Date of Award

2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Erika L. Holzbaur

Abstract

Dynamic interactions with the cytoskeleton are essential to move and anchor nuclei during tissue development, and defects resulting in nuclear mispositioning are often associated with human disease, such as muscular dystrophy and myopathy. Skeletal muscle cells are large syncytia formed by fusion of myoblasts, and contain hundreds of nuclei positioned regularly along the length the cell. During muscle cell development, nuclear movement in myotubes requires microtubules, but the mechanisms involved have not been elucidated. Here, we find that nuclei actively translocate through myotubes. As they translocate, they also rotate in three-dimensions. These movements require an intact microtubule cytoskeleton, which forms a dynamic bipolar network around the nuclei, and are driven by the microtubule motor proteins, kinesin-1 and cytoplasmic dynein. Depletion of the plus-end directed motor kinesin-1 abolishes nuclear rotation and significantly inhibits nuclear translocation, resulting in the abnormal aggregation of nuclei near the midline of the myotube. Loss of the minus-end directed dynein motor also inhibits nuclear dynamics, but to a lesser extent, leading to altered spacing between adjacent nuclei. The motors are found throughout the cytoplasm, but also decorate the nuclear envelope. To test whether kinesin-1 on the nucleus is essential for nuclear distribution, we controlled the recruitment of truncated, constitutively active kinesin-1 motors to the nuclear envelope. We show that nuclear-based kinesin-1 is necessary to prevent nuclear aggregation. Additionally, we show that kinesin-1 localization to the nuclear envelope in myotubes is mediated at least in part by interaction with the nuclear envelope protein, nesprin-2. We identify a conserved kinesin light chain-binding motif in nesprin-2 and show that recruitment of the motor complex to the nucleus via this motif is essential for proper nuclear distribution. Thus, our work indicates that oppositely directed motors acting from the surface of the nucleus drive nuclear motility in myotubes. The variable dynamics observed for individual nuclei within a single myotube likely result from the stochastic activity of competing motors interacting with a complex bipolar microtubule cytoskeleton. The three-dimensional rotation of myotube nuclei may facilitate their motility through the complex and crowded cellular environment of the developing muscle cell, allowing for proper myonuclear positioning.

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