Central-spindle microtubules are strongly coupled to chromosomes during both anaphase A and anaphase B

Abstract

Spindle microtubules, whose dynamics vary over time and at different locations, cooperatively drive chromosome segregation. Measurements of microtubule dynamics and spindle ultrastructure can provide insight into the behaviors of microtubules, helping elucidate the mechanism of chromosome segregation. Much work has focused on the dynamics and organization of kinetochore microtubules, that is, on the region between chromosomes and poles. In comparison, microtubules in the central-spindle region, between segregating chromosomes, have been less thoroughly characterized. Here, we report measurements of the movement of central-spindle microtubules during chromosome segregation in human mitotic spindles and Caenorhabditis elegans mitotic and female meiotic spindles. We found that these central-spindle microtubules slide apart at the same speed as chromosomes, even as chromosomes move toward spindle poles. In these systems, damaging central-spindle microtubules by laser ablation caused an immediate and complete cessation of chromosome motion, suggesting a strong coupling between central-spindle microtubules and chromosomes. Electron tomographic reconstruction revealed that the analyzed anaphase spindles all contain microtubules with both ends between segregating chromosomes. Our results provide new dynamical, functional, and ultrastructural characterizations of central-spindle microtubules during chromosome segregation in diverse spindles and suggest that central-spindle microtubules and chromosomes are strongly coupled in anaphase.

Force-generating mechanisms of anaphase in human cells

“…We found bridging fibers to be essential for the separation of displaced kinetochores, and continuous cutting of most interpolar MTs in the central spindle also decreased spindle elongation rates (Vukušićet al, 2017). In agreement with the latter result, a single-hit laser ablation of all interpolar MTs in human cells immediately stopped anaphase chromosome motion for a short period of time (Yu et al, 2019). This is reminiscent of the pioneering ablation experiments showing that chromosomes continue to move poleward after centrosome removal as long as the k-fiber stub is longer than 1 µm (Nicklas, 1989;Nicklas et al, 1982), as previously mentioned.…”

“…By using a laser ablation assay (Buđa et al, 2017), we observed that these bridging fibers moved together with sister kinetochores and their k-fibers, indicating strong crosslinking between these structures during metaphase (Kajtez et al, 2016;Milas and Tolic, 2016;Tolic, 2018; and anaphase (Vukušićet al, 2017). In agreement with the observation of bridging MTs in anaphase (Vukušićet al, 2017), recent large-scale electron tomography reconstructions of spindles in human cells reported different classes of interpolar MTs that were often tightly associated into bundles contacting k-fibers (Yu et al, 2019). It will be interesting to identify the crosslinkers that keep k-fibers and bridging fibers laterally linked, with the kinesins Eg5 (also known as Kif11) and Kif15 being good candidates as they are involved in the regulation of neighboring k-fiber coupling during metaphase (Vladimirou et al, 2013).…”

“…In agreement, our laser ablation of astral MTs in U2OS cells did not slow down chromatid segregation and spindle elongation (Vukušićet al, 2017) ( Fig. 3B) and there was no acceleration of spindle pole separation after cutting the spindle midzone region during early anaphase (Vukušićet al, 2017;Yu et al, 2019). Thus, in contrast to what is seen for C. elegans and PtK2 cells, where astral MTs drive spindle elongation (Aist et al, 1993;Grill et al, 2001), cortical pulling forces seem dispensable for fast spindle elongation and chromosome segregation during early anaphase in human cells.…”

“…2B,C). Accordingly, interpolar MTs originate mostly away from the pole in human cells (Yu et al, 2019), arguing for pushing through k-fibers. The velocity of interpolar MT sliding is greater than the velocity of spindle elongation (2.1 µm/min and 1.3 µm/min, respectively; Fig.…”

Chromosome segregation is driven by joint microtubule sliding action of kinesins KIF4A and EG5

“…4E). Sliding forces generated in the midzone antiparallel region by these modules are then transmitted to spindle poles most probably through their lateral connections to k-fibers (4,31). Finally, it would be interesting to study the details of signalling cascade that regulates spindle elongation in human cells where Aurora kinases are promising candidates that could separately regulate both sliding modules (30,32,33).…”