Knowledge of the basic mechanisms involved in neurite outgrowth is important both for the understanding of nervous system development and for the design and implementation of therapies for regeneration of damaged nervous tissue. Investigators of neurite outgrowth have hypothesized that nonconventional myosins are responsible for many of the amoeboid and organelle movements observed in the growth cone of the neurite. Our recent discovery that the mouse dilute locus encodes a nonconventional heavy chain is consistent with these proposed mechanisms, since mice without a functional dilute gene have neurological defects. Preliminary evidence suggests that morphology has been altered in only a small subset of neurons. These results strongly suggest that dilute is a member of a family of nonconventional myosin heavy chains with overlapping functions. To more fully investigate this hypothesis, cDNAs for other members of the dilute- related and nonconventional (type I) myosin heavy chain families expressed in the nervous system will be cloned by two methods: 1) polymerase chain reaction (PCR) amplification of brain cDNA with degenerate oligonucleotides representing conserved amino acid sequences in myosin heavy chains and 2) screening of brain cDNA libraries with similar degenerate oligonucleotides as probes. In preliminary experiments, two clones that, by the criterion of sequence similarity, represent myosin I heavy chains have been isolated and sequenced. These are most similar in sequence to the brush border myosin I. These cDNAs and others cloned by this technique will be mapped on the mouse (and by extension, human) genome, to determine if they correspond to known mouse or human loci associated with neurological defects. At the same time, spatial and temporal analyses of RNA and protein expression will be performed to determine if these new myosin heavy chains play a role in neurite outgrowth. Functional studies will be performed in cell culture systems to elucidate the roles that dilute and other nonconventional myosin heavy chains may play in cell motility and morphogenesis. The motility of cells from mice lacking dilute function will be studied by computer-assisted morphology analysis, and function of the nonconventional myosin heavy chains characterized above (including dilute) will be inhibited by treatment of cultured cells with antisense oligonucleotides and microinjection of cells with antibodies, again followed by analysis of changes in motility or morphology.
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