Oocytes of many animals form bipolar spindles in the absence of centrosomes, and how these acentrosomal spindles establish bipolarity remains unclear. In C. elegans oocyte meiosis I, spindle microtubules first form a cage-like structure peripheral to the chromosomes, with multiple small and peripheral spindle poles appearing as assembly progresses. These small poles later merge together and ultimately form two mature poles on the opposite sites of the aligned chromosomes (Wolff et al., 2016). Previous studies have documented defects in oocyte meiotic spindle structure in ZYG-9 or TAC-1 depleted oocytes (Matthews LR et al., 1998; Bellanger JM and Gonczy P, 2003; Yang et al., 2003), but when these assembly defects first appear is not known. Using live imaging, we are investigating the temporal requirements for ZYG-9 and TAC-1 during meiotic spindle assembly. Here we show that ZYG-9 and TAC-1 are required very early in spindle formation, with defects that then further interfere with the establishment of spindle bipolarity. Using GFP and mCherry fusions to mark microtubules and chromosomes, respectively, in
zyg-9 (-) oocytes, we have detected defects very early in meiotic spindle assembly. Rather than forming a cage-like structure with all microtubules restricted to the periphery, we instead observed a cage-like structure with some microtubules extending across the interior, projecting through the volume occupied by chromosomes. This abnormal microtubule scaffold subsequently assembled into a multi-polar spindle network, with individual bivalents sometimes surrounded by discrete small bipolar spindles. Ultimately chromosomes often segregated toward more than two poles. Similarly, using GFP fused to ASPM-1 to mark spindle poles, we found that wild-type oocytes had two stable ASPM-1 foci established by metaphase, while
zyg-9 (-) oocytes had multiple ASPM-1 foci that dynamically coalesced and dispersed throughout most of meiosis I. ZYG-9 and TAC-1 form a complex that promotes microtubule assembly (Bellanger JM and Gonczy P, 2003; Le Bot N et al., 2003; Srayko M et al., 2003), and TAC-1 depleted oocytes showed similar phenotypes to the
zyg-9 (-) oocytes. We are currently investigating the localization of the ZYG-9/TAC-1 complex during early meiotic spindle assembly to further advance our understanding of how these proteins contribute to the acentrosomal assembly of bipolar spindles during oocyte meiotic cell division. References: Wolff et al., Mol Biol Cell, 2016; Matthews LR et al., J Cell Biol., 1998; Bellanger JM and Gonczy P, J Cell Biol., 2003; Yang et al., Dev Biol., 2003; Le Bot N et al., Curr Biol., 2003; Srayko M et al., Curr Biol., 2003.