Date of Award

2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular Physiology and Biophysics

First Advisor

Christopher L. Berger

Abstract

The unique relationship between neuronal structure and function is paramount for the complexity of the human nervous system. This relationship allows neurons to receive, process, and transmit information through many inter- and intracellular mechanisms. Axonal transport is an essential intracellular mechanism for neuronal health and viability. This process involves the transport of cellular cargo in the anterograde and retrograde direction along the axon, relaying materials between the soma and axon terminals, respectively. The necessity of an expedited form of transport becomes clear when one considers the magnitude of distance cargo must travel; in some cases, the axon can be up to a meter in length. While axonal transport increases the efficiency of cargo delivery, it is imperative that this process be regulated both spatially and temporally.

The choreography of axonal transport is mediated by molecular motor proteins that carry neuronal cargo along microtubule tracks within the axon. Kinesin proteins, a class of molecular motors, transport cargo in the anterograde direction. KIF1A is a kinesin-3 family member that is responsible for the transport of certain critical intracellular cargo. However, the mechanisms for KIF1A regulation remain largely misunderstood. A known regulatory mechanism of kinesin motors is via the presence of microtubule associated proteins (MAPs) that bind to and crowd microtubule roadways. Specifically, the neuronal MAP Tau has been shown to differentially regulate kinesin families found in the neuron, such as kinesin-1 and kinesin-2. Furthermore, these regulatory capabilities are directly related to the behavioral binding state of Tau, of which it can bind statically or diffusively to the microtubule. While the potential regulatory relationship between KIF1A and Tau is unknown, both of these players exhibit pathological behavior in neurodegenerative diseases such as Alzheimer’s disease and frontotemporal dementia. These perturbations in KIF1A transport, in tandem with Tau dysfunction, present compelling evidence of important relationship between these two proteins.

To investigate and define the relationship between KIF1A and Tau, a single-molecule in vitro reconstituted system approach was employed. In doing so, an unexpected finding occurred in the early stages of experimentation. It was revealed that KIF1A exhibits a unique pausing behavior between segments of processive movement on the microtubule surface. This behavior, which had been unreported and uncharacterized, contradicts the canonical behavior of other well studied kinesin proteins. KIF1A pausing was found to be mediated by the C-terminal tail (CTT) structure of tubulin, the building blocks of microtubules. Further exploration revealed that KIF1A pausing was reliant upon the level of polyglutamylation, a post-translational modification enriched on neuronal tubulin, of the CTTs. Lastly, it was determined that polyglutamylated CTTs allow for an electrostatic tethering mechanism with the K-loop, a motor domain surface loop of KIF1A, that allows the motor to pause. Like KIF1A, Tau also relies on the tubulin CTTs to exhibit its characteristic diffusive binding behavior. In considering this fact, KIF1A regulation via Tau’s behavioral binding state was investigated. Ultimately, it was discovered that the diffusive binding state of Tau regulates KIF1A, not the static binding state. This regulation occurs when KIF1A tries to engage in a pause, but cannot due to diffusive Tau occupying the CTTs. This work provides a new mechanism of Tau-mediated kinesin motor regulation and the first direct link between KIF1A and Tau function, expanding our knowledge of the spatiotemporal regulation of axonal transport.

Language

en

Number of Pages

157 p.

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