Nonmuscle myosin IIb is involved in the guidance of fibroblast migration.

Although myosin II is known to play an important role in cell migration, little is known about its specific functions. We have addressed the function of one of the isoforms of myosin II, myosin IIB, by analyzing the movement and mechanical characteristics of fibroblasts where this protein has been ablated by gene disruption. Myosin IIB null cells displayed multiple unstable and disorganized protrusions, although they were still able to generate a large fraction of traction forces when cultured on flexible polyacrylamide substrates. However, the traction forces were highly disorganized relative to the direction of cell migration. Analysis of cell migration patterns indicated an increase in speed and decrease in persistence, which were likely responsible for the defects in directional movements as demonstrated with Boyden chambers. In addition, unlike control cells, mutant cells failed to respond to mechanical signals such as compressing forces and changes in substrate rigidity. Immunofluorescence staining indicated that myosin IIB was localized preferentially along stress fibers in the interior region of the cell. Our results suggest that myosin IIB is involved not in propelling but in directing the cell movement, by coordinating protrusive activities and stabilizing the cell polarity.

[1]  Ehud Goldin,et al.  Identification and Characterization of Nonmuscle Myosin II-C, a New Member of the Myosin II Family* , 2004, Journal of Biological Chemistry.

[2]  M. Sheetz,et al.  Cell migration: regulation of force on extracellular-matrix-integrin complexes. , 1998, Trends in cell biology.

[3]  M. Yanagisawa,et al.  Two distinct nonmuscle myosin-heavy-chain mRNAs are differentially expressed in various chicken tissues. Identification of a novel gene family of vertebrate non-sarcomeric myosin heavy chains. , 1989, European journal of biochemistry.

[4]  S. Kawamoto,et al.  Gene dosage affects the cardiac and brain phenotype in nonmuscle myosin II-B-depleted mice. , 2000, The Journal of clinical investigation.

[5]  S. R. Wylie,et al.  Separate but linked functions of conventional myosins modulate adhesion and neurite outgrowth , 2000, Nature Cell Biology.

[6]  W. Loomis,et al.  Cell motility and chemotaxis in Dictyostelium amebae lacking myosin heavy chain. , 1988, Developmental biology.

[7]  J. Kolega,et al.  Cytoplasmic dynamics of myosin IIA and IIB: spatial 'sorting' of isoforms in locomoting cells. , 1998, Journal of cell science.

[8]  J. Wolenski.  Regulation of calmodulin-binding myosins. , 1995, Trends in cell biology.

[9]  Yu-Li Wang,et al.  The Small GTP-binding Protein Rho Regulates Cortical Activities in Cultured Cells during Division , 1999, The Journal of cell biology.

[10]  J. Sellers,et al.  Xenopus nonmuscle myosin heavy chain isoforms have different subcellular localizations and enzymatic activities [published erratum appears in J Cell Biol 1997 Jul 14;138(1):215] , 1996, Journal of Cell Biology.

[11]  K. Burridge,et al.  Focal adhesions, contractility, and signaling. , 1996, Annual review of cell and developmental biology.

[12]  R. Adelstein,et al.  Localization of myosin II A and B isoforms in cultured neurons. , 1995, Journal of cell science.

[13]  J. Small.  Organization of actin in the leading edge of cultured cells: influence of osmium tetroxide and dehydration on the ultrastructure of actin meshworks , 1981, The Journal of cell biology.

[14]  M. Dembo,et al.  Traction force microscopy of migrating normal and H-ras transformed 3T3 fibroblasts. , 2001, Biophysical journal.

[15]  M. Dembo,et al.  Stresses at the cell-to-substrate interface during locomotion of fibroblasts. , 1999, Biophysical journal.

[16]  T. Mitchison,et al.  Actin-Based Cell Motility and Cell Locomotion , 1996, Cell.

[17]  K. Beningo,et al.  Nascent Focal Adhesions Are Responsible for the Generation of Strong Propulsive Forces in Migrating Fibroblasts , 2001, The Journal of cell biology.

[18]  R. Singer,et al.  A Rho-dependent signaling pathway operating through myosin localizes β-actin mRNA in fibroblasts , 2001, Current Biology.

[19]  Gary G. Borisy,et al.  Analysis of the Actin–Myosin II System in Fish Epidermal Keratocytes: Mechanism of Cell Body Translocation , 1997, The Journal of cell biology.

[20]  D. Lauffenburger,et al.  Cell Migration: A Physically Integrated Molecular Process , 1996, Cell.

[21]  S. Kawamoto,et al.  Human nonmuscle myosin heavy chains are encoded by two genes located on different chromosomes. , 1991, Circulation research.

[22]  Y. Wang,et al.  Cell locomotion and focal adhesions are regulated by substrate flexibility. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[23]  F. Maxfield,et al.  Ca2+-dependent myosin II activation is required for uropod retraction during neutrophil migration. , 2000, Journal of cell science.

[24]  Micah Dembo,et al.  Measurements of cell-generated deformations on flexible substrata using correlation-based optical flow. , 2003, Methods in enzymology.

[25]  R. Adelstein,et al.  Myosin IIB Is Required for Growth Cone Motility , 2001, The Journal of Neuroscience.

[26]  T. Pollard,et al.  Differential localization of myosin-II isozymes in human cultured cells and blood cells. , 1994, Journal of cell science.

[27]  K. Kaibuchi,et al.  Stress fiber organization regulated by MLCK and Rho-kinase in cultured human fibroblasts. , 2001, American journal of physiology. Cell physiology.

[28]  P. Bridgman,et al.  Retrograde flow rate is increased in growth cones from myosin IIB knockout mice , 2003, Journal of Cell Science.

[29]  J. Condeelis,et al.  The physiological significance of β-actin mRNA localization in determining cell polarity and directional motility , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[30]  D. Buxton,et al.  Induction of Nonmuscle Myosin Heavy Chain II-C by Butyrate in RAW 264.7 Mouse Macrophages* , 2003, The Journal of Biological Chemistry.

[31]  J. Small,et al.  Coordination of protrusion and translocation of the keratocyte involves rolling of the cell body , 1996, The Journal of cell biology.

[32]  Y. Wang,et al.  Preparation of a flexible, porous polyacrylamide substrate for mechanical studies of cultured cells. , 1998, Methods in enzymology.

[33]  Dunn Ga,et al.  Characterising a kinesis response: time averaged measures of cell speed and directional persistence. , 1983 .

[34]  Jacquelyn A. Brown,et al.  Role of Myosin II in Axon Outgrowth , 2003, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[35]  Fumio Matsumura,et al.  Distinct Roles of Rock (Rho-Kinase) and Mlck in Spatial Regulation of Mlc Phosphorylation for Assembly of Stress Fibers and Focal Adhesions in 3t3 Fibroblasts , 2000, The Journal of cell biology.

[36]  Y. Wang,et al.  High resolution detection of mechanical forces exerted by locomoting fibroblasts on the substrate. , 1999, Molecular biology of the cell.

[37]  S. R. Wylie,et al.  A conventional myosin motor drives neurite outgrowth. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[38]  J. Berg,et al.  A millennial myosin census. , 2001, Molecular biology of the cell.

[39]  V. Ferrans,et al.  Nonmuscle myosin II-B is required for normal development of the mouse heart. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[40]  G. Dunn,et al.  Characterising a kinesis response: time averaged measures of cell speed and directional persistence. , 1983, Agents and actions. Supplements.

[41]  M. Dembo,et al.  Cell movement is guided by the rigidity of the substrate. , 2000, Biophysical journal.

[42]  H. Haga,et al.  Differential localization of non‐muscle myosin II isoforms and phosphorylated regulatory light chains in human MRC‐5 fibroblasts , 2001, FEBS letters.

[43]  E. Elson,et al.  A mechanical function of myosin II in cell motility. , 1995, Journal of cell science.

[44]  S. Carter,et al.  Haptotaxis and the Mechanism of Cell Motility , 1967, Nature.

[45]  R. Adelstein,et al.  Conditional expression of a truncated fragment of nonmuscle myosin II-A alters cell shape but not cytokinesis in HeLa cells. , 2000, Molecular biology of the cell.

[46]  J. Spudich,et al.  Towards a molecular understanding of cytokinesis. , 2000, Trends in cell biology.