Figure 1.

Direct Investigation of Rho-Dependent GEF-H1 Plasma Membrane Recruitment

(A) TIRF image of a U2OS cell that expresses the Rho activity sensor mCherry-Rhotekin-GBD. Rho activity dynamics were stimulated by releasing GEF-H1 (ARHGEF2) from microtubules into the cytosol via nocodazole.

(B) Kymographs corresponding to the yellow box in (A), which represent irregular, Rho activity dynamics before and after nocodazole application.

(C) Schematic representation of acute chemo-optogenetic plasma membrane recruitment of active Rho. NvocTMP-Cl: photocaged chemical dimerizer; HaloTag/eDHFR: dimerization domains for chemo-optogenetic perturbation.

(D) Representative TIRF images of chemo-optogenetic mTurquoise2-eDHFR-Rho Q63L plasma membrane recruitment and co-recruitment of Rho activity sensor (mCitrine-Rhotekin-GBD) and cytosolic, microtubule-binding deficient GEF-H1(C53R) mutant fused to mCherry (see also Video S1).

(E and F) Quantification of co-recruitment of RhoA constructs, Rho activity or control sensors, and GEF-H1(C53R) or GEF-H1 PH domain (percentage increase above average intensity before photoactivation at t = 0 s with standard error of the mean (SEM); n ≥ 10 (E) or n ≥ 15 (F) cells from 3 experiments; ^∗∗p ≤ 0.01; ^∗∗∗p ≤ 0.001; ^∗∗∗∗p ≤ 0.0001; paired t test before and 10 s after photoactivation.

Scale bars: 10 μm.

See also Figure S1.