title: Supervillin slows cell spreading by facilitating myosin II activation at the cell periphery.
abstract: During cell migration, myosin II modulates adhesion, cell protrusion and actin organization at the leading edge. We show that an F-actin- and membrane-associated scaffolding protein, called supervillin (SV, p205), binds directly to the subfragment 2 domains of nonmuscle myosin IIA and myosin IIB and to the N-terminus of the long form of myosin light chain kinase (L-MLCK). SV inhibits cell spreading via an MLCK- and myosin II-dependent mechanism. Overexpression of SV reduces the rate of cell spreading, and RNAi-mediated knockdown of endogenous SV increases it. Endogenous and EGFP-tagged SV colocalize with, and enhance the formation of, cortical bundles of F-actin and activated myosin II during early cell spreading. The effects of SV are reversed by inhibition of myosin heavy chain (MHC) ATPase (blebbistatin), MLCK (ML-7) or MEK (U0126), but not by inhibiting Rho-kinase with Y-27632. Flag-tagged L-MLCK co-localizes in cortical bundles with EGFP-SV, and kinase-dead L-MLCK disorganizes these bundles. The L-MLCK- and myosin-binding site in SV, SV1-171, rearranges and co-localizes with mono- and di-phosphorylated myosin light chain and with L-MLCK, but not with the short form of MLCK (S-MLCK) or with myosin phosphatase. Thus, the membrane protein SV apparently contributes to myosin II assembly during cell spreading by modulating myosin II regulation by L-MLCK.
authors.affiliation: Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA 01605, USA. Norio.Takizawa@umassmed.edu
authors.forename: Norio
authors.initials: N
authors.lastname: Takizawa
authors.forename: Reiko
authors.initials: R
authors.lastname: Ikebe
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Ikebe
authors.forename: Elizabeth J
authors.initials: EJ
authors.lastname: Luna
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D002448:Cell Adhesion / Q000502:physiology*; D002465:Cell Movement / Q000502:physiology*; D002522:Chlorocebus aethiops; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism*; D024462:Myosin Type II / Q000235:genetics / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000199:Actins; D007527:Isoenzymes; D008565:Membrane Proteins; D008840:Microfilament Proteins; D011993:Recombinant Fusion Proteins; D009219:Myosin-Light-Chain Kinase; D024462:Myosin Type II