Abstract
The mechanism of actin incorporation and turnover in the nerve growth cone was examined by immunoelectron microscopy and low-light-level video microscopy of cultured neurons injected with biotin-labeled actin or fluorescently labeled actin. We first determined the sites of actin incorporation into the cytoskeleton of growth cones by immunoelectron microscopy of cultured neurons injected with biotin-labeled actin and reacted with an anti-biotin antibody and a gold-labeled secondary antibody. Shortly after the injection, biotin-actin molecules incorporated into the cytoskeleton were localized in the distal part of actin bundles in the filopodia and at the membrane-associated fringe of the actin filament network. With longer incubation, most actin polymers in the growth cones were labeled uniformly, suggesting that actin subunits are added preferentially at the membrane-associated ends of preexisting actin filaments. We then determined whether actin filaments translocate within the growth cones by low-light-level video microscopy of living neurons injected with fluorescently labeled actin and photobleached with a laser beam. When actin fluorescence at the leading edge of a growth cone was bleached, a rearward translocation of the bleached spot toward the base of the growth cone was observed. This observation suggests the presence of a rearward flow of actin polymers within growth cones. Taken together, these results indicate that there is a continuous addition of actin monomers at the leading edge of the growth cone and a successive rearward translocation of the assembled filaments.