Localization of Unconventional Myosins V and VI in Neuronal Growth Cones Daniel M. Suter, 1 Foued S. Espindola, 1,4, * Chi-Hung Lin, 1,† Paul Forscher, 1 Mark S. Mooseker 1,2,3 1 Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06520 2 Department of Cell Biology, Yale University, New Haven, Connecticut 06520 3 Department of Pathology, Yale University, New Haven, Connecticut 06520 4 Departments of Genetics and Biochemistry, University Federal of Uberlandia, Uberlandia, MG 38400-982, Brazil Received 20 August 1999; accepted 27 September 1999 ABSTRACT: Class V and VI myosins, two of the six known classes of actin-based motor genes expressed in vertebrate brain (Class I, II, V, VI, IX, and XV), have been suggested to be organelle motors. In this report, the neu- ronal expression and subcellular localization of chicken brain myosin V and myosin VI is examined. Both myosins are expressed in brain during embryogenesis. In cultured dorsal root ganglion (DRG) neurons, immunolocalization of myosin V and myosin VI revealed a similar distribution for these two myosins. Both are present within cell bodies, neurites and growth cones. Both of these myosins exhibit punctate labeling patterns that are found in the same subcellular region as microtubules in growth cone central domains. In peripheral growth cone domains, where indi- vidual puncta are more readily resolved, we observe a similar number of myosin V and myosin VI puncta. How- ever, less than 20% of myosin V and myosin VI puncta colocalize with each other in the peripheral domains. After live cell extraction, punctate staining of myosin V and myosin VI is reduced in peripheral domains. However, we do not detect such changes in the central domains, suggest- ing that these myosins are associated with cytoskeletal/ organelle structures. In peripheral growth cone domains myosin VI exhibits a higher extractability than myosin V. This difference between myosin V and VI was also found in a biochemical growth cone particle preparation from brain, suggesting that a significant portion of these two motors has a distinct subcellular distribution. © 2000 John Wiley & Sons, Inc. J Neurobiol 42: 370 –382, 2000 Keywords: myosin V; myosin VI; neuronal growth cone; dorsal root ganglia neurons; cytoskeleton; subcellular localization The myosin superfamily consists of at least 15 struc- turally distinct classes of actin-based molecular mo- tors (Mermall et al., 1998; Probst et al., 1998; Wang et al., 1998). In addition to the well-defined roles of conventional or class II myosins in mediating actin- based contractile phenomena in muscle and non- muscle cells, a wide range of functions have been proposed for various “unconventional” members of Correspondence to: D. M. Suter (daniel.suter@yale.edu). * Present address: Department of Genetics and Biochemistry, University Federal of Uberlandia, Uberlandia, MG 38400-982, Brazil † Present address: Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan Contract grant sponsor: MDA (M.S.M.). Contract grant sponsor: National Institutes of Health; contract grant number: DK-25387 and PPG 1-P01-DK55389-01 (M.S.M.) and NS-28695 (P.F.). © 2000 John Wiley & Sons, Inc. CCC 0022-3034/00/030370-13 Contract grant sponsor: Swiss National Science Foundation (D.M.S.). Contract grant sponsor: Pew Charitable Trust—Latin American Fellows Program (F.S.E.). Contract grant sponsor: Conselho Nacional de Desenvolvi- mento Cientifico e Tecnologico (F.S.E.). 370