C. elegans XMAP215/ZYG-9 and TACC/TAC-1 act at multiple times throughout oocyte meiotic spindle assembly to promote both the coalescence of pole foci into a bipolar structure and the stability of coalesced poles during oocyte meiotic cell division

Abstract

The conserved two-component XMAP215/TACC modulator of microtubule stability is required in multiple animal phyla for acentrosomal spindle assembly during oocyte meiotic cell division, with C. elegans XMAP215/zyg-9 and TACC/tac-1 mutant oocytes exhibiting multiple and indistinguishable defects beginning early in meiosis I. To determine if these defects represent one or more early requirements with additional later and indirect consequences, or multiple temporally distinct and more direct requirements, we have used live cell imaging and fast-acting temperature-sensitive zyg-9 and tac-1 alleles to dissect at high temporal resolution their oocyte meiotic spindle assembly requirements. Our results from temperature upshift and downshift experiments indicate that the ZYG-9/TAC-1 complex has multiple temporally distinct and separable requirements throughout oocyte meiotic cell division. First, we show that during prometaphase ZYG-9 and TAC-1 promote the coalescence of early pole foci into a bipolar structure both by stabilizing pole foci as they grow and by limiting their growth rate, with these requirements being independent of an earlier defect in microtubule organization. Second, during metaphase, ZYG-9 and TAC-1 maintain spindle bipolarity by suppressing ectopic pole formation, and this pole stability is important for maintaining chromosome congression. Finally, we show that ZYG-9 and TAC-1 also are required for the proper coalescence of pole foci during meiosis II, independently of their requirements during meiosis I. Our findings highlight the value of fast-acting temperature-sensitive alleles for high resolution temporal dissection of gene requirements, and we discuss how negative regulation of microtubule stability by ZYG-9/TAC-1 during oocyte meiotic cell division might account for the observed defects in spindle pole coalescence and stability. Author SummaryWhen most animal cells divide, organizing centers called centrosomes nucleate and organize protein filaments called microtubules into a dynamic bipolar structure called the spindle that equally partitions the duplicated genome between two daughter cells. However, female egg cells, called oocytes, lack centrosomes but still assemble bipolar spindles that separate chromosomes. Using the nematode C. elegans as a model system, and taking advantage of fast-acting temperature-sensitive alleles of both ZYG-9 and TAC-1 that rapidly inactivate or reactivate upon temperature upshifts or downshifts, respectively, we show that a complex of two regulators of microtubule stability, called ZYG-9 and TAC-1, has multiple and separable requirements during the process of acentrosomal oocyte spindle assembly. These requirements include promoting the coalescence of early pole foci into a bipolar structure, and the maintenance of pole stability after the assembly of a bipolar structure, both of which are essential for proper chromosome separation. Furthermore, oocyte meiosis involves two consecutive cell divisions, and we show that ZYG-9/TAC-1 are required for pole coalescence during both the first and second meiotic cell divisions. Our findings provide a high resolution view of the distinct and separable temporal requirements for these widely conserved regulators of microtubule stability during acentrosomal oocyte spindle assembly.