Abstract
The small GTPase RhoA is involved in cell morphology and migration. RhoA activity is tightly regulated in time and space and depends on guanine exchange factors (GEFs). However, the kinetics and subcellular localization of GEF activity towards RhoA are poorly defined. To study the mechanism underlying the spatiotemporal control of RhoA activity by GEFs, we performed single cell imaging with an improved FRET sensor reporting on the nucleotide loading state of RhoA. By employing the FRET sensor we show that a plasma membrane located RhoGEF, p63RhoGEF, can rapidly activate RhoA through endogenous GPCRs and that localized RhoA activity at the cell periphery correlates with actin polymerization. Moreover, synthetic recruitment of the catalytic domain derived from p63RhoGEF to the plasma membrane, but not to the Golgi apparatus, is sufficient to activate RhoA. The synthetic system enables local activation of endogenous RhoA and effectively induces actin polymerization and changes in cellular morphology. Together, our data demonstrate that GEF activity at the plasma membrane is sufficient for actin polymerization via local RhoA signaling.
Highlights
Rho GTPases belong to the Ras superfamily of small G proteins and are involved in a variety of cellular processes, such as the dynamic regulation of the actin cytoskeleton and cell morphology, cell cycle progression, and gene transcription[1,2]
To examine whether the plasma membrane location is important for its function, we investigated the effect of multiple p63RhoGEF deletion constructs on actin polymerization
Using the Dimerization Optimized Reporter for Activation (DORA)-RhoA FRET based biosensor, we demonstrate the kinetics of GTP loading of RhoA by p63RhoGEF via the Gα q class of heterotrimeric G proteins at the plasma membrane in living cells
Summary
Rho GTPases belong to the Ras superfamily of small G proteins and are involved in a variety of cellular processes, such as the dynamic regulation of the actin cytoskeleton and cell morphology, cell cycle progression, and gene transcription[1,2]. Like most typical G proteins, Rho GTPases function as molecular switches by cycling between an inactive GDP-bound state and an active GTP-bound state[7]. Three classes of accessory proteins that control the molecular switch kinetics and the location of Rho GTPases in cells have been identified[8,9]. Rho guanine exchange factors (Rho GEFs) stimulate the exchange of GDP for GTP, resulting in Rho GTPase activation. Rho GTPase-activating proteins (Rho GAPs) accelerate the hydrolysis of bound GTP to GDP, which abrogates Rho GTPase signaling. The signaling output of Rho GTPases is dictated by spatiotemporal control of GEF and GAP activity and the subcellular location of the Rho GTPase itself. The DH domain interacts directly with the Rho GTPase and is responsible for the catalytic activity that accelerates the exchange of GDP for GTP on the Rho GTPase[7]. The catalytic DH domain of p63RhoGEF was shown to be necessary and sufficient for its downstream signaling function[15], as is the case for many other GEFs
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