Two-step interphase microtubule disassembly aids spindle morphogenesis.

BACKGROUND: Entry into mitosis triggers profound changes in cell shape and cytoskeletal organisation. Here, by studying microtubule remodelling in human flat mitotic cells, we identify a two-step process of interphase microtubule disassembly. RESULTS: First, a microtubule-stabilising protein, Ensconsin/MAP7, is inactivated in prophase as a consequence of its phosphorylation downstream of Cdk1/cyclin B. This leads to a reduction in interphase microtubule stability that may help to fuel the growth of centrosomally nucleated microtubules. The peripheral interphase microtubules that remain are then rapidly lost as the concentration of tubulin heterodimers falls following dissolution of the nuclear compartment boundary. Finally, we show that a failure to destabilise microtubules in prophase leads to the formation of microtubule clumps, which interfere with spindle assembly. CONCLUSIONS: This analysis highlights the importance of the step-wise remodelling of the microtubule cytoskeleton and the significance of permeabilisation of the nuclear envelope in coordinating the changes in cellular organisation and biochemistry that accompany mitotic entry.

Meeting report--Getting Into and Out of Mitosis.

The Company of Biologists Workshop 'Getting Into and Out of Mitosis' was held 10-13 May 2015 at Wiston House in West Sussex, UK. The workshop brought together researchers from wide-ranging disciplines and provided a forum to discuss their latest work on the control of cell division from mitotic entry to exit. This report highlights the main topics and summarises the discussion around the key themes and questions that emerged from the meeting.

Myo19 ensures symmetric partitioning of mitochondria and coupling of mitochondrial segregation to cell division.

During animal cell division, an actin-based ring cleaves the cell into two. Problems with this process can cause chromosome missegregation and defects in cytoplasmic inheritance and the partitioning of organelles, which in turn are associated with human diseases. Although much is known about how chromosome segregation is coupled to cell division, the way organelles coordinate their inheritance during partitioning to daughter cells is less well understood. Here, using a high-content live-imaging small interfering RNA screen, we identify Myosin-XIX (Myo19) as a novel regulator of cell division. Previously, this actin-based motor was shown to control the interphase movement of mitochondria. Our analysis shows that Myo19 is indeed localized to mitochondria and that its silencing leads to defects in the distribution of mitochondria within cells and in mitochondrial partitioning at division. Furthermore, many Myo19 RNAi cells undergo stochastic division failure--a phenotype that can be mimicked using a treatment that blocks mitochondrial fission and rescued by decreasing mitochondrial fusion, implying that mitochondria can physically interfere with cytokinesis. Strikingly, using live imaging we also observe the inappropriate movement of mitochondria to the poles of spindles in cells depleted for Myo19 as they enter anaphase. Since this phenocopies the results of an acute loss of actin filaments in anaphase, these data support a model whereby the Myo19 actin-based motor helps to control mitochondrial movement to ensure their faithful segregation during division. The presence of DNA within mitochondria makes their inheritance an especially important aspect of symmetrical cell division.

Nonautonomous movement of chromosomes in mitosis.

Kinetochores are the central force-generating machines that move chromosomes during cell division. It is generally assumed that kinetochores move in an autonomous manner. However, we reveal here that movements of neighboring sister-kinetochore pairs in metaphase are correlated in a distance-dependent manner. This correlation increases in the absence of kinetochore oscillations or stable end-on attachments. This suggests that periodic movements of bioriented chromosomes limit the correlated motion of nonsisters. Computer simulations show that these correlated movements can occur when elastic crosslinks are placed between the K-fibers of oscillating kinetochores. Strikingly, inhibition of the microtubule crosslinking motor kinesin-5 Eg5 leads to an increase in nonsister correlation and impairs periodic oscillations. These phenotypes are partially rescued by codepletion of the kinesin-12 Kif15, demonstrating a function for kinesin-5 and kinesin-12 motors in driving chromosome movements, possibly as part of a crosslinking structure that correlates the movements of nonsister kinetochores.

Kinetochores accelerate centrosome separation to ensure faithful chromosome segregation.

At the onset of mitosis, cells need to break down their nuclear envelope, form a bipolar spindle and attach the chromosomes to microtubules via kinetochores. Previous studies have shown that spindle bipolarization can occur either before or after nuclear envelope breakdown. In the latter case, early kinetochore-microtubule attachments generate pushing forces that accelerate centrosome separation. However, until now, the physiological relevance of this prometaphase kinetochore pushing force was unknown. We investigated the depletion phenotype of the kinetochore protein CENP-L, which we find to be essential for the stability of kinetochore microtubules, for a homogenous poleward microtubule flux rate and for the kinetochore pushing force. Loss of this force in prometaphase not only delays centrosome separation by 5-6 minutes, it also causes massive chromosome alignment and segregation defects due to the formation of syntelic and merotelic kinetochore-microtubule attachments. By contrast, CENP-L depletion has no impact on mitotic progression in cells that have already separated their centrosomes at nuclear envelope breakdown. We propose that the kinetochore pushing force is an essential safety mechanism that favors amphitelic attachments by ensuring that spindle bipolarization occurs before the formation of the majority of kinetochore-microtubule attachments.

Kinetochore alignment within the metaphase plate is regulated by centromere stiffness and microtubule depolymerases.

During mitosis in most eukaryotic cells, chromosomes align and form a metaphase plate halfway between the spindle poles, about which they exhibit oscillatory movement. These movements are accompanied by changes in the distance between sister kinetochores, commonly referred to as breathing. We developed a live cell imaging assay combined with computational image analysis to quantify the properties and dynamics of sister kinetochores in three dimensions. We show that baseline oscillation and breathing speeds in late prometaphase and metaphase are set by microtubule depolymerases, whereas oscillation and breathing periods depend on the stiffness of the mechanical linkage between sisters. Metaphase plates become thinner as cells progress toward anaphase as a result of reduced oscillation speed at a relatively constant oscillation period. The progressive slowdown of oscillation speed and its coupling to plate thickness depend nonlinearly on the stiffness of the mechanical linkage between sisters. We propose that metaphase plate formation and thinning require tight control of the state of the mechanical linkage between sisters mediated by centromeric chromatin and cohesion.