A force-balance model for centrosome positioning and spindle elongation during Interphase and Anaphase B

A computational model in one dimension is proposed to position a single centrosome using astral microtubules (MTs) interacting with the cell cortex. The mechanism exploits mutually antagonistic pulling and pushing forces arising from the astral MTs' binding to cortical dynein motors in the actin-rich cell cortex and their buckling while growing against the cell cortex, respectively. The underlying mechanism of astral MTs is also extended to account for the elongation and positioning of the bipolar spindle during mitotic anaphase B. The model for bipolar spindle involves both IPMTs and astral MTs, can predict its elongation and positioning under various circumstances. The model reveals that the bipolar spindle elongation, weakened by decreasing overlap between the antiparallel interpolar microtubules (IPMTs) in the spindle mid-zone, is recovered by the astral MTs.

Cdc42 reactivation at growth sites is regulated by local cell-cycle-dependent loss of its GAP Rga4

In fission yeast, polarized cell growth stops during division and resumes after cytokinesis completes and cells separate. It is unclear how growth reactivation is timed to occur immediately after cell separation. We uncoupled these sequential events by delaying cytokinesis with a temporary Latrunculin A treatment. Mitotic cells recovering from treatment initiate end growth during septation, displaying a polar elongation simultaneous with septation (PrESS) phenotype. PrESS cell ends reactivate Cdc42, a major regulator of polarized growth, during septation, but at a fixed time after anaphase B. A candidate screen implicates Rga4, a negative regulator of Cdc42, in this process. We show that Rga4 appears punctate at the cell sides during G2, but is diffuse during mitosis, extending to the ends. While the Morphogenesis Orb6 (MOR) pathway is known to promote cell separation and growth by activating protein synthesis, we find that for polarized growth, removal of Rga4 from the ends is also necessary. Therefore, we propose that growth resumes after division once the MOR pathway is activated and the ends lose Rga4 in a cell-cycle-dependent manner.

Microtubule rescue at midzone edges promotes overlap stability and prevents spindle collapse during anaphase B

During anaphase B, molecular motors slide interpolar microtubules to elongate the mitotic spindle, contributing to the separation of chromosomes. However, sliding of antiparallel microtubules reduces their overlap, which may lead to spindle breakage, unless microtubules grow to compensate sliding. How sliding and growth are coordinated is still poorly understood. In this study, we have used the fission yeast S. pombe to measure microtubule dynamics during anaphase B. We report that the coordination of microtubule growth and sliding relies on promoting rescues at the midzone edges. This makes microtubules stable from pole to midzone, while their distal parts including the plus ends alternate between assembly and disassembly. Consequently, the midzone keeps a constant length throughout anaphase, enabling sustained sliding without the need for a precise regulation of microtubule growth speed. Additionally, we found that in S. pombe, which undergoes closed mitosis, microtubule growth speed decreases when the nuclear membrane wraps around the spindle midzone.

Kinesin-6 Klp9 orchestrates spindle elongation by regulating microtubule sliding and growth

Mitotic spindle function depends on the precise regulation of microtubule dynamics and microtubule sliding. Throughout mitosis, both processes have to be orchestrated to establish and maintain spindle stability. We show that during anaphase B spindle elongation in S. pombe, the sliding motor Klp9 (kinesin-6) also promotes microtubule growth in vivo. In vitro, Klp9 can enhance and dampen microtubule growth, depending on the tubulin concentration. This indicates that the motor is able to promote and block tubulin subunit incorporation into the microtubule lattice in order to set a well-defined microtubule growth velocity. Moreover, Klp9 recruitment to spindle microtubules is dependent on its dephosphorylation mediated by XMAP215/Dis1, a microtubule polymerase, creating a link between the regulation of spindle length and spindle elongation velocity. Collectively, we unravel the mechanism of anaphase B, from Klp9 recruitment to the motors dual-function in regulating microtubule sliding and microtubule growth, allowing an inherent coordination of both processes.

Kinesin-6 Klp9 orchestrates spindle elongation by regulating microtubule sliding and growth

Mitotic spindle function depends on the precise regulation of microtubule dynamics and microtubule sliding. Throughout mitosis, both processes have to be orchestrated to establish and maintain spindle stability. We show that during anaphase B spindle elongation in S. pombe, the sliding motor Klp9 (kinesin-6) also promotes microtubule growth in vivo. In vitro, Klp9 can enhance and dampen microtubule growth, depending on the tubulin concentration. This indicates that the motor is able to promote and block tubulin subunit incorporation into the microtubule lattice in order to set a well-defined microtubule growth velocity. Moreover, Klp9 recruitment to spindle microtubules is dependent on its dephosphorylation mediated by XMAP215/Dis1, a microtubule polymerase, to link the regulation of spindle length and spindle elongation velocity. Collectively, we unravel the mechanism of anaphase B, from Klp9 recruitment to the motors dual-function in regulating microtubule sliding and microtubule growth, allowing an inherent coordination of both processes.

Evidence for anaphase pulling forces during C. elegans meiosis

Anaphase chromosome movement is thought to be mediated by pulling forces generated by end-on attachment of microtubules to the outer face of kinetochores. However, it has been suggested that during C. elegans female meiosis, anaphase is mediated by a kinetochore-independent pushing mechanism with microtubules only attached to the inner face of segregating chromosomes. We found that the kinetochore proteins KNL-1 and KNL-3 are required for preanaphase chromosome stretching, suggesting a role in pulling forces. In the absence of KNL-1,3, pairs of homologous chromosomes did not separate and did not move toward a spindle pole. Instead, each homolog pair moved together with the same spindle pole during anaphase B spindle elongation. Two masses of chromatin thus ended up at opposite spindle poles, giving the appearance of successful anaphase.

Male meiotic spindle features that efficiently segregate paired and lagging chromosomes

Chromosome segregation during male meiosis is tailored to rapidly generate multitudes of sperm. Little is known about mechanisms that efficiently partition chromosomes to produce sperm. Using live imaging and tomographic reconstructions of spermatocyte meiotic spindles in Caenorhabditis elegans, we find the lagging X chromosome, a distinctive feature of anaphase I in C. elegans males, is due to lack of chromosome pairing. The unpaired chromosome remains tethered to centrosomes by lengthening kinetochore microtubules, which are under tension, suggesting that a ‘tug of war’ reliably resolves lagging. We find spermatocytes exhibit simultaneous pole-to-chromosome shortening (anaphase A) and pole-to-pole elongation (anaphase B). Electron tomography unexpectedly revealed spermatocyte anaphase A does not stem solely from kinetochore microtubule shortening. Instead, movement of autosomes is largely driven by distance change between chromosomes, microtubules, and centrosomes upon tension release during anaphase. Overall, we define novel features that segregate both lagging and paired chromosomes for optimal sperm production.

Two S. pombe septation phases differ in ingression rate, septum structure, and response to F-actin loss

In fission yeast, cytokinesis requires a contractile actomyosin ring (CR) coupled to membrane and septum ingression. Septation proceeds in two phases. In anaphase B, the septum ingresses slowly. During telophase, the ingression rate increases, and the CR becomes dispensable. Here, we explore the relationship between the CR and septation by analyzing septum ultrastructure, ingression, and septation proteins in cells lacking F-actin. We show that the two phases of septation correlate with septum maturation and the response of cells to F-actin removal. During the first phase, the septum is immature and, following F-actin removal, rapidly loses the Bgs1 glucan synthase from the membrane edge and fails to ingress. During the second phase, the rapidly ingressing mature septum can maintain a Bgs1 ring and septum ingression without F-actin, but ingression becomes Cdc42 and exocyst dependent. Our results provide new insights into fungal cytokinesis and reveal the dual function of CR as an essential landmark for the concentration of Bgs1 and a contractile structure that maintains septum shape and synthesis.

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

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.