Interplay between microtubule bundling and sorting factors ensures acentriolar spindle stability during C. elegans oocyte meiosis

Abstract

In many species, oocyte meiosis is carried out in the absence of centrioles. As a result, microtubule organization, spindle assembly, and chromosome segregation proceed by unique mechanisms. Here, we report insights into the principles underlying this specialized form of cell division, through studies of C. elegans KLP-15 and KLP-16, 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 we found that during anaphase, microtubules are able to reorganize into a bundled array that facilitates chromosome segregation. This phenotype therefore enabled us to identify factors promoting microtubule organization during anaphase, whose contributions are normally undetectable in wild-type worms; we 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. Therefore, our studies have revealed an interplay between distinct mechanisms that together promote spindle formation and chromosome segregation in the absence of structural cues such as centrioles.

Microtubule Assembly and Pole Coalescence: Early Steps inC. elegansOocyte Meiosis I Spindle Assembly

“…To further characterize spindle assembly, we imaged live control oocytes using a GFP fusion to the pole-marker ASPM-1 (GFP::ASPM-1) and the mCherry::H2B fusion (Figure 1B, Supplemental Figure 2). As described previously (Gigant et al, 2017; Mullen and Wignall, 2017), GFP::ASPM-1 initially localized along microtubules during cage assembly but then became restricted to multiple small pole foci that coalesced into two poles by metaphase. These GFP::ASPM-1-marked poles subsequently broadened and faded during anaphase.…”

“…While the early microtubule cage assembles in close proximity to the nuclear lamina, how these microtubules are nucleated and whether the nuclear lamina is required for their assembly are not known. RNAi-mediated knockdown of two nearly identical minus-end directed C. elegans kinesin-14 family members, KLP-15 and −16, destabilizes the cage-like structure (Mullen and Wignall, 2017), consistent with the demonstrated ability of kinesin-14 family members to bundle parallel microtubules (Fink et al, 2009). The small GTPase Ran has been implicated in chromosome-mediated nucleation of microtubule assembly during meiosis and mitosis in some organisms (Clarke and Zhang, 2008), but not in C. elegans (Askjaer et al, 2002).…”

“…Because the microtubule cage that assembles after the initiation of NEBD is adjacent to the nuclear lamina (Mullen and Wignall, 2017), we first asked if the nuclear lamina is required for cage assembly. After using RNAi in the GFP::TBB-2, mCherry::H2B background to knock down the single C. elegans lamin LMN-1, we did not detect any peripheral microtubule bundles in 8 of 10 oocytes, and in 2 oocytes we detected only a few relatively short bundles (Figure 2A, Supplemental Figure 3).…”

“…Recent studies have defined a sequence of four steps that assemble bipolar spindles in C. elegans oocytes (Gigant et al, 2017; Mullen and Wignall, 2017) (Fig. 1A).…”

Sumoylation regulates central spindle protein dynamics during chromosome segregation in oocytes

“…Additionally, BUB-1 and CPC components are also present within the central spindle. The limited evidence on the dynamics of these proteins during meiosis I suggests that they do not necessarily occupy the same domains throughout anaphase (Dumont et al, 2010;Davis-Roca et al, 2017;Mullen and Wignall, 2017;Davis-Roca et al, 2018). Considering that i) the CPC and CLS-2 are essential for chromosome segregation and ii) BUB-1 also plays a role during chromosome segregation, we sought to focus our attention on these proteins and characterise their dynamics during anaphase I.…”