title: Fibronectin matrix assembly requires distinct contributions from Rho kinases I and -II.
abstract: Extracellular matrix is integral to tissue architecture and regulates many aspects of cell behavior. Fibronectin matrix assembly involves the actin cytoskeleton and the small GTPase RhoA, but downstream signaling is not understood. Here, down-regulation of either rho kinase isoform (ROCK I or -II) by small interfering RNA treatment blocked fibronectin matrix assembly, although the phenotypes were distinct and despite persistence of the alternate kinase. Remnant fibronectin on ROCK-deficient fibroblasts was mostly punctate and more deoxycholate soluble compared with controls. Fibronectin matrix assembly defects in ROCK-deficient cells did not result from decreased synthesis/secretion, altered fibronectin mRNA splicing, metalloproteinase activity, or alpha5beta1 integrin dysfunction. Rescue could be effected by ROCK protein restoration or phosphomimetic myosin light chain expression. However, the effect of ROCK I deficiency on fibronectin matrix assembly was secondary to altered cell surface morphology, rich in filopodia, resulting from high GTP-Cdc42 levels. Total internal reflection microscopy revealed that a submembranous pool of myosin light chain in control cells was missing in ROCK II-deficient cells and replaced by stress fibers. Together, two rho kinases contribute to fibronectin matrix assembly in a different manner and cortical myosin II-driven contractility, but not stress fibers, may be critical in this activity.
authors.affiliation: Division of Biomedical Sciences, Imperial College London, London SW7 2AZ, United Kingdom.
authors.forename: Atsuko
authors.initials: A
authors.lastname: Yoneda
authors.forename: Dmitriy
authors.initials: D
authors.lastname: Ushakov
authors.forename: Hinke A B
authors.initials: HA
authors.lastname: Multhaupt
authors.forename: John R
authors.initials: JR
authors.lastname: Couchman
mesh_terms: D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003840:Deoxycholic Acid / Q000494:pharmacology; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects; D005353:Fibronectins / Q000096:biosynthesis / Q000378:metabolism*; D017353:Gene Deletion; D006801:Humans; D039081:Integrin alpha5beta1 / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000172:deficiency / Q000378:metabolism*; D007527:Isoenzymes / Q000172:deficiency / Q000378:metabolism; D008840:Microfilament Proteins / Q000378:metabolism; D018994:Myosin Light Chains / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000172:deficiency / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D022502:Stress Fibers / Q000187:drug effects / Q000378:metabolism; D000070876:Tensins; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D054460:rho-Associated Kinases
chemical_list: D005353:Fibronectins; D039081:Integrin alpha5beta1; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D008840:Microfilament Proteins; D018994:Myosin Light Chains; D034741:RNA, Small Interfering; D000070876:Tensins; C000603372:Tns1 protein, rat; D003840:Deoxycholic Acid; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D020764:cdc42 GTP-Binding Protein