GAP-43 in growth cones is associated with areas of membrane that are tightly bound to substrate and is a component of a membrane skeleton subcellular fraction

Abstract

To ascertain the subcellular localization of the growth-associated protein GAP-43 in growth cones, we isolated growth cones from the forebrains of neonatal rats. Anti-GAP-43 immunoreactivity in these isolated growth cones (IGCs) resembled that seen in cultured growth cones in 2 respects: First, in substrate-attached IGCs, as in cultured growth cones, immunoreactivity was intracellular, punctate, and extended throughout the IGCs and their processes. Second, when IGCs were dislodged from this substrate, patches of membrane that were highly immunoreactive for GAP-43 were left behind. Extracting IGCs in nonionic detergents revealed that almost all the particulate GAP-43 colocalized with a membrane skeleton fraction: It was not present in the cytoskeleton. The association of GAP-43 with the membrane skeleton was not due to nonspecific aggregation, nor was it calcium dependent. Examination of this fraction under the electron microscope showed it to consist of membrane fragments associated with amorphous material that could be visualized with tannic acid. Immunoelectron microscopy showed that anti-GAP-43 immunoreactivity was localized to this amorphous material. Fodrin, talin, and a-actinin immunoreactivity could be detected in the membrane skeleton fraction, and actin and tubulin were also identifiable from SDS gels. The association of GAP-43 with the membrane skeleton, which by analogy with other cell types is involved in the dynamic regulation of cell shape, implies that GAP-43 in growth cones may be involved in this function.