent Slc13a5 a high-affinity/low-capacity transporter. In the liver, SLC13A5 is expressed exclusively in the sinusoidal membrane of the hepatocytes, where it plays a role in the uptake of circulating citrate from the sinusoidal blood for metabolic use. In the testis, the transporter is expressed only in spermatozoa, which is also only in the mid piece where mitochondria are located; the likely function of the transporter in spermatozoa is to mediate the uptake of citrate present at high levels in the seminal fluid for subsequent metab. in the sperm mitochondria to generate biol. energy, thereby supporting sperm motility. In the brain, the transporter is expressed mostly in neurons. As astrocytes secrete citrate into extracellular medium, the potential function of SLC13A5 in neurons is to mediate the uptake of circulating citrate and astrocyte-released citrate for subsequent metab. Slc13a5-knockout mice have been generated; these mice do not have any overt phenotype but are resistant to exptl. induced metabolic syndrome. Recently however, loss-of-function mutations in human SLC13A5 have been found to cause severe epilepsy and encephalopathy early in life. Interestingly, there is no evidence of epilepsy or encephalopathy in Slc13a5-knockout mice, underlining the significant differences in clin. consequences of the loss of function of this transporter between humans and mice. The markedly different biochem. features of human SLC13A5 and mouse Slc13a5 likely contribute to these differences between humans and mice with regard to the metabolic consequences of the transporter deficiency. The exact mol. mechanisms by which the functional deficiency of the citrate transporter causes epilepsy and impairs neuronal development and function remain to be elucidated, but available literature implicate both dysfunction of GABA (γ-aminobutyrate) signaling and hyperfunction of NMDA (N-methyl-D-aspartate) receptor signaling. Plausible synaptic mechanisms linking loss-of-function mutations in SLC13A5 to epilepsy are discussed.
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