Functional midbody assembly in the absence of a central spindle

Abstract

Contractile ring constriction during cytokinesis is thought to compact central spindle microtubules to form the midbody, an antiparallel microtubule bundle at the intercellular bridge. In Caenorhabditis elegans, central spindle microtubule assembly requires targeting of the CLASP family protein CLS-2 to the kinetochores in metaphase and spindle midzone in anaphase. CLS-2 targeting is mediated by the CENP-F–like HCP-1/2, but their roles in cytokinesis and midbody assembly are not known. We found that although HCP-1 and HCP-2 mostly function cooperatively, HCP-1 plays a more primary role in promoting CLS-2–dependent central spindle microtubule assembly. HCP-1/2 codisrupted embryos did not form central spindles but completed cytokinesis and formed functional midbodies capable of supporting abscission. These central spindle–independent midbodies appeared to form via contractile ring constriction–driven bundling of astral microtubules at the furrow tip. This work suggests that, in the absence of a central spindle, astral microtubules can support midbody assembly and that midbody assembly is more predictive of successful cytokinesis than central spindle assembly.

Synergistic stabilization of microtubules by BUB-1, HCP-1 and CLS-2 controls meiotic spindle assembly in C. elegans oocytes

“…In mammals, CENP-F kinetochore targeting involves physical interaction between a specific C-terminal CENP-F targeting domain and the kinase domain of Bub1 46 . HCP-1 and 2 are long coiled-coil proteins that only share 27.5% and 24.7% similarity respectively with CENP-F, which precluded identification of conserved functional domains 76 . To identify the domains of HCP-1 responsible for its localization, we thus generated a series of transgenic strains expressing RNAi-resistant GFP-fused truncations of HCP-1 (Figure 2D, figure 2 – Figure supplement 1A).…”

“…To identify the domains of HCP-1 responsible for its localization, we thus generated a series of transgenic strains expressing RNAi-resistant GFP-fused truncations of HCP-1 (Figure 2D, figure 2 – Figure supplement 1A). We decided to specifically focus on HCP-1 because, although HCP-1 and 2 are functionally redundant and can compensate for each other to support embryonic development, HCP-1 plays a more primary role in early embryos 76 . We introduced the HCP-1 transgenes in an hcp-2 deletion mutant (hereafter hcp-2Δ ) and analyzed embryonic viability after endogenous HCP-1 depletion by RNAi 39 .…”

“…In contrast in C. elegans, the two CENP-F-like proteins HCP-1/2 are essential for embryonic viability 22 . Together with their downstream partner CLS-2, kinetochore-localized HCP-1/2 control kinetochore-microtubule dynamics to prevent sister chromatid co-segregation to the same spindle pole in mitosis, and promote midzone microtubule assembly for central spindle formation in anaphase 22,[74][75][76] . During meiosis in C. elegans oocytes, HCP-1/2 and CLS-2 localize along spindle microtubules, at the cup-shaped kinetochores and on the ring domains 34,35 .…”

A back-up source of microtubules for the midbody during cytokinesis

“…Because the contractile ring closes around the central spindle, it has long been assumed that the midbody must require the gathering and compaction of these central spindle microtubules. However, a new study by Canman and colleagues using the one-cell Caenorhabditis elegans embryo suggests that this widely held view is not the full story ( 4 ). Using genetic manipulations and quantitative live-cell microscopy, they present evidence that midbodies can in fact still form without any preceding central spindle, using astral microtubules as their source.…”

“… Schematic depicting the contributions of different populations of microtubules to midbody formation. Upper cartoons depict a wild-type C. elegans zygote, while the lower cartoons depict the midbody assembly pathway uncovered by Hirsch et al ( 4 ) upon loss of the HCP-1/2–dependent central spindle. …”