? 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. ? ?
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|Yap, Chan Choo; Nokes, Rita L; Wisco, Dolora et al. (2008) Pathway selection to the axon depends on multiple targeting signals in NgCAM. J Cell Sci 121:1514-25|
|Yap, Chan Choo; Wisco, Dolora; Kujala, Pekka et al. (2008) The somatodendritic endosomal regulator NEEP21 facilitates axonal targeting of L1/NgCAM. J Cell Biol 180:827-42|
|Thompson, Anthony; Nessler, Randy; Wisco, Dolora et al. (2007) Recycling endosomes of polarized epithelial cells actively sort apical and basolateral cargos into separate subdomains. Mol Biol Cell 18:2687-97|
|Boiko, Tatiana; Vakulenko, Max; Ewers, Helge et al. (2007) Ankyrin-dependent and -independent mechanisms orchestrate axonal compartmentalization of L1 family members neurofascin and L1/neuron-glia cell adhesion molecule. J Neurosci 27:590-603|
|Chang, Michael C; Wisco, Dolora; Ewers, Helge et al. (2006) Inhibition of sphingolipid synthesis affects kinetics but not fidelity of L1/NgCAM transport along direct but not transcytotic axonal pathways. Mol Cell Neurosci 31:525-38|
|Wisco, Dolora; Anderson, Eric D; Chang, Michael C et al. (2003) Uncovering multiple axonal targeting pathways in hippocampal neurons. J Cell Biol 162:1317-28|