Localization of myosin II A and B isoforms in cultured neurons.

Tension generated by growth cones regulates both the rate and the direction of neurite growth. The most likely effectors of tension generation are actin and myosins. We are investigating the role of conventional myosin in growth cone advance. In this paper we report the localization of the two most prominent isoforms of brain myosin II in growth cones, neurites and cell bodies of rat superior cervical ganglion neurons. Affinity purified polyclonal antibodies were prepared against unique peptide sequences from human and rat A and B isoforms of myosin heavy chain. Although each of these antibodies brightly stained nonneuronal cells, antibodies to myosin heavy chain B stained neurons with greater intensity than antibodies to myosin heavy chain A. In growth cones, myosin heavy chain B was most concentrated in the margin bordering the thickened, organelle-rich central region and the thin, actin-rich peripheral region. The staining colocalized with actin bundles proximal and distal to the marginal zone, though the staining was more prominent proximally. The trailing edge of growth cones and the distal portion of the neurite often had a rimmed appearance, but more proximal regions of neurites had cytoplasmic labelling. Localizing MHC-B in growth cones previously monitored during advance (using differential interference contrast microscopy) revealed a positive correlation with edges at which retraction had just occurred and a negative correlation with lamellipodia that had recently undergone protrusion. Cell bodies were brightly labelled for myosin heavy chain B. Myosin heavy chain A staining was dimmer and its colocalization with filamentous actin bundles in growth cones was less striking than that of myosin heavy chain B. Growth cones stained for both myosin heavy chain A and B revealed that the two antigens overlapped frequently, but not exclusively, and that myosin heavy chain A lacked the elevation in the marginal zone that was characteristic of myosin heavy chain B. The pattern of staining we observed is consistent with a prominent role for myosin heavy chain B in either generating tension between widely separated areas of the growth cone, or bundling of actin filaments, which would enable other motors to effect this tension. These data support the notion that conventional myosin is important in growth cone advance and turning.

[1]  T. Svitkina,et al.  Myosin II filament assemblies in the active lamella of fibroblasts: their morphogenesis and role in the formation of actin filament bundles , 1995, The Journal of cell biology.

[2]  R. Adelstein,et al.  Neuronal Cell Expression of Inserted Isoforms of Vertebrate Nonmuscle Myosin Heavy Chain II-B (*) , 1995, The Journal of Biological Chemistry.

[3]  K. Jacobson,et al.  Traction forces generated by locomoting keratocytes , 1994, The Journal of cell biology.

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

[5]  P. Forscher,et al.  Brain myosin-V is a two-headed unconventional myosin with motor activity , 1993, Cell.

[6]  M. Bähler,et al.  Identification, characterization and cloning of myr 1, a mammalian myosin-I , 1993, The Journal of cell biology.

[7]  D. Burgess,et al.  Golgi-derived vesicles from developing epithelial cells bind actin filaments and possess myosin-I as a cytoplasmically oriented peripheral membrane protein , 1993, The Journal of cell biology.

[8]  P. De Camilli,et al.  Primary structure and cellular localization of chicken brain myosin-V (p190), an unconventional myosin with calmodulin light chains , 1992, The Journal of cell biology.

[9]  P. Bridgman,et al.  Nerve growth cone lamellipodia contain two populations of actin filaments that differ in organization and polarity , 1992, The Journal of cell biology.

[10]  M. Elzinga,et al.  Localization of myosin IIB at the leading edge of growth cones from rat dorsal root ganglionic cells , 1992, FEBS letters.

[11]  P. Levitt,et al.  Myosin II distribution in neurons is consistent with a role in growth cone motility but not synaptic vesicle mobilization , 1992, Neuron.

[12]  T. Pollard,et al.  Myosin-I moves actin filaments on a phospholipid substrate: implications for membrane targeting , 1992, The Journal of cell biology.

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

[14]  G. C. Johnston,et al.  The Saccharomyces cerevisiae MYO2 gene encodes an essential myosin for vectorial transport of vesicles , 1991, The Journal of cell biology.

[15]  S. Kawamoto,et al.  Chicken nonmuscle myosin heavy chains: differential expression of two mRNAs and evidence for two different polypeptides , 1991, The Journal of cell biology.

[16]  P. Chantler.  A unique cellular myosin II exhibiting differential expression in the cerebral cortex. , 1991, Biochemical and biophysical research communications.

[17]  R. Buxbaum,et al.  On the cytomechanics and fluid dynamics of growth cone motility , 1991, Journal of Cell Science.

[18]  M. Elzinga,et al.  Studies on the distribution of cellular myosin with antibodies to isoform‐specific synthetic peptides , 1991, FEBS letters.

[19]  N. Copeland,et al.  Molecular analysis of two mouse dilute locus deletion mutations: spontaneous dilute lethal20J and radiation-induced dilute prenatal lethal Aa2 alleles , 1990, Molecular and cellular biology.

[20]  J. C. Myers,et al.  Human nonmuscle myosin heavy chain mRNA: generation of diversity through alternative polyadenylylation. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[21]  R. Waterston,et al.  The minor myosin heavy chain, mhcA, of Caenorhabditis elegans is necessary for the initiation of thick filament assembly. , 1989, The EMBO journal.

[22]  Y. Wang,et al.  Formation and movement of myosin-containing structures in living fibroblasts , 1989, The Journal of cell biology.

[23]  Robert E. Buxbaum,et al.  Direct evidence that growth cones pull , 1989, Nature.

[24]  J. Small,et al.  Microfilament-based motility in non-muscle cells. , 1989, Current opinion in cell biology.

[25]  M. Dailey,et al.  The organization of myosin and actin in rapid frozen nerve growth cones , 1989, The Journal of cell biology.

[26]  B. Jockusch,et al.  Modulation of cellular morphology and locomotory activity by antibodies against myosin , 1988, The Journal of cell biology.

[27]  S. J. Smith,et al.  Actions of cytochalasins on the organization of actin filaments and microtubules in a neuronal growth cone , 1988, The Journal of cell biology.

[28]  D A Knecht,et al.  Antisense RNA inactivation of myosin heavy chain gene expression in Dictyostelium discoideum. , 1987, Science.

[29]  J. Spudich,et al.  Disruption of the Dictyostelium myosin heavy chain gene by homologous recombination. , 1987, Science.

[30]  H. Berg,et al.  A miniature flow cell designed for rapid exchange of media under high-power microscope objectives. , 1984, Journal of general microbiology.

[31]  I. Mabuchi,et al.  The effect of myosin antibody on the division of starfish blastomeres , 1977, The Journal of cell biology.

[32]  D. Bray,et al.  Purification and structural analysis of myosins from brain and other non-muscle tissues. , 1975, Journal of molecular biology.

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

[34]  M. Elzinga,et al.  Immunohistochemical studies on the distribution of cellular myosin II isoforms in brain and aorta. , 1992, Cell motility and the cytoskeleton.

[35]  J. Spudich,et al.  Myosin structure and function in cell motility. , 1987, Annual review of cell biology.