This project uses structural methods to study neurons grown in vitro with the goal of understanding the molecular mechanisms involved in neurite outgrowth and pathfinding. Current work focuses on two problems: (1) Cytoskeletal mechanisms and substrate interactions involved in initial neurite outgrowth. The sequence of changes in cell shape and movements of the cytoskeleton accompanying neurite outgrowth from peripheral ganglion neurons grown in vitro was examined with time-lapse videomicroscopy and immunofluorescence labeling methods. Neurons initially form multiple filopodia which contain actin microfilaments, while microtubules and neurofilaments are restricted to the cell center. Formation of a neurite entails protrusion of cytoplasm containing microtubules and neurofilaments into a filopodium. Entry of microtubules does not appear to require polymerization of tubulin because microtubules enter filopodia during inhibition of tubulin polymerization. Transformation of a filopodium into a neurite is promoted by particular spatial patterns of substrate adhesion but does not require a specific cell adhesion molecule. (2) Influence of purified cell adhesion molecules on growth cone behavior. Growth cones of retinal neurons growing on substrates coated with either a purified cell adhesion molecule (laminin, L1, N-cadherin, or merosin) or polylysine was examined using time-lapse laser scanning interference reflection microscopy (LS-IRM) to show the spatial separation of the cell membrane from the substrate. There were marked differences between substrates in the overall extent of growth cone adhesion and in the variation in adhesion levels of individual membrane patches over time. Rate of growth cone migration did not bear a consistent relationship to either the overall level or temporal dynamics of substrate adhesion.
