Hypertonicity triggers RhoA-dependent assembly of myosin-containing striated polygonal actin networks in endothelial cells.
Endothelial cells respond to mechanical stresses of the circulation with cytoskeletal rearrangements such as F-actin stress fiber alignment along the axis of fluid flow. Endothelial cells are exposed to hypertonic stress in the renal medulla or during mannitol treatment of cerebral edema. We report here that arterial endothelial cells exposed to hypertonic stress rearranged F-actin into novel actin-myosin II fibers with regular 0.5-microm striations, in which alpha-actinin colocalizes with actin. These striated fibers assembled over hours into three-dimensional, irregular, polygonal actin networks most prominent at the cell base, and occasionally surrounding the nucleus in a geodesic-like structure. Hypertonicity-induced assembly of striated polygonal actin networks was inhibited by cytochalasin D, blebbistatin, cell ATP depletion, and intracellular Ca(2+) chelation but did not require intact microtubules, regulatory volume increase, or de novo RNA or protein synthesis. Striated polygonal actin network assembly was insensitive to inhibitors of MAP kinases, tyrosine kinases, or phosphatidylinositol 3-kinase, but was prevented by C3 exotoxin, by the RhoA kinase inhibitor Y-27632, and by overexpressed dominant-negative RhoA. In contrast, overexpression of dominant-negative Rac or of dominant-negative cdc42 cDNAs did not prevent striated polygonal actin network assembly. The actin networks described here are novel in structure, as striated actin-myosin structures in nonmuscle cells, as a cellular response to hypertonicity, and as a cytoskeletal regulatory function of RhoA. Endothelial cells may use RhoA-dependent striated polygonal actin networks, possibly in concert with cytoskeletal load-bearing elements, as a contractile, tension-generating component of their defense against isotropic compressive forces.
Malek
Adel M
AM
Molecular and Vascular Medicine Unit, Beth Israel Deaconess Medical Center, 330 Brookline Ave, Boston, MA 02215, USA. amalek@tufts-nemc.org
Xu
Chang
C
Kim
Edward S
ES
Alper
Seth L
SL
D036002:ADP Ribose Transferases / Q000494:pharmacology; D000185:Actinin / Q000378:metabolism; D000199:Actins / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000577:Amides / Q000494:pharmacology; D000818:Animals; D001905:Botulinum Toxins / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D048430:Cell Shape; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D022001:Focal Adhesions / Q000378:metabolism; D008353:Mannitol / Q000378:metabolism; D024462:Myosin Type II / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D012462:Saline Solution, Hypertonic / Q000378:metabolism*; D022502:Stress Fibers / Q000187:drug effects / Q000378:metabolism*; D013997:Time Factors; D020742:rhoA GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000199:Actins; D000577:Amides; D047428:Protein Kinase Inhibitors; D011725:Pyridines; D012462:Saline Solution, Hypertonic; D000185:Actinin; C108830:Y 27632; D008353:Mannitol; D000255:Adenosine Triphosphate; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D001905:Botulinum Toxins; D024462:Myosin Type II; D020742:rhoA GTP-Binding Protein; D002118:Calcium