Our overall goal is to determine the role of microtubule-associated proteins (MAPs) in the establishment of nerve cell polarity, including the segregation of MAPs in dendrites and axons, and the localization of organelle transport within the neuron. Recently, we have shown that the mechanism of equilibrium binding of MAP2 to microtubules (MTs) is a highly cooperative process that leads to the segregation of MAP, producing MAP-containing and MAP-free MTs in vitro. We hypothesize that the MAP binding mechanism itself maintains biochemically and functionally distinct compartments in axons and dendrites, that tau in the axon permits kinesin- and dynein-mediated transport which is inhibited by MAP2 in the cell body and dendrites, and that these biochemical and physiological properties are consequences of the basic MAP-MT binding mechanism. Our specific goals are to: (1) Analyze equilibrium binding between MAP2 and MTs by immuno-EM and by sedimentation of determine if the binding of MAP2 is a multiphasic process and is influenced by the presence of other MAPs such as MAP1A and tau, and map the cooperativity site on the MAP2 molecule. We will determine if cooperative binding results in the segregation of MAP2 and tau on MTs in vitro; (2) Establish if cooperative binding occurs in neurons by perfusing permeabilized cultures of mouse spinal cord macro-neurons with biotin-labeled MAP2 and tau. We will also examine the mechanism of binding of MAPs to MTs in vivo by photoactivating living macro-neurons that have been microinjected with MAP2 labeled with caged fluorescein; (3) Determine if tau-containing MTs promote, but MAP2 containing MTs inhibits, dynein-and kinesin- dependent bead and organelle movement on MTs both in vitro and in vivo. We especially want to know if beads coated with kinesin or dynein can undergo transport in axons containing tau but are inhibited in dendrites containing MAP2. We will also examine the consequences of the phosphorylation state of MAP2 for kinesin- and dynein-mediated organelle transport on MT substrates in vitro. This work will relate cooperative binding to the segregation of MAPs in neurons and determine if the significance of segregation is to create special environments for organelle transport which would otherwise be inhibited by certain MAPs.
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