Genetically encoded biosensors for visualizing live-cell biochemical activity at super-resolution

Abstract

Compartmentalized biochemical activities are essential to all cellular processes, but there is no generalizable method to visualize dynamic protein activities in living cells at a resolution commensurate with their compartmentalization. Here we introduce a new class of fluorescent biosensors that detect biochemical activities in living cells at a resolution up to three-fold better than the diffraction limit. Utilizing specific, binding-induced changes in protein fluorescence dynamics, these biosensors translate kinase activities or protein-protein interactions into changes in fluorescence fluctuations, which are quantifiable through stochastic optical fluctuation imaging. A Protein Kinase A (PKA) biosensor allowed us to resolve minute PKA activity microdomains on the plasma membrane of living cells and uncover the role of clustered anchoring proteins in organizing these activity microdomains. Together, these findings suggest that biochemical activities of the cell are spatially organized into an activity architecture, whose structural and functional characteristics can be revealed by these new biosensors.

Visualizing Dynamics of Cell Signaling In Vivo with a Phase Separation-Based Kinase Reporter

“…These FRET reporters are valuable tools for imaging kinase signaling in living cells, including protein kinase A (PKA) (Zhang et al, 2001, 2005) and ERK (Harvey et al, 2008). While FRET reporters achieve subcellular resolution and have recently unveiled compartmentalized kinase signaling in cultured single cells (Mo et al, 2017), in vivo use of the FRET reporters is problematic because of their small fluorescence ratio change of acceptor over donor fluorophores (Kardash et al, 2011). Thus, a robust fluorescent reporter for imaging dynamic kinase activity in living animals is much needed.…”

Spatially compartmentalized phase regulation of a Ca2+-cAMP-PKA oscillatory circuit

“…Indeed, how cAMP, a rapidly diffusing small molecule, is spatially compartmentalized in cells is not yet clearly understood, especially given the low catalytic efficiency of a single cAMP-producing AC and degrading PDE (Conti et al 2014;Lohse et al 2017). Given that AKAP79/150 exists in nanoclusters at the plasma membrane in multiple cell types (Mo et al 2017;Zhang et al 2016) and associates with AC8 in β cells (Willoughby et al 2010), we hypothesized that AC8 could form nanoclusters on the plasma membrane of MIN6 cells and compartmentalize cAMP dynamics. To test this hypothesis, we examined the spatial organization of AC8 and AKAP150 at the membrane using Stochastic Optical Reconstruction Microscopy (STORM).…”

SOFIevaluator: a strategy for the quantitative quality assessment of SOFI data

“…Super-resolution optical fluctuation imaging (SOFI) [1] is a super-resolution fluorescence imaging approach that uses fluctuations in fluorescence dye emission to calculate an image that has a resolution that is better than the diffraction limit. Its main distinguishing feature is that it is relatively insensitive to background signal, and can be performed over a broad range of labelling densities and probe brightness [2,3]. This robustness has also made it possible to use the technique for the first sub-diffraction observation of kinase activity [4].…”