Lovelace MD, Powter EE, Coleman PR et al.
Centre for the Endothelium, Vascular Biology Program, Centenary Institute, The University of Sydney, Sydney, New South Wales, 2050 Australia.
Molecular biology of the cell. Mar 2017.
RhoGTPases are important regulators of the cell cytoskeleton, controlling cell shape, migration and proliferation. Previously, we showed ARHGAP18 in endothelial cells is important in cell junctions. Here we show using structured illumination microscopy (SIM), ground state depletion (GSD) and total internal reflection fluorescence (TIRF) that a proportion of ARHGAP18 localises to microtubules in endothelial cells as well as non-endothelial cells, an association confirmed biochemically. In endothelial cells some ARHGAP18 puncta also colocalised to Weibel-Palade Bodies on the microtubules. Depletion of ARHGAP18 by siRNA or analysis of endothelial cells isolated from ARHGAP18 knockout mice showed microtubule destabilisation as evidenced by altered morphology and decreased acetylated α-tubulin and glu-tubulin. The destabilisation was rescued by inhibition of ROCK, histone deacetylase 6 but not by a GAP mutant form of ARHGAP18. Depletion of ARHGAP18 resulted in a failure to secrete endothelin-1 and a reduction in neutrophil transmigration, both events known to be microtubule-dependent. Thrombin, a critical regulator of the Rho-mediated barrier function of endothelial cells through microtubule destabilisation, enhanced the plasma membrane bound fraction of ARHGAP18. Thus, in endothelial cells ARHGAP18 may act as a significant regulator of vascular homeostasis.