Immobilization of Recombinant Fluorescent Biosensors Permits Imaging of Extracellular Ion Signals

Given the importance of ion gradients and fluxes in biology, monitoring ions locally at the exterior of the plasma membrane of intact cells in a noninvasive manner is highly desirable but challenging. Classical targeting of genetically encoded biosensors at the exterior of cell surfaces would be a suitable approach; however, it often leads to intracellular accumulation of the tools in vesicular structures and adverse modifications, possibly impairing sensor functionality. To tackle these issues, we generated recombinant fluorescent ion biosensors fused to traptavidin (TAv) specifically coupled to a biotinylated AviTag expressed on the outer cell surface of cells. We show that purified chimeras of TAv and pH-Lemon or GEPII 1.0, Förster resonance energy transfer-based pH and K+ biosensors, can be immobilized directly and specifically on biotinylated surfaces including glass platelets and intact cells, thereby remaining fully functional for imaging of ion dynamics. The immobilization of recombinant TAv–GEPII 1.0 on the extracellular cell surface of primary cortical rat neurons allowed imaging of excitotoxic glutamate-induced K+ efflux in vitro. We also performed micropatterning of purified TAv biosensors using a microperfusion system to generate spatially separated TAv–pH-Lemon and TAv–GEPII 1.0 spots for simultaneous pH and K+ measurements on cell surfaces. Our results suggest that the approach can be greatly expanded by immobilizing various biosensors on extracellular surfaces to quantitatively visualize microenvironmental transport and signaling processes in different cell culture models and other experimental settings.

A n elegant approach to monitor intra-and extracellular signaling events is to use genetically encoded fluorescent biosensors that can be well targeted to cell organelles. 1,2 Targeting fluorescent protein (FP)-based biosensors to the outer leaflet of the plasma membrane is usually achieved by fusing them with N-terminal secretion signals and C-terminal plasma membrane anchoring domains. 2,3 Such targeting strategies have been used to successfully target biosensors for small-molecule (neuro)transmitters including γ-aminobutyric acid, 4,5 glutamate, 6 ATP, 2 or serotonin 7 to the cell surface. In a previous study, we used such a strategy to target pH-Lemon, a pH reporter, to the exterior of the plasma membrane using an N-terminal secretory leading sequence and a glycosylphosphatidyl (GPI)-anchored moiety. 3,8,9 However, such targeting signals induce the entering of biosensors into the endoplasmatic reticulum (ER), further sorting in the Golgi apparatus and traveling to the cell surface within secretory vesicles. Endocytosis can then bring the plasma membrane-targeted biosensor back into intracellular vesicular structures. Thus, the use of secretion signals and plasma membrane anchoring domains results in strong labeling of intracellular vesicles, 3 which can complicate the selective investigation of extracellular activities on cell surfaces. Here, we tested cell surface-targeted pH-Lemon and GEPII 1.0, a Forster resonance energy transfer (FRET)-based pH and K + biosensor, 3,10 respectively, which we have developed recently. As expected, both GPI-anchored biosensors were also found predominately in intracellular structures. 3,8 Moreover, while pH-Lemon−GPI remained pHsensitive, we here found that the GPI-anchored K + biosensor completely lost its functionality. Thus, we devised an alternative strategy for the application of both biosensors on the outer leaflet of the plasma membrane for extracellular cell surface pH and K + imaging, which might be important and interesting in cancer research 11,12 and when using excitable cells such as neurons. 13 A quite distinct strategy to bring biosensors to the extracellular microenvironment is to immobilize the purified, fluorescent tools on the cell surface, thereby omitting sensors traveling through the secretory pathway. This might allow the usage of biosensors on cell surfaces without unwanted intracellular accumulations and modifications. Such an approach has indeed already been used successfully to immobilize biotinylated fluorescent amino acid biosensors on biotinylated cell surfaces via streptavidin, creating a biotin− streptavidin−biotin sandwich. 14−16 Here, we worked out another simplified biotinylation-based technique to directly, selectively, and functionally anchor recombinant biosensor constructs on biotinylated surfaces. 8 Instead of fusing a biotin tag to the biosensor, we directly fused it with traptavidin (TAv), 17 an improved streptavidin variant. We here show that the newly engineered recombinant biosensors for pH 3 and K +10 fused with TAv could be directly coupled to biotinylated glass surfaces and remain functional. 8 The biosensor constructs could also be selectively and directly immobilized on cells of interest that expressed a cell surface-targeted biotinylated AviTag. 8,18,19 Our study suggests that this strategy can easily be expanded to other biosensors in various applications.

■ EXPERIMENTAL METHODS
Substances and Buffers. All substances and buffers are mentioned in the Supporting Information.
Plasmids, Targeting and Sequences. Plasmids, targeting strategies and sequences are mentioned and explained in the Supporting Information.
Characterization of Recombinant Proteins. A detailed description of the protocol can be found in the Supporting Information.
Fluorescence Microscopy. Table S3 gives an overview of microscopes used for the different measurements.
Cell Culture and Transfection. Cell culture and transfection methods are explained in the Supporting Information.
Immobilization of TAv−pH-Lemon and TAv−GEPII 1.0. The biotinylated glass slides (Microsurfaces Inc., New Jersey, USA) were incubated with the sensor solution for 20 min and washed with phosphate-buffered saline (PBS) after incubation. Alternatively, a 2 μL drop containing the sensor solution was added to the glass slide and air-dried for approximately 15−20 min. The glass slide was washed with PBS to remove unbound sensors. Cells were washed thrice with PBS. For coupling the whole well, 500 μL of the protein solution (8−10 μM final concentration) was added to the cells and incubated at 25°C for 20 min in the dark. The cells were washed thrice with PBS and kept in physiological buffer for imaging. For micropatterning, a BioPen 20 (Fluicell, Molndal, Sweden) was used for  Figure 1e). 3 Importantly, although GPI anchoring did not affect the sensor functionality of pH-Lemon 3 (Figure 1e), GEPII 1.0−GPI completely lost its K + sensitivity ( Figure 1f). 8 We hypothesized that N-glycosylation, a frequent posttranslational modification within the secretory pathway, 21 of GEPII 1.0−GPI rendered it K + -insensitive and confirmed the glycosylation of the construct by western blot (Figure S2a−c).
Also, while cytosolic GEPII 1.0 responded to K + changes as expected ( Figure S2d,e), targeting GEPII 1.0 into the ER lumen, the entry gate of the secretory pathway, already impaired K + sensing ( Figure S2f,g). Considering that the functionality of pH-Lemon−GPI (Figure 1e) but not of GEPII 1.0−GPI (Figure 1f) was maintained, we hypothesized that glycosylation, possibly within the K + binding domain at N36 and N76 ( Figure S2a,b), impaired the functionality of the GPIanchored K + biosensor. 22 The vesicular abundance of plasma membrane-targeted biosensors did not appear problematic for other sensors and experimental settings. 2,23 However, we here aimed to tackle these issues in the case of both biosensors, pH-Lemon and GEPII 1.0, to accomplish pH and K + measurements at the extracellular surface of the plasma membrane of cells of interest. Thus, we came up with an improved biotinylationbased approach to immobilize the recombinant constructs specifically to cell surfaces.
Recombinant TAv Biosensors Dissolved in Solution and Immobilized on Glass Remain Functional. To omit intracellular sensor traveling but localize the biosensors to the cell surface as recombinant proteins, we designed and purified recombinant biosensors. Recombinant pH-Lemon and GEPII 1.0 were directly fused with TAv, a mutated and improved streptavidin analogue, 17  Plasma Membrane-Coupled TAv Biosensors Are Functional and Report Glutamate-Induced K + Efflux from Neurons. We next aimed to immobilize the TAv biosensors on the surface of living cells (Figure 3a). To selectively express a biotinylated tag on cell surfaces of cells of interest, we modified an established approach 18,19 and generated two separate mCherry-labeled constructs. One consisted of the plasma membrane-targeted AviTag 24 (AviTag-mCherry-GPI) ( Figure S4a,c,e), and the other consisted of ER-BirA-mCherry, the ER-targeted biotin ligase 18 ( Figure S4b,d,e). The co-expression of the two constructs should result in a biotinylation of the AviTag by BirA when the AviTag traversed the ER lumen upon membrane targeting ( Figure S4c−e).
Expectedly, the biotinylated AviTag efficiently coupled with the purified recombinant TAv−pH-Lemon (Figure 3b) and TAv−GEPII 1.0 (Figure 3d) with minimal intracellular background fluorescence under these conditions ( Figure  3c,e). Notably, we here used cells for less than 1 h at room temperature and directly after coupling the biosensors to the biotinylated cell surface. However, the internalization of cell surface-coupled biosensors by endocytosis at higher temperatures in in vitro and in vivo application over time is The same TAv biosensor fluorescence was found on the cell periphery after 10 min of rinsing the cell with buffer using a perfusion system ( Figure  S5e), pointing again to the stable interaction between the recombinant sensor and the biotinylated cell surface tag. Although K + sensing of GEPII 1.0 relies on a conformational rearrangement of the K + sensing domain 10 (Figure 1a), immobilization did not hamper the K + measurements ( Figure  4c,d). However, the sensitivity (EC 50 ) and dynamic range of the TAv biosensors were significantly reduced upon immobilization (Table S4). The immobilization of TAv−GEPII 1.0 on cells also increased basal FRET ratio values even in the absence of extracellular K + in the buffer (Table S4). This observation might point to K + -insensitive intermolecular FP interactions due to the spatially close sensor coupling. The mean pKa of coupled TAv−pH-Lemon on cells was 7.12 (range: pH 7.03− pH 7.20) (Figure 4b and Table S4), and the mean EC 50 of cell surface-coupled TAv−GEPII 1.0 was 3.46 mM (range: 2.59− 4.62 mM) (Figure 4d and Table S4). According to previous studies, the pKa values of TAv−pH-Lemon and the EC 50 of TAv−GEPII 1.0 seem to be well suitable for the detection of extracellular pH and K + alterations. 27,28 Because K + plays a major role in neuronal homeostasis, 29 we next tested our approach for monitoring K + efflux from primary neurons in response to glutamate. Considering the effect of high glutamate levels being accompanied by strong K + efflux, 30 we examined whether this glutamate-induced K + efflux can be optically detected using TAv−GEPII 1.0 coupled to the cell surface of primary neurons (Figure 4e,f) expressing ER-BirA and AviTag-mCherry-GPI ( Figure S6a). We, therefore, used a high-pressure microperfusion system 20 to load selected mCherry-positive cells with purified TAv−GEPII 1.0 directly on the fluorescence microscope ( Figures S6b,c and S7a). To prevent fast dilution of extracellular K + , we stopped the perfusion and added a cytotoxic glutamate bolus to the neurons by injection (Figure 4e,f). The first injection of glutamate instantly resulted in a rapid increase in the FRET ratio signal of the cell surface-coupled K + biosensor, indicating massive K + efflux from neurons in response to the excitatory neurotransmitter (Figures 4e,f and S7b). A second injection only slightly further increased the FRET ratio signal, indicating that the primary neurons were already strongly stimulated ( Figure 4f). As expected, a subsequent start of the perfusion reversed the effect (Figure 4f). Subsequent K + addition and removal rapidly and strongly changed the FRET ratio signal of the cell surface-coupled K + biosensor, confirming its functionality (Figure 4f).
Micropatterning of TAv Biosensors Allows for Simultaneous Imaging of pH and K + in the Same Experimental Setting. To co-image pH and K + with the spectrally identical biosensors in one experimental setting, we next used a micropatterning approach. The usage of the highpressure microperfusion 20 device allowed us to "paint" spatially separated microspots of TAv−pH-Lemon and TAv−GEPII 1.0 on cells expressing the biotinylated GPI-anchored AviTag. (Figures 5a and S6b,d,e). FRET ratio signals from both spots containing the two different FRET-based biosensors were recorded simultaneously over time to test the suitability for coimaging of pH and K + alterations (Figure 5b). Upon addition of buffers with different [K + ] (but the same pH), TAv−GEPII showed fast ratiometric changes, whereas TAv−pH-Lemon was only minimally affected (Figure 5b). Vice versa, with the addition of buffers with different pH values (but the same

■ CONCLUSIONS AND OUTLOOK
While purified biosensors have mainly been used for sensor characterization purposes, 3,10,32,33 we describe a novel biotinylation-based procedure to use genetically encoded sensors for the recording of extracellular ions locally on cell surfaces and glass slides upon their immobilization using three workflows ( Figure S8): first, purification of the recombinant TAv biosensor, second, expression of the biotinylated cell surface tag or using a biotinylated glass slide, and third, coupling of the TAv biosensor to the biotinylated surface. Considering the remaining functionality and precise targeting of recombinant TAv biosensor constructs with different working principles (conformational rearrangement vs intrinsic sensitivity), this technique might be expanded to immobilize a variety of different ratiometric and intensiometric biosensors. The precise application using microperfusion might furthermore allow for the immobilization of sensor protein on selected cells (types) (e.g., on tumor slices). While extracellular ion alterations might be hard to capture in a big extracellular volume in vitro, this technique might be further used in interesting experimental settings with reduced volume such as in the extracellular area of mouse brains. However, the meaningful usage of the recombinant fluorescent biosensors in vivo might be challenging and requires further research. primary rat neurons in response to glutamate injections. First, the perfusion was stopped, and two glutamate boli were injected into 0 mM K + buffer to reach a final concentration of 500 μM and 1 mM as indicated (arrows). Subsequently, the sensor functionality was checked by starting perfusion to transiently elevate extracellular K + from 0 to 140 mM K + . The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssensors.1c01369.
Substances, protein purification buffers, protein purification, buffers for in vitro fluorimetry, cell culture and transfection, buffers for fluorescence microscopy, fluorescence microscopy, western blot analysis, glutamate addition to primary rat neurons, data processing, statistical analysis, list of all plasmids/vectors used for mammalian and bacterial expression, sequences of targeting signals and constructs used, overview of the microscopes used for measuring GEPII and pH-Lemon (fusion) constructs, comparison of targeted and immobilized pH-Lemon and GEPII 1.0 variants, GPI targeting of biosensors leads to sensor accumulation within the secretory pathway, biosensor localization within the secretory pathway impacts sensor functionality, possibly due to glycosylation, characterization of TAv-fused sensors in solution, constructs and scheme for the immobilization of TAv-fused biosensors, functionality of TAv-pH-Lemon and TAv−GEPII 1.0 after immobilization, local immobilization of TAv sensors using microperfusion, visualization of glutamate-induced K + efflux from primary neurons using surface-coupled TAv−GEPII 1.0, and cell and sensor preparation and application to living cells (PDF)