Investigating the Mechanisms of Acentriolar Spindle Assembly in Caenorhabditis elegans Oocytes

Mullen, Timothy James

doi:https://doi.org/10.21985/n2-md1r-r151

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Investigating the Mechanisms of Acentriolar Spindle Assembly in Caenorhabditis elegans Oocytes

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When a cell divides, it must assemble a microtubule-based structure called a spindle, which provides the forces that physically segregate the chromosomes. In most cell types the microtubules that comprise the spindle are nucleated and organized by centriole-containing centrosomes. In many species, however, oocyte meiosis is carried out in the absence of centrioles. As a result, microtubule organization, spindle assembly, and chromosome segregation proceed by unique mechanisms. In this dissertation, I report insights into the principles underlying this specialized form of cell division using C. elegans oocyte meiosis as a model system. First, I identify two microtubule motor proteins, KLP-15 and KLP-16, which are two highly homologous members of the kinesin-14 family of minus-end-directed kinesins. These proteins localize to the acentriolar oocyte spindle and promote microtubule bundling during spindle assembly; following KLP-15/16 depletion, microtubule bundles form but then collapse into a disorganized array. Surprisingly, despite this defect I found that during anaphase, microtubules are able to reorganize into a bundled array that facilitates chromosome segregation. This phenotype therefore enabled me to identify factors promoting microtubule organization during anaphase, whose contributions are normally undetectable in wild-type worms; I found that SPD-1 (PRC1) bundles microtubules and KLP-18 (kinesin-12) likely sorts those bundles into a functional orientation capable of mediating chromosome segregation. In the second part of this dissertation, I investigate the role of the microtubule stabilizing protein, ZYG-9 (XMAP215) during oocyte meiosis, and found that it functions to maintain the integrity of the acentriolar spindle poles. When I acutely depleted this protein from the bipolar spindle, the spindle poles lose stability and begin to fragment. The discoveries described here therefore deepen our understanding of the molecular mechanisms of acentriolar spindle assembly, spindle stability, and chromosome segregation in oocytes.

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