Date of Award

2018

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Cellular, Molecular and Biomedical Sciences

First Advisor

Christopher Berger

Abstract

Neurons are among the most highly polarized cells in the human body. This polarization allows the neuron to participate in the transfer of chemical and electrical signals which are crucial to the survival of the organism. As part of polarization, each neuron develops a dendritic arbor and an axon. To ensure the survival of the cell, materials synthesized in the cell body must be trafficked through the axon for delivery throughout ultimately ending at the synaptic termini. The bulk of this cargo transport is microtubule-based fast axonal transport which is molecular motor mediated and tightly regulated though many pathways. Motor based transport is established early in development and maintained for the life of the cell. The kinesin motor protein family plays an integral role in fast axonal transport and the regulation of these motors is essential to proper cargo delivery. Regulation occurs through auto-inhibition, motor interactions with microtubule associated proteins (MAPs) and complex signaling pathways which control the post-translational modification of MAPs, the microtubule track and the motors.

The disruption of cargo transport is linked to neurodegeneration and disease state development. Of particular interest in this process is the MAP Tau which has been implicated in a number of neurodegenerative diseases including Alzheimer’s Disease. Tau is expressed at all stages of neural development and has been shown to participate in signaling cascades, modulate microtubule dynamics and preferentially inhibit kinesin-1 motility. Though Tau is involved in these processes, the non-disease state regulation of this MAP and it’s inhibition of kinesin-1 is not well understood. Tau has been shown to bind the microtubule surface in a static-diffusive state equilibrium which differs with isoform and lattice. Previous work demonstrates that the static state is more inhibitory to kinesin-1 than the diffusive state. These different binding behaviors with their different effects on kinesin-1 motility, suggest that cellular regulation of Tau’s static-diffusive binding equilibrium may control inhibition of kinesin-1 and that structural changes may underlie Tau binding to the microtubule surface. Cellular regulation of Tau’s structure and therefore its behavior on the microtubule surface points to a means by which Tau is regulated in the non-disease state. Additionally, this would highlight how early changes lead to disease state development.

Using a combination of molecular biology, biochemical techniques and imaging strategies including Total Internal Reflection Fluorescence, single molecule Fluorescence Resonance Energy Transfer (smFRET) and Alternating Laser Excitation, we show that Tau’s static-diffusive state equilibrium is regulated by non-disease state phosphorylation at tyrosine 18. Phospho-mimetics are shifted towards diffusive binding and have decreased affinity for the microtubule surface which in turn reduces inhibition of kinesin-1 motility. These results further demonstrate that Tau undergoes long range structural change while bound to the microtubule surface. We performed smFRET assays and found that Tau binds the microtubule surface in distinct conformations which underlie static and diffusive binding. This work ties the regulation of Tau’s structure and binding behavior to its function and paves the way for our understanding of how cellular regulation acts on multiple levels to fine tune axonal transport.

Language

en

Number of Pages

176 p.

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