Chromosome clustering in mitosis by the nuclear protein Ki-67

Ki-67 is highly expressed in proliferating cells, a characteristic that made the protein a very important proliferation marker widely used in the clinic. However, the molecular functions and properties of Ki-67 remained quite obscure for a long time. Only recently important discoveries have shed some light on its function and shown that Ki-67 has a major role in the formation of mitotic chromosome periphery compartment, it is associated with protein phosphatase one (PP1) and regulates chromatin function in interphase and mitosis. In this review, we discuss the role of Ki-67 during cell division. Specifically, we focus on the importance of Ki-67 in chromosome individualisation at mitotic entry (prometaphase) and its contribution to chromosome clustering and nuclear remodelling during mitotic exit.

Mitotic inactivation of the cGAS‒MITA/STING pathways

The cyclic GMP‒AMP synthase (cGAS)‒mediator of interferon response factor 3 activation/stimulator of interferon genes (MITA/STING) axis has emerged as a major pathway, which senses microbial or mis-located cellular DNA in the cytosol to trigger innate immune responses. cGAS senses cytosolic DNA without a preference of self or non-self DNA. How the cGAS‒MITA/STING axis is inactivated upon nuclear membrane breakdown (NEBD) at mitotic entry in vertebrate cells to avoid self DNA sensing remains unclear until very recently. In this review, we summarize the recent advances on how cGAS responds to chromosomes upon NEBD and the mechanisms involved in the inactivation of the cGAS‒MITA/STING pathways in mitosis.

Mechanosensitive control of mitotic entry

As cells prepare to divide, they must ensure that enough space is available to assemble the mitotic machinery without perturbing tissue homeostasis. To do so, cells undergo a series of biochemical reactions regulated by cyclin B1-CDK1 that trigger the reorganization of the actomyosin cytoskeleton and ensure the coordination of cytoplasmic and nuclear events. Along with the biochemical events that control mitotic entry, mechanical forces have recently emerged as important players in the regulation of cell cycle events. However, the exact link between mechanical forces and the biochemical events that control mitotic progression remains to be established. Here, we identify a mechanical signal on the nucleus that sets the time for nuclear envelope permeabilization and mitotic entry. This signal relies on nuclear unfolding during the G2-M transition, which activates the stretch-sensitive cPLA2 on the nuclear envelope. This activation upregulates actomyosin contractility, determining the spatiotemporal translocation of cyclin B1 in the nucleus. Our data demonstrate how the mechanosensitive behaviour of cyclin B1 ensures timely and efficient mitotic spindle assembly and prevents chromosomal instability.

Zika virus induces mitotic catastrophe in human neural progenitors by triggering unscheduled mitotic entry in the presence of DNA damage while functionally depleting nuclear PNKP

Vertical transmission of Zika virus (ZIKV) leads with high frequency to congenital ZIKV syndrome (CZS), whose worse outcome is microcephaly. However, the mechanisms of congenital ZIKV neurodevelopmental pathologies, including direct cytotoxicity to neural progenitor cells (NPC), placental insufficiency, and immune responses, remain incompletely understood. At the cellular level, microcephaly typically results from death or insufficient proliferation of NPC or cortical neurons. NPCs replicate fast, requiring efficient DNA damage responses to ensure genome stability. Like congenital ZIKV infection, mutations in the polynucleotide 5’-kinase 3’-phosphatase (PNKP) gene, which encodes a critical DNA damage repair enzyme, results in recessive syndromes often characterized by congenital microcephaly with seizures (MCSZ). We thus tested whether there were any links between ZIKV and PNKP. Here we show that a PNKP phosphatase inhibitor inhibits ZIKV replication. PNKP relocalized from the nucleus to the cytoplasm in infected cells, co-localizing with the marker of ZIKV replication factories (RF) NS1 and resulting in functional nuclear PNKP depletion. Although infected NPC accumulated DNA damage, they failed to activate the DNA damage checkpoint kinases Chk1 and Chk2. ZIKV also induced activation of cytoplasmic CycA/CDK1 complexes, which trigger unscheduled mitotic entry. Inhibition of CDK1 activity inhibited ZIKV replication and the formation of RF, supporting a role of cytoplasmic CycA/CDK1 in RF morphogenesis. In brief, ZIKV infection induces mitotic catastrophe resulting from unscheduled mitotic entry in the presence of DNA damage. PNKP and CycA/CDK1 are thus host factors participating in ZIKV replication in NPC, and probably pathogenesis.

Aurora A kinase activation: Different means to different ends

Aurora A is a serine/threonine kinase essential for mitotic entry and spindle assembly. Recent molecular studies have revealed the existence of multiple, distinct mechanisms of Aurora A activation, each occurring at specific subcellular locations, optimized for cellular context, and primed by signaling events including phosphorylation and oxidation.

Mapping the invisible chromatin transactions of prophase chromosome remodelling

We have used a combination of chemical genetics, chromatin proteomics and imaging to map the earliest chromatin transactions during vertebrate cell entry into mitosis. Chicken DT40 CDK1as cells undergo synchronous mitotic entry within 15 minutes following release from a 1NM-PP1-induced arrest in late G2. In addition to changes in chromatin association with nuclear pores and the nuclear envelope, earliest prophase is dominated by changes in the association of ribonucleoproteins with chromatin, particularly in the nucleolus, where pre-rRNA processing factors leave chromatin significantly before RNA polymerase I. Nuclear envelope barrier function is lost early in prophase and cytoplasmic proteins begin to accumulate on the chromatin. As a result, outer kinetochore assembly appears complete by nuclear envelope breakdown (NEBD). Most interphase chromatin proteins remain associated with chromatin until NEBD, after which their levels drop sharply. An interactive proteomic map of chromatin transactions during mitotic entry is available as a resource at https://mitoChEP.bio.ed.ac.uk.

Interphase microtubule disassembly is a signaling cue that drives cell rounding at mitotic entry.

Reorganization of the cortical actin cytoskeleton at mitotic entry is essential to increase membrane tension for cell rounding. This spherical shape is necessary for the biogenesis and organization of the mitotic spindle. Proteins of the Ezrin, Radixin, Moesin (ERM) family play essential roles in mitotic morphogenesis by linking actomyosin forces to the plasma membrane. While ERMs drive metaphase cell rounding, the cell-cycle signals that prompt their conformational activation in mitosis are unknown. We screened a library of small molecules using novel ERM biosensors and we unexpectedly found that drugs that disassemble microtubules promote ERM activation. Remarkably, cells disassemble their interphase microtubules while entering mitosis. We further discovered that this disassembly of microtubules acts as a cell-cycle signal that directs ERM activation and metaphase cell rounding. We show that GEF-H1, a Rho-GEF inhibited by microtubule binding, acts downstream of microtubule disassembly to activate ERMs via RhoA and its kinase effector SLK. In addition, we demonstrate that GEF-H1 and Ect2, another Rho-GEF responsible for the generation of mitotic actomyosin forces, act together to drive metaphase ERM activation and cell rounding. In summary, we report microtubule disassembly as a cell cycle signal that triggers a signaling network ensuring that actomyosin forces are efficiently integrated at the plasma membrane to promote cell rounding at mitotic entry.

TRIM37 prevents formation of condensate-organized ectopic spindle poles to ensure mitotic fidelity

Centrosomes are composed of a centriolar core surrounded by pericentriolar material that nucleates microtubules. The ubiquitin ligase TRIM37 localizes to centrosomes, but its centrosomal roles are not yet defined. We show that TRIM37 does not control centriole duplication, structure, or the ability of centrioles to form cilia but instead prevents assembly of an ectopic centrobin-scaffolded structured condensate that forms by budding off of centrosomes. In ∼25% of TRIM37-deficient cells, the condensate organizes an ectopic spindle pole, recruiting other centrosomal proteins and acquiring microtubule nucleation capacity during mitotic entry. Ectopic spindle pole–associated transient multipolarity and multipolar segregation in TRIM37-deficient cells are suppressed by removing centrobin, which interacts with and is ubiquitinated by TRIM37. Thus, TRIM37 ensures accurate chromosome segregation by preventing the formation of centrobin-scaffolded condensates that organize ectopic spindle poles. Mutations in TRIM37 cause the disorder mulibrey nanism, and patient-derived cells harbor centrobin condensate-organized ectopic poles, leading us to propose that chromosome missegregation is a pathological mechanism in this disorder.