Signaling Microdomains in the Spotlight: Visualizing Compartmentalized Signaling Using Genetically Encoded Fluorescent Biosensors

Abstract

How cells muster a network of interlinking signaling pathways to faithfully convert diverse external cues to specific functional outcomes remains a central question in biology. Through their ability to convert dynamic biochemical activities to rapid and precise optical readouts, genetically encoded fluorescent biosensors have become instrumental in unraveling the molecular logic controlling the specificity of intracellular signaling. In this review, we discuss how the use of genetically encoded fluorescent biosensors to visualize dynamic signaling events within their native cellular context is elucidating the different strategies employed by cells to organize signaling activities into discrete compartments, or signaling microdomains, to ensure functional specificity.

Quantifying the Kinetics of Signaling and Arrestin Recruitment by Nervous System G-Protein Coupled Receptors

“…They provide us with exciting, real time views of signaling that until now were studied with end point assays. They also reveal entirely new cellular phenomena such as the recent discovery of phase transitions in the cytosol (Hyman and Simons, 2012 ; Watanabe et al, 2017 ; Zhang et al, 2021a ). These new views come with new challenges.…”

“…The sensor can be delivered into cells via a suitable viral or plasmid vector or incorporated into the germline in genetically-manipulated animals. The sensors can be targeted to specific locations within the cell with the incorporation of localization sequences, enabling spatial resolution of signaling events (Vilardaga et al, 2014 ; Moore et al, 2016 ; Halls and Canals, 2018 ; Hilgendorf et al, 2019 ; Lobingier and Von Zastrow, 2019 ; Jullie et al, 2020 ; Zhang et al, 2021a ). The time course of signaling is typically measured by default in these experiments, which has stimulated an explosion in the kinetic quantification of GPCR signaling.…”

Protocol for reading and imaging live-cell PKA activity using ExRai-AKAR2

“…The reversible phosphorylation of proteins on Ser, Thr, or Tyr residues by protein kinases is of particular importance in signaling, regulating almost all cellular functions. Understanding the architecture of kinase signaling has been greatly improved by the development of optical tools such as genetically encoded fluorescent biosensors, which enable direct visualization of the spatial and temporal dynamics of kinase activities in live cells ( Zhang et al., 2021b ). Among these, fluorescence resonance energy transfer (FRET)-based kinase biosensors are the most widely used tools to visualize kinase activities.…”

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

“…These compartments are often formed through the assembly of various signaling proteins, such as a scaffolding proteins, signaling enzymes, and receptors. Genetically encoded fluorescence-based biosensors are instrumental in measuring the unique spatiotemporal dynamics associated with these signaling microdomains ( Zhang et al., 2021 ). Fluorescent sensors typically contain one or more fluorescent protein(s) coupled to a molecular switch that is sensitive to a molecule of interest such as a signaling molecule.…”

“…FluoSTEPs follow the design of existing FRET-based sensors ( Greenwald et al., 2018 ; Zhang et al., 2021 ). For instance, FluoSTEP indicator of cAMP using Epac (FluoSTEP-ICUE; Figure 1 ) contains the same Epac1-based molecular switch found in several previous FRET-based cAMP indicators ( DiPilato and Zhang, 2009 ; DiPilato et al., 2004 ).…”