Fig. 1: A general method for rapid perturbation of Rho GTPase activity in living cells. | Nature Communications

Fig. 1: A general method for rapid perturbation of Rho GTPase activity in living cells.

From: Rho GTPase activity crosstalk mediated by Arhgef11 and Arhgef12 coordinates cell protrusion-retraction cycles

Fig. 1

a Schematic of rapid, reversible Rho GTPase activity perturbation via chemically-induced dimerization and readout of cell morphology and cytoskeletal organization by an actin-reporter. b Representative frames from TIRF microscopy time series of mCherry-Actin obtained 30 s before, 24 min during and 24 min after Rho GTPase activation in Neuro-2a neuroblastoma cells (see also Supplementary Movie 1). Yellow arrows point to cell areas that reversibly generate protrusions during Rac1 or Cdc42 activation, and yellow arrowheads point to areas that undergo reversible retraction during RhoA activation. Observations are representative for 3 independent repetitions with a total of at least 40 cells per condition (exact numbers of cells are indicated in individual panels). Numbers in middle panels indicate percentage ± standard error of the mean of cells that initiate protrusion (Rac1/Cdc42) or retraction (RhoA) after addition of dimerizer. Numbers in panels indicate percentage of reacting cells that showed a phenotypic reversal. Scale bar: 10 μm; 0.26 μm/pixel; CFP cyan fluorescent protein, BFP blue fluorescent protein, RFP red fluorescent protein, FKBP’ FK506-binding protein with F36V mutation, eDHFR E. coli dihydrofolate reductase, SLF’ synthetic ligand of FKBP’, TMP eDHFR interacting small molecule trimethoprim.

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