C. elegans chromosomes connect to centrosomes by anchoring into the spindle network

Abstract

The mitotic spindle ensures the faithful segregation of chromosomes. Here we combine the first large-scale serial electron tomography of whole mitotic spindles in early C. elegans embryos with live-cell imaging to reconstruct all microtubules in 3D and identify their plus- and minus-ends. We classify them as kinetochore (KMTs), spindle (SMTs) or astral microtubules (AMTs) according to their positions, and quantify distinct properties of each class. While our light microscopy and mutant studies show that microtubules are nucleated from the centrosomes, we find only a few KMTs directly connected to the centrosomes. Indeed, by quantitatively analysing several models of microtubule growth, we conclude that minus-ends of KMTs have selectively detached and depolymerized from the centrosome. In toto, our results show that the connection between centrosomes and chromosomes is mediated by an anchoring into the entire spindle network and that any direct connections through KMTs are few and likely very transient.

Mapping Load-Bearing in the Mammalian Spindle Reveals Local Kinetochore Fiber Anchorage that Provides Mechanical Isolation and Redundancy

“…Notably, the ability of the spindle to locally bear load implies that force generation on kinetochores, thought to for example stabilize kinetochore-microtubule attachments [44], is robust to distant changes in spindle architecture or forces. Thus, local and redundant load-bearing could allow the mammalian spindle to dynamically reorganize itself while preserving its ability to bear the load of chromosome movement, and is well-suited to support chromosome movement in species where few microtubules directly connect the kinetochore and spindle pole [45]. …”

Microtubule Dynamics Scale with Cell Size to Set Spindle Length and Assembly Timing

“…Specifically, 1- and 2-cell stage sea urchin blastomeres deviated from the linear scaling trend followed by other cells, and assembled spindles that were shorter than predicted by values of <L> . Several non-exclusive causes could account for the discrepancy between <L> and spindle length: microtubules may bend, detach from the centrosomes or be severed (Brangwynne et al, 2006; Crowder et al, 2015; Dumont and Mitchison, 2009a; Gadde and Heald, 2004; Goshima et al, 2005a; Maiato et al, 2004; McBeath and Fujiwara, 1990; Reber and Hyman, 2015; Redemann et al, 2017; Wuhr et al, 2008). We also note that theoretically, the microtubule growth rate should regulate microtubule mass within the spindle rather than the spindle length itself (Mitchison et al, 2015; Reber et al, 2013).…”

“…Quantitative western blotting allowed us to estimate the tubulin concentration to approximately 22 μM in C. elegans embryos, which is similar to Xenopus embryos (Figure S5 and Methods) (Belmont et al., 1990). Based on a recent study providing the first complete electron tomographic reconstruction of the mitotic spindle in the C. elegans zygote (Redemann et al, 2017), we estimated that the total amount of tubulin heterodimers should be sufficient to assemble 2–3 zygotic mitotic spindles (see Methods). Thus, tubulin seems unlikely to be the only limiting factor for spindle length scaling.…”

Physical Limits on the Precision of Mitotic Spindle Positioning by Microtubule Pushing forces

“…In reality, microtubules can associate with one another within cells via cross-linkers and form complex architectures such as bundles (seen fission yeast [91] and mammalian cells [92] ) or branches (seen in Xenopus egg extracts [93,94] ). In C. elegans , microtubules in the inner spindle do not form bundles [95] . However, microtubules in the asters have been reported to form bundles during anaphase [96] , though this needs to be confirmed by electron microscopy.…”