Localized cdc42 activation, detected using a novel assay, mediates microtubule organizing center positioning in endothelial cells in response to fluid shear stress.
Fluid flow regulates morphology, physiology, and pathophysiology of vascular endothelial cells (reviewed in Ref. 1). The small GTPase Cdc42 mediates polarity in several systems including migrating cells and early embryos, which involve reorientation of the microtubule organizing center (MTOC) and Golgi apparatus toward the direction of movement. Here, we show that Cdc42 is activated by fluid shear stress and that activation is a consequence of integrins binding to extracellular matrix. A novel fluorescence energy transfer assay to visualize Cdc42 activation in single cells shows that Cdc42 activity is polarized in the direction of flow. Localized activation of Cdc42 as well as the activity of Par6 and protein kinase Czeta direct the reorientation of the MTOC to a position on the downstream side of the nucleus relative to the direction of flow. Thus, shear-stimulated integrin dynamics induce polarized Cdc42 activity, which induces MTOC localization through the Par6-protein kinase Czeta complex.
Tzima
Eleni
E
Department of Cell Biology, Division of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Kiosses
William B
WB
del Pozo
Miguel A
MA
Schwartz
Martin Alexander
MA
D000818:Animals; D001011:Aorta / Q000166:cytology; D002417:Cattle; D016764:Cell Polarity / Q000502:physiology; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000378:metabolism; D016023:Integrins / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D022101:Microtubule-Organizing Center / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013314:Stress, Mechanical; D020764:cdc42 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*; D020741:rho GTP-Binding Proteins / Q000378:metabolism; D000099221:Protein Kinase C zeta
D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents; D016023:Integrins; D008164:Luminescent Proteins; D011493:Protein Kinase C; D011993:Recombinant Fusion Proteins; D020764:cdc42 GTP-Binding Protein; D020741:rho GTP-Binding Proteins; D000099221:Protein Kinase C zeta