Nearly every aspect of neuronal function depends on the correct polarization of membrane proteins to axons or dendrites. Following exit from the Golgi complex, membrane proteins destined for different cellular domains are sorted into carrier vesicles and transported along microtubules to their destinations, where they are delivered to the plasma membrane by exocytosis. The long-term goal of our research is to uncover the mechanisms that govern the selectivity of these trafficking pathways.We have developed novel methods that allow protein trafficking to be visualized in living hippocampal neurons, permitting each step along the exocytic pathway -- sorting, transport, and delivery -- to be assessed independently. In addition, we have generated a catalog of GFP tagged protein markers designed to label a representative subset of the different carriers that deliver proteins to dendrites and axons. We propose to use these tools to address three specific aspects of the trafficking of polarized proteins in nerve cells. First, we will estimate the number of different carriers that convey dendritically polarized proteins and identify the motifs in dendritic proteins that govern their sorting into these carriers. Second, we will test the hypothesis that interaction of carriers with minus-end directed motors prevents the transport of dendritic proteins into axons, thereby ensuring their delivery only to the correct domain. Third, we will define the principal populations of carriers that convey axonally polarized proteins, identify the motifs that govern the sorting of axonal proteins into these carriers, and confirm that these carriers deliver axonal proteins exclusively to the axonal membrane.The complexity of neuronal protein trafficking combined with the extreme dimensions of nerve cells contribute to their selective vulnerability to injury and degenerative disease. Improved understanding of these aspects of neuronal biology may significantly advance our understanding of the etiology of these diseases and suggest potential avenues for therapy.
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