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.

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.

Chromodomain-peptide recognition specificity is decided by physiochemical properties defined by posttranslational modifications.

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Signaling networks are spatiotemporally organized to sense diverse inputs, process information, and carry out specific cellular tasks. In β 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 β 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.

The biochemical activities involved in signal transduction in cells are under tight spatiotemporal regulation. To study the effects of the spatial patterning and temporal dynamics of biochemical activities on downstream signaling, researchers require methods to manipulate signaling pathways acutely and rapidly. In this review, we summarize recent developments in the design of three broad classes of molecular tools for perturbing signal transduction, classified by their type of input signal: chemically induced, optically induced, and magnetically induced.

9In single-molecule localization based super-resolution microscopy (SMLM), a fluorophore stochastically 10 switches between fluorescent-and dark-states, leading to intermittent emission of fluorescence. Inter-11 mittent emissions create multiple localizations belonging to the same molecule, a phenomenon known as 12 blinking. Blinking distorts SMLM images and confound quantitative interpretations by forming artificial 13 nanoclusters, which are often interpreted as true biological assemblies. Multiple methods have been de-14 veloped to eliminate these artifacts, but they either require additional experiments, arbitrary thresholds, 15 or specific photo-kinetic models. Here we present a method, termed Distance Distribution Correction 16 (DDC), to eliminate fluorophore blinking in superresolution imaging without any additional calibrations. 17 The approach relies on the finding that the true pairwise distance distribution of different fluorophores 18 in an SMLM image can be naturally obtained from the imaging sequence by using the distances between 19 localizations separated by a time much longer than the average fluorescence survival time. We show that 20 using the true pairwise distribution we can define and then maximize the likelihood of obtaining a partic-21 ular set of localizations without blinking and generate an accurate reconstruction of the true underlying 22 cellular structure. Using both simulated and experimental data, we show that DDC surpasses all previous 23 existing blinking correction methodologies, resulting in drastic improvements in obtaining the closest esti-24 mate of the true spatial organization and number of fluorescent emitters. The simplicity and robustness of 25 DDC will enable its wide application in SMLM imaging, providing the most accurate reconstruction and 26 quantification of SMLM images to date. Introduction 28In recent years the development of superresolution fluorescence microscopy has enabled the probing of 29 macromolecular assemblies in cells with nanometer resolutions. Amongst different superresolution imaging 30 techniques, single-molecule localization superresolution microscopy (SMLM) has gained wide popularity 31 due to its relatively simple implementation, which is based on post-imaging analysis of single-molecule 32 detection. 33 34SMLM reconstructs a superresolution image by stochastic photo-activation of individual fluorophores and 35 subsequent accurate post-imaging localization determination (1-3). One major advantage of SMLM is that 36 due to its single-molecule detection nature, one can determine the number of molecules in a macromolec-37 ular assembly quantitatively, allowing the investigation of both the molecular composition and spatial 38 arrangement at a level unmatched by other ensemble imaging-based superresolution imaging techniques. 39In the past few years SMLM has led to novel discoveries and quantitative characterizations of numerous 40 biological assemblies (4, 5) such as those composed of RNA polymerase (6-8), membrane proteins (9), bac-41 teria...

Enzymes are well known for their catalytic abilities, some even reaching “catalytic perfection” in the sense that the reaction they catalyze has reached the physical bound of the diffusion rate. However, our growing understanding of enzyme superfamilies has revealed that only some share a catalytic chemistry while others share a substrate‐handle binding motif, for example, for a particular phosphate group. This suggests that some families emerged through a “substrate‐handle‐binding‐first” mechanism (“binding‐first” for brevity) instead of “chemistry‐first” and we are, therefore, left to wonder what the role of non‐catalytic binders might have been during enzyme evolution. In the last of their eight seminal, back‐to‐back articles from 1976, John Albery and Jeremy Knowles addressed the question of enzyme evolution by arguing that the simplest mode of enzyme evolution is what they defined as “uniform binding” (parallel stabilization of all enzyme‐bound states to the same degree). Indeed, we show that a uniform‐binding proto‐catalyst can accelerate a reaction, but only when catalysis is already present, that is, when the transition state is already stabilized to some degree. Thus, we sought an alternative explanation for the cases where substrate‐handle‐binding preceded any involvement of a catalyst. We find that evolutionary starting points that exhibit negative catalysis can redirect the reaction's course to a preferred product without need for rate acceleration or product release; that is, if they do not stabilize, or even destabilize, the transition state corresponding to an undesired product. Such a mechanism might explain the emergence of “binding‐first” enzyme families like the aldolase superfamily.