The mitotic spindle protein CKAP2 potently increases formation and stability of microtubules

Cells increase microtubule dynamics to make large rearrangements to their microtubule cytoskeleton during cell division. Changes in microtubule dynamics are essential for the formation and function of the mitotic spindle, and misregulation can lead to aneuploidy and cancer. Using in vitro reconstitution assays we show that the mitotic spindle protein Cytoskeleton-Associated Protein 2 (CKAP2) has a strong effect on nucleation of microtubules by lowering the critical tubulin concentration 100-fold. CKAP2 increases the apparent rate constant ka of microtubule growth by 50-fold and increases microtubule growth rates. In addition, CKAP2 strongly suppresses catastrophes. Our results identify CKAP2 as the most potent microtubule growth factor to date. These finding help explain CKAP2's role as an important spindle protein, proliferation marker, and oncogene.

Spatial and Temporal Scaling of Microtubules and Mitotic Spindles

During cell division, the mitotic spindle, a macromolecular structure primarily comprised of microtubules, drives chromosome alignment and partitioning between daughter cells. Mitotic spindles can sense cellular dimensions in order to adapt their length and mass to cell size. This scaling capacity is particularly remarkable during early embryo cleavage when cells divide rapidly in the absence of cell growth, thus leading to a reduction of cell volume at each division. Although mitotic spindle size scaling can occur over an order of magnitude in early embryos, in many species the duration of mitosis is relatively short, constant throughout early development and independent of cell size. Therefore, a key challenge for cells during embryo cleavage is not only to assemble a spindle of proper size, but also to do it in an appropriate time window which is compatible with embryo development. How spatial and temporal scaling of the mitotic spindle is achieved and coordinated with the duration of mitosis remains elusive. In this review, we will focus on the mechanisms that support mitotic spindle spatial and temporal scaling over a wide range of cell sizes and cellular contexts. We will present current models and propose alternative mechanisms allowing cells to spatially and temporally coordinate microtubule and mitotic spindle assembly.

Structure of the human inner kinetochore bound to a centromeric CENP-A nucleosome

Accurate chromosome segregation, controlled by kinetochore-mediated chromatid attachments to the mitotic spindle, ensures the faithful inheritance of genetic information. Kinetochores assemble onto specialized CENP-A nucleosomes (CENP-ANuc) of centromeric chromatin. In humans, this is mostly organized as thousands of copies of an ~171 bp α-satellite repeat. Here, we describe the cryo-EM structure of the human inner kinetochore CCAN (Constitutive Centromere Associated Network) complex bound to CENP-ANuc reconstituted onto α-satellite DNA. CCAN forms edge-on contacts with CENP-ANuc, while a linker DNA segment of the α-satellite repeat emerges from the fully-wrapped end of the nucleosome to thread through the central CENP-LN channel which tightly grips the DNA. The CENP-TWSX histone-fold module, together with CENP-HIKHead, further augments DNA binding and partially wraps the linker DNA in a manner reminiscent of canonical nucleosomes. Our study suggests that the topological entrapment of the α-satellite repeat linker DNA by CCAN provides a robust mechanism by which the kinetochore withstands the pushing and pulling of centromeres associated with chromosome congression and segregation forces.

Separate to operate: the centriole-free inner core of the centrosome regulates the assembly of the intranuclear spindle in Toxoplasma gondii

Centrosomes are the main microtubule-organizing center of the cell. They are normally formed by two centrioles, embedded in a cloud of proteins known as pericentriolar material. The PCM ascribes centrioles with their microtubule nucleation capacity. Toxoplasma gondii, the causative agent of toxoplasmosis, divides by endodyogeny. Successful cell division is critical for pathogenesis. The centrosome, plays central roles in orchestrating the temporal and physical coordination of major organelle segregation and daughter cell formation. The T. gondii centrosome is formed by two domains; an outer core, distal from the nucleus, and an inner core, proximal to the nucleus. This dual organization has been proposed to underlie T. gondii’s cell division plasticity. However, the role of the inner core remains undeciphered. Here, we focus on the role of its only known molecular marker; TgCEP250L1. We show that upon conditional degradation of TgCEP250L1, parasites exhibit nuclear segregation defects, whilst normally forming daughter cells. In addition, the centrioles, disconnect from the nucleus. We explore the structural defects underlying these phenotypes by high resolution microscopy. We show that TgCEP250L1’s location is dynamic and encompasses the formation of the mitotic spindle. Moreover, we show that in the absence of TgCEP250L1, the microtubule binding protein TgEB1, fails to translocate from the nucleus to the mitotic spindle, while polyploid nuclei accumulate. Overall, our data supports a model in which the inner core of the T. gondii centrosome critically participates in cell division by directly impacting the formation or stability of the mitotic spindle.

Drosophila centrocortin is dispensable for centriole duplication but contributes to centrosome separation

Centrosomes are microtubule-organizing centers that duplicate exactly once to organize the bipolar mitotic spindle required for error-free mitosis. Prior work indicated that Drosophila centrocortin (cen) is required for normal centrosome separation, although a role in centriole duplication was not closely examined. Through time-lapse recordings of rapid syncytial divisions, we monitored centriole duplication and the kinetics of centrosome separation in control vs. cen null embryos. Our data suggest that although cen is dispensable for centriole duplication, it contributes to centrosome separation.

More than Two Populations of Microtubules Comprise the Dynamic Mitotic Spindle

The microtubules of the mitotic spindle mediate chromosome alignment to the metaphase plate, then sister chromatid segregation to the spindle poles in anaphase. Previous analyses of spindle microtubule kinetics utilizing fluorescence dissipation after photoactivation described two main populations, a slow and a fast turnover population, and these were ascribed to reflect kinetochore versus non-kinetochore microtubules, respectively. Here, we test this categorization by disrupting kinetochores through depletion of the Ndc80 complex. In the absence of functional kinetochores, microtubule dynamics still exhibit slow and fast turnover populations, though the proportion of each population and the timings of turnover are altered. Importantly, the data obtained following Hec1/Ndc80 depletion suggests other sub-populations, in addition to kinetochore microtubules, contribute to the slow turnover population. Further manipulation of spindle microtubules revealed a complex landscape. For example, while Aurora B kinase functions to destabilize kinetochore bound microtubules it may also stabilize certain slow turnover, non-kinetochore microtubules. Dissection of the dynamics of microtubule populations provides a greater understanding of mitotic spindle kinetics and insight into their roles in facilitating chromosome attachment, movement, and segregation during mitosis.

Combined effect of cell geometry and polarity domains determines the orientation of unequal division

Cell division orientation is thought to result from a competition between cell geometry and polarity domains controlling the position of the mitotic spindle during mitosis. Depending on the level of cell shape anisotropy or the strength of the polarity domain, one dominates the other and determines the orientation of the spindle. Whether and how such competition is also at work to determine unequal cell division (UCD), producing daughter cells of different size, remains unclear. Here, we show that cell geometry and polarity domains cooperate, rather than compete, in positioning the cleavage plane during UCDs in early ascidian embryos. We found that the UCDs and their orientation at the ascidian third cleavage rely on the spindle tilting in an anisotropic cell shape, and cortical polarity domains exerting different effects on spindle astral microtubules. By systematically varying mitotic cell shape, we could modulate the effect of attractive and repulsive polarity domains and consequently generate predicted daughter cell size asymmetries and position. We therefore propose that the spindle position during UCD is set by the combined activities of cell geometry and polarity domains, where cell geometry modulates the effect of cortical polarity domain(s).

The APC/C targets the Cep152-Cep63 complex at the centrosome to regulate mitotic spindle assembly

The control of protein abundance is a fundamental regulatory mechanism during mitosis. The anaphase promoting complex/cyclosome (APC/C) is the main protein ubiquitin ligase responsible for the temporal regulation of mitotic progression. It has been proposed that the APC/C might fulfil other functions including assembly of the mitotic spindle. Here, we show that the APC/C localizes to centrosomes, the organizers of the eukaryotic microtubule cytoskeleton, specifically during mitosis. Recruitment of the APC/C to spindle poles requires the centrosomal protein Cep152, and we identified Cep152 as both an APC/C interaction partner and as an APC/C substrate. Previous studies showed that Cep152 forms a complex with Cep57 and Cep63. The APC/C-mediated ubiquitination of Cep152 at the centrosome releases Cep57 from this inhibitory complex and enables its interaction with pericentrin, a critical step in promoting microtubule nucleation. Thus, our study extends the function of the APC/C from being a regulator of mitosis to also acting as a positive governor of spindle assembly. The APC/C thereby integrates control of these two important processes in a temporal manner.