Characterization of Genetically Encoded FRET Biosensors for Rho-Family GTPases

Donnelly, Sara K.; Miskolci, Veronika; Garrastegui, Alice M.; Cox, Dianne; Hodgson, Louis

doi:10.1007/978-1-4939-8612-5_7

Cited by 5 publications

(4 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Enzyme activation biosensors often incorporate the targets of interest within their design architecture ( 32 ), which can cause side effects from overexpression ( 33 ). Thus, we hypothesized that FluoSTEPs could help untangle this dependence by uncoupling the expression of the target of interest from the rest of the sensor.…”

Section: Resultsmentioning

confidence: 99%

FluoSTEPs: Fluorescent biosensors for monitoring compartmentalized signaling within endogenous microdomains

et al. 2021

View full text Add to dashboard Cite

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.

show abstract

Section: Resultsmentioning

confidence: 99%

FluoSTEPs: Fluorescent biosensors for monitoring compartmentalized signaling within endogenous microdomains

et al. 2021

View full text Add to dashboard Cite

show abstract

“…Such an approach might be also used to co-detect and correct for pH fluctuations, which have the potency to perturb the performance of most FP-based biosensors. 31 ■ CONCLUSIONS AND OUTLOOK While purified biosensors have mainly been used for sensor characterization purposes, 3,10,32,33 we describe a novel biotinylation-based procedure to use genetically encoded sensors for the recording of extracellular ions locally on cell surfaces and glass slides upon their immobilization using three workflows (Figure S8): first, purification of the recombinant TAv biosensor, second, expression of the biotinylated cell surface tag or using a biotinylated glass slide, and third, coupling of the TAv biosensor to the biotinylated surface.…”

Section: ■ Experimental Methodsmentioning

confidence: 99%

Immobilization of Recombinant Fluorescent Biosensors Permits Imaging of Extracellular Ion Signals

et al. 2021

View full text Add to dashboard Cite

Given the importance of ion gradients and fluxes in biology, monitoring ions locally at the exterior of the plasma membrane of intact cells in a noninvasive manner is highly desirable but challenging. Classical targeting of genetically encoded biosensors at the exterior of cell surfaces would be a suitable approach; however, it often leads to intracellular accumulation of the tools in vesicular structures and adverse modifications, possibly impairing sensor functionality. To tackle these issues, we generated recombinant fluorescent ion biosensors fused to traptavidin (TAv) specifically coupled to a biotinylated AviTag expressed on the outer cell surface of cells. We show that purified chimeras of TAv and pH-Lemon or GEPII 1.0, Forster resonance energy transferbased pH and K + biosensors, can be immobilized directly and specifically on biotinylated surfaces including glass platelets and intact cells, thereby remaining fully functional for imaging of ion dynamics. The immobilization of recombinant TAv−GEPII 1.0 on the extracellular cell surface of primary cortical rat neurons allowed imaging of excitotoxic glutamate-induced K + efflux in vitro. We also performed micropatterning of purified TAv biosensors using a microperfusion system to generate spatially separated TAv−pH-Lemon and TAv−GEPII 1.0 spots for simultaneous pH and K + measurements on cell surfaces. Our results suggest that the approach can be greatly expanded by immobilizing various biosensors on extracellular surfaces to quantitatively visualize microenvironmental transport and signaling processes in different cell culture models and other experimental settings.

show abstract

“…Since their development, FRET biosensors have improved with technological advancements. The design of these biosensors allows for the detection of activity (protein and binding domain) and for the detection of binding of proteins within the same complex at proximal distances (Donnelly et al, 2018). Lately, the FRET technique has been getting a lot of attention in the field due to its capacity to detect the spatiotemporal activation of RhoGTPases and their localization inside the cell.…”

Section: Discussionmentioning

confidence: 99%

“…In turn, this results in an increase in the FRET signal, allowing us to distinguish the temporal and spatial activation of the GTPase (Donnelly et al, 2014;Sin et al, 2020). The development of the RhoGTPases family biosensors has helped understand, like never before, the activation pattern of these proteins during cell migration and invasion and the important role they play in actin polymerization (Donnelly et al, 2018;S. J. Hanna et al, 2017;Itoh et al, 2002;Kraynov et al, 2000).…”

Section: Overviewmentioning

confidence: 99%

The Activation Kinetics and Crosstalk of RhoGTPases. (c2020)

Haddad¹

View full text Add to dashboard Cite

Fluorescence or Förster resonance energy transfer (FRET) is a quantitative transfer of energy between two fluorescent moieties in proximal distance, between 10Å and 100Å. The energy transfer is useful in studying direct molecular interactions and activation. Hence, FRET led to the design of biosensors. Biosensors are two chromophores linked to 2 entities, joined together by a linker which determines their juxtaposition relative to each other, and thus the amount of energy transfer. By exciting one chromophore and observing the emission of the other, one can determine the activation and binding of certain proteins. Using this technique, we are studying the crosstalk of Rho GTPases in different cell lines, with a focus on RhoA, RhoC, Cdc42 and Rac1. We first showed how RhoGTPase activity is concentrated at the cell periphery. We also found Cdc42 activation to be concentrated at invadopodia and membrane ruffles. Then, we investigated the interplay between Cdc42 and RhoA by knocking down one target and studying the activation of the other in brain cancer cells, and vice versa. The results indicate an antagonistic relationship between the two in one glioblastoma cell line and a cooperative relation in the other. As for the interplay of RhoA and RhoC with Rac1, we found out that RhoA regulates the expression of Rac1 in cells, whereas RhoC has no effect on Rac1 expression or activity levels. Additionally, we wanted to use FRET so test the regulation of RhoGTPases by upstream effectors such as STARD13. We checked for the effect of STARD13 knockdown on Cdc42 activation in lung cancer cells. In the A549 lung cancer cells, the activation of Cdc42 increased as we hypothesized. Altogether these experiments show the efficiency of using FRET as a tool for studying the activation kinetics and dynamics of Rho GTPases.

show abstract

Characterization of Genetically Encoded FRET Biosensors for Rho-Family GTPases

Cited by 5 publications

References 43 publications

FluoSTEPs: Fluorescent biosensors for monitoring compartmentalized signaling within endogenous microdomains

FluoSTEPs: Fluorescent biosensors for monitoring compartmentalized signaling within endogenous microdomains

Immobilization of Recombinant Fluorescent Biosensors Permits Imaging of Extracellular Ion Signals

The Activation Kinetics and Crosstalk of RhoGTPases. (c2020)

Contact Info

Product

Resources

About