? Proper functioning of all cell types depends on correctly targeting a large number of proteins to specific cellular sites of action, incorrect protein targeting has been linked to a variety of disease states and abnormalities, including neurological disorders. In polarized vertebrate neurons, for instance, proteins need to be trafficked correctly to axons and dendrites. Some progress has been made in elucidating the trafficking of somatodendritic proteins, but targeting of axonal proteins is poorly understood. The goal of the proposed work is to elucidate the membrane traffic pathways into the axon of polarized neurons and to uncover their molecular underpinnings. Specifically, we will analyze the axonal targeting of the cell adhesion molecule L1/NgCAM, which mediates axonal pathfinding and fasciculation during brain development. The proposed work is based on results from my laboratory that demonstrate that two distinct post-Golgi transport routes to the axon exist for L1/NgCAM. One is an indirect transcytotic route via the somatodendritic plasma membrane and endosomes, the other a direct route without intermediate stops on the somatodendritic plasma membrane. Our major hypothesis is that whether L1/NgCAM travels to the axon directly or transcytotically depends on the reading and execution of distinct sorting signals contained within it. In order to uncover the molecular principles that govern this pathway selection, we will pursue three specific aims:
(Aim 1) Map in detail the targeting signals and the specific order of their execution required for LI/NgCAM to reach the axon via transcytosis.
(Aim 2) Characterize the molecular machinery for recognizing L1/NgCAM's targeting signals.
(Aim 3) Dissect the organization of endocytic pathways. Our approaches include expression of mutant molecules from adenovirus vectors in cultured hippocampal and other neurons (Aim 1), novel approaches to study the kinetics of axonal transport (Aim 1), live-cell imaging of fluorescent molecules to dissect pathways (Aim 3) and two-hybrid screens and biochemical approaches to identify molecular components (Aim 2). The overall goal of our work is to specify the mechanisms producing the polarized architecture of neurons. ? ?
