Protocol for using fluorescent sensors targeted to endogenous proteins (FluoSTEPs) to measure microdomain-specific signaling events.

Fluorescence-based sensors are powerful molecular tools for studying the spatiotemporal regulation of cell signaling, which is often organized into discrete microdomains. Here, we present a protocol for using fluorescent sensors targeted to endogenous proteins (FluoSTEPs), a new class of fluorescent sensors in which the functional probe is exclusively reconstituted at an endogenously expressed protein of interest associated with a specific microdomain. FluoSTEPs allow microdomain-specific signaling activities to be measured with high selectivity without perturbing the native stoichiometry of signaling components. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2020) and Tenner et al. (2021).

A pairwise distance distribution correction (DDC) algorithm to eliminate blinking-caused artifacts in SMLM.

Single-molecule localization microscopy (SMLM) relies on the blinking behavior of a fluorophore, which is the stochastic switching between fluorescent and dark states. Blinking creates multiple localizations belonging to the same fluorophore, confounding quantitative analyses and interpretations. Here we present a method, termed distance distribution correction (DDC), to eliminate blinking-caused repeat localizations without any additional calibrations. The approach relies on obtaining the true pairwise distance distribution of different fluorophores naturally from the imaging sequence by using distances between localizations separated by a time much longer than the average fluorescence survival time. We show that, using the true pairwise distribution, we can define and maximize the likelihood, obtaining a set of localizations void of blinking artifacts. DDC results in drastic improvements in obtaining the closest estimate of the true spatial organization and number of fluorescent emitters in a wide range of applications, enabling accurate reconstruction and quantification of SMLM images.

FluoSTEPs: Fluorescent biosensors for monitoring compartmentalized signaling within endogenous microdomains.

Growing evidence suggests that many essential intracellular signaling events are compartmentalized within kinetically distinct microdomains in cells. Genetically encoded fluorescent biosensors are powerful tools to dissect compartmentalized signaling, but current approaches to probe these microdomains typically rely on biosensor fusion and overexpression of critical regulatory elements. Here, we present a novel class of biosensors named FluoSTEPs (fluorescent sensors targeted to endogenous proteins) that combine self-complementing split green fluorescent protein, CRISPR-mediated knock-in, and fluorescence resonance energy transfer biosensor technology to probe compartmentalized signaling dynamics in situ. We designed FluoSTEPs for simultaneously highlighting endogenous microdomains and reporting domain-specific, real-time signaling events including kinase activities, guanosine triphosphatase activation, and second messenger dynamics in live cells. A FluoSTEP for 3',5'-cyclic adenosine monophosphate (cAMP) revealed distinct cAMP dynamics within clathrin microdomains in response to stimulation of G protein-coupled receptors, showcasing the utility of FluoSTEPs in probing spatiotemporal regulation within endogenous signaling architectures.

An ultrasensitive biosensor for high-resolution kinase activity imaging in awake mice.

Protein kinases control nearly every facet of cellular function. These key signaling nodes integrate diverse pathway inputs to regulate complex physiological processes, and aberrant kinase signaling is linked to numerous pathologies. While fluorescent protein-based biosensors have revolutionized the study of kinase signaling by allowing direct, spatiotemporally precise kinase activity measurements in living cells, powerful new molecular tools capable of robustly tracking kinase activity dynamics across diverse experimental contexts are needed to fully dissect the role of kinase signaling in physiology and disease. Here, we report the development of an ultrasensitive, second-generation excitation-ratiometric protein kinase A (PKA) activity reporter (ExRai-AKAR2), obtained via high-throughput linker library screening, that enables sensitive and rapid monitoring of live-cell PKA activity across multiple fluorescence detection modalities, including plate reading, cell sorting and one- or two-photon imaging. Notably, in vivo visual cortex imaging in awake mice reveals highly dynamic neuronal PKA activity rapidly recruited by forced locomotion.

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

Signaling networks are spatiotemporally organized to sense diverse inputs, process information, and carry out specific cellular tasks. In ß cells, Ca2+, cyclic adenosine monophosphate (cAMP), and Protein Kinase A (PKA) exist in an oscillatory circuit characterized by a high degree of feedback. Here, we describe a mode of regulation within this circuit involving a spatial dependence of the relative phase between cAMP, PKA, and Ca2+. We show that in mouse MIN6 ß cells, nanodomain clustering of Ca2+-sensitive adenylyl cyclases (ACs) drives oscillations of local cAMP levels to be precisely in-phase with Ca2+ oscillations, whereas Ca2+-sensitive phosphodiesterases maintain out-of-phase oscillations outside of the nanodomain. Disruption of this precise phase relationship perturbs Ca2+ oscillations, suggesting the relative phase within an oscillatory circuit can encode specific functional information. This work unveils a novel mechanism of cAMP compartmentation utilized for localized tuning of an oscillatory circuit and has broad implications for the spatiotemporal regulation of signaling networks.

Phase Separation of a PKA Regulatory Subunit Controls cAMP Compartmentation and Oncogenic Signaling.

The fidelity of intracellular signaling hinges on the organization of dynamic activity architectures. Spatial compartmentation was first proposed over 30 years ago to explain how diverse G protein-coupled receptors achieve specificity despite converging on a ubiquitous messenger, cyclic adenosine monophosphate (cAMP). However, the mechanisms responsible for spatially constraining this diffusible messenger remain elusive. Here, we reveal that the type I regulatory subunit of cAMP-dependent protein kinase (PKA), RIα, undergoes liquid-liquid phase separation (LLPS) as a function of cAMP signaling to form biomolecular condensates enriched in cAMP and PKA activity, critical for effective cAMP compartmentation. We further show that a PKA fusion oncoprotein associated with an atypical liver cancer potently blocks RIα LLPS and induces aberrant cAMP signaling. Loss of RIα LLPS in normal cells increases cell proliferation and induces cell transformation. Our work reveals LLPS as a principal organizer of signaling compartments and highlights the pathological consequences of dysregulating this activity architecture.

Single-fluorophore biosensors for sensitive and multiplexed detection of signalling activities.

Unravelling the dynamic molecular interplay behind complex physiological processes such as neuronal plasticity requires the ability to both detect minute changes in biochemical states in response to physiological signals and track multiple signalling activities simultaneously. Fluorescent protein-based biosensors have enabled the real-time monitoring of dynamic signalling processes within the native context of living cells, yet most commonly used biosensors exhibit poor sensitivity (for example, due to low dynamic range) and are limited to imaging signalling activities in isolation. Here, we address this challenge by developing a suite of excitation ratiometric kinase activity biosensors that offer the highest reported dynamic range and enable the detection of subtle changes in signalling activity that could not be reliably detected previously, as well as a suite of single-fluorophore biosensors that enable the simultaneous tracking of as many as six distinct signalling activities in single living cells.

Single-color, ratiometric biosensors for detecting signaling activities in live cells.

Genetically encoded fluorescent biosensors have revolutionized the study of signal transduction by enabling the real-time tracking of signaling activities in live cells. Investigating the interaction between signaling networks has become increasingly important to understanding complex cellular phenomena, necessitating an update of the biosensor toolkit to allow monitoring and perturbing multiple activities simultaneously in the same cell. We therefore developed a new class of fluorescent biosensors based on homo-FRET, deemed FLuorescence Anisotropy REporters (FLAREs), which combine the multiplexing ability of single-color sensors with a quantitative, ratiometric readout. Using an array of color variants, we were able to demonstrate multiplexed imaging of three activity reporters simultaneously in the same cell. We further demonstrate the compatibility of FLAREs for use with optogenetic tools as well as intravital two-photon imaging.

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

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 cellular compartmentalization. Here, we introduce a new class of fluorescent biosensors that detect biochemical activities in living cells at a resolution up to threefold better than the diffraction limit. These 'FLINC' biosensors use binding-induced changes in protein fluorescence dynamics to 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 membranes of living cells and to 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.

Optical sensors to gain mechanistic insights into signaling assemblies.

Protein complexes play a major role in transducing information from outside the cell into instructions for growth and survival, and understanding how these complexes relay and shape intracellular signals has been a central question in signaling biology. Fluorescent proteins have proven paramount in opening windows for researchers to peer into the architecture and inner workings of signaling assemblies within the living cell and in real-time. In this review, we will provide readers with a current perspective on the development and use of genetically encoded optical probes to dissect the function of signaling complexes.

Biphasic kinetic behavior of E. coli WrbA, an FMN-dependent NAD(P)H:quinone oxidoreductase.

The E. coli protein WrbA is an FMN-dependent NAD(P)H:quinone oxidoreductase that has been implicated in oxidative defense. Three subunits of the tetrameric enzyme contribute to each of four identical, cavernous active sites that appear to accommodate NAD(P)H or various quinones, but not simultaneously, suggesting an obligate tetramer with a ping-pong mechanism in which NAD departs before oxidized quinone binds. The present work was undertaken to evaluate these suggestions and to characterize the kinetic behavior of WrbA. Steady-state kinetics results reveal that WrbA conforms to a ping-pong mechanism with respect to the constancy of the apparent Vmax to Km ratio with substrate concentration. However, the competitive/non-competitive patterns of product inhibition, though consistent with the general class of bi-substrate reactions, do not exclude a minor contribution from additional forms of the enzyme. NMR results support the presence of additional enzyme forms. Docking and energy calculations find that electron-transfer-competent binding sites for NADH and benzoquinone present severe steric overlap, consistent with the ping-pong mechanism. Unexpectedly, plots of initial velocity as a function of either NADH or benzoquinone concentration present one or two Michaelis-Menten phases depending on the temperature at which the enzyme is held prior to assay. The effect of temperature is reversible, suggesting an intramolecular conformational process. WrbA shares these and other details of its kinetic behavior with mammalian DT-diaphorase, an FAD-dependent NAD(P)H:quinone oxidoreductase. An extensive literature review reveals several other enzymes with two-plateau kinetic plots, but in no case has a molecular explanation been elucidated. Preliminary sedimentation velocity analysis of WrbA indicates a large shift in size of the multimer with temperature, suggesting that subunit assembly coupled to substrate binding may underlie the two-plateau behavior. An additional aim of this report is to bring under wider attention the apparently widespread phenomenon of two-plateau Michaelis-Menten plots.