Abstract
ABSTRACTHow oocytes assemble bipolar meiotic spindles in the absence of centrosomes as microtubule organizing centers remains poorly understood. We have used live cell imaging in Caenorhabditis elegans to investigate requirements for the nuclear lamina and for conserved regulators of microtubule dynamics during oocyte meiosis I spindle assembly, assessing these requirements with respect to recently identified spindle assembly steps. We show that the nuclear lamina is required for microtubule bundles to form a peripheral cage-like structure that appears shortly after oocyte nuclear envelope breakdown and surrounds the oocyte chromosomes, although bipolar spindles still assembled in its absence. Although two conserved regulators of microtubule nucleation, RAN-1 and γ-tubulin, are not required for bipolar spindle assembly, both contribute to normal levels of spindle-associated microtubules and spindle assembly dynamics. Finally, the XMAP215 ortholog ZYG-9 and the nearly identical minus-end directed kinesins KLP-15/16 are required for proper assembly of the early cage-like structure of microtubule bundles, and for early spindle pole foci to coalesce into a bipolar structure. Our results provide a framework for assigning molecular mechanisms to recently described steps in C. elegans oocyte meiosis I spindle assembly.
Highlights
Oocyte meiotic spindles in many animals form in the absence of centrosomes, and reduce the duplicated diploid genome to a haploid content through two rounds of cell division called meiosis I and II (Dumont and Desai, 2012; Mullen et al, 2019; Ohkura, 2015; Severson et al, 2016)
To assess progression through meiosis I, we designated the time at which the value for mCherry::H2B intensity in the nucleoplasm became equal to the value for the cytoplasm, which marks the initiation of nuclear envelope breakdown (NEBD), as t=zero and collected z-stacks encompassing the oocyte volume every 5 s
We have examined the requirements for several factors involved in C. elegans oocyte meiotic spindle assembly, with the goal of assessing their roles during a sequence of four recently described assembly steps that generate these acentrosomal and yet bipolar spindles during meiosis I
Summary
Oocyte meiotic spindles in many animals form in the absence of centrosomes, and reduce the duplicated diploid genome to a haploid content through two rounds of cell division called meiosis I and II (Dumont and Desai, 2012; Mullen et al, 2019; Ohkura, 2015; Severson et al, 2016). The microtubule cage becomes organized such that multiple small foci of microtubule ends form in association with the pole-marker ASPM-1, which initially is present along the entire length of the microtubule bundles that form the early cage-like structure. These small pole foci coalesce to form a bipolar spindle as chromosomes congress to a metaphase plate (Connolly et al, 2015)
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