Stephenson RE and Miller AL (2017), Tools for live imaging of active Rho GTPases in...

XB-ART-53030

Genesis 2017 Jan 01;551-2:. doi: 10.1002/dvg.22998.

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Tools for live imaging of active Rho GTPases in Xenopus.

Stephenson RE , Miller AL .

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Rho family GTPases are signaling molecules that orchestrate cytoskeletal dynamics in a variety of cellular processes. Because they effect localized changes to the cytoskeleton only in their active (GTP-bound) conformation, the ability to monitor the active state of Rho GTPases in space and time is critical for understanding their function. Here, we summarize popular tools used for live imaging of active Rho GTPases, outlining advantages and drawbacks of these approaches. Additionally, we highlight key features of the Xenopus laevis embryo that make it well-suited for epithelial cell biology and discuss how application of Rho activity reporters in the Xenopus laevis embryo led to the discovery of a novel phenomenon, junctional Rho flares.

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Species referenced: Xenopus laevis
Genes referenced: rho rho.2 rhoa

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	Figure 1. Rho GTPase activity is tightly regulated to create distinct zones of activation. a. Typical Rho family GTPases cycle between an active, GTP-bound state and an inactive, GDP-bound state. Guanine nucleotide exchange factors (GEFs) promote the active state by exchanging GDP for GTP, while GTPase activating proteins (GAPs) inactivate GTPases by stimulating GTP hydrolysis. Rho guanine nucleotide dissociation inhibitor (Rho GDI) sequesters Rho-GDP in the cytoplasm, protecting it from degradation and preventing its activation. In the active conformation, Rho GTPases activate effectors through direct binding, usually by relieving an autoinhibited conformation, allowing effectors to act on their downstream targets. b. RhoA is active in distinct zones in epithelia: at cellâ€“cell junctions and at the contractile ring of dividing cells.
	Figure 2. Approaches for studying active Rho GTPases. a. In the GBD affinity pull-down approach, the active GTPase is pulled down with a GST-tagged effector GBD specific for the GTPase of interest (rGBDâ€‰=â€‰Rhotekin GBD, which binds RhoA, B, and C). The amount of GTPase in the pull-down is compared with total GTPase in the sample to approximate the pool of active GTPase in the sample. b. In the effector translocation (GBD probe) approach, the effector GBD is fluorescently-tagged (with GFP) and binds to the endogenous active GTPase. Local increase in fluorescence intensity over background is interpreted as increased active GTPase. c. In the GTPase-effector FRET biosensor (FRET biosensor) approach, the GTPase is tagged with a donor fluorophore (CFP) and the effector GBD is tagged with an acceptor fluorophore (YFP). A unimolecular Rho biosensor is shown here. When the GTPase is inactive, the donor fluorophore emits light. When the GTPase is active, the donor fluorophore excites the acceptor fluorophore.
	Figure 3. Rho zones and Rho flares in X. laevis embryos. a. GFP-rGBD (GBD probe for active Rho) in the large blastomeres of the early X. laevis embryo. A distinct zone of active Rho specifies the position of the contractile ring (yellow arrowhead). b. GFP-rGBD in epithelial cells of the gastrula-stage X. laevis embryo. Zones of active Rho encircle the perimeter of each epithelial cell. c. A montage depicting a Rho flare over time. These transient accumulations of active Rho at cellâ€“cell junctions were first observed in the X. laevis embryo (Reyes et al., 2014).

References [+] :

Alan, Mutationally activated Rho GTPases in cancer. 2013, Pubmed