This project is aimed at characterizing differences in the adhesive patterns and associated growth characteristics of neuronal growth cones on defined substrates. Neurite formation by embryonic retinal ganglion cell neurons was observed using a technique (time~lapse laser scanning interference reflection microscopy) to show local distances of the cell membrane from substrates composed of purified, bio~logically relevant cell and substrate adhesion molecules. Members of the three major classes of adhesion molecules were tested ~ the calcium~dependent, calcium~independent, and the immunoglobulin super~family of molecules. Previously observed differences in the overall degree to which growth cones ad~hered to different molecular substrates were minimized when we took into account potential variability in our optical method. We, therefore, cannot conclude whether a critical level of attachment is necessary for migration. However, the patterns of attachment on different substrates remained highly distinct even when variability was accounted for. Growth cones on all substrates had some areas of close apposition to the substrate but none of them exhibited dark intensities that were comparable to the focal contacts seen in fibroblasts. Analysis of the dynamic changes in the adhesion patterns also showed wide variation among substrates. Growth cones on L1, for example, exhibited a complicated pattern of close and distant attachment that varied greatly in both temporal and spatial domains. Growth cones on laminin, on the other hand, exhibited a roughly uniform level of distant attachment with a few areas of close attachment at the leading edge. Dynamic changes in adhesion over time and between numerous points on the growth cone were relatively small by comparison. Since growth cones in vivo may encounter all of these molecules, and others at the same time, it seems reasonable to suggest that they must be able to integrate these many signals into a response suitable for directing them to their appropriate targets. The question remains why the patterns of adhesion of growth cones to different substrates differ and how those distinct patterns may be involved in the secondary signaling mechanisms that promote and direct neurite outgrowth. Experiments will continue to analyze adhesive interactions with molecules in combination and in patterned arrays. We will also begin to investigate the role of cytoskeletal interactions with adhesion molecules on the cell surface to understand how signals in the growth cone's environment may be transduced into behavioral responses.
