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. One initiative focuses on the initial outgrowth of neurites from isolated chick peripheral ganglion neurons, using time-lapse video microscopy with conventional and laser scanning microscopes. Neurite formation begins when a filopodium is invaded by a bulge of perinuclear cytoplasm, which contains microtubules, neurofilaments and organelles and is surrounded by a cortex of actin microfilaments. These cytoplasmic components invade the filopodium, converting it into a definitive neurite with a growth cone at its distal end. Experiments in which neurons were grown in the presence of drugs that inhibit the dynamic turnover of microtubules showed that the initial steps in neurite formation do not require assembly of microtubules and provided evidence that microtubules assembled in the cell body can be translocated into neurites as they emerge. These findings were used to formulate a model of the cytoskeletal mechanisms and substrate interactions that lead to the initiation of neurite outgrowth. Current work tests predictions of this model. A second initiative concerns mechanisms of growth cone migration. In order for a growth cone to pull itself across a substrate, its actin cytoskeleton must become linked to molecules in the plasma membrane that mediate adhesion to that substrate. Several proteins that fibroblasts use to link their actin cytoskeleton to the substrate at focal contacts have been studied using immunofluorescence techniques to determine whether these proteins aggregate in growth cones at sites of contact with the substrate, as identified by interference reflection microscopy. Knowing the identity of the linkage proteins and how their interactions with actin filaments are regulated is essential to understanding growth cone migration.