Supervillin slows cell spreading by facilitating myosin II activation at the cell periphery.
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.
Takizawa
Norio
N
Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA 01605, USA. Norio.Takizawa@umassmed.edu
Ikebe
Reiko
R
Ikebe
Mitsuo
M
Luna
Elizabeth J
EJ
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
D000199:Actins; D007527:Isoenzymes; D008565:Membrane Proteins; D008840:Microfilament Proteins; D011993:Recombinant Fusion Proteins; D009219:Myosin-Light-Chain Kinase; D024462:Myosin Type II