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Development of a Dual Reporter System to Simultaneously Visualize Ca2+ Signals and AMPK Activity
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  • Yusuf C. Erdoğan
    Yusuf C. Erdoğan
    Gottfried Schatz Research Center, Molecular Biology and Biochemistry, Medical University of Graz, Neue Stiftingtalstraße 6, Graz 8010, Austria
    BioTechMed Graz, Mozartgasse 12/2, Graz 8010, Austria
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  • Johannes Pilic
    Johannes Pilic
    Gottfried Schatz Research Center, Molecular Biology and Biochemistry, Medical University of Graz, Neue Stiftingtalstraße 6, Graz 8010, Austria
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  • Benjamin Gottschalk
    Benjamin Gottschalk
    Gottfried Schatz Research Center, Molecular Biology and Biochemistry, Medical University of Graz, Neue Stiftingtalstraße 6, Graz 8010, Austria
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  • Esra N. Yiğit
    Esra N. Yiğit
    Regenerative and Restorative Medicine Research Center (REMER), Research Institute for Health Sciences and Technologies (SABITA), Istanbul Medipol University, Istanbul 34810, Turkey
    Department of Physiology, International School of Medicine, İstanbul Medipol University, İstanbul 34810, Türkiye
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  • Asal G. Zaki
    Asal G. Zaki
    Regenerative and Restorative Medicine Research Center (REMER), Research Institute for Health Sciences and Technologies (SABITA), Istanbul Medipol University, Istanbul 34810, Turkey
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  • Gürkan Öztürk
    Gürkan Öztürk
    Regenerative and Restorative Medicine Research Center (REMER), Research Institute for Health Sciences and Technologies (SABITA), Istanbul Medipol University, Istanbul 34810, Turkey
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    Orcidhttps://orcid.org/0000-0003-0352-1947
  • Emrah Eroğlu
    Emrah Eroğlu
    Regenerative and Restorative Medicine Research Center (REMER), Research Institute for Health Sciences and Technologies (SABITA), Istanbul Medipol University, Istanbul 34810, Turkey
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  • Begüm Okutan
    Begüm Okutan
    Department of Orthopedics and Traumatology, Medical University of Graz, Auenbruggerplatz 5, Graz 8036, Austria
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    Orcidhttps://orcid.org/0000-0002-4951-5940
  • Nicole G. Sommer
    Nicole G. Sommer
    Department of Orthopedics and Traumatology, Medical University of Graz, Auenbruggerplatz 5, Graz 8036, Austria
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  • Annelie M. Weinberg
    Annelie M. Weinberg
    Department of Orthopedics and Traumatology, Medical University of Graz, Auenbruggerplatz 5, Graz 8036, Austria
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  • Rainer Schindl
    Rainer Schindl
    Gottfried Schatz Research Center, Biophysics, Medical University of Graz, Neue Stiftingtalstrasse 6, Graz 8010, Austria
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  • Wolfgang F. Graier
    Wolfgang F. Graier
    Gottfried Schatz Research Center, Molecular Biology and Biochemistry, Medical University of Graz, Neue Stiftingtalstraße 6, Graz 8010, Austria
    BioTechMed Graz, Mozartgasse 12/2, Graz 8010, Austria
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    Orcidhttps://orcid.org/0000-0003-1871-3298
  • Roland Malli*
    Roland Malli
    BioTechMed Graz, Mozartgasse 12/2, Graz 8010, Austria
    Center for Medical Research, Bioimaging, Medial University of Graz, Neue Stiftingtalstrasse 6, Graz 8010, Austria
    *Email: [email protected]
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    Orcidhttps://orcid.org/0000-0001-6327-8729
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ACS Sensors

Cite this: ACS Sens. 2024, 9, 9, 4680–4689
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https://doi.org/10.1021/acssensors.4c01058
Published August 21, 2024

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Abstract

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In this study, we introduce a new separation of phases-based activity reporter of kinase (SPARK) for AMP-activated kinase (AMPK), named AMPK-SPARK, which reports the AMPK activation by forming bright fluorescent clusters. Furthermore, we introduce a dual reporter system, named GCaMP-AMPK-SPARK, by incorporating a single-fluorescent protein (FP)-based Ca2+ biosensor, GCaMP6f, into our initial design, enabling simultaneous monitoring of Ca2+ levels and AMPK activity. This system offers the essential quality of information by single-channel fluorescence microscopy without the need for coexpression of different biosensors and elaborate filter layouts to overcome spectral limitations. We used AMPK-SPARK to map endogenous AMPK activity in different cell types and visualized the dynamics of AMPK activation in response to various stimuli. Using GCaMP-AMPK-SPARK, we revealed cell-to-cell heterogeneities in AMPK activation by Ca2+ mobilization. We anticipate that this dual reporter strategy can be employed to study the intricate interplays between different signaling networks and kinase activities.

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This paper was published ASAP on August 21, 2024, with distorted versions of Figure 1 and Figure 3. These were corrected in the version published ASAP on August 22, 2024.

Protein kinases play a crucial role in regulating vital cellular processes by orchestrating the phosphorylation of proteins, a widely recognized and prevalent form of posttranslational modification that profoundly impacts signal transduction, cell metabolism, cell cycle progression, and gene expression. (1) Measuring kinase activity at the single-cell level helps capture cellular heterogeneity, reveal dynamic responses, and assess drug responses accurately. FP-based biosensor technologies enabled the development of tools to help monitor real-time kinase activities in live cells with a high spatiotemporal resolution. (2)
AMPK is a crucial regulator of energy homeostasis in eukaryotic cells. (3) Various genetic AMPK biosensors have been developed based on FPs to study AMPK activity at the single-cell level. (4−8) However, these sensors demonstrate limited fluorescence changes upon kinase activation. In this study, we introduce a novel separation of phases-based activity reporter (SPARK) for AMPK, named AMPK-SPARK. The phase separation-based kinase reporter approach, introduced initially for visualizing the activity of protein kinase A (PKA) and extracellular signal-regulated kinase (ERK), uses a different physical principle than the available single FP- and Förster Resonance Energy Transfer (FRET)-based kinase biosensors. (9) SPARK allows a high dynamic range in terms of the fluorescent change upon kinase activation. Drawing an analogy, we employed an AMPK-specific substrate peptide, previously utilized in developing single FP- and FRET-based genetic AMPK reporters, (4,7) to design AMPK-SPARK.
AMPK activation is a multifaceted process that involves various signaling pathways in response to cellular stresses. These stresses include energy-related challenges such as hypoxia, ischemia, and heat shock, where fluctuations in AMP/ATP ratios and fructose-1,6-bisphosphate (FBP) levels play pivotal roles in sensing energy stress by AMPK. (10,11) Cells respond to increases in AMP/ADP to ATP ratio and decrease in FBP by activating AMPK. The activation cascade involves two essential upstream kinases, liver kinase B1 (LKB1) and calcium/calmodulin-dependent protein kinase 2 (CaMKK2). (12) The direct phosphorylation of AMPK by CAMKK2, triggered by a rise in intracellular Ca2+ levels, thus adds another layer of complexity to the regulatory network of noncanonical AMPK activation. Deciphering Ca2+-mediated regulation of AMPK activity at the single-cell level would require coimaging spectrally distinct AMPK and Ca2+ biosensors. However, such coimaging may be challenging due to complex microscopy setups and spectral limitations. To circumvent these challenges, we built on a recent report that established signaling reporter islands (SiRIs) with clustered biosensors to monitor signaling networks, demonstrating that single FP-based biosensors remain functional within clusters. (13) Motivated by these findings, we replaced the enhanced green FP (EGFP) in AMPK-SPARK with a single FP-based Ca2+ biosensor, GCaMP6f, (14) to create a dual reporter capable of simultaneously tracking intracellular Ca2+ levels and AMPK activity. Named GCaMP-AMPK- SPARK, this dual reporter operates solely through one channel (GFP), effectively overcoming the spectral challenges. We demonstrate that imaging with GCaMP-AMPK-SPARK offers comprehensive insights into the nuanced regulation of AMPK activation by Ca2+, shedding light on the variability of Ca2+ signals in triggering AMPK activation.

Results and Discussion

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Design of EGFP-Based AMPK-SPARK

To achieve visualization of AMPK activity at the single-cell level with an enhanced dynamic range in terms of fluorescence change, we developed a multivalent interaction system based on phosphorylation-inducible phase separation, inspired by the recently established SPARK technology. (9) Like other phase separation-based kinase reporters, (15,16) AMPK-SPARK comprises two components (Figure 1A). In the first component, we fused the validated AMPK-specific substrate peptide sequence (4) with an enhanced GFP (EGFP) and a coiled-coil domain (a hexameric tag homo-oligomeric tag 3, (HOTag3)), as shown in Figure 1A. In the second component of the reporter, we fused a phospho-threonine-binding domain, the forkhead-associated domain 1 (FHA1), to another coiled-coil domain, the tetrameric tag homo-oligomeric tag 6 (HOTag6, Figure 1A). A self-cleaving T2A sequence (17) was used to link the two parts of the reporter to allow coexpression of both components. In cells where both components of SPARK-AMPK are present, activation of AMPK leads to multivalent protein–protein interactions, inducing phase separation resulting in intensely bright green fluorescent clusters, which, in turn, can reversibly dissolve upon activation of phosphatases (18) (Figure 1A).

Figure 1

Figure 1. Mapping endogenous AMPK activity in different cell types using SPARK technology. (A) Schematic shows two components of AMPK-SPARK and their working principle. The first component consists of the AMPK-specific substrate sequence, EGFP, and hexameric HOTag3. The second component consists of the phosphobinder FHA1 sequence and tetrameric HOTag6. Upon AMPK activation, the two components cluster and form bright fluorescent droplets due to phase separation. Phosphatases work in the opposite direction, and they work to dissolve the clusters formed. (B) Confocal images show different types of HEK293D cells based on the diameter (d) of the largest cluster observed, transiently expressing AMPK-SPARK. (C) Bar graphs represent the proportion of cells for the presence of clusters in HEK293D (n = 3/259), EA.hy926 (n = 2/85), and HeLa (n = 3/130) upon transient expression of AMPK-SPARK. (D) Graph displays the type distribution with respect to cells described in panel C.

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AMPK-SPARK Reveals Cell-to-Cell Variations in Endogenous AMPK Activity

Next, we assessed the relative energy stress status of different cell lines in vitro using AMPK-SPARK. Therefore, we transfected human embryonic kidney (HEK) 293 cells, the well-known cervical cancer cell line, HeLa, and immortalized human umbilical vein endothelial cells, the EA.hy926 cells, with the new SPARK reporter system. The cells were then imaged 2 days after transfection. Interestingly, while some cells expressing AMPK-SPARK had uniform fluorescence, others had droplets of different sizes, inferring varying states of cellular energy stress among the cells within the same population under these conditions (Figure 1B–D). In HEK 293 cells expressing SPARK-AMPK, 81% displayed droplets; while in EA.hy926 and HeLa cells, the proportions of cells with droplets were 58 and 44%, respectively (Figure 1C). Notably, these proportions exhibited marked variations when PKA-SPARK, the SPARK-based PKA reporter, (9) was expressed in the same cell lines (Figure S1A). Moreover, the phospho-null mutant reporter, AMPK(T>A)-SPARK, in which we substituted phospho-threonine with alanine to prevent phosphorylation by AMPK, demonstrated exclusively homogeneous fluorescence in the same cell lines (Figure S2). Based on these, we presumed that droplets formed upon AMPK-SPARK expression are intrinsic to AMPK activity and that they qualitatively offer insights into the on–off status of AMPK at the single-cell resolution. Exploiting the simple signal pattern of AMPK-SPARK, we classified three cases defined by the diameter of the largest droplet observed in a single cell. Suppose the diameter of the largest droplet observed is (i) smaller than 1.0 μm, (ii) between 1.0–3.6 μm, or (iii) larger than 3.6 μm, the cell was classified as type 1, type 2, or type 3, respectively (Figure 1B). Interestingly, we observed distinct distribution patterns of these AMPK-SPARK droplet types across the three cell types (Figure 1D), suggesting potential variations in endogenous AMPK and phosphatase activities or expression levels. Moreover, the observed heterogeneity within the same cell population and among different cell types may arise from the differential restriction of diffusion based on cell shape or macromolecular crowding levels between individual cells, which might impact both the kinase activities (19) or the phase separation. (20) Thus, the comparative interpretation of SPARK data should be done with caution. Furthermore, the expression of SPARK-PKA showed entirely different profiles of these types for the same cell lines (Figure S1B), suggesting inherent differences between endogenous AMPK and PKA activities.
Next, we investigated the impact of expression levels of AMPK-SPARK on cluster presence, count, volume, and sphericity (Figure S3) using high-resolution volumetric z-scan imaging of HeLa cells and compared the control and energy-stressed conditions. Independently of the cluster presence, we have cells over a wide spectrum of expression (Figure S3A). HeLa cells were pretreated with a glucose-free medium supplemented with 2-DG (2-Deoxy-d-glucose), a glucose analog that inhibits glycolysis and strongly reduces cellular ATP levels, (21,22) to induce severe energy stress. Such energy stress yielded a higher cluster count (Figure S3B), total cluster volume (Figure S3C), and mean cluster volume (Figure S3D) compared to the control condition. We observed no significant difference in cluster sphericity under these two conditions (Figure S3E). Moreover, we did not find any correlations between expression levels and cluster counts or sphericity. However, under energy stress, we determined a moderate correlation between expression levels and cluster volume (Figure S3C,D). Although these results indicate that the expression level has a minimal impact on cluster formation and morphology, we recommend using cells with similar expression levels for comparative analyses. If possible, then normalization to the expression level is advised. Additionally, we think that SPARK technology should be primarily utilized for qualitative rather than quantitative investigations.
Previous studies reported that the dissociation of Mg2+ from ATP influences cellular energy metabolism and response to energy stress. (23,24) However, the impact of the extracellular Mg2+ concentration on cellular energy stress remains unexplored. This aspect is particularly relevant in assessing the biodegradation of magnesium implants used in bone fixation surgeries. (25) To investigate this, we employed AMPK-SPARK in EA.hy926 cells cultured with varying Mg2+ concentrations (Figure S4). Without adding Mg2+ to the culture medium, we observed an increase of 14% of endothelial cells showing no AMPK-SPARK clusters, indicating that a reduction in Mg2+ might lower cellular energy stress. AMP binding to the γ subunit of the AMPK facilitates activation of AMPK, which is counteracted by the presence of Mg2+-unbound free ATP. (26,27) We think that reduced levels of Mg2+ (0 mM) may increase the levels of Mg2+-unbound free ATP, leading to lower AMPK activity. Surprisingly, high levels of extracellular Mg2+ (8 mM) showed no significant effect on the presence or size of AMPK-SPARK clusters compared to basal Mg2+ levels (0.8 mM) (Figure S4B). These findings suggest that high extracellular Mg2+ levels have a minimal impact on AMPK activity.
Delving deeper into the cells, we noted a distinct difference in the condensate morphology between AMPK-SPARK and PKA-SPARK clusters. Specifically, the clusters of AMPK-SPARK were predominantly spheroid in the three cell types tested (Figure S5A,B). In contrast, those of PKA-SPARK appeared to take on an amorphous structure in a considerable proportion across various cell types (Figure S5C,D). While the origin of these morphological differences remains unclear, it is intriguing to hypothesize that the morphology of SPARK clusters may indicate temporal variations in the interplay between the kinase and the phosphatase activities. (28) Furthermore, we also identified notable differences in how SPARK clusters are spatially arranged within the cell, which may reflect the cellular organization of kinases to regulate signaling. (29) While the spherical clusters of AMPK-SPARK are typically scattered over the cytosol (Figure S6A), the PKA-SPARK clusters were sporadically spotted at the subplasmalemmal space (Figure S6B) or around vesicular structures (Figure S6C,D). There is growing interest in investigating the biophysical principles and properties of biomolecular condensates. (30−32) We think a more comprehensive examination of the morphology and subcellular localization of SPARK clusters may help gain deeper insights into the spatial and temporal dynamics of kinase activity alterations with this technology.

AMPK-SPARK Dynamically Forms Clusters in Response to Canonical and Noncanonical Ways of Activation

The activation of AMPK occurs through distinct pathways: the canonical pathway, induced by changes in the ATP/ADP/AMP ratios, facilitated by the upstream kinase LKB1, and noncanonical pathways, including activation via elevated cytosolic Ca2+ levels, mediated by CaMKK2 (Figure 2A). (12) To achieve rapid activation of endogenous AMPK activity, we thus subjected HEK293 cells to glucose deprivation to trigger energy stress while simultaneously stimulating them with a mixture of compounds (ATP and BHQ) that efficiently mobilize Ca2+ to elevate cytosolic Ca2+ levels. The combination of canonical and noncanonical routes of AMPK activation resulted in the swift formation (1.56 ± 0.30 min, 8 cells) of numerous AMPK-SPARK droplets in multiple cells (Supporting Information, Videos 1 and 2). In some cells, the stimulation led to droplets that gradually increased in size and persisted even after stimulation, while smaller droplets swiftly dissipated (Supporting Information, Videos 1 and 2). This observation aligns with our findings of AMPK-SPARK droplets of varying sizes across different cell types in the culture (Figure 1B,D). However, most droplets (86% ± 5.62 of counted clusters in 8 cells) dispersed upon glucose reintroduction and withdrawal of the Ca2+-mobilizing compounds, pointing to the fundamental reversibility of the AMPK-SPARK tool following transient stimulation of the AMPK pathway (Supporting Information, Videos 1 and 2). To quantitatively illustrate the dynamic AMPK activity over time, utilizing the novel AMPK-SPARK tool, throughout the study, we opted for X–Y diagrams plotting the number of droplets of a single cell against the time. Upon induction of energy stress through glucose removal and simultaneous elevation of cytosolic Ca2+ by depleting Ca2+ stored in the endoplasmic reticulum (ER), a substantial and rapid increase in the count of individual AMPK-SPARK droplets was observed (Figure S7A,B). Following an initial peak, the droplet counts stabilized, gradually decreasing only after the end of stimulation (Figure S7B). The initial peak observed in the AMPK-SPARK curve is due to the emergence of small clusters. The signal then dropped as clusters coalesced over time (Supporting Information, Video 1, the middle-very left cell). Cells expressing AMPKAR, a FRET-based AMPK reporter, (4) immediately responded (1.07 ± 0.17 min, 7 cells) to the same strong stimulus. Overall, under identical conditions, we observed comparable readouts for the activation and the deactivation of endogenous AMPK in HEK293D cells between AMPKAR and AMPK-SPARK (Figure S7B,C). It should also be noted that while FRET-based or single FP-based AMPK reporters have a unimolecular design, AMPK-SPARK has a bimolecular design. Unimolecular biosensors go through a conformational change as fast as nanoseconds, (33) whereas bimolecular biosensors can be slowed down due to diffusion. (34) Moreover, to achieve a detectable signal, AMPK-SPARK needs to concentrate enough protein domains. Together, these all may delay the response time of the SPARK-based kinase biosensors (35) and make AMPK-SPARK less sensitive than unimolecular AMPK reporters.

Figure 2

Figure 2. AMPK-SPARK dynamically forms clusters in response to various stresses. (A) Schematic shows the fundamental mechanisms for the activation of AMPK, which consists of 3 subunits (α, β, γ). The confocal images demonstrate (B) AMPK-SPARK or (C) phospho-null AMPKT>A-SPARK expressing single EA.hy926 cells at indicated time points and their respective time-course traces of cluster formation upon perfusion with glucose-free imaging buffer supplemented with 5 mM 2-deoxyglucose (2-DG). (D) Three adjacent HEK293D cells (cell 1, 2, 3) have been subjected to Ca2+ mobilization from ER achieved by the administration of 100 μM carbachol and 15 μM BHQ. The confocal images and the corresponding time-course traces of cluster count of the indicated cells are presented in the top and bottom rows, respectively. The last panel in the bottom row depicts the comparative cluster count of the cells with a close-up time scale. Scale bars are 10 μm. All images were taken at 5 s intervals, with no binning, in confocal mode.

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Axotomy is an in vitro neuronal injury experimental model system for investigating cellular responses involving significant fluctuations in cellular ATP and Ca2+ levels. (36,37) To visualize AMPK activation during axotomy on the single-cell level, we introduced the AMPK-SPARK construct into freshly isolated primary mouse cortical neurons and monitored its behavior following axotomy (Figure S8). Out of 23 single axotomized neurons, 9 cells exhibited a clear AMPK-SPARK response that persisted throughout the observation period (Figure S8). It is noteworthy that not all neurons exhibited an AMPK-SPARK response to axonal injury, reflecting the inherent heterogeneity of neuronal responses. Collectively, these experiments underscore the applicability of SPARK technology for visualizing AMPK activation in neuronal cells.
Next, we treated endothelial cells with 2-DG, which induces robust energy stress. Following the substitution of glucose with 2-DG, a progressive elevation in the formation of AMPK-SPARK clusters was observed (Figure 2B), indicating gradual AMPK activation upon energy stress. Notably, no clusters emerged in 2-DG treated cells expressing the AMPK-insensitive variant AMPK(T>A)-SPARK (Figure 2C). These findings emphasize that SPARK-AMPK can also report the activation of AMPK solely by instigating energy stress through ATP reduction in the endothelial cell line. We then monitored the formation of AMPK-SPARK clusters induced exclusively via Ca2+ elevation by ER Ca2+ depletion in three adjacent HEK293 cells (Figure 2D). Despite the simultaneous treatment of the cells using a gravity-driven perfusion system with Ca2+-mobilizing compounds, the clusters did not emerge at the same time across the three cells; instead, they formed with noticeable temporal delays. These delays may imply variances in Ca2+ signal dynamics (including variances in amplitude, frequency, duration, delay, or cumulative level). (38) Moreover, different cellular responses to the induction of AMPK activation may stem from different capacities of ATP production and turnover, (39) elusive upstream mechanisms, (40) or metabolic history of the single cells. (40) Following Ca2+ mobilization, the number of clusters per cell declined significantly (Figure 2D and Supporting Information, Video 3), showing the reversibility of Ca2+-triggered AMPK activation. We hypothesize that the remaining clusters reflect a memory of the previous AMPK activation (Figure 2D and Supporting Information, Video 3).

Design of a Dual Ca2+ AMPK-SPARK Reporter System Integrating GCaMP6f

To shed light on Ca2+-mediated AMPK activity, we developed a dual reporter system that allows for simultaneous monitoring of intracellular Ca2+ levels and AMPK activity. To accomplish this, we replaced EGFP in AMPK-SPARK with a single FP-based Ca2+ biosensor, GCaMP6f. We named this new dual reversible reporter GCaMP-AMPK-SPARK, which enables the simultaneous monitoring of two parameters through a single GFP channel, providing a comprehensive view of Ca2+-coupled AMPK activation. While an increase in cytosolic fluorescence reflects a rise in intracellular Ca2+ levels, cluster formation reports an increase in AMPK activity (Figure 3A, Graphical Abstract).

Figure 3

Figure 3. GCaMP-AMPK-SPARK reported variances in Ca2+-mediated AMPK activation. (A) Cartoons depict clustered GCaMP-AMPK-SPARK reporters under AMPK-activated conditions; glucose deprivation, and Ca2+ elevation. Single units of component 1 under these two conditions are zoomed in for a better comparison. The cartoon at the very right depicts the components of the GCaMP-AMPK-SPARK construct. (B) EA.hy926 cells transiently transfected to express GCaMP-AMPK-SPARK. The first (binarized images to emphasize cluster count) and second (raw images to emphasize intensity changes) rows of images show a single EA.hy926 cell that has been subjected to glucose deprivation, glucose reintroduction, and Ca2+ elevation by 100 μM ATP and 15 μM BHQ at indicated time points. Cell outline was defined using raw images. The top graph shows the time-course quantification of cluster count, reporting AMPK activity over time (curve with green circles). The red curve in the bottom graph shows intensity changes in integrated GCaMP6f, reporting changes in cytosolic Ca2+ levels. Images were taken at 1 min intervals with 2 × 2 binning in confocal mode. (C) The graph showcases the overall intensity change of this cell in pink and the respective changes in AMPK activity with cluster count in green circles. Cells are subjected to Ca2+ elevation by perfusion of different buffers (including 100 μM ATP, 15 μM BHQ, or a combination of the two) at designated time points. Cells are subjected to Ca2+ removal by the perfusion of 0 mM Ca2+ imaging buffer with 1 mM EGTA at indicated time points. Single-cell heatmaps illustrate the changes in GCaMP-AMPK-SPARK intensity, i.e., cytosolic Ca2+ signals, and cluster count, i.e., AMPK-activation, to the given protocol in the graph. Images were taken at 3 s intervals with no binning in confocal mode.

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To test the functionality of the dual reporter, we expressed GCaMP-AMPK-SPARK in the EA.hy926 cells. We observed cluster formation without intensity elevation upon glucose removal, indicating Ca2+-independent AMPK activation. With glucose reprovision, the clusters dissolved, indicating the reversibility of GCaMP-AMPK-SPARK. Then, we further stressed the cell by using Ca2+ mobilizing agents and observed a significant increase in the number of clusters that emerged (Figure 3B, top row and Supporting Information, Video 4) and in the fluorescence intensity of the cell (Figure 3B, bottom row and Supporting Information, Video 5), indicating a concurrent rise in AMPK activity and cytosolic Ca2+ levels, respectively. The reduction in intensity observed (Figure 3B, bottom row) during cluster formation is likely due to the accumulation of FP out of the focal plane.

Visualizing Cell-to-Cell Heterogeneities of Ca2+-AMPK Coupling, Employing the Dual Reporter System

Next, we conducted simultaneous visualization of Ca2+ signals and AMPK activation of individual HeLa cells expressing the dual reporter system (Figure 3C). To induce varied Ca2+ signals, we treated cells sequentially with Ca2+-mobilizing compounds. Intriguingly, not all cytosolic Ca2+ signals led to a notable cluster formation. In Cell 1, initial ATP treatment did not induce cluster formation despite triggering a prominent Ca2+ elevation. Interestingly, the subsequent re-elevation of Ca2+ signals yielded conspicuous cluster formation, demonstrating discrepancies in Ca2+-coupled AMPK activation. We employed a color code to illustrate the outcomes of the dual reporter system in several cells from the same imaging region (Figure 3C, heatmaps). In all cells imaged, we observed similar temporal patterns of Ca2+ elevation upon administration of Ca2+ mobilizing agents, yet not all of these signals have translated into a parallel increase in AMPK activity (Figure 3C, Supporting Information, Video 6). Notably, the cluster formation of Cell 6 may not be determined due to its low expression of GCaMP-AMPK-SPARK. These experiments imply a cell-to-cell heterogeneity related to the implications of Ca2+ signals for AMPK activation. Additionally, we detected a similar variance in Ca2+-coupled AMPK activation using the Ca2+ ionophore, ionomycin (Figure S9). With ionomycin, intracellular Ca2+ levels are elevated to supraphysiological levels, minimizing the potential variances in Ca2+ signals. (38) Under these conditions, we observed adjacent cells exhibiting apparent differences in the temporal linkage between the Ca2+ signal and cluster formation, indicating heterogeneities in AMPK activation (Figure S9B,C). The heterogeneity among single cells is a common attribute of dynamic metabolic processes, (41,42) which has been reported for various aspects of subcellular AMPK activity as well as for other kinases. (5,7−9,39,40,43−45) Our results with cell lines and primary cultures of cortical neurons add to the expanding body of evidence supporting the metabolic heterogeneity at the single-cell level.

Conclusions

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The new phase-separation-based AMPK activity reporter, AMPK-SPARK, expands the toolkit of researchers who study AMPK signaling. This reporter works reversibly in a single fluorescent channel with a high signal-to-noise ratio. The high dynamic range in terms of fluorescent change amplifies the signal, making AMPK-SPARK less prone to artifacts, such as autofluorescence or light scattering effects. This makes AMPK-SPARK suitable for high-throughput drug screening and tissue applications. The simple signal pattern of SPARK-based sensors provides a clear and direct qualitative readout of kinase activity. On the other hand, while FRET-based or single FP-based AMPK biosensors are quantitative, AMPK-SPARK rather falls semiquantitative. AMPK-SPARK relies on the qualitative imaging of single cells, which restricts its quantitative use to heterogeneous cell populations. To quantify the relative status of AMPK activity between single cells using the SPARK constructs, one could choose to calculate the sum of the fluorescence intensity of clusters over the whole cell intensity, (9,15,16) the cluster count, (35) or size (46) or define cells based on the size of SPARK clusters. Moreover, the very small AMPK-SPARK clusters that lack sufficient contrast (against the nearby nonclustered fluorescence) cannot be determined. Therefore, the response of AMPK-SPARK biosensors may be delayed by protein recruitment, rendering the sensors less sensitive to minor changes in AMPK activity or when the sensor has low levels of expression. Furthermore, it should be noted that AMPK-SPARK lacks the high spatial resolution of targeted unimolecular AMPK biosensors.
The GCaMP-AMPK-SPARK combines SPARK technology with a single FP-based Ca2+ biosensor, demonstrating a proof of concept of a new dual reporter system. GCaMP-AMPK-SPARK can report Ca2+ levels, providing a comprehensive look into Ca2+-mediated AMPK activity. The dual reporter system is a refined method to monitor an intracellular messenger (i.e., Ca2+) and kinase activity (i.e., AMPK) in a single fluorescence channel, at the same time.
Imaging with GCaMP-AMPK-SPARK is thus advantageous, as it yields two layers of information simultaneously. However, integrating GCaMP6f, which has very large changes in fluorescence upon Ca2+ elevations, complicates cluster analysis based on intensities. For this reason, we opted for cluster counting throughout the study. While cluster counting can report the relative changes in AMPK activity in dynamic measurements, we acknowledge that it lacks the cluster size metrics, as well as the reciprocal information on nonclustered components in the cytosol. Although we intentionally utilized only one fluorescence channel in this study, our dual reporter approach, merging intensiometric single FP-based biosensors with SPARK technology, can be expanded to include additional colors. We anticipate that this expansion will enable the visualization of several signaling processes and their corresponding kinase activities in a multiplexing mode.

Experimental Section

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Buffers and Solutions

Cell culture materials were obtained from Greiner Bio-One (Kremsmünster, Austria). During z-stack imaging and before time-course imaging, cells were equilibrated and incubated in physiological storage solution (in mM): 2 CaCl2, 135 NaCl, 1 MgCl2, 5 KCl, 10 HEPES, 2.6 NaHCO3, 0.44 KH2PO4, 0.34 Na2HPO4, 1x amino acids, 1x vitamins, 10 glucose, and 2 l-glutamine with a pH of 7.45. The physiological imaging buffer used during time-course microscopy experiments contains (in mM): 138 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, and 10 d-glucose, pH adjusted to 7.4 with NaOH. The modifications on the imaging buffer are indicated in the figures as well as in the figure legends. The Ca2+-free imaging buffer contains (in mM): 138 NaCl, 5 KCl, 1 EGTA, 1 MgCl2, and 10 d-glucose, pH adjusted to 7.4 with NaOH. Adenosine 5′-triphosphate disodium salt (ATP) was purchased from Carl Roth (Graz, Austria). 2,5-di(tert-butyl)-1,4-hydroquinone (BHQ) was purchased from Sigma-Aldrich (Massachusetts, United States). 2-Deoxy-d-glucose (2-DG) was purchased from Thermo Fisher Scientific (Massachusetts, United States).

Construct Design

The SPARK-AMPK was generated by the substitution of kinase substrate sequence in the previously designed SPARK-PKA biosensor. (9) From the N-terminus, SPARK-AMPK insert consists of AMPK substrate peptide, (4) EGFP, the homohexameric coiled coil HOTag3 domain, self-cleavage sequence (T2A), phosphothreonine-binding forkhead-associated domain 1 (FHA1), and the homotetrameric coiled HOTag6 domain. For GCaMP-AMPK-SPARK, EGFP in SPARK-AMPK was replaced with GCaMP6f. (14) For AMPK(T>A)-SPARK, the phospho-threonine within the AMPK substrate sequence was replaced with an alanine. Inserts were synthesized and cloned into the pTwist CMV backbone by Twist Biosciences (California, United States).

Cell Culture and Transfection

HEK 293D, HeLa, and EA.hy926 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM D5523, Sigma-Aldrich) supplemented with 10% FCS, 10 mM sodium bicarbonate, 50 U/mL penicillin–streptomycin, 1.25 μg/mL amphotericin B, and 25 mM HEPES; pH was calibrated to 7.45 with NaOH. Cells were grown in a humified incubator (5% CO2, 37 °C). Cells were seeded on 30 mm glass coverslips (Co. KG, Lauda-Königshofen) in 6-well plates (Paul Marienfeld GmbH, Germany). For transient transfection, a transfection reagent PolyJet (SignaGen Laboratories, Rockville, USA) was used according to the manufacturer’s instructions. Live-cell imaging was performed 24–48 h after transfection.
For investigating the Mg2+ effect on cellular energy status, EA.hy926 cells were incubated in modified custom media (0MgCl2, 0NaCl) by Cytvia (Marlborough, United States) supplemented with 10% FCS, 10 mM sodium bicarbonate, 50 U/mL penicillin–streptomycin, 1.25 μg/mL amphotericin B, and 25 mM HEPES; pH was calibrated to 7.45 with NaOH. For different concentrations of MgCl2, NaCl levels were modified accordingly to maintain isoosmotic solutions.
Primary neuron cultures were established following previously described protocols and slightly adapted in this study to cortical neuron cultures. (36) Mice at the postnatal stage of p3 were humanely euthanized by using CO2, and their brains were carefully isolated. The cortex was dissected and sliced into small pieces under a stereo microscope (Zeiss Discovery V8) in a solution containing 1% antibiotic-antimycotic (Sigma, A5955) and L15 medium (ThermoFisher Scientific, A1247501). Tissue digestion was carried out with papain (Sigma, P4762) at 4 °C for 45 min. Subsequently, DNase (Sigma, D5025) was introduced, and the tissues were homogenized through serial pipetting. The enzymatic activity was halted by adding 10% FBS at 4 °C for 15 min, and the medium was removed via centrifugation at 180g for 5 min. The isolated cells were resuspended in Neurobasal-A (Gibco, 10888022) supplemented with 1% antibiotic-antimycotic solution, 1% GlutaMAX (Gibco, 35050061), and 2% B27 (Gibco, 17504044). These cells were then plated on glass-bottom dishes coated with poly-d-lysine and maintained at 37 °C with 5% CO2. Following a 48 h incubation period, cells were transfected using lipofectamine (Invitrogen, 11668019) at a plasmid-to-lipofectamine ratio of 1:3, following the manufacturer’s instructions.

Animal Experiments

The animal experiments conducted in this study received approval from the Istanbul Medipol University Animal Experimentation Ethical Committee. All procedures were performed in strict accordance and full compliance with European Council Directive 2010/63/EU.

Live Cell Imaging

Before imaging, cells were preincubated in a physiological storage solution for at least 20 min at room temperature. For high-resolution live cell imaging, an array confocal laser scanning microscope (Axiovert 200 M, Zeiss), equipped with a 100×/1.45 NA oil immersion objective (Plan-Fluor, Zeiss) and a Nipkow-based confocal scanner unit (CSU-X1, Yokogawa Electric Corporation), was used. The fluorescent proteins within the biosensors were illuminated with an F488 diode laser (Visitron Systems, Puchheim, Germany). Emission light was collected with a CoolSNAP HQ2 CCD Camera (Photometrics Tucson, Arizona, USA) using the ET525/36m emission filter (Chroma Technology Corporation). Alternatively, a Nikon Eclipse Ti2 microscope was used for time-lapse imaging. The microscope was equipped with a 40 ×/1.15 NA water immersion objective (CFI Apochromat, Nikon), 100×/1.45 NA oil objective (CFI Apochromat, Nikon) standard filter sets, and two Kinetix Scientific CMOS cameras (Photometrics). Excitation of reporters was achieved with 476 nm laser light (Celesta, Light Engine) and emission was collected at 501–521 nm.
For FRET-imaging of AMPKAR, we employed an NGFI AnglerFish C–Y7G widefield microscope equipped with a 40× oil immersion objective (Plan Apochromat 1,3 NA Oil DIC (UV) VISIR, Carl Zeiss GmbH, Vienna, Austria) and a standard CFP/YFP filter cube. The biosensor was excited at 440 nm (440AF21, Omega Optical, Brattleboro, VT, USA), and emissions were collected at 480 and 535 nm (480AF30 and 535AF26, Omega Optical, Brattleboro, VT, USA) using a 505 dcxr beam splitter on two sides of the camera (CCD camera, CoolSNAP Dyno, Photometrics, Tucson, AZ, USA).
During the time-course measurements, cells were continuously perfused via a gravity-based perfusion system (NGFI, Austria).
For laser axotomy, neurons were preincubated in a physiological salt solution (pH 7.42) for 1 h at room temperature before imaging. Continuous time-lapse images were acquired using a confocal microscope (Zeiss LSM 780) equipped with a 40x/1.4 oil objective and a 488 nm excitation laser. The detector was tuned to capture emissions ranging from 493 to 598 nm, and the pixel dwell time was set at 1.27 μs. Laser axotomy was performed during the time-lapse imaging using a femtosecond laser system (Coherent) tuned to 790 nm with a 690+ MBS filter. The laser was operated at 100% power with 3 iterations.

Image Analysis

For cluster quantification, images were processed in ImageJ. For AMPK-SPARK, the stack of time-lapsed images was analyzed using a macro script to run the “Find Maxima/Count” function by manual thresholding.
GCaMP-AMPK-SPARK cluster quantification was done by using the same function. Here, two concurrent thresholding were done; a high threshold to capture clusters that emerged under increased Ca2+ levels and a lower threshold to capture clusters when Ca2+ levels are low. These two data points were then merged manually.
For analyses in Figure S3, confocal images were background subtracted using a background ROI. Using the corrected z-stack, the mean intensity of each cell was determined. To evaluate the cluster count and morphology, the original z-stacks were background corrected using a rolling ball background subtraction. To increase the contrast and simplify segmentation, the background corrected image was pixel-wise multiplicated with itself and subsequently scaled down to an 8-bit format. A Triangle auto threshold using the z-stack intensity histogram was applied to segment single clusters. The Fiji plugins 3D Volume, 3D Compactness, and 3D Ellipsoid were used to measure cluster count, cluster volume, and cluster sphericity, respectively.
For Figure 3B and Supporting Information,Video 4, images were subjected to background subtraction with a rolling stack and each frame multiplied with itself to create a 32-bit stack. Then, the minimum and maximum displayed values were set to each frame’s mean and maximal fluorescence, respectively. Images were converted to 16 bit. Autothresholding with the Max Entropy function was applied to the stack images. The stack was then inverted. The Analyze Particles function was then applied to obtain the mask with clusters. The outline of the cell was generated using raw cell images through the Binary/Outline function. The cluster mask and outline mask were then merged to make the cellular boundary apparent.

Data and Statistical Analysis

The data obtained were analyzed via Excel (Microsoft), MetaMorph (Molecular devices), and GraphPad Prism 5 Software (GraphPad Software, Inc., La Jolla, CA, USA).
To depict the single-cell heatmaps of AMPK activity and cytosolic Ca2+ levels in Figure 3D, we used the Conditional Formatting function in Excel. The manually corrected cluster count data and background subtracted fluorescence intensity data from image analysis were used for the first set of heatmaps (Figure 3D, left). For the second set of heatmaps (Figure 3D, right), we normalized the data to the maximal cluster count and maximal fluorescence of the individual cell.
The number of independent experiments is given as ‘X’, and the number of single cells is ‘x’. Each figure legend contains this statistical information as n = X/x. For instance, n = 3/120 indicates 3 biological replicates and a total of 120 cells that were analyzed.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssensors.4c01058.

  • Mapping endogenous PKA activity in different cell types using the SPARK technology; phospho-null mutant of AMPK-SPARK not showing any clusters upon its expression; expression level of AMPK-SPARK not correlating with cluster count or sphericity; elevated extracellular Mg2+ levels show higher AMPK activity than reduced extracellular Mg2+ levels; cluster morphology difference between AMPK-SPARK and PKA-SPARK; clusters of AMPK-SPARK and PKA-SPARK with different spatial arrangements; AMPK-SPARK and FRET-biosensor AMPKAR show analogous readouts; primary cortical neurons exhibit heterogeneous AMPK responses to axotomy injury; and dual reporter unveils variances in Ca2+-mediated AMPK activation under supraphysiological Ca2+ levels (PDF)

  • Canonical and noncanonical activation of AMPK in HEK293 cells expressing AMPK-SPARK (Position 1) (AVI)

  • Canonical and noncanonical activation of AMPK in HEK293 cells expressing AMPK-SPARK (Position 2); (AVI)

  • Ca2+ elevation mediated AMPK activation in HEK293 cells expressing AMPK-SPARK (AVI)

  • Cluster emergence and dissolution upon glucose removal, glucose reintroduction, and Ca2+ elevation in a EA.hy926 cell expressing GCaMP-AMPK-SPARK (AVI)

  • Intensity changes upon glucose removal, glucose reintroduction, and Ca2+ elevation in an EA.hy926 cell expressing GCaMP-AMPK-SPARK (AVI)

  • HeLa cells expressing GCaMP-AMPK-SPARK in response to sequential increase and reduction of cytosolic Ca2+ (MP4)

Terms & Conditions

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 Author
  • Authors
    • Yusuf C. Erdoğan - Gottfried Schatz Research Center, Molecular Biology and Biochemistry, Medical University of Graz, Neue Stiftingtalstraße 6, Graz 8010, Austria;  BioTechMed Graz, Mozartgasse 12/2, Graz 8010, Austria
    • Johannes Pilic - Gottfried Schatz Research Center, Molecular Biology and Biochemistry, Medical University of Graz, Neue Stiftingtalstraße 6, Graz 8010, Austria
    • Benjamin Gottschalk - Gottfried Schatz Research Center, Molecular Biology and Biochemistry, Medical University of Graz, Neue Stiftingtalstraße 6, Graz 8010, Austria
    • Esra N. Yiğit - Regenerative and Restorative Medicine Research Center (REMER), Research Institute for Health Sciences and Technologies (SABITA), Istanbul Medipol University, Istanbul 34810, Turkey;  Department of Physiology, International School of Medicine, İstanbul Medipol University, İstanbul 34810, Türkiye
    • Asal G. Zaki - Regenerative and Restorative Medicine Research Center (REMER), Research Institute for Health Sciences and Technologies (SABITA), Istanbul Medipol University, Istanbul 34810, Turkey
    • Gürkan Öztürk - Regenerative and Restorative Medicine Research Center (REMER), Research Institute for Health Sciences and Technologies (SABITA), Istanbul Medipol University, Istanbul 34810, Turkey;  Orcidhttps://orcid.org/0000-0003-0352-1947
    • Emrah Eroğlu - Regenerative and Restorative Medicine Research Center (REMER), Research Institute for Health Sciences and Technologies (SABITA), Istanbul Medipol University, Istanbul 34810, Turkey
    • Begüm Okutan - Department of Orthopedics and Traumatology, Medical University of Graz, Auenbruggerplatz 5, Graz 8036, Austria;  Orcidhttps://orcid.org/0000-0002-4951-5940
    • Nicole G. Sommer - Department of Orthopedics and Traumatology, Medical University of Graz, Auenbruggerplatz 5, Graz 8036, Austria
    • Annelie M. Weinberg - Department of Orthopedics and Traumatology, Medical University of Graz, Auenbruggerplatz 5, Graz 8036, Austria
    • Rainer Schindl - Gottfried Schatz Research Center, Biophysics, Medical University of Graz, Neue Stiftingtalstrasse 6, Graz 8010, Austria
    • Wolfgang F. Graier - Gottfried Schatz Research Center, Molecular Biology and Biochemistry, Medical University of Graz, Neue Stiftingtalstraße 6, Graz 8010, Austria;  BioTechMed Graz, Mozartgasse 12/2, Graz 8010, Austria;  Orcidhttps://orcid.org/0000-0003-1871-3298
  • Author Contributions

    Y.C.E. designed the AMPK-SPARK, AMPK(T>A)-SPARK, and GCaMP-AMPK-SPARK constructs, performed experiments, and analyzed data. J.P and B.G. assisted in-image analysis. B.O. and N.G.S. assisted in the experimental design of the Mg2+ experiment. E.N.Y, A.G.Z, G.Ö, and E.E designed and performed the neuronal experiments. Y.C.E., E.E., R.S., A.M.W., R.M., and W.F.G contributed to the interpretation of results and the experimental design. R.M. supervised the project. Y.C.E. and R.M. wrote the first draft of the manuscript. All authors contributed to the final version.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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We would like to acknowledge Anna Schreilechner and Rene Rost for their technical support in cell culture. We appreciate Fatih Kuru for his computational assistance. AMPKAR was obtained from Addgene (Plasmid #35097) and was a kind gift from Lewis Cantley (Harvard Medical School). The research was funded by the Ph.D. program Molecular Medicine (MOLMED) of the Medical University of Graz, by Nikon Austria within the Nikon-Center of Excellence, Graz, the Austrian Science Fund (FWF) projects I3716–B27 to R.M. and I5474 to A.M.W, the doctoral program Metabolic and Cardiovascular Disease (DK-W1226), and P27070 to W.F.G. The Nikon Center of Excellence, Graz, is supported by the Austrian infrastructure program 2013/2014, Nikon Austria Inc., and BioTechMed, Graz.

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    Cell Metabolism (2018), 27 (2), 299-313CODEN: CMEEB5; ISSN:1550-4131. (Elsevier Inc.)
    Mammalian AMPK is known to be activated by falling cellular energy status, signaled by rising AMP/ATP and ADP/ATP ratios. We review recent information about how this occurs but also discuss new studies suggesting that AMPK is able to sense glucose availability independently of changes in adenine nucleotides. The glycolytic intermediate fructose-1,6-bisphosphate (FBP) is sensed by aldolase, which binds to the v-ATPase on the lysosomal surface. In the absence of FBP, interactions between aldolase and the v-ATPase are altered, allowing formation of an AXIN-based AMPK-activation complex contg. the v-ATPase, Ragulator, AXIN, LKB1, and AMPK, causing increased Thr172 phosphorylation and AMPK activation. This nutrient-sensing mechanism activates AMPK but also primes it for further activation if cellular energy status subsequently falls. Glucose sensing at the lysosome, in which AMPK and other components of the activation complex act antagonistically with another key nutrient sensor, mTORC1, may have been one of the ancestral roles of AMPK.
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  11. 11
    Zhang, C. S.; Hawley, S. A.; Zong, Y.; Li, M.; Wang, Z.; Gray, A.; Ma, T.; Cui, J.; Feng, J. W.; Zhu, M. Fructose-1,6-bisphosphate and aldolase mediate glucose sensing by AMPK. Nature 2017, 548, 112– 116,  DOI: 10.1038/nature23275
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    11
    Fructose-1,6-bisphosphate and aldolase mediate glucose sensing by AMPK
    Zhang, Chen-Song; Hawley, Simon A.; Zong, Yue; Li, Mengqi; Wang, Zhichao; Gray, Alexander; Ma, Teng; Cui, Jiwen; Feng, Jin-Wei; Zhu, Mingjiang; Wu, Yu-Qing; Li, Terytty Yang; Ye, Zhiyun; Lin, Shu-Yong; Yin, Huiyong; Piao, Hai-Long; Hardie, D. Grahame; Lin, Sheng-Cai
    Nature (London, United Kingdom) (2017), 548 (7665), 112-116CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    The major energy source for most cells is glucose, from which ATP is generated via glycolysis and/or oxidative metab. Glucose deprivation activates AMP-activated protein kinase (AMPK), but it is unclear whether this activation occurs solely via changes in AMP or ADP, the classical activators of AMPK. Here, we describe an AMP/ADP-independent mechanism that triggers AMPK activation by sensing the absence of fructose-1,6-bisphosphate (FBP), with AMPK being progressively activated as extracellular glucose and intracellular FBP decrease. When unoccupied by FBP, aldolases promote the formation of a lysosomal complex contg. at least v-ATPase, regulator, axin, liver kinase B1 (LKB1) and AMPK, which has previously been shown to be required for AMPK activation. Knockdown of aldolases activates AMPK even in cells with abundant glucose, whereas the catalysis-defective D34S aldolase mutant, which still binds FBP, blocks AMPK activation. Cell-free reconstitution assays show that addn. of FBP disrupts the assocn. of axin and LKB1 with v-ATPase and regulator. Importantly, in some cell types AMP/ATP and ADP/ATP ratios remain unchanged during acute glucose starvation, and intact AMP-binding sites on AMPK are not required for AMPK activation. These results establish that aldolase, as well as being a glycolytic enzyme, is a sensor of glucose availability that regulates AMPK.
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  12. 12
    Steinberg, G. R.; Hardie, D. G. New insights into activation and function of the AMPK. Nat. Rev. Mol. Cell Biol. 2023, 24, 255– 272,  DOI: 10.1038/s41580-022-00547-x
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    12
    New insights into activation and function of the AMPK
    Steinberg, Gregory R.; Hardie, D. Grahame
    Nature Reviews Molecular Cell Biology (2023), 24 (4), 255-272CODEN: NRMCBP; ISSN:1471-0072. (Nature Portfolio)
    A review. Abstr.: The classical role of AMP-activated protein kinase (AMPK) is as a cellular energy sensor activated by falling energy status, signalled by increases in AMP to ATP and ADP to ATP ratios. Once activated, AMPK acts to restore energy homeostasis by promoting ATP-producing catabolic pathways while inhibiting energy-consuming processes. In this Review, we provide an update on this canonical (AMP/ADP-dependent) activation mechanism, but focus mainly on recently described non-canonical pathways, including those by which AMPK senses the availability of glucose, glycogen or fatty acids and by which it senses damage to lysosomes and nuclear DNA. We also discuss new findings on the regulation of carbohydrate and lipid metab., mitochondrial and lysosomal homeostasis, and DNA repair. Finally, we discuss the role of AMPK in cancer, obesity, diabetes, nonalcoholic steatohepatitis (NASH) and other disorders where therapeutic targeting may exert beneficial effects.
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    Linghu, C.; Johnson, S. L.; Valdes, P. A.; Shemesh, O. A.; Park, W. M.; Park, D.; Piatkevich, K. D.; Wassie, A. T.; Liu, Y.; An, B. Spatial Multiplexing of Fluorescent Reporters for Imaging Signaling Network Dynamics. Cell 2020, 183, 1682– 1698.e24,  DOI: 10.1016/j.cell.2020.10.035
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    Chen, T. W.; Wardill, T. J.; Sun, Y.; Pulver, S. R.; Renninger, S. L.; Baohan, A.; Schreiter, E. R.; Kerr, R. A.; Orger, M. B.; Jayaraman, V. 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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    Li, X.; Chung, C. I.; Yang, J. J.; Chaudhuri, S.; Munster, P. N.; Shu, X. ATM-SPARK: A GFP phase separation-based activity reporter of ATM. Sci. Adv. 2023, 9, 1– 14,  DOI: 10.1126/sciadv.ade3760
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    Li, X.; Combs, J. D.; Salaita, K.; Shu, X. Polarized focal adhesion kinase activity within a focal adhesion during cell migration. Nat. Chem. Biol. 2023, 19, 1458– 1468,  DOI: 10.1038/s41589-023-01353-y
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    De Felipe, P.; Luke, G. A.; Hughes, L. E.; Gani, D.; Halpin, C.; Ryan, M. D. E unum pluribus: Multiple proteins from a self-processing polyprotein. Trends Biotechnol. 2006, 24, 68– 75,  DOI: 10.1016/j.tibtech.2005.12.006
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    E unum pluribus: multiple proteins from a self-processing polyprotein
    De Felipe, Pablo; Luke, Garry A.; Hughes, Lorraine E.; Gani, David; Halpin, Claire; Ryan, Martin D.
    Trends in Biotechnology (2006), 24 (2), 68-75CODEN: TRBIDM; ISSN:0167-7799. (Elsevier Ltd.)
    A review. Many applications of genetic engineering require transformation with multiple (trans)genes, although to achieve these using conventional techniques can be challenging. The 2A oligopeptide is emerging as a highly effective new tool for the facile co-expression of multiple proteins in a single transformation step, whereby a gene encoding multiple proteins, linked by 2A sequences, is transcribed from a single promoter. The polyprotein self-processes co-translationally such that each constituent protein is generated as a discrete translation product. 2A functions in all the eukaryotic systems tested to date and has already been applied, with great success, to a broad range of biotechnol. applications: from plant metabolome engineering to the expression of T-cell receptor complexes, monoclonal antibodies or heterodimeric cytokines in animals.
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    Jeon, S. M. Regulation and function of AMPK in physiology and diseases. Exp. Mol. Med. 2016, 48, e245  DOI: 10.1038/EMM.2016.81
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    Sang, D.; Shu, T.; Pantoja, C. F.; Ibáñez de Opakua, A.; Zweckstetter, M.; Holt, L. J. Condensed-phase signaling can expand kinase specificity and respond to macromolecular crowding. Mol. Cell 2022, 82, 3693– 3711 e10,  DOI: 10.1016/j.molcel.2022.08.016
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    Condensed-phase signaling can expand kinase specificity and respond to macromolecular crowding
    Sang, Dajun; Shu, Tong; Pantoja, Christian F.; Ibanez de Opakua, Alain; Zweckstetter, Markus; Holt, Liam J.
    Molecular Cell (2022), 82 (19), 3693-3711.e10CODEN: MOCEFL; ISSN:1097-2765. (Elsevier Inc.)
    Phase sepn. can conc. biomols. and accelerate reactions. However, the mechanisms and principles connecting this mesoscale organization to signaling dynamics are difficult to dissect because of the pleiotropic effects assocd. with disrupting endogenous condensates. To address this limitation, we engineered new phosphorylation reactions within synthetic condensates. We generally found increased activity and broadened kinase specificity. Phosphorylation dynamics within condensates were rapid and could drive cell-cycle-dependent localization changes. High client concn. within condensates was important but not the main factor for efficient phosphorylation. Rather, the availability of many excess client-binding sites together with a flexible scaffold was crucial. Phosphorylation within condensates was also modulated by changes in macromol. crowding. Finally, the phosphorylation of the Alzheimer's-disease-assocd. protein Tau by cyclin-dependent kinase 2 was accelerated within condensates. Thus, condensates enable new signaling connections and can create sensors that respond to the biophys. properties of the cytoplasm.
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    Bonucci, M.; Shu, T.; Holt, L. J. How it feels in a cell. Trends Cell Biol. 2023, 33, 924– 938,  DOI: 10.1016/j.tcb.2023.05.002
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    How it feels in a cell
    Bonucci, Martina; Shu, Tong; Holt, Liam J.
    Trends in Cell Biology (2023), 33 (11), 924-938CODEN: TCBIEK; ISSN:0962-8924. (Elsevier Ltd.)
    A review. Life emerges from thousands of biochem. processes occurring within a shared intracellular environment. We have gained deep insights from in vitro reconstitution of isolated biochem. reactions. However, the reaction medium in test tubes is typically simple and dild. The cell interior is far more complex: macromols. occupy more than a third of the space, and energy-consuming processes agitate the cell interior. Here, we review how this crowded, active environment impacts the motion and assembly of macromols., with an emphasis on mesoscale particles (10-1000 nm diam.). We describe methods to probe and analyze the biophys. properties of cells and highlight how changes in these properties can impact physiol. and signaling, and potentially contribute to aging, and diseases, including cancer and neurodegeneration.
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    Rauter, T.; Burgstaller, S.; Gottschalk, B.; Ramadani-Muja, J.; Bischof, H.; Hay, J. C.; Graier, W. F.; Malli, R. ER-to-Golgi transport in hela cells displays high resilience to Ca2+ and energy stresses. Cells 2020, 9, 2311– 2326,  DOI: 10.3390/cells9102311
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    Pilic, J.; Gottschalk, B.; Bourgeois, B.; Habisch, H.; Koshenov, Z.; Oflaz, F. E.; Erdogan, Y. C.; Miri, S. M.; Yiğit, E. N.; Aydın, M. Ş. Hexokinase 1 forms rings that regulate mitochondrial fission during energy stress. Mol. Cell 2024, 84, 2732– 2746.e5,  DOI: 10.1016/j.molcel.2024.06.009
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    Yamanaka, R.; Tabata, S.; Shindo, Y.; Hotta, K.; Suzuki, K.; Soga, T.; Oka, K. Mitochondrial Mg2+ homeostasis decides cellular energy metabolism and vulnerability to stress. Sci. Rep. 2016, 6, 30027,  DOI: 10.1038/srep30027
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    Mitochondrial Mg2+ homeostasis decides cellular energy metabolism and vulnerability to stress
    Yamanaka, Ryu; Tabata, Sho; Shindo, Yutaka; Hotta, Kohji; Suzuki, Koji; Soga, Tomoyoshi; Oka, Kotaro
    Scientific Reports (2016), 6 (), 30027CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)
    Cellular energy prodn. processes are composed of many Mg2+ dependent enzymic reactions. In fact, dysregulation of Mg2+ homeostasis is involved in various cellular malfunctions and diseases. Recently, mitochondria, energy-producing organelles, have been known as major intracellular Mg2+ stores. Several biol. stimuli alter mitochondrial Mg2+ concn. by intracellular redistribution. However, in living cells, whether mitochondrial Mg2+ alteration affect cellular energy metab. remains unclear. Mg2+ transporter of mitochondrial inner membrane MRS2 is an essential component of mitochondrial Mg2+ uptake system. Here, we comprehensively analyzed intracellular Mg2+ levels and energy metab. in Mrs2 knockdown (KD) cells using fluorescence imaging and metabolome anal. Dysregulation of mitochondrial Mg2+ homeostasis disrupted ATP prodn. via shift of mitochondrial energy metab. and morphol. Moreover, Mrs2 KD sensitized cellular tolerance against cellular stress. These results indicate regulation of mitochondrial Mg2+via MRS2 critically decides cellular energy status and cell vulnerability via regulation of mitochondrial Mg2+ level in response to physiol. stimuli.
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    Pilchova, I.; Klacanova, K.; Tatarkova, Z.; Kaplan, P.; Racay, P. The Involvement of Mg2+ in Regulation of Cellular and Mitochondrial Functions. Oxid. Med. Cell. Longevity 2017, 2017, 6797460,  DOI: 10.1155/2017/6797460
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    The Involvement of Mg(2+) in Regulation of Cellular and Mitochondrial Functions
    Pilchova Ivana; Klacanova Katarina; Tatarkova Zuzana; Kaplan Peter; Racay Peter
    Oxidative medicine and cellular longevity (2017), 2017 (), 6797460 ISSN:.
    Mg(2+) is an essential mineral with pleotropic impacts on cellular physiology and functions. It acts as a cofactor of several important enzymes, as a regulator of ion channels such as voltage-dependent Ca(2+) channels and K(+) channels and on Ca(2+)-binding proteins. In general, Mg(2+) is considered as the main intracellular antagonist of Ca(2+), which is an essential secondary messenger initiating or regulating a great number of cellular functions. This review examines the effects of Mg(2+) on mitochondrial functions with a particular focus on energy metabolism, mitochondrial Ca(2+) handling, and apoptosis.
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    Suljevic, O.; Fischerauer, S. F.; Weinberg, A. M.; Sommer, N. G. Immunological reaction to magnesium-based implants for orthopedic applications. What do we know so far? A systematic review on in vivo studies. Mater. Today Bio 2022, 15, 100315,  DOI: 10.1016/j.mtbio.2022.100315
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    Xiao, B.; Sanders, M. J.; Underwood, E.; Heath, R.; Mayer, F. V.; Carmena, D.; Jing, C.; Walker, P. A.; Eccleston, J. F.; Haire, L. F. Structure of mammalian AMPK and its regulation by ADP. Nature 2011, 472, 230– 233,  DOI: 10.1038/nature09932
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    Structure of mammalian AMPK and its regulation by ADP
    Xiao, Bing; Sanders, Matthew J.; Underwood, Elizabeth; Heath, Richard; Mayer, Faith V.; Carmena, David; Jing, Chun; Walker, Philip A.; Eccleston, John F.; Haire, Lesley F.; Saiu, Peter; Howell, Steven A.; Aasland, Rein; Martin, Stephen R.; Carling, David; Gamblin, Steven J.
    Nature (London, United Kingdom) (2011), 472 (7342), 230-233CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    The heterotrimeric AMP-activated protein kinase (AMPK) has a key role in regulating cellular energy metab.; in response to a fall in intracellular ATP levels it activates energy-producing pathways and inhibits energy-consuming processes. AMPK has been implicated in a no. of diseases related to energy metab. including type 2 diabetes, obesity and, most recently, cancer. AMPK is converted from an inactive form to a catalytically competent form by phosphorylation of the activation loop within the kinase domain: AMP binding to the γ-regulatory domain promotes phosphorylation by the upstream kinase, protects the enzyme against dephosphorylation, as well as causing allosteric activation. Here we show that ADP binding to just one of the two exchangeable AXP (AMP/ADP/ATP) binding sites on the regulatory domain protects the enzyme from dephosphorylation, although it does not lead to allosteric activation. Our studies show that active mammalian AMPK displays significantly tighter binding to ADP than to Mg-ATP, explaining how the enzyme is regulated under physiol. conditions where the concn. of Mg-ATP is higher than that of ADP and much higher than that of AMP. We have detd. the crystal structure of an active AMPK complex. The structure shows how the activation loop of the kinase domain is stabilized by the regulatory domain and how the kinase linker region interacts with the regulatory nucleotide-binding site that mediates protection against dephosphorylation. From our biochem. and structural data we develop a model for how the energy status of a cell regulates AMPK activity.
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    Hardie, D. G. AMP-activated protein kinase-an energy sensor that regulates all aspects of cell function. Genes Dev. 2011, 25, 1895– 1908,  DOI: 10.1101/gad.17420111
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    AMP-activated protein kinase-an energy sensor that regulates all aspects of cell function
    Hardie, D. Grahame
    Genes & Development (2011), 25 (18), 1895-1908CODEN: GEDEEP; ISSN:0890-9369. (Cold Spring Harbor Laboratory Press)
    A review. AMP-activated protein kinase (AMPK) is a sensor of energy status that maintains cellular energy homeostasis. It arose very early during eukaryotic evolution, and its ancestral role may have been in the response to starvation. Recent work shows that the kinase is activated by increases not only in AMP, but also in ADP. Although best known for its effects on metab., AMPK has many other functions, including regulation of mitochondrial biogenesis and disposal, autophagy, cell polarity, and cell growth and proliferation. Both tumor cells and viruses establish mechanisms to down-regulate AMPK, allowing them to escape its restraining influences on growth.
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    Söding, J.; Zwicker, D.; Sohrabi-Jahromi, S.; Boehning, M.; Kirschbaum, J. Mechanisms for Active Regulation of Biomolecular Condensates. Trends Cell Biol. 2020, 30, 4– 14,  DOI: 10.1016/j.tcb.2019.10.006
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    Mechanisms for Active Regulation of Biomolecular Condensates
    Soding Johannes; Zwicker David; Kirschbaum Jan; Sohrabi-Jahromi Salma; Boehning Marc
    Trends in cell biology (2020), 30 (1), 4-14 ISSN:.
    Liquid-liquid phase separation is a key organizational principle in eukaryotic cells, on par with intracellular membranes. It allows cells to concentrate specific proteins into condensates, increasing reaction rates and achieving switch-like regulation. We propose two active mechanisms that can explain how cells regulate condensate formation and size. In both, the cell regulates the activity of an enzyme, often a kinase, that adds post-translational modifications to condensate proteins. In enrichment inhibition, the enzyme enriches in the condensate and weakens interactions, as seen in stress granules (SGs), Cajal bodies, and P granules. In localization-induction, condensates form around immobilized enzymes that strengthen interactions, as observed in DNA repair, transmembrane signaling, and microtubule assembly. These models can guide studies into the many emerging roles of biomolecular condensates.
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    Gormal, R. S.; Martinez-Marmol, R.; Brooks, A. J.; Meunier, F. A. Location, location, location: Protein kinase nanoclustering for optimised signalling output. elife 2024, 13, 1– 21,  DOI: 10.7554/eLife.93902
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    Banani, S. F.; Lee, H. O.; Hyman, A. A.; Rosen, M. K. Biomolecular condensates: Organizers of cellular biochemistry. Nat. Rev. Mol. Cell Biol. 2017, 18, 285– 298,  DOI: 10.1038/nrm.2017.7
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    Biomolecular condensates: organizers of cellular biochemistry
    Banani, Salman F.; Lee, Hyun O.; Hyman, Anthony A.; Rosen, Michael K.
    Nature Reviews Molecular Cell Biology (2017), 18 (5), 285-298CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)
    A review. Biomol. condensates are micron-scale compartments in eukaryotic cells that lack surrounding membranes but function to conc. proteins and nucleic acids. These condensates are involved in diverse processes, including RNA metab., ribosome biogenesis, the DNA damage response and signal transduction. Recent studies have shown that liq.-liq. phase sepn. driven by multivalent macromol. interactions is an important organizing principle for biomol. condensates. With this phys. framework, it is now possible to explain how the assembly, compn., phys. properties and biochem. and cellular functions of these important structures are regulated.
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    Alberti, S.; Gladfelter, A.; Mittag, T. Considerations and Challenges in Studying Liquid-Liquid Phase Separation and Biomolecular Condensates. Cell 2019, 176, 419– 434,  DOI: 10.1016/j.cell.2018.12.035
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    Considerations and Challenges in Studying Liquid-Liquid Phase Separation and Biomolecular Condensates
    Alberti, Simon; Gladfelter, Amy; Mittag, Tanja
    Cell (Cambridge, MA, United States) (2019), 176 (3), 419-434CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
    A review. Evidence is now mounting that liq.-liq. phase sepn. (LLPS) underlies the formation of membraneless compartments in cells. This realization has motivated major efforts to delineate the function of such biomol. condensates in normal cells and their roles in contexts ranging from development to age-related disease. There is great interest in understanding the underlying biophys. principles and the specific properties of biol. condensates with the goal of bringing insights into a wide range of biol. processes and systems. The explosion of physiol. and pathol. contexts involving LLPS requires clear stds. for their study. Here, we propose guidelines for rigorous exptl. characterization of LLPS processes in vitro and in cells, discuss the caveats of common exptl. approaches, and point out exptl. and theor. gaps in the field.
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    Wingreen, N. S.; Brangwynne, C. P.; Panagiotopoulos, A. Z.; Wingreen, N. S. Interfacial Exchange Dynamics of Biomolecular Condensates are Highly Sensitive to Client Interactions. J. Chem. Phys. 2024, 160, 145102,  DOI: 10.1063/5.0188461
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    Bu, Z.; Callaway, D. J. E. Proteins move! Protein dynamics and long-range allostery in cell signaling. In Advances in Protein Chemistry and Structural Birology, 1st ed.; Elsevier Inc., 2011; Vol. 83, pp 163– 221.  DOI: 10.1016/B978-0-12-381262-9.00005-7 .
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    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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    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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  • Abstract

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    Figure 1

    Figure 1. Mapping endogenous AMPK activity in different cell types using SPARK technology. (A) Schematic shows two components of AMPK-SPARK and their working principle. The first component consists of the AMPK-specific substrate sequence, EGFP, and hexameric HOTag3. The second component consists of the phosphobinder FHA1 sequence and tetrameric HOTag6. Upon AMPK activation, the two components cluster and form bright fluorescent droplets due to phase separation. Phosphatases work in the opposite direction, and they work to dissolve the clusters formed. (B) Confocal images show different types of HEK293D cells based on the diameter (d) of the largest cluster observed, transiently expressing AMPK-SPARK. (C) Bar graphs represent the proportion of cells for the presence of clusters in HEK293D (n = 3/259), EA.hy926 (n = 2/85), and HeLa (n = 3/130) upon transient expression of AMPK-SPARK. (D) Graph displays the type distribution with respect to cells described in panel C.

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    Figure 2

    Figure 2. AMPK-SPARK dynamically forms clusters in response to various stresses. (A) Schematic shows the fundamental mechanisms for the activation of AMPK, which consists of 3 subunits (α, β, γ). The confocal images demonstrate (B) AMPK-SPARK or (C) phospho-null AMPKT>A-SPARK expressing single EA.hy926 cells at indicated time points and their respective time-course traces of cluster formation upon perfusion with glucose-free imaging buffer supplemented with 5 mM 2-deoxyglucose (2-DG). (D) Three adjacent HEK293D cells (cell 1, 2, 3) have been subjected to Ca2+ mobilization from ER achieved by the administration of 100 μM carbachol and 15 μM BHQ. The confocal images and the corresponding time-course traces of cluster count of the indicated cells are presented in the top and bottom rows, respectively. The last panel in the bottom row depicts the comparative cluster count of the cells with a close-up time scale. Scale bars are 10 μm. All images were taken at 5 s intervals, with no binning, in confocal mode.

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    Figure 3

    Figure 3. GCaMP-AMPK-SPARK reported variances in Ca2+-mediated AMPK activation. (A) Cartoons depict clustered GCaMP-AMPK-SPARK reporters under AMPK-activated conditions; glucose deprivation, and Ca2+ elevation. Single units of component 1 under these two conditions are zoomed in for a better comparison. The cartoon at the very right depicts the components of the GCaMP-AMPK-SPARK construct. (B) EA.hy926 cells transiently transfected to express GCaMP-AMPK-SPARK. The first (binarized images to emphasize cluster count) and second (raw images to emphasize intensity changes) rows of images show a single EA.hy926 cell that has been subjected to glucose deprivation, glucose reintroduction, and Ca2+ elevation by 100 μM ATP and 15 μM BHQ at indicated time points. Cell outline was defined using raw images. The top graph shows the time-course quantification of cluster count, reporting AMPK activity over time (curve with green circles). The red curve in the bottom graph shows intensity changes in integrated GCaMP6f, reporting changes in cytosolic Ca2+ levels. Images were taken at 1 min intervals with 2 × 2 binning in confocal mode. (C) The graph showcases the overall intensity change of this cell in pink and the respective changes in AMPK activity with cluster count in green circles. Cells are subjected to Ca2+ elevation by perfusion of different buffers (including 100 μM ATP, 15 μM BHQ, or a combination of the two) at designated time points. Cells are subjected to Ca2+ removal by the perfusion of 0 mM Ca2+ imaging buffer with 1 mM EGTA at indicated time points. Single-cell heatmaps illustrate the changes in GCaMP-AMPK-SPARK intensity, i.e., cytosolic Ca2+ signals, and cluster count, i.e., AMPK-activation, to the given protocol in the graph. Images were taken at 3 s intervals with no binning in confocal mode.

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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXkslelsQ%253D%253D&md5=62917ca05b9b1699fb248d6a951736a3
    10. 10
      Lin, S. C.; Hardie, D. G. AMPK: Sensing Glucose as well as Cellular Energy Status. Cell Metab. 2018, 27, 299– 313,  DOI: 10.1016/j.cmet.2017.10.009
      10
      AMPK: Sensing Glucose as well as Cellular Energy Status
      Lin, Sheng-Cai; Hardie, D. Grahame
      Cell Metabolism (2018), 27 (2), 299-313CODEN: CMEEB5; ISSN:1550-4131. (Elsevier Inc.)
      Mammalian AMPK is known to be activated by falling cellular energy status, signaled by rising AMP/ATP and ADP/ATP ratios. We review recent information about how this occurs but also discuss new studies suggesting that AMPK is able to sense glucose availability independently of changes in adenine nucleotides. The glycolytic intermediate fructose-1,6-bisphosphate (FBP) is sensed by aldolase, which binds to the v-ATPase on the lysosomal surface. In the absence of FBP, interactions between aldolase and the v-ATPase are altered, allowing formation of an AXIN-based AMPK-activation complex contg. the v-ATPase, Ragulator, AXIN, LKB1, and AMPK, causing increased Thr172 phosphorylation and AMPK activation. This nutrient-sensing mechanism activates AMPK but also primes it for further activation if cellular energy status subsequently falls. Glucose sensing at the lysosome, in which AMPK and other components of the activation complex act antagonistically with another key nutrient sensor, mTORC1, may have been one of the ancestral roles of AMPK.
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    11. 11
      Zhang, C. S.; Hawley, S. A.; Zong, Y.; Li, M.; Wang, Z.; Gray, A.; Ma, T.; Cui, J.; Feng, J. W.; Zhu, M. Fructose-1,6-bisphosphate and aldolase mediate glucose sensing by AMPK. Nature 2017, 548, 112– 116,  DOI: 10.1038/nature23275
      11
      Fructose-1,6-bisphosphate and aldolase mediate glucose sensing by AMPK
      Zhang, Chen-Song; Hawley, Simon A.; Zong, Yue; Li, Mengqi; Wang, Zhichao; Gray, Alexander; Ma, Teng; Cui, Jiwen; Feng, Jin-Wei; Zhu, Mingjiang; Wu, Yu-Qing; Li, Terytty Yang; Ye, Zhiyun; Lin, Shu-Yong; Yin, Huiyong; Piao, Hai-Long; Hardie, D. Grahame; Lin, Sheng-Cai
      Nature (London, United Kingdom) (2017), 548 (7665), 112-116CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      The major energy source for most cells is glucose, from which ATP is generated via glycolysis and/or oxidative metab. Glucose deprivation activates AMP-activated protein kinase (AMPK), but it is unclear whether this activation occurs solely via changes in AMP or ADP, the classical activators of AMPK. Here, we describe an AMP/ADP-independent mechanism that triggers AMPK activation by sensing the absence of fructose-1,6-bisphosphate (FBP), with AMPK being progressively activated as extracellular glucose and intracellular FBP decrease. When unoccupied by FBP, aldolases promote the formation of a lysosomal complex contg. at least v-ATPase, regulator, axin, liver kinase B1 (LKB1) and AMPK, which has previously been shown to be required for AMPK activation. Knockdown of aldolases activates AMPK even in cells with abundant glucose, whereas the catalysis-defective D34S aldolase mutant, which still binds FBP, blocks AMPK activation. Cell-free reconstitution assays show that addn. of FBP disrupts the assocn. of axin and LKB1 with v-ATPase and regulator. Importantly, in some cell types AMP/ATP and ADP/ATP ratios remain unchanged during acute glucose starvation, and intact AMP-binding sites on AMPK are not required for AMPK activation. These results establish that aldolase, as well as being a glycolytic enzyme, is a sensor of glucose availability that regulates AMPK.
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    12. 12
      Steinberg, G. R.; Hardie, D. G. New insights into activation and function of the AMPK. Nat. Rev. Mol. Cell Biol. 2023, 24, 255– 272,  DOI: 10.1038/s41580-022-00547-x
      12
      New insights into activation and function of the AMPK
      Steinberg, Gregory R.; Hardie, D. Grahame
      Nature Reviews Molecular Cell Biology (2023), 24 (4), 255-272CODEN: NRMCBP; ISSN:1471-0072. (Nature Portfolio)
      A review. Abstr.: The classical role of AMP-activated protein kinase (AMPK) is as a cellular energy sensor activated by falling energy status, signalled by increases in AMP to ATP and ADP to ATP ratios. Once activated, AMPK acts to restore energy homeostasis by promoting ATP-producing catabolic pathways while inhibiting energy-consuming processes. In this Review, we provide an update on this canonical (AMP/ADP-dependent) activation mechanism, but focus mainly on recently described non-canonical pathways, including those by which AMPK senses the availability of glucose, glycogen or fatty acids and by which it senses damage to lysosomes and nuclear DNA. We also discuss new findings on the regulation of carbohydrate and lipid metab., mitochondrial and lysosomal homeostasis, and DNA repair. Finally, we discuss the role of AMPK in cancer, obesity, diabetes, nonalcoholic steatohepatitis (NASH) and other disorders where therapeutic targeting may exert beneficial effects.
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    13. 13
      Linghu, C.; Johnson, S. L.; Valdes, P. A.; Shemesh, O. A.; Park, W. M.; Park, D.; Piatkevich, K. D.; Wassie, A. T.; Liu, Y.; An, B. Spatial Multiplexing of Fluorescent Reporters for Imaging Signaling Network Dynamics. Cell 2020, 183, 1682– 1698.e24,  DOI: 10.1016/j.cell.2020.10.035
      There is no corresponding record for this reference.
    14. 14
      Chen, T. W.; Wardill, T. J.; Sun, Y.; Pulver, S. R.; Renninger, S. L.; Baohan, A.; Schreiter, E. R.; Kerr, R. A.; Orger, M. B.; Jayaraman, V. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 2013, 499, 295– 300,  DOI: 10.1038/nature12354
      14
      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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    15. 15
      Li, X.; Chung, C. I.; Yang, J. J.; Chaudhuri, S.; Munster, P. N.; Shu, X. ATM-SPARK: A GFP phase separation-based activity reporter of ATM. Sci. Adv. 2023, 9, 1– 14,  DOI: 10.1126/sciadv.ade3760
      There is no corresponding record for this reference.
    16. 16
      Li, X.; Combs, J. D.; Salaita, K.; Shu, X. Polarized focal adhesion kinase activity within a focal adhesion during cell migration. Nat. Chem. Biol. 2023, 19, 1458– 1468,  DOI: 10.1038/s41589-023-01353-y
      There is no corresponding record for this reference.
    17. 17
      De Felipe, P.; Luke, G. A.; Hughes, L. E.; Gani, D.; Halpin, C.; Ryan, M. D. E unum pluribus: Multiple proteins from a self-processing polyprotein. Trends Biotechnol. 2006, 24, 68– 75,  DOI: 10.1016/j.tibtech.2005.12.006
      17
      E unum pluribus: multiple proteins from a self-processing polyprotein
      De Felipe, Pablo; Luke, Garry A.; Hughes, Lorraine E.; Gani, David; Halpin, Claire; Ryan, Martin D.
      Trends in Biotechnology (2006), 24 (2), 68-75CODEN: TRBIDM; ISSN:0167-7799. (Elsevier Ltd.)
      A review. Many applications of genetic engineering require transformation with multiple (trans)genes, although to achieve these using conventional techniques can be challenging. The 2A oligopeptide is emerging as a highly effective new tool for the facile co-expression of multiple proteins in a single transformation step, whereby a gene encoding multiple proteins, linked by 2A sequences, is transcribed from a single promoter. The polyprotein self-processes co-translationally such that each constituent protein is generated as a discrete translation product. 2A functions in all the eukaryotic systems tested to date and has already been applied, with great success, to a broad range of biotechnol. applications: from plant metabolome engineering to the expression of T-cell receptor complexes, monoclonal antibodies or heterodimeric cytokines in animals.
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    18. 18
      Jeon, S. M. Regulation and function of AMPK in physiology and diseases. Exp. Mol. Med. 2016, 48, e245  DOI: 10.1038/EMM.2016.81
      There is no corresponding record for this reference.
    19. 19
      Sang, D.; Shu, T.; Pantoja, C. F.; Ibáñez de Opakua, A.; Zweckstetter, M.; Holt, L. J. Condensed-phase signaling can expand kinase specificity and respond to macromolecular crowding. Mol. Cell 2022, 82, 3693– 3711 e10,  DOI: 10.1016/j.molcel.2022.08.016
      19
      Condensed-phase signaling can expand kinase specificity and respond to macromolecular crowding
      Sang, Dajun; Shu, Tong; Pantoja, Christian F.; Ibanez de Opakua, Alain; Zweckstetter, Markus; Holt, Liam J.
      Molecular Cell (2022), 82 (19), 3693-3711.e10CODEN: MOCEFL; ISSN:1097-2765. (Elsevier Inc.)
      Phase sepn. can conc. biomols. and accelerate reactions. However, the mechanisms and principles connecting this mesoscale organization to signaling dynamics are difficult to dissect because of the pleiotropic effects assocd. with disrupting endogenous condensates. To address this limitation, we engineered new phosphorylation reactions within synthetic condensates. We generally found increased activity and broadened kinase specificity. Phosphorylation dynamics within condensates were rapid and could drive cell-cycle-dependent localization changes. High client concn. within condensates was important but not the main factor for efficient phosphorylation. Rather, the availability of many excess client-binding sites together with a flexible scaffold was crucial. Phosphorylation within condensates was also modulated by changes in macromol. crowding. Finally, the phosphorylation of the Alzheimer's-disease-assocd. protein Tau by cyclin-dependent kinase 2 was accelerated within condensates. Thus, condensates enable new signaling connections and can create sensors that respond to the biophys. properties of the cytoplasm.
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    20. 20
      Bonucci, M.; Shu, T.; Holt, L. J. How it feels in a cell. Trends Cell Biol. 2023, 33, 924– 938,  DOI: 10.1016/j.tcb.2023.05.002
      20
      How it feels in a cell
      Bonucci, Martina; Shu, Tong; Holt, Liam J.
      Trends in Cell Biology (2023), 33 (11), 924-938CODEN: TCBIEK; ISSN:0962-8924. (Elsevier Ltd.)
      A review. Life emerges from thousands of biochem. processes occurring within a shared intracellular environment. We have gained deep insights from in vitro reconstitution of isolated biochem. reactions. However, the reaction medium in test tubes is typically simple and dild. The cell interior is far more complex: macromols. occupy more than a third of the space, and energy-consuming processes agitate the cell interior. Here, we review how this crowded, active environment impacts the motion and assembly of macromols., with an emphasis on mesoscale particles (10-1000 nm diam.). We describe methods to probe and analyze the biophys. properties of cells and highlight how changes in these properties can impact physiol. and signaling, and potentially contribute to aging, and diseases, including cancer and neurodegeneration.
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    21. 21
      Rauter, T.; Burgstaller, S.; Gottschalk, B.; Ramadani-Muja, J.; Bischof, H.; Hay, J. C.; Graier, W. F.; Malli, R. ER-to-Golgi transport in hela cells displays high resilience to Ca2+ and energy stresses. Cells 2020, 9, 2311– 2326,  DOI: 10.3390/cells9102311
      There is no corresponding record for this reference.
    22. 22
      Pilic, J.; Gottschalk, B.; Bourgeois, B.; Habisch, H.; Koshenov, Z.; Oflaz, F. E.; Erdogan, Y. C.; Miri, S. M.; Yiğit, E. N.; Aydın, M. Ş. Hexokinase 1 forms rings that regulate mitochondrial fission during energy stress. Mol. Cell 2024, 84, 2732– 2746.e5,  DOI: 10.1016/j.molcel.2024.06.009
      There is no corresponding record for this reference.
    23. 23
      Yamanaka, R.; Tabata, S.; Shindo, Y.; Hotta, K.; Suzuki, K.; Soga, T.; Oka, K. Mitochondrial Mg2+ homeostasis decides cellular energy metabolism and vulnerability to stress. Sci. Rep. 2016, 6, 30027,  DOI: 10.1038/srep30027
      23
      Mitochondrial Mg2+ homeostasis decides cellular energy metabolism and vulnerability to stress
      Yamanaka, Ryu; Tabata, Sho; Shindo, Yutaka; Hotta, Kohji; Suzuki, Koji; Soga, Tomoyoshi; Oka, Kotaro
      Scientific Reports (2016), 6 (), 30027CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)
      Cellular energy prodn. processes are composed of many Mg2+ dependent enzymic reactions. In fact, dysregulation of Mg2+ homeostasis is involved in various cellular malfunctions and diseases. Recently, mitochondria, energy-producing organelles, have been known as major intracellular Mg2+ stores. Several biol. stimuli alter mitochondrial Mg2+ concn. by intracellular redistribution. However, in living cells, whether mitochondrial Mg2+ alteration affect cellular energy metab. remains unclear. Mg2+ transporter of mitochondrial inner membrane MRS2 is an essential component of mitochondrial Mg2+ uptake system. Here, we comprehensively analyzed intracellular Mg2+ levels and energy metab. in Mrs2 knockdown (KD) cells using fluorescence imaging and metabolome anal. Dysregulation of mitochondrial Mg2+ homeostasis disrupted ATP prodn. via shift of mitochondrial energy metab. and morphol. Moreover, Mrs2 KD sensitized cellular tolerance against cellular stress. These results indicate regulation of mitochondrial Mg2+via MRS2 critically decides cellular energy status and cell vulnerability via regulation of mitochondrial Mg2+ level in response to physiol. stimuli.
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    24. 24
      Pilchova, I.; Klacanova, K.; Tatarkova, Z.; Kaplan, P.; Racay, P. The Involvement of Mg2+ in Regulation of Cellular and Mitochondrial Functions. Oxid. Med. Cell. Longevity 2017, 2017, 6797460,  DOI: 10.1155/2017/6797460
      24
      The Involvement of Mg(2+) in Regulation of Cellular and Mitochondrial Functions
      Pilchova Ivana; Klacanova Katarina; Tatarkova Zuzana; Kaplan Peter; Racay Peter
      Oxidative medicine and cellular longevity (2017), 2017 (), 6797460 ISSN:.
      Mg(2+) is an essential mineral with pleotropic impacts on cellular physiology and functions. It acts as a cofactor of several important enzymes, as a regulator of ion channels such as voltage-dependent Ca(2+) channels and K(+) channels and on Ca(2+)-binding proteins. In general, Mg(2+) is considered as the main intracellular antagonist of Ca(2+), which is an essential secondary messenger initiating or regulating a great number of cellular functions. This review examines the effects of Mg(2+) on mitochondrial functions with a particular focus on energy metabolism, mitochondrial Ca(2+) handling, and apoptosis.
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    25. 25
      Suljevic, O.; Fischerauer, S. F.; Weinberg, A. M.; Sommer, N. G. Immunological reaction to magnesium-based implants for orthopedic applications. What do we know so far? A systematic review on in vivo studies. Mater. Today Bio 2022, 15, 100315,  DOI: 10.1016/j.mtbio.2022.100315
      There is no corresponding record for this reference.
    26. 26
      Xiao, B.; Sanders, M. J.; Underwood, E.; Heath, R.; Mayer, F. V.; Carmena, D.; Jing, C.; Walker, P. A.; Eccleston, J. F.; Haire, L. F. Structure of mammalian AMPK and its regulation by ADP. Nature 2011, 472, 230– 233,  DOI: 10.1038/nature09932
      26
      Structure of mammalian AMPK and its regulation by ADP
      Xiao, Bing; Sanders, Matthew J.; Underwood, Elizabeth; Heath, Richard; Mayer, Faith V.; Carmena, David; Jing, Chun; Walker, Philip A.; Eccleston, John F.; Haire, Lesley F.; Saiu, Peter; Howell, Steven A.; Aasland, Rein; Martin, Stephen R.; Carling, David; Gamblin, Steven J.
      Nature (London, United Kingdom) (2011), 472 (7342), 230-233CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      The heterotrimeric AMP-activated protein kinase (AMPK) has a key role in regulating cellular energy metab.; in response to a fall in intracellular ATP levels it activates energy-producing pathways and inhibits energy-consuming processes. AMPK has been implicated in a no. of diseases related to energy metab. including type 2 diabetes, obesity and, most recently, cancer. AMPK is converted from an inactive form to a catalytically competent form by phosphorylation of the activation loop within the kinase domain: AMP binding to the γ-regulatory domain promotes phosphorylation by the upstream kinase, protects the enzyme against dephosphorylation, as well as causing allosteric activation. Here we show that ADP binding to just one of the two exchangeable AXP (AMP/ADP/ATP) binding sites on the regulatory domain protects the enzyme from dephosphorylation, although it does not lead to allosteric activation. Our studies show that active mammalian AMPK displays significantly tighter binding to ADP than to Mg-ATP, explaining how the enzyme is regulated under physiol. conditions where the concn. of Mg-ATP is higher than that of ADP and much higher than that of AMP. We have detd. the crystal structure of an active AMPK complex. The structure shows how the activation loop of the kinase domain is stabilized by the regulatory domain and how the kinase linker region interacts with the regulatory nucleotide-binding site that mediates protection against dephosphorylation. From our biochem. and structural data we develop a model for how the energy status of a cell regulates AMPK activity.
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    27. 27
      Hardie, D. G. AMP-activated protein kinase-an energy sensor that regulates all aspects of cell function. Genes Dev. 2011, 25, 1895– 1908,  DOI: 10.1101/gad.17420111
      27
      AMP-activated protein kinase-an energy sensor that regulates all aspects of cell function
      Hardie, D. Grahame
      Genes & Development (2011), 25 (18), 1895-1908CODEN: GEDEEP; ISSN:0890-9369. (Cold Spring Harbor Laboratory Press)
      A review. AMP-activated protein kinase (AMPK) is a sensor of energy status that maintains cellular energy homeostasis. It arose very early during eukaryotic evolution, and its ancestral role may have been in the response to starvation. Recent work shows that the kinase is activated by increases not only in AMP, but also in ADP. Although best known for its effects on metab., AMPK has many other functions, including regulation of mitochondrial biogenesis and disposal, autophagy, cell polarity, and cell growth and proliferation. Both tumor cells and viruses establish mechanisms to down-regulate AMPK, allowing them to escape its restraining influences on growth.
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    28. 28
      Söding, J.; Zwicker, D.; Sohrabi-Jahromi, S.; Boehning, M.; Kirschbaum, J. Mechanisms for Active Regulation of Biomolecular Condensates. Trends Cell Biol. 2020, 30, 4– 14,  DOI: 10.1016/j.tcb.2019.10.006
      28
      Mechanisms for Active Regulation of Biomolecular Condensates
      Soding Johannes; Zwicker David; Kirschbaum Jan; Sohrabi-Jahromi Salma; Boehning Marc
      Trends in cell biology (2020), 30 (1), 4-14 ISSN:.
      Liquid-liquid phase separation is a key organizational principle in eukaryotic cells, on par with intracellular membranes. It allows cells to concentrate specific proteins into condensates, increasing reaction rates and achieving switch-like regulation. We propose two active mechanisms that can explain how cells regulate condensate formation and size. In both, the cell regulates the activity of an enzyme, often a kinase, that adds post-translational modifications to condensate proteins. In enrichment inhibition, the enzyme enriches in the condensate and weakens interactions, as seen in stress granules (SGs), Cajal bodies, and P granules. In localization-induction, condensates form around immobilized enzymes that strengthen interactions, as observed in DNA repair, transmembrane signaling, and microtubule assembly. These models can guide studies into the many emerging roles of biomolecular condensates.
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    29. 29
      Gormal, R. S.; Martinez-Marmol, R.; Brooks, A. J.; Meunier, F. A. Location, location, location: Protein kinase nanoclustering for optimised signalling output. elife 2024, 13, 1– 21,  DOI: 10.7554/eLife.93902
      There is no corresponding record for this reference.
    30. 30
      Banani, S. F.; Lee, H. O.; Hyman, A. A.; Rosen, M. K. Biomolecular condensates: Organizers of cellular biochemistry. Nat. Rev. Mol. Cell Biol. 2017, 18, 285– 298,  DOI: 10.1038/nrm.2017.7
      30
      Biomolecular condensates: organizers of cellular biochemistry
      Banani, Salman F.; Lee, Hyun O.; Hyman, Anthony A.; Rosen, Michael K.
      Nature Reviews Molecular Cell Biology (2017), 18 (5), 285-298CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)
      A review. Biomol. condensates are micron-scale compartments in eukaryotic cells that lack surrounding membranes but function to conc. proteins and nucleic acids. These condensates are involved in diverse processes, including RNA metab., ribosome biogenesis, the DNA damage response and signal transduction. Recent studies have shown that liq.-liq. phase sepn. driven by multivalent macromol. interactions is an important organizing principle for biomol. condensates. With this phys. framework, it is now possible to explain how the assembly, compn., phys. properties and biochem. and cellular functions of these important structures are regulated.
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    31. 31
      Alberti, S.; Gladfelter, A.; Mittag, T. Considerations and Challenges in Studying Liquid-Liquid Phase Separation and Biomolecular Condensates. Cell 2019, 176, 419– 434,  DOI: 10.1016/j.cell.2018.12.035
      31
      Considerations and Challenges in Studying Liquid-Liquid Phase Separation and Biomolecular Condensates
      Alberti, Simon; Gladfelter, Amy; Mittag, Tanja
      Cell (Cambridge, MA, United States) (2019), 176 (3), 419-434CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      A review. Evidence is now mounting that liq.-liq. phase sepn. (LLPS) underlies the formation of membraneless compartments in cells. This realization has motivated major efforts to delineate the function of such biomol. condensates in normal cells and their roles in contexts ranging from development to age-related disease. There is great interest in understanding the underlying biophys. principles and the specific properties of biol. condensates with the goal of bringing insights into a wide range of biol. processes and systems. The explosion of physiol. and pathol. contexts involving LLPS requires clear stds. for their study. Here, we propose guidelines for rigorous exptl. characterization of LLPS processes in vitro and in cells, discuss the caveats of common exptl. approaches, and point out exptl. and theor. gaps in the field.
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    32. 32
      Wingreen, N. S.; Brangwynne, C. P.; Panagiotopoulos, A. Z.; Wingreen, N. S. Interfacial Exchange Dynamics of Biomolecular Condensates are Highly Sensitive to Client Interactions. J. Chem. Phys. 2024, 160, 145102,  DOI: 10.1063/5.0188461
      There is no corresponding record for this reference.
    33. 33
      Bu, Z.; Callaway, D. J. E. Proteins move! Protein dynamics and long-range allostery in cell signaling. In Advances in Protein Chemistry and Structural Birology, 1st ed.; Elsevier Inc., 2011; Vol. 83, pp 163– 221.  DOI: 10.1016/B978-0-12-381262-9.00005-7 .
      There is no corresponding record for this reference.
    34. 34
      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
      34
      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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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXisFSmtLbO&md5=433efbe52ec9f7e15265246eb839939e
    35. 35
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      Aydın, M. Ş.; Bay, S.; Yiğit, E. N.; Özgül, C.; Oğuz, E. K.; Konuk, E. Y.; Ayşit, N.; Cengiz, N.; Erdoğan, E.; Him, A. Active shrinkage protects neurons following axonal transection. iScience 2023, 26, 107715,  DOI: 10.1016/j.isci.2023.107715
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      Ghaffari Zaki, A.; Yiğit, E. N.; Aydın, M. Ş.; Vatandaslar, E.; Öztürk, G.; Eroglu, E. Genetically Encoded Biosensors Unveil Neuronal Injury Dynamics via Multichromatic ATP and Calcium Imaging. ACS Sensors 2024, 9, 1261– 1271,  DOI: 10.1021/acssensors.3c02111
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      Encoding and Decoding Cellular Information through Signaling Dynamics
      Purvis, Jeremy E.; Lahav, Galit
      Cell (Cambridge, MA, United States) (2013), 152 (5), 945-956CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      A review. A growing no. of studies are revealing that cells can send and receive information by controlling the temporal behavior (dynamics) of their signaling mols. In this Review, we discuss what is known about the dynamics of various signaling networks and their role in controlling cellular responses. We identify general principles that are emerging in the field, focusing specifically on how the identity and quantity of a stimulus is encoded in temporal patterns, how signaling dynamics influence cellular outcomes, and how specific dynamical patterns are both shaped and interpreted by the structure of mol. networks. We conclude by discussing potential functional roles for transmitting cellular information through the dynamics of signaling mols. and possible applications for the treatment of disease.
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      Islam, M. T.; Holland, W. L.; Lesniewski, L. A. Multicolor fluorescence biosensors reveal a burning need for diversity in the single-cell metabolic landscape. Trends Endocrinol. Metab. 2021, 32, 537– 539,  DOI: 10.1016/j.tem.2021.04.002
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      Evers, T. M. J.; Hochane, M.; Tans, S. J.; Heeren, R. M. A.; Semrau, S.; Nemes, P.; Mashaghi, A. Deciphering Metabolic Heterogeneity by Single-Cell Analysis. Anal. Chem. 2019, 91, 13314– 13323,  DOI: 10.1021/acs.analchem.9b02410
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      Deciphering Metabolic Heterogeneity by Single-Cell Analysis
      Evers, Tom M. J.; Hochane, Mazene; Tans, Sander J.; Heeren, Ron M. A.; Semrau, Stefan; Nemes, Peter; Mashaghi, Alireza
      Analytical Chemistry (Washington, DC, United States) (2019), 91 (21), 13314-13323CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      A review. Single-cell anal. provides insights into cellular heterogeneity and dynamics of individual cells. This feature highlights recent developments in key anal. techniques suited for single-cell metabolic anal. with a special focus on mass spectrometry-based anal. platforms and RNA-seq, as well as imaging techniques that reveal stochasticity in metab.
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      Depaoli, M. R.; Karsten, F.; Madreiter-Sokolowski, C. T.; Klec, C.; Gottschalk, B.; Bischof, H.; Eroglu, E.; Waldeck-Weiermair, M.; Simmen, T.; Graier, W. F. Real-Time Imaging of Mitochondrial ATP Dynamics Reveals the Metabolic Setting of Single Cells. Cell Rep. 2018, 25, 501– 512 e3,  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
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      Hung, Y. P.; Teragawa, C.; Kosaisawe, N.; Gillies, T. E.; Pargett, M.; Minguet, M.; Distor, K.; Rocha-Gregg, B. L.; Coloff, J. L.; Keibler, M. A. Akt regulation of glycolysis mediates bioenergetic stability in epithelial cells. elife 2017, 6, 1– 25,  DOI: 10.7554/eLife.27293
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      Zhang, H.; Zhao, T.; Huang, P.; Wang, Q.; Tang, H.; Chu, X.; Jiang, J. Spatiotemporally Resolved Protein Detection in Live Cells Using Nanopore Biosensors. ACS Nano 2022, 16, 5752– 5763,  DOI: 10.1021/acsnano.1c10796
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      Spatiotemporally Resolved Protein Detection in Live Cells Using Nanopore Biosensors
      Zhang, Hongshuai; Zhao, Tao; Huang, Peifeng; Wang, Qingsong; Tang, Hao; Chu, Xia; Jiang, Jianhui
      ACS Nano (2022), 16 (4), 5752-5763CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
      Spatiotemporal detection of proteins in living cells is a persistent challenge but is the key to understanding their cellular biol. and developing theranostic technologies. We develop a dual-nanopore biosensor using affinity-tunable peptide probes, which enables label-free and spatiotemporal monitoring of protein abundance and its concn. change in single live cells. We demonstrate that by screening for peptide probes with tunable affinities, the nanopore modified with a medium-affinity peptide allowed reversible and sensitive detection of the protein kinase A (PKA) catalytic subunit with a detection limit of 0.04 nM. The sensor is shown to have the ability to effectively eliminate interferences from cell membrane resistance and coexisting species in live cell detection. Moreover, our sensor is successfully implemented in monitoring of dynamic PKA activity changes (PKA catalytic subunit dynamic concn. changes) under different stimulations in single live cells. Our design may provide a paradigm for developing nanopore biosensors for spatiotemporally resolved protein anal. in live cells.
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  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssensors.4c01058.

    • Mapping endogenous PKA activity in different cell types using the SPARK technology; phospho-null mutant of AMPK-SPARK not showing any clusters upon its expression; expression level of AMPK-SPARK not correlating with cluster count or sphericity; elevated extracellular Mg2+ levels show higher AMPK activity than reduced extracellular Mg2+ levels; cluster morphology difference between AMPK-SPARK and PKA-SPARK; clusters of AMPK-SPARK and PKA-SPARK with different spatial arrangements; AMPK-SPARK and FRET-biosensor AMPKAR show analogous readouts; primary cortical neurons exhibit heterogeneous AMPK responses to axotomy injury; and dual reporter unveils variances in Ca2+-mediated AMPK activation under supraphysiological Ca2+ levels (PDF)

    • Canonical and noncanonical activation of AMPK in HEK293 cells expressing AMPK-SPARK (Position 1) (AVI)

    • Canonical and noncanonical activation of AMPK in HEK293 cells expressing AMPK-SPARK (Position 2); (AVI)

    • Ca2+ elevation mediated AMPK activation in HEK293 cells expressing AMPK-SPARK (AVI)

    • Cluster emergence and dissolution upon glucose removal, glucose reintroduction, and Ca2+ elevation in a EA.hy926 cell expressing GCaMP-AMPK-SPARK (AVI)

    • Intensity changes upon glucose removal, glucose reintroduction, and Ca2+ elevation in an EA.hy926 cell expressing GCaMP-AMPK-SPARK (AVI)

    • HeLa cells expressing GCaMP-AMPK-SPARK in response to sequential increase and reduction of cytosolic Ca2+ (MP4)


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