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McVicker, Derrick Paul

Cell and Molecular Biology Program

2012

Ph. D.

Neurons are complex polar cells consisting ofa cell body, numerous processes known as dendrites, and a single extremely long process known as an axon. These cells receive, transmit, and interpret chemical and electrical signals throughout the body. The ability to accurately transmit materials from a neuron to its target cells is vital to its function and the operation of the entire organism. Most of the materials needed to sustain these cells, and to communicate with connecting cells, are produced in the cell body but needed at other cellular locations. To facilitate the movement of these materials to their target cellular locations, neurons take advantage of a class of molecular motor proteins, known as kinesin, which can pick up cargos produced in the cell body and actively move them along cytoskeletal tracks known as microtubules. The ability of kinesin to actively transport specific cargos to unique destinations within the cell suggests that there must be multiple mechanisms by which to regulate this process. Posttranslational modification of microtubules, as well as multiple isoforrns of tubulin, kinesin, and microtubule associated proteins (MAPs) have all been proposed as regulatory mechanisms for axonal transport in the neuron.
Of recent interest is the MAP tau, known for its role in Alzheimer's disease (AD), which has been shown to strongly inhibit kinesin mediated transport. This inhibition is isoform specific with the 3-repeat short tau isoform (3RS-tau) inhibiting kinesin at a much higher level than the longest 4-repeat-Iong tau isoform (4RL-tau). Because there is a large quantity of tau in the axon, it is unclear how transport proceeds even under normal physilogical conditions. To date the mechanism of tau's ability to regulate kinesin, and how tau is functioning physiologically in neurons, has not been elucidated.
Using a variety oftechniques including total internal reflection fluorescence (TIRF) microscopy for single molecule imaging and steady state kinetic analyses, we demonstrate that tau is sensitive to the nucleotide state ofthe microtubule, and can bind in a non-inhibitory conformation on microtubules in the GTP-state. Additionally, we show tau binds microtubulesin two distinct populations: diffusing and static with the diffusing population being favored on GTP-microtubules. We also demonstrate that static tau can exist as a single molecule or complexes of 2-3 tau molecules on GDP microtubules, but only as a single tau molecule on GTP-molecules.
In addition, 3RS-tau favors both the static conformation and has a higher propensity to form multiple tau complexes on GDP-microtubules as compared to 4RL-tau or on GTP-microtubules. These isoform and microtubule specific differences explain why 3RS-tau is a more potent inhibitor of kinesin than 4RL-tau and why neither isoform affects kinesin on GTP microtubules. Given the recent discovery of large populations of GTP-microtubules in axons, we have developed a novel model that can explain how tau may regulate axonal transport while still allowing transport to proceed unhindered in the axon. We also demonstrate the versatile nature of tau; which may help to explain how a single protein can perform multiple functions within the cell.