Mechanisms of Acentrosomal Spindle Assembly and Maintenance in C. elegans Oocytes

Wolff, Ian

doi:https://doi.org/10.21985/n2-my2d-pf82

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Mechanisms of Acentrosomal Spindle Assembly and Maintenance in C. elegans Oocytes

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Although centrosomes nucleate and organize microtubules in mitotically-dividing cells, spindles in female reproductive cells (oocytes) form in their absence. In some organisms acentrosomal spindle assembly is mediated by acentriolar microtubule organizing centers (MTOCs) that are thought to functionally replace centrosomes. However, spindle assembly in human oocytes does not require MTOCs; little is known about the molecular mechanisms underlying this MTOC-independent pathway. In this dissertation, I demonstrate that acentrosomal spindle assembly in C. elegans oocytes is also MTOC-independent, establishing it as powerful model system to investigate this process. High resolution imaging of acentrosomal spindle formation revealed that following nuclear envelope breakdown, microtubules of mixed polarity surround the chromosomes in a cage-like structure adjacent to the disassembling nuclear envelope. Microtubules are then sorted so that minus ends are forced to the periphery of the array where they coalesce into multiple nascent poles before achieving bipolarity. I characterized how two essential proteins, KLP-18/kinesin-12 and MESP-1 (meiotic spindle 1), act to promote acentrosomal spindle bipolarity. Following KLP-18 or MESP-1 depletion, the microtubule cage forms but then minus-ends rapidly converge, bypassing the multipolar stage and instead forming a monopolar spindle. BMK-1/Kinesin-5, the essential bipolarity- generating motor in many organisms, is not essential for spindle assembly in this system. Therefore, KLP-18/kinesin-12 and MESP-1 are likely the primary force generators that sort microtubule minus ends away from the chromosomes. However, the biochemical mechanism of how these proteins generate force was unknown. To gain insight into this important problem I employed a combination of in vitro and in vivo approaches. First, I purified recombinant truncations of the KLP-18 coiled-coil stalk domain along with full length MESP-1 to use in microtubule binding experiments in vitro. I identified a non-motor microtubule binding site at the C-terminus of the KLP-18 stalk and found that this microtubule binding site is activated through MESP-1 interaction with an adjacent region of the stalk. I then tested the importance of the KLP-18 C-terminal microtubule binding site in vivo using a temperature sensitive mutant strain containing two amino acid substitutions in the mapped domain. Prolonged incubation at the restrictive temperature caused spindle assembly defects that are identical to those observed following depletion of KLP-18 by RNAi; monopolar instead of bipolar spindles formed. In addition, I found that short incubation of this mutant at the restrictive temperature caused the collapse of already formed bipolar spindles into monopoles. In both cases, KLP-18 still localized to the aberrant spindles, indicating that the protein is present but non-functional. These results demonstrate that the C-terminal microtubule binding site we identified in vitro is required for both spindle assembly and for the maintenance of spindle bipolarity in vivo. Altogether, this work sets the basis for further investigation into how microtubule associated proteins govern spindle assembly and maintenance, specifically in a system lacking centrosomes.

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