N.Anilkumar and M.Parsons contributed equally to this workCoordination of protrusive and contractile cell±matrix contacts is important for cell adhesion and migration, but the mechanisms involved are not well understood. We report an unexpected direct association between fascin, an actin-bundling component of ®lopodia, microspikes and lamellipodial ribs, and protein kinase Ca (PKCa), a regulator of focal adhesions. The association is detectable by protein±protein binding in vitro, by coimmunoprecipitation from cell extracts, and in live cells as¯uorescence resonance energy transfer detected by¯uorescence imaging lifetime microscopy. The interaction is physiologically regulated by the extracellular matrix context of cells, depends on activation of PKCa and is mediated by the C1B domain of PKCa. Strikingly, a fascin mutant, fascin S39D, associates constitutively with PKCa. Through use of a newly developed set of membranepermeable peptides that separately inhibit either fascin/PKCa or fascin/actin binding, we have uncovered that speci®c blockade of the fascin/PKCa interaction increases cell migration on ®bronectin in conjunction with increased fascin protrusions and remodeling of focal adhesions. These results identify the fascin±PKCa interaction as an important novel intersection in the regulation and networking of cell± matrix contacts.

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Much current knowledge of oligodendrocyte biology, the myelin-forming cells in the central nervous system (CNS), comes from cell culture studies mainly from postnatal rat tissue but mouse cells have been much more difficult to produce in large quantities. We have developed a high yield protocol for production of oligodendrocyte precursor cells from mouse embryonic neural progenitors grown as neurospheres. Neurospheres can be maintained and expanded for long periods in culture in the presence of EGF. When floating neurospheres were plated on substrate-coated dishes in media supplemented with PDGF and bFGF, the spheres attached and generated migrating cells that were predominantly oligodendrocyte-lineage cells. Furthermore, cells in spheres could be shifted to the oligodendrocyte phenotype prior to plating on substrate, by incubation in suspension with PDGF/bFGF. Single cell suspensions plated after dissociation of either EGF-treated neurospheres or PDGF/bFGF-treated oligospheres had the bipolar, elongated morphology characteristic of oligodendrocyte precursor cells. mRNA and protein expression analysis of the cells generated by this method confirmed their oligodendrocyte lineage. Oligodendrocyte precursors generated by this method matured in response to ciliary neurotrophic factor treatment, producing cells with multiple processes and myelin-like membranes. The most important aspect of this protocol is the ability to generate very high numbers of relatively pure mouse oligodendrocyte progenitor cells, which can be easily transfected. These studies open up many kinds of investigations on transgenic and mutant mouse oligodendrocytes, thereby providing a valuable tool to study oligodendrocyte biology and development.