Date of Award

2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Neuroscience

First Advisor

Erika L. Holzbaur

Abstract

Neurons are specialized cells that extend polarized processes called dendrites and axons in order to maintain synaptic connections over long distances. Consequently, neuronal homeostasis requires axonal transport of organelles, such as mitochondria, synaptic vesicles, and autophagosomes. The microtubule-based motors responsible for long-distance fast axonal transport are the anterograde kinesin motors and the retrograde dynein motors. Two cargos that exhibit robust axonal transport characterized by high speeds with few directional switches are APP- (amyloid precursor protein) positive vesicles and autophagosomes. While APP-positive vesicles transport occurs in both anterograde and retrograde directions, autophagosomes move unidirectionally in the retrograde direction. Here, we demonstrate that processive transport of both these cargos requires coordination of opposing motor activity by the scaffolding protein JIP1 (c-jun N-terminal kinase- interacting protein). We identify novel interactions between JIP1 and kinesin heavy chain (KHC), which are sufficient to relieve KHC autoinhibition and activate motor function in single molecule assays. In addition, the direct binding of the dynactin subunit p150Glued to JIP1 competitively inhibits KHC activation in vitro and disrupts the transport of APP in neurons. Together with coimmunoprecipitation results, these experiments support a model whereby JIP1 coordinates transport by switching between anterograde and retrograde motile complexes. Furthermore, we find that mutations in the JNK-dependent phosphorylation site S421 in JIP1 alter both KHC activation in vitro and the directionality of APP and autophagosome transport in neurons. In knockdown and rescue experiments, the phosphomimetic JIP1-S421D promotes anterograde APP transport and disrupts retrograde autophagosome transport while the phosphodeficient JIP1-S421A promotes retrograde APP transport and rescues retrograde autophagosome transport. Thus, post-translational modification of a scaffolding protein can serve as a molecular switch that coordinates opposing motor activity in order to regulate the direction of vesicular transport of various organelles in the axon.

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