Abstract
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.
Highlights
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
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]
While cytosolic GEPII 1.0 responded to K+ changes as expected (Figure S2d,e), targeting GEPII 1.0 into the endoplasmatic reticulum (ER)
Summary
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; 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) coupled to a biotinylated AviTag expressed on the outer cell surface of cells. 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. The biosensor constructs could be selectively and directly immobilized on cells of interest that expressed a cell surface-targeted biotinylated
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