title: The RhoGAP protein ARHGAP18/SENEX localizes to microtubules and regulates their stability in endothelial cells.
abstract: RhoGTPases are important regulators of the cell cytoskeleton, controlling cell shape, migration and proliferation. Previously we showed that 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 microscopy (TIRF) that a proportion of ARHGAP18 localizes to microtubules in endothelial cells, as well as in nonendothelial cells, an association confirmed biochemically. In endothelial cells, some ARHGAP18 puncta also colocalized to Weibel-Palade bodies on the microtubules. Depletion of ARHGAP18 by small interfering RNA or analysis of endothelial cells isolated from ARHGAP18-knockout mice showed microtubule destabilization, as evidenced by altered morphology and decreased acetylated α-tubulin and glu-tubulin. The destabilization was rescued by inhibition of ROCK and 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 known to be microtubule dependent. Thrombin, a critical regulator of the Rho-mediated barrier function of endothelial cells through microtubule destabilization, enhanced the plasma membrane-bound fraction of ARHGAP18. Thus, in endothelial cells, ARHGAP18 may act as a significant regulator of vascular homeostasis.
authors.affiliation: Centre for the Endothelium, Vascular Biology Program, University of Sydney, Sydney, NSW 2050, Australia.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Lovelace
authors.affiliation: Centre for the Endothelium, Vascular Biology Program, University of Sydney, Sydney, NSW 2050, Australia.
authors.forename: Elizabeth E
authors.initials: EE
authors.lastname: Powter
authors.affiliation: Centre for the Endothelium, Vascular Biology Program, University of Sydney, Sydney, NSW 2050, Australia.
authors.forename: Paul R
authors.initials: PR
authors.lastname: Coleman
authors.affiliation: Centre for the Endothelium, Vascular Biology Program, University of Sydney, Sydney, NSW 2050, Australia.
authors.forename: Yang
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Tumour Biology and Targeting Program, Children's Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW 2052, Australia.
authors.forename: Amelia
authors.initials: A
authors.lastname: Parker
authors.affiliation: Centre for the Endothelium, Vascular Biology Program, University of Sydney, Sydney, NSW 2050, Australia.
authors.forename: Garry H
authors.initials: GH
authors.lastname: Chang
authors.affiliation: Centre for the Endothelium, Vascular Biology Program, University of Sydney, Sydney, NSW 2050, Australia.
authors.forename: Angelina J
authors.initials: AJ
authors.lastname: Lay
authors.affiliation: Centre for the Endothelium, Vascular Biology Program, University of Sydney, Sydney, NSW 2050, Australia.
authors.forename: Julie
authors.initials: J
authors.lastname: Hunter
authors.affiliation: Structural Biology Laboratory, University of Sydney, Sydney, NSW 2050, Australia.
authors.forename: Aaron P
authors.initials: AP
authors.lastname: McGrath
authors.affiliation: Structural Biology Laboratory, University of Sydney, Sydney, NSW 2050, Australia.
authors.forename: Mika
authors.initials: M
authors.lastname: Jormakka
authors.affiliation: Liver Immunology Laboratory, University of Sydney, Sydney, NSW 2050, Australia.
authors.forename: Patrick
authors.initials: P
authors.lastname: Bertolino
authors.affiliation: Liver Biology and Cancer Laboratory, Centenary Institute, University of Sydney, Sydney, NSW 2050, Australia.
authors.forename: Geoffrey
authors.initials: G
authors.lastname: McCaughan
authors.affiliation: Tumour Biology and Targeting Program, Children's Cancer Institute, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW 2052, Australia.|ARC Centre of Excellence in Convergent Bio-Nano Science and Technology and Australian Centre for NanoMedicine, University of New South Wales, Sydney, NSW 2052, Australia.
authors.forename: Maria
authors.initials: M
authors.lastname: Kavallaris
authors.affiliation: Centre for the Endothelium, Vascular Biology Program, University of Sydney, Sydney, NSW 2050, Australia j.gamble@centenary.org.au m.vadas@centenary.org.au.
authors.forename: Mathew A
authors.initials: MA
authors.lastname: Vadas
authors.affiliation: Centre for the Endothelium, Vascular Biology Program, University of Sydney, Sydney, NSW 2050, Australia j.gamble@centenary.org.au m.vadas@centenary.org.au.
authors.forename: Jennifer R
authors.initials: JR
authors.lastname: Gamble
mesh_terms: D000107:Acetylation; D000199:Actins / Q000378:metabolism; D000818:Animals; D002465:Cell Movement / Q000502:physiology; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D020690:GTPase-Activating Proteins / Q000378:metabolism / Q000502:physiology*; D006367:HeLa Cells; D000073864:Histone Deacetylase 6; D006655:Histone Deacetylases / Q000378:metabolism; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D007365:Intercellular Junctions / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D008870:Microtubules / Q000378:metabolism / Q000502:physiology*; D014404:Tubulin / Q000378:metabolism; D054460:rho-Associated Kinases / Q000378:metabolism
chemical_list: C542464:ARHGAP18 protein, human; C000599629:ARHGAP18 protein, mouse; D000199:Actins; D020690:GTPase-Activating Proteins; D014404:Tubulin; D054460:rho-Associated Kinases; C433811:HDAC6 protein, human; D000073864:Histone Deacetylase 6; D006655:Histone Deacetylases