Genetically encoded biosensors based on fluorescent proteins (FPs) allow for the real-time monitoring of molecular dynamics in space and time, which are crucial for the proper functioning and regulation of complex cellular processes. Depending on the types of molecular events to be monitored, different sensing strategies need to be applied for the best design of FP-based biosensors. Here, we review genetically encoded biosensors based on FPs with various sensing strategies, for example, translocation, fluorescence resonance energy transfer (FRET), reconstitution of split FP, pH sensitivity, maturation speed, and so on. We introduce general principles of each sensing strategy and discuss critical factors to be considered if available, then provide representative examples of these FP-based biosensors. These will help in designing the best sensing strategy for the successful development of new genetically encoded biosensors based on FPs.

Optobiochemical control of protein activities allows the investigation of protein functions in living cells with high spatiotemporal resolution. Over the last two decades, numerous natural photosensory domains have been characterized and synthetic domains engineered and assembled into photoregulatory systems to control protein function with light. Here, we review the field of optobiochemistry, categorizing photosensory domains by chromophore, describing photoregulatory systems by mechanism of action, and discussing protein classes frequently investigated using optical methods. We also present examples of how spatial or temporal control of proteins in living cells has provided new insights not possible with traditional biochemical or cell biological techniques.

Platelet-derived growth factor receptor (PDGFR) senses extracellular growth factors and transfer the signals inside the cells regulating cell proliferation, migration and survival. It has been controversial at which membrane microdomains PDGFRs reside and how they control such diverse intracellular signaling pathways. Here, we developed a novel PDGFR biosensor based on fluorescence resonance energy transfer (FRET), which can detect the real-time PDGFR activity in live cells with high spatiotemporal resolutions. To study subcellular PDGFR activity at membrane microdomains, this PDGFR biosensor was further targeted in or outside lipid rafts via different lipid modification signals. The results suggest that, in response to PDGF stimulation, PDGFR activity is evenly distributed at different membrane microdomains, while integrin-mediated signaling events have inhibitory effects on the activation of PDGFR specifically located in lipid rafts but not outside rafts, implying the role of lipid microdomains as segregated signaling platforms.

Despite the diverse pharmacological activities of berberine, including anti-cancer and anti-inflammatory effects, the direct proteomic targets of berberine have remained largely unknown. Here, we have identified actin as a direct proteomic target of berberine using an affinity-based chemical probe. In addition, we found that actin assembly was significantly modulated by berberine in vitro at the biochemical level and cellular level.

G protein-coupled receptors (GPCRs) regulate a wide range of physiological and pathophysiological cellular processes, thus it is important to understand how GPCRs are activated and function in various cellular contexts. In particular, the activation process of GPCRs is dynamically regulated upon various extracellular stimuli, and emerging evidence suggests the subcellular functions of GPCRs at endosomes and other organelles. Therefore, precise monitoring of the GPCR activation process with high spatiotemporal resolution is required to investigate the underlying molecular mechanisms of GPCR functions. In this review, we will introduce genetically encoded fluorescent biosensors that can precisely monitor the real-time GPCR activation process in live cells. The process includes the binding of extracellular GPCR ligands, conformational change of GPCR, recruitment of G proteins or β-arrestin, GPCR internalization and trafficking, and the GPCR-related downstream signaling events. We will introduce fluorescent GPCR biosensors based on a variety of strategies such as fluorescent resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), circular permuted fluorescent protein (cpFP), and nanobody. We will discuss the pros and cons of these GPCR biosensors as well as their applications in GPCR research.

How protein signaling networks respond to different input strengths is an important but poorly understood problem in cell biology. For example, RhoA can promote focal adhesion (FA) growth or disassembly, but how RhoA activity mediates these opposite outcomes is not clear. Here, we develop a photoswitchable RhoA guanine nucleotide exchange factor (GEF), psRhoGEF, to precisely control endogenous RhoA activity. Using this optical tool, we discover that peak FA disassembly selectively occurs upon activation of RhoA to submaximal levels. We also find that Src activation at FAs selectively occurs upon submaximal RhoA activation, identifying Src as an amplitude-dependent RhoA effector. Finally, a pharmacological Src inhibitor reverses the direction of the FA response to RhoA activation from disassembly to growth, demonstrating that Src functions to suppress FA growth upon RhoA activation. Thus, rheostatic control of RhoA activation by psRhoGEF reveals that cells can use signal amplitude to produce multiple responses to a single biochemical signal.