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Microscopy and genomic approaches provide detailed descriptions of the three-dimensional folding of chromosomes and nuclear organization. The fundamental question is how activity of molecules at the nanometer scale can lead to complex and orchestrated spatial organization at the scale of chromosomes and the whole nucleus. At least three key mechanisms can bridge across scales: (1) tethering of specific loci to nuclear landmarks leads to massive reorganization of the nucleus; (2) spatial compartmentalization of chromatin, which is driven by molecular affinities, results in spatial isolation of active and inactive chromatin; and (3) loop extrusion activity of SMC (structural maintenance of chromosome) complexes can explain many features of interphase chromatin folding and underlies key phenomena during mitosis. Interestingly, many features of chromosome organization ultimately result from collective action and the interplay between these mechanisms, and are further modulated by transcription and topological constraints. Finally, we highlight some outstanding questions that are critical for our understanding of nuclear organization and function. We believe many of these questions can be answered in the coming years.

SummaryUnderstanding how the nuclear pore complex (NPC) assembles is of fundamental importance to grasp the mechanisms behind its essential function and understand its role during evolution of eukaryotes1–4. While at least two NPC assembly pathways exist, one during exit from mitosis and one during nuclear growth in interphase, we currently lack a quantitative map of the molecular assembly events. Here, we use fluorescence correlation spectroscopy (FCS) calibrated live imaging of endogenously fluorescently-tagged nucleoporins to map the changes in composition and stoichiometry of seven major modules of the human NPC during its assembly in single dividing cells. This systematic quantitative map reveals that the two assembly pathways employ strikingly different molecular mechanisms, inverting the order of addition of two large structural components, the central ring complex and nuclear filaments. The dynamic stoichiometry data underpinned integrative spatiotemporal modeling of the NPC assembly pathway, predicting the structures of postmitotic NPC assembly intermediates.

Cholangiocarcinoma (CCA) is characterized by resistance to chemotherapy and a poor prognosis. Therefore, treatments that can effectively suppress tumor growth are urgently needed. Aberrant activation of hedgehog (HH) signaling has been implicated in several cancers, including those of the hepatobiliary tract. However, the role of HH signaling in intrahepatic CCA (iCCA) has not been completely elucidated. In this study, we addressed the function of the main transducer Smoothened (SMO) and the transcription factors (TFs) GLI1 and GLI2 in iCCA. In addition, we evaluated the potential benefits of the combined inhibition of SMO and the DNA damage kinase WEE1. Transcriptomic analysis of 152 human iCCA samples showed increased expression of GLI1, GLI2, and Patched 1 (PTCH1) in tumor tissues compared with nontumor tissues. Genetic silencing of SMO, GLI1, and GLI2 inhibited the growth, survival, invasiveness, and self-renewal of iCCA cells. Pharmacologic inhibition of SMO reduced iCCA growth and viability in vitro, by inducing double-strand break DNA damage, leading to mitotic arrest and apoptotic cell death. Importantly, SMO inhibition resulted in the activation of the G2–M checkpoint and DNA damage kinase WEE1, increasing the vulnerability to WEE1 inhibition. Hence, the combination of MRT-92 with the WEE1 inhibitor AZD-1775 showed increased antitumor activity in vitro and in iCCA xenografts compared with single treatments. These data indicate that combined inhibition of SMO and WEE1 reduces tumor burden and may represent a strategy for the clinical development of novel therapeutic approaches in iCCA. Cholangiocarcinoma (CCA) is characterized by resistance to chemotherapy and a poor prognosis. Therefore, treatments that can effectively suppress tumor growth are urgently needed. Aberrant activation of hedgehog (HH) signaling has been implicated in several cancers, including those of the hepatobiliary tract. However, the role of HH signaling in intrahepatic CCA (iCCA) has not been completely elucidated. In this study, we addressed the function of the main transducer Smoothened (SMO) and the transcription factors (TFs) GLI1 and GLI2 in iCCA. In addition, we evaluated the potential benefits of the combined inhibition of SMO and the DNA damage kinase WEE1. Transcriptomic analysis of 152 human iCCA samples showed increased expression of GLI1, GLI2, and Patched 1 (PTCH1) in tumor tissues compared with nontumor tissues. Genetic silencing of SMO, GLI1, and GLI2 inhibited the growth, survival, invasiveness, and self-renewal of iCCA cells. Pharmacologic inhibition of SMO reduced iCCA growth and viability in vitro, by inducing double-strand break DNA damage, leading to mitotic arrest and apoptotic cell death. Importantly, SMO inhibition resulted in the activation of the G2-M checkpoint and DNA damage kinase WEE1, increasing the vulnerability to WEE1 inhibition. Hence, the combination of MRT-92 with the WEE1 inhibitor AZD-1775 showed increased antitumor activity in vitro and in iCCA xenografts compared with single treatments. These data indicate that combined inhibition of SMO and WEE1 reduces tumor burden and may represent a strategy for the clinical development of novel therapeutic approaches in iCCA.

Wnt signalling is an essential player in tissue formation, notably in the regulation of stem cell function. Wnt signalling is best known for its roles in G1/S progression. However, a complex Wnt programme that also mediates mitotic progression and asymmetric cell division (ACD) is emerging. Recent developments in this area have provided mechanistic insights as well as tools to engineer or target Wnt signalling for translational and therapeutic purposes. Here, we discuss the bidirectional relationship between Wnt activity and mitosis. We emphasise how various Wnt-dependent mechanisms control spindle dynamics, chromosome segregation, and ACD. Finally, we illustrate how knowledge about these mechanisms has been successfully employed in tissue engineering for regenerative medicine applications.

Cell cycle-dependent gene transcription is tightly controlled by the retinoblastoma (RB):E2F and DREAM complexes, which repress all cell cycle genes during quiescence. Cyclin-dependent kinase (CDK) phosphorylation of RB and DREAM allows for the expression of two gene sets. The first set of genes, with peak expression in G1/S, is activated by E2F transcription factors (TFs) and is required for DNA synthesis. The second set, with maximum expression during G2/M, is required for mitosis and is coordinated by the MuvB complex, together with B-MYB and Forkhead box M1 (FOXM1). In this review, we summarize the key findings that established the distinct control mechanisms regulating G1/S and G2/M gene expression in mammals and discuss recent advances in the understanding of the temporal control of these genes.

Kinetochores are molecular machines that power chromosome segregation during the mitotic and meiotic cell divisions of all eukaryotes. Aristotle explains how we think we have knowledge of a thing only when we have grasped its cause. In our case, to gain understanding of the kinetochore, the four causes correspond to questions that we must ask: ( a) What are the constituent parts, ( b) how does it assemble, ( c) what is the structure and arrangement, and ( d) what is the function? Here we outline the current blueprint for the assembly of a kinetochore, how functions are mapped onto this architecture, and how this is shaped by the underlying pericentromeric chromatin. The view of the kinetochore that we present is possible because an almost complete parts list of the kinetochore is now available alongside recent advances using in vitro reconstitution, structural biology, and genomics. In many organisms, each kinetochore binds to multiple microtubules, and we propose a model for how this ensemble-level architecture is organized, drawing on key insights from the simple one microtubule–one kinetochore setup in budding yeast and innovations that enable meiotic chromosome segregation.

The Regulator of Chromosome Condensation 2 (RCC2) is an important gene that regulates mitosis and cytoplasmic division in the cell cycle. Although there have been reported in several individual tumors, an integrative analysis of

The midbody is a transient structure forming out of the central spindle at late telophase. Both the midbody and central spindle have important functions ensuring completion of cytokinesis and defects in this process may lead to genetic diseases, including cancer. Thus, understanding the mechanisms that control cytokinesis during mitosis can reveal the key components taking part in some of the processes that promote accurate cell division. Our previous study showed that overexpression of FLJ25439 causes cytokinesis defect with midbody arrest and induces tetraploids with prolonged cell growth/cell cycle progression (Pan et al., 2015). Here, we extend our investigation with regard to the expression profile/regulation and cellular localization/function of FLJ25439 during mitosis/cytokinesis. Using a monoclonal antibody 2A4 we found that FLJ25439 expression is cell cycle-dependent and subjected to APC/C complex regulation. Furthermore, it is a novel substrate for the APC/C-Cdc20 complex and its degradation is proteasome-dependent through D-box recognition during mitotic exit. Immunofluorescence microscopy showed it is distributed at the central spindle and midbody, two structures considered important for completion of cell division, in telophase and cytokinesis, respectively, during cell cycle progression. Depletion of FLJ25439 expression revealed defects in chromosome alignment/segregation and delayed mitosis/cytokinesis progression. We thus conclude that FLJ25439 is a hitherto undiscovered factor involved in cytokinesis regulation.

Ulceration and high mitosis are considered among the major unfavorable prognostic factors in the survival of cutaneous melanoma patients. The aim of this study was to investigate the clinical significance of these parameters and to compare them to see which one is superior to predicting prognosis across all clinical stages of melanoma. A total of 1,074 melanomas were analyzed retrospectively. Tumor ulceration was found to be limited to the local stage for predicting survival, whereas, mitosis maintained its prognostic strength for predicting survival across all clinical stages. Furthermore, no survival differences were observed between ulceration and mitosis across clinical stages.

Cilia and mitotic spindles are microtubule (MT)-based, macromolecular machines that consecutively assemble and disassemble during interphase and M phase of the cell cycle, respectively, and play fundamental roles in how eukaryotic cells swim through a fluid, sense their environment, and divide to reproduce themselves. The formation and function of these structures depend on several types of cytoskeletal motors, notably MT-based kinesins and dyneins, supplemented by actin-based myosins, which may function independently or collaboratively during specific steps in the pathway of mitosis or ciliogenesis. System-specific differences in these pathways occur because, instead of conforming to a simple one motor–one function rule, ciliary and mitotic motors can be deployed differently by different cell types. This reflects the well-known influence of natural selection on basic molecular processes, creating diversity at subcellular scales. Here we review our current understanding of motor function and cooperation during the assembly–disassembly, maintenance, and functions of cilia and mitotic spindles.

Polyploidization and polyploidy reversal (depolyploidization) are crucial pathways to conversely alter genomic contents in organisms. Understanding the mechanisms switching between polyploidization and polyploidy reversal should broaden our knowledge of the generation of pathological polyploidy and pave a new path to prevent related diseases.

The number of mitotic cells present in histopathological slides is an important predictor of tumor proliferation in the diagnosis of breast cancer. However, the current approaches can hardly perform precise pixel-level prediction for mitosis datasets with only weak labels (i.e., only provide the centroid location of mitotic cells), and take no account of the large domain gap across histopathological slides from different pathology laboratories. In this work, we propose a Domain adaptive Box-supervised Instance segmentation Network (DBIN) to address the above issues. In DBIN, we propose a high-performance Box-supervised Instance-Aware (BIA) head with the core idea of redesigning three box-supervised mask loss terms. Furthermore, we add a Pseudo-Mask-supervised Semantic (PMS) head for enriching characteristics extracted from underlying feature maps. Besides, we align the pixel-level feature distributions between source and target domains by a Cross-Domain Adaptive Module (CDAM), so as to adapt the detector learned from one lab can work well on unlabeled data from another lab. The proposed method achieves state-of-the-art performance across four mainstream datasets. A series of analysis and experiments show that our proposed BIA and PMS head can accomplish mitosis pixel-wise localization under weak supervision, and we can boost the generalization ability of our model by CDAM.

Podocytes are unique, highly specialized, terminally differentiated cells, which are restricted in a post-mitotic state with limited ability to repair or regenerate. Re-entering the mitotic phase causes podocyte mitotic catastrophe, thereby leading to podocyte death and glomerular injury. Myeloid-derived growth factor (MYDGF) is a novel secreted protein and plays an important role in the regulation of cardiovascular function. However, whether MYDGF is expressed in kidney parenchymal cells and whether it has biological functions in the kidney remain unknown. Here, we found that MYDGF was expressed in kidney parenchymal cells and was significantly reduced in podocytes from mice with models of focal segmental glomerulosclerosis and diabetic kidney disease. Podocyte-specific deletion of Mydgf in mice exacerbated podocyte injury and proteinuria in both disease models. Functionally, MYDGF protected podocytes against mitotic catastrophe by reducing accumulation of podocytes in the S phase, a portion of the cell cycle in which DNA is replicated. Mechanistically, MYDGF regulates the expression of the transcription factor RUNX2 which mediates some MYDGF effects. Importantly, a significant reduction of MYDGF was found in glomeruli from patients with glomerular disease due to focal segmental glomerulosclerosis and diabetic kidney disease and the level of MYDGF was correlated with glomerular filtration rate, serum creatinine and podocyte loss. Thus, our studies indicate that MYDGF may be an attractive therapeutic target for glomerular disease.

Inflammatory, proliferative and remodeling phases constitute a cutaneous wound healing program. Therapeutic applications and medication are available; however, they commonly are comprised of fortified preservatives that might prolong the healing process. Chick early amniotic fluids (ceAF) contain native therapeutic factors with balanced chemokines, cytokines and growth-related factors; their origins in principle dictate no existence of harmful agents that would otherwise hamper embryo development. Instead, they possess a spectrum of molecules driving expeditious mitotic divisions and possibly exerting other functions. Employing both in vitro and in vivo models, we examined ceAF's therapeutic potentials in wound healing and found intriguing involvement of transient senescence, known to be intimately intermingled with Senescence Associated Secretory Phenotypes (SASP) that function in addition to or in conjunction with ceAF to facilitate wound healing. In our cutaneous wound healing models, a low dose of ceAF exhibited the best efficacies; however, higher doses attenuated the wound healing presumably by inducing p16 expression over a threshold. Our studies thus link an INK4/ARF locus-mediated signaling cascade to cutaneous wound healing, suggesting therapeutic potentials of ceAF exerting functions likely by driving transient senescence, expediting cellular proliferation, migration, and describing a homeostatic and balanced dosage strategy in medical intervention.

Virtually all cell types have the same DNA, yet each type exhibits its own cell-specific pattern of gene expression. During the brief period of mitosis, the chromosomes exhibit changes in protein composition and modifications, a marked condensation, and a consequent reduction in transcription. Yet as cells exit mitosis, they reactivate their cell-specific programs with high fidelity. Initially, the field focused on the subset of transcription factors that are selectively retained in, and hence bookmark, chromatin in mitosis. However, recent studies show that many transcription factors can be retained in mitotic chromatin and that, surprisingly, such retention can be due to nonspecific chromatin binding. Here, we review the latest studies focusing on low-level transcription via promoters, rather than enhancers, as contributing to mitotic memory, as well as new insights into chromosome structure dynamics, histone modifications, cell cycle signaling, and nuclear envelope proteins that together ensure the fidelity of gene expression through a round of mitosis.

The kinetochore is a large proteinaceous structure assembled on the centromeres of chromosomes. The complex machinery links chromosomes to the mitotic spindle and is essential for accurate chromosome segregation during cell division. The kinetochore is composed of two submodules: the inner and outer kinetochore. The inner kinetochore is assembled on centromeric chromatin and persists with centromeres throughout the cell cycle. The outer kinetochore attaches microtubules to the inner kinetochore, and assembles only during mitosis. The review focuses on recent advances in our understanding of the mechanisms governing the proper assembly of the outer kinetochore during mitosis and highlights open questions for future investigation.

Chromosome engineering has been attempted successfully in yeast but remains challenging in higher eukaryotes, including mammals. Here, we report programmed chromosome ligation in mice that resulted in the creation of new karyotypes in the lab. Using haploid embryonic stem cells and gene editing, we fused the two largest mouse chromosomes, chromosomes 1 and 2, and two medium-size chromosomes, chromosomes 4 and 5. Chromatin conformation and stem cell differentiation were minimally affected. However, karyotypes carrying fused chromosomes 1 and 2 resulted in arrested mitosis, polyploidization, and embryonic lethality, whereas a smaller fused chromosome composed of chromosomes 4 and 5 was able to be passed on to homozygous offspring. Our results suggest the feasibility of chromosome-level engineering in mammals.

SUMMARYThe anaphase-promoting complex/cyclosome (APC/C) coordinates advancement through mitosis via temporally controlled polyubiquitination of effector proteins. Despite the long-appreciated spatial organization of key events in mitosis mediated largely by cytoskeletal networks, the spatial regulation of APC/C, the major mitotic E3 ligase, is poorly understood. Here, we describe a microtubule-resident protein, PLEKHA5, as an interactor of APC/C and spatial regulator of its activity in mitosis. PLEKHA5 knockdown delayed mitotic progression, causing accumulation of APC/C substrates dependent upon the PLEKHA5–APC/C interaction. To assess the spatial importance of this interaction, we developed microtubule-localized proximity biotinylation tools, which revealed that depletion of PLEKHA5 decreased the extent of APC/C association with the microtubule cytoskeleton. This decreased APC/C microtubule-localization in turn prevented efficient loading of APC/C with its co-activator CDC20, leading to defects in E3 ligase catalytic activity. We propose that PLEKHA5 functions as an adaptor of APC/C that promotes its subcellular localization to microtubules and facilitates its activation by CDC20, thus ensuring the timely turnover of key mitotic APC/C substrates and proper progression through mitosis.

Aneuploidy is a pervasive feature of cancer cells that results from chromosome missegregation. Several transcription factors have been associated with aneuploidy; however, no studies to date have demonstrated that mammalian transcription factors directly regulate chromosome segregation during mitosis. Here, we demonstrate that the ubiquitously expressed transcription factor specificity protein 1 (Sp1), which we have previously linked to aneuploidy, has a mitosis-specific role regulating chromosome segregation. We find that Sp1 localizes to mitotic centromeres and auxin-induced rapid Sp1 degradation at mitotic onset results in chromosome segregation errors and aberrant mitotic progression. Furthermore, rapid Sp1 degradation results in anomalous mitotic chromosome assembly characterized by loss of condensin complex I localization to mitotic chromosomes and chromosome condensation defects. Consistent with these defects, Sp1 degradation results in reduced chromosome passenger complex activity and histone H3 serine 10 phosphorylation during mitosis, which is essential for condensin complex I recruitment and chromosome condensation. Together, these data provide the first evidence of a mammalian transcription factor acting specifically during mitosis to regulate chromosome segregation.

Chemoresistance of cancer cells is a major problem in treating cancer. Knowledge of how cancer cells may die or resist cancer drugs is critical to providing certain strategies to overcome tumour resistance to treatment. Paclitaxel is known as a chemotherapy drug that can suppress the proliferation of cancer cells by inducing cell cycle arrest and induction of mitotic catastrophe. However, today, it is well known that paclitaxel can induce multiple kinds of cell death in cancers. Besides the induction of mitotic catastrophe that occurs during mitosis, paclitaxel has been shown to induce the expression of several pro-apoptosis mediators. It also can modulate the activity of anti-apoptosis mediators. However, certain cell-killing mechanisms such as senescence and autophagy can increase resistance to paclitaxel. This review focuses on the mechanisms of cell death, including apoptosis, mitotic catastrophe, senescence, autophagic cell death, pyroptosis, etc., following paclitaxel treatment. In addition, mechanisms of resistance to cell death due to exposure to paclitaxel and the use of combinations to overcome drug resistance will be discussed.

Pre-mRNA splicing is an indispensable mechanism for eukaryotic gene expression. Splicing inhibition causes cell cycle arrest at the G(1) and G(2)/M phases, and this is thought to be one of the reasons for the potent antitumor activity of splicing inhibitors. However, the molecular mechanisms underlying the cell cycle arrest have many unknown aspects. In particular, the mechanism of G(2)/M-phase arrest caused by splicing inhibition is completely unknown. Here, we found that lower and higher concentrations of pladienolide B caused M-phase and G(2)-phase arrest, respectively. We analyzed protein levels of cell cycle regulators and found that a truncated form of the p27 cyclin-dependent kinase inhibitor, named p27*, accumulated in G(2)-arrested cells. Overexpression of p27* caused partial G(2)-phase arrest. Conversely, knockdown of p27* accelerated exit from G(2)/M phase after washout of splicing inhibitor. These results suggest that p27* contributes to G(2)/M-phase arrest caused by splicing inhibition. We also found that p27* bound to and inhibited M-phase cyclins, although it is well known that p27 regulates the G(1)/S transition. Intriguingly, p27*, but not full-length p27, was resistant to proteasomal degradation and remained in G(2)/M phase. These results suggest that p27*, which is a very stable truncated protein in G(2)/M phase, contributes to G(2)-phase arrest caused by splicing inhibition.

Funder: Regione Lazio; Grant(s): Progetti Gruppi di Ricerca 2020 ID: A0375-2020-36597, Funder: AstraZeneca, Funder: Associazione Italiana per la Ricerca sul Cancro; Grant(s): MFAG-2017 ID:20447, IG-2021 ID: 25648, The AurkA kinase is a well-known mitotic regulator, frequently overexpressed in tumors. The microtubule-binding protein TPX2 controls AurkA activity, localization, and stability in mitosis. Non-mitotic roles of AurkA are emerging, and increased nuclear localization in interphase has been correlated with AurkA oncogenic potential. Still, the mechanisms leading to AurkA nuclear accumulation are poorly explored. Here, we investigated these mechanisms under physiological or overexpression conditions. We observed that AurkA nuclear localization is influenced by the cell cycle phase and nuclear export, but not by its kinase activity. Importantly, AURKA overexpression is not sufficient to determine its accumulation in interphase nuclei, which is instead obtained when AURKA and TPX2 are co-overexpressed or, to a higher extent, when proteasome activity is impaired. Expression analyses show that AURKA, TPX2, and the import regulator CSE1L are co-overexpressed in tumors. Finally, using MCF10A mammospheres we show that TPX2 co-overexpression drives protumorigenic processes downstream of nuclear AurkA. We propose that AURKA/TPX2 co-overexpression in cancer represents a key determinant of AurkA nuclear oncogenic functions.

The spindle is a dynamic intracellular structure self-organized from microtubules and microtubule-associated proteins. The spindle’s bipolar morphology is essential for the faithful segregation of chromosomes during cell division, and it is robustly maintained by multifaceted mechanisms. However, abnormally shaped spindles, such as multipolar spindles, can stochastically arise in a cell population and cause chromosome segregation errors. The physical basis of how microtubules fail in bipolarization and occasionally favor nonbipolar assembly is poorly understood. Here, using live fluorescence imaging and quantitative shape analysis in Xenopus egg extracts, we find that spindles of varied shape morphologies emerge through nonrandom, bistable self-organization paths, one leading to a bipolar and the other leading to a multipolar phenotype. The bistability defines the spindle’s unique morphological growth dynamics linked to each shape phenotype and can be promoted by a locally distorted microtubule flow that arises within premature structures. We also find that bipolar and multipolar spindles are stable at the steady-state in bulk but can infrequently switch between the two phenotypes. Our microneedle-based physical manipulation further demonstrates that a transient force perturbation applied near the assembled pole can trigger the phenotypic switching, revealing the mechanical plasticity of the spindle. Together with molecular perturbation of kinesin-5 and augmin, our data propose the physical and molecular bases underlying the emergence of spindle-shape variation, which influences chromosome segregation fidelity during cell division.

A functional centrosome is vital for the development and physiology of animals. Among numerous regulatory mechanisms of the centrosome, ubiquitin-mediated proteolysis is known to be critical for the precise regulation of centriole duplication. However, its significance beyond centrosome copy number control remains unclear. Using an in vitro screen for centrosomal substrates of the APC/C ubiquitin ligase in Drosophila, we identify several conserved pericentriolar material (PCM) components, including the inner PCM protein Spd2. We show that Spd2 levels are controlled by the interphase-specific form of APC/C, APC/CFzr , in cultured cells and developing brains. Increased Spd2 levels compromise neural stem cell-specific asymmetric PCM recruitment and microtubule nucleation at interphase centrosomes, resulting in partial randomisation of the division axis and segregation patterns of the daughter centrosome in the following mitosis. We further provide evidence that APC/CFzr -dependent Spd2 degradation restricts the amount and mobility of Spd2 at the daughter centrosome, thereby facilitating the accumulation of Polo-dependent Spd2 phosphorylation for PCM recruitment. Our study underpins the critical role of cell cycle-dependent proteolytic regulation of the PCM in stem cells.

Cell divisions are accurately positioned to generate cells of the correct size and shape. In plant cells, the new cell wall is built in the middle of the cell by vesicles trafficked along an antiparallel microtubule and a microfilament array called the phragmoplast. The phragmoplast expands toward a specific location at the cell cortex called the division site, but how it accurately reaches the division site is unclear. We observed microtubule arrays that accumulate at the cell cortex during the telophase transition in maize (Zea mays) leaf epidermal cells. Before the phragmoplast reaches the cell cortex, these cortical-telophase microtubules transiently interact with the division site. Increased microtubule plus end capture and pausing occur when microtubules contact the division site-localized protein TANGLED1 or other closely associated proteins. Microtubule capture and pausing align the cortical microtubules perpendicular to the division site during telophase. Once the phragmoplast reaches the cell cortex, cortical-telophase microtubules are incorporated into the phragmoplast primarily by parallel bundling. The addition of microtubules into the phragmoplast promotes fine-tuning of the positioning at the division site. Our hypothesis is that division site-localized proteins such as TANGLED1 organize cortical microtubules during telophase to mediate phragmoplast positioning at the final division plane.

How do very large cells coordinate their entry into mitosis? A new study shows that the bistability of the Cdk/Cyclin system allows cells to generate either 'trigger waves' or 'sweep waves' that drive cells into mitosis in different ways with distinct consequences.

Although counting mitoses is part of breast cancer grading, concordance studies showed low agreement. Refining the criteria for mitotic counting can improve concordance, particularly when using whole slide images (WSIs). This study aims to refine the methodology for optimal mitoses counting on WSI. Digital images of 595 hematoxylin and eosin stained sections were evaluated. Several morphological criteria were investigated and applied to define mitotic hotspots. Reproducibility, representativeness, time, and association with outcome were the criteria used to evaluate the best area size for mitoses counting. Three approaches for scoring mitoses on WSIs (single and multiple annotated rectangles and multiple digital high-power (×40) screen fields (HPSFs)) were evaluated. The relative increase in tumor cell density was the most significant and easiest parameter for identifying hotspots. Counting mitoses in 3 mm

We have presented an in vitro trackable model system, atavistic induced from conservation in our genome, which strongly is applicable to tumorigenesis start and evolution. The inducing factor was death signals to proliferating normal human cells (primary cell strains), which respon-ded by a special type of tetraploidization, chromosomes with 4-chromatids (diplochromosomes, earlier described in cancer cells). The response included cell cycle stress, which prolonged S-period with result of mitotic slippage process, forming the special 4n cells by re-replication of diploid cells, which showed cell division capability to unexpected, genome reduced diploid cells which remarkably, showed fitness gain. This unique response through cell cycle stress and mitotic slippage process was further discovered to be linked to a rather special characteristic of the, 4n nucleus. The nucleus turned, self-inflicted, 90° perpendicular to the cell's cytoskeleton axis, importantly, before the special 4n-division system produced genome reduce diploid cells, we call "first cells", because of fitness gain. These 2n cells also showed the nuclear dependent 90° turn, which in both cases was associated with cells gaining cell shape changes, herein illustrated from normal fibroblastic cells changing to roundness cells, indistinguishable from todays' diagnostic cancer cell morphology. This 3-D ball-like cell shape, in metastasis, sque-ezing in and out between (?) endothelial cells in the lining of blood veins during disbursement, would be advantageous.

During mitosis, the allocation of genetic material concurs with organelle transformation and distribution. The coordination of genetic material inheritance with organelle dynamics directs accurate mitotic progression, cell fate determination, and organismal homeostasis. Small GTPases belonging to the Ras superfamily regulate various cell organelles during division. Being the key regulators of membrane dynamics, the dysregulation of small GTPases is widely associated with cell organelle disruption in neoplastic and non-neoplastic diseases, such as cancer and Alzheimer's disease. Recent discoveries shed light on the molecular properties of small GTPases as sophisticated modulators of a remarkably complex and perfect adaptors for rapid structure reformation. This review collects current knowledge on small GTPases in the regulation of cell organelles during mitosis and highlights the mediator role of small GTPase in transducing cell cycle signaling to organelle dynamics during mitosis.

Melanomas and their precursors, the melanocytes, are frequently exposed to UV due to their anatomic location, leading to DNA damage and reactive oxygen stress related harm. Such damage can result in multinucleation or polyploidy, in particularly in presence of mitotic or cell division failure. As a consequence, the cell encounters either of two fates: mitotic catastrophe, resulting in cell death, or survival and recovery, the latter occurring less frequently. However, when cells manage to recover in an polyploid state, they have often acquired new features, which allow them to tolerate and adapt to oncogene- or therapy induced stress. This review focuses on polyploidy inducers in melanoma and their effects on transcriptional reprogramming and phenotypic adaptation as well as the relevance of polyploid melanoma cells for therapy resistance.

Mitotic catastrophe (MC) is a suppressive mechanism that mediates the elimination of mitosis-deficient cells through apoptosis, necrosis or senescence after M phase block. SIRT1 is involved in the regulation of several cellular processes, including autophagy. However, the relationship between SIRT1 and MC has been largely obscure. Our study highlights that SIRT1 might be involved in the regulation of MC. We have shown that degradation of the SIRT1 protein via proteasome and lysosomal pathway was accompanied by MC induced via BMH-21. Overexpression of SIRT1 alleviated MC by decreasing the proportion of apoptotic and multinuclear cells induced by G2/M block and triggered autophagy whereas knockdown of SIRT1 aggravated MC and repressed autophagy. Furthermore, we found that serum starvation triggered autophagy evidently generated lower MC whereas siRNA of ATG5/7 suppressed autophagy leading to higher MC. ChIP analysis revealed that SIRT1 could bind to the promoter of BubR1, a component of spindle assembly checkpoint (SAC), to upregulate its expression. Overexpression of BubR1 decreased MC whereas knockdown of BubR1 increased it. These results reveal that SIRT1 regulates MC through autophagy and BubR1 signaling, and provide evidence for SIRT1, autophagy and BubR1 being the potential cancer therapeutic targets.

Introduction Mitosis-karyorrhexis index (MKI) is important for risk stratification workup of neuroblastic tumors. MKI is calculated by estimating the denominator (5000 tumor cells). We hypothesized that whole slide image (WSI) with appropriate digital image analytical software could provide an objective aid to pathologist’s MKI workup. Materials & Methods With IRB approval, sixteen cases of neuroblastic tumors as convenient cases were used. H&E slides were scanned at 40X using an Aperio Scanscope AT2 scanner and stored in SVS format. Digital photos were also taken and stored in TIFF format. Qupath, an open source image analytical software, was used to annotate, define region of interest (ROI) and automatically count the cells within ROI. Results With selected parameters, Qupath was able to provide cell count using both WSI (.svs) and digital images (.TIFF). Comparison of automated count and eyeball manual count generated precision above .96, recall above .96, F1 scores above .98, with false positive rate ranging from .6 to 3.7%, and false negative rate from .6 to 3.8%. Compared to original pathological report, automated tumor cell count led to lower MKI in 3 of 16 cases (18.8%) and change of “unfavorable histology” to “favorable” in one case (1/16, 6.3%). Conclusion Combination of WSI (or digital images) with Qupath is able to provide an automated, objective and consistent way for cell count to facilitate pathologist’s MKI determination in neuroblastic tumors’ workup and research.

SMC protein complexes are molecular machines that provide structure to chromosomes. These complexes bridge DNA elements and by doing so build DNA loops in cis and hold together the sister chromatids in trans. We discuss how drastic conformational changes allow SMC complexes to build such intricate DNA structures. The tight regulation of these complexes controls fundamental chromosomal processes such as transcription, recombination, repair, and mitosis.

XBP1 variant 1 (Xv1) is the most abundant XBP1 variant and is highly enriched across cancer types but nearly none in normal tissues. Its expression is associated with poor patients’ survival and is specifically required for survival of malignant cells, but the underlying mechanism is not known. Here we report that Xv1 upregulates the polyglutamylase tubulin tyrosine ligase-like 6 (TTLL6) and promotes mitosis of cancer cells. Like the canonical XBP1, Xv1 mRNA undergoes unconventional splicing by IRE1α under endoplasmic reticulum stress, but it is also constitutively spliced by IRE1β. The spliced Xv1 mRNA encodes the active form of Xv1 protein (Xv1s). RNA sequencing in HeLa cells revealed that Xv1s overexpression regulates expression of genes that are not involved in the canonical unfolded protein response, including TTLL6 as a highly upregulated gene. Gel shift assay and chromatin immunoprecipitation revealed that Xv1s bind to the TTLL6 promoter region. Knockdown of TTLL6 caused death of cancer cells but not benign and normal cells, similar to the effects of knocking down Xv1. Moreover, overexpression of TTLL6 partially rescued BT474 cells from apoptosis induced by either TTLL6 or Xv1 knockdown, supporting TTLL6 as an essential downstream effector of Xv1 in regulating cancer cell survival. TTLL6 is localized in the mitotic spindle of cancer cells. Xv1 or TTLL6 knockdown resulted in decreased spindle polyglutamylation and interpolar spindle, as well as congression failure, mitotic arrest and cell death. These findings suggest that Xv1 is essential for cancer cell mitosis, which is mediated, at least in part, by increasing TTLL6 expression.

Objective This study aims to compare the atypical epithelial changes of oral inflammatory lesions with the dysplastic epithelial changes of oral squamous cell carcinomas and to examine the underlying pathogenetic mechanisms that cause the changes by transmission electron microscopy (TEM). Methods Eight cases of parulides (oral inflammatory lesions) and 10 cases of oral squamous cell carcinomas were studied. Each specimen was cut into two pieces: one piece was fixed in a neutral buffered 10 % formaldehyde solution and prepared for light microscopy, and the other was fixed in a cold, 2.5 % glutaraldehyde solution and prepared for TEM. Results Based on the six histopathologic criteria in this study, both types of epithelia showed similar ultrastructural features, but some significant differences are observed as follows: In parulides, the epithelial cells with features similar to abnormal mitoses showed nuclear fragments with chromatin condensation (karyorrhexis), and no discernible organelles in the cytoplasm, indicating that these are necrotic cells. In contrast, in oral squamous cell carcinomas, the epithelial cells with abnormal mitotic figures showed clumping of chromosomes with abnormal mitotic spindles, indicating that these are abnormally dividing cells. Abnormal keratinization was also seen. The nuclei of the abnormally keratinized cells were irregular with coarse and clumped chromatin. Conclusions The atypical epithelial changes in oral inflammatory lesions are a result of necrotic changes in the epithelia due to underlying inflammatory processes and are not true epithelial dysplasia. In contrast, the dysplastic epithelial changes in oral squamous cell carcinomas are dysplastic changes due to premalignant neoplastic processes.

Stratification of the epidermis is essential for the barrier function of the skin. However, the molecular mechanisms governing epidermal stratification are not fully understood. Herein, we demonstrate that enkurin domain-containing protein 1 (ENKD1) contributes to epidermal stratification by modulating the cell-division orientation of basal keratinocytes. The epidermis of Enkd1 knockout mice is thinner than that of wild-type mice due to reduced generation of suprabasal cells from basal keratinocytes through asymmetric division. Depletion of ENKD1 impairs proper orientation of the mitotic spindle and delays mitotic progression in cultured cells. Mechanistic investigation further reveals that ENKD1 is a novel microtubule-binding protein that promotes the stability of astral microtubules. Introduction of the microtubule-binding domain of ENKD1 can largely rescue the spindle orientation defects in ENKD1-depleted cells. These findings establish ENKD1 as a critical regulator of astral microtubule stability and spindle orientation that stimulates epidermal stratification in mammalian cells.

The nuclear envelope (NE) separates genomic DNA from the cytoplasm in eukaryotes. The structure of the NE is dynamically altered not only in mitotic disassembly and reassembly but also during interphase. Recent studies have shown that the NE is frequently damaged by various cellular stresses that degenerate NE components and/or disrupt their functional interactions. These stresses are referred to as ‘NE stress’. Accumulating evidence has demonstrated that NE stress potentially causes severe cellular dysfunctions, such as cell death and genome instability. In this review, the concept of NE stress, the processes repairing damage of the NE caused by NE stress, and the molecular mechanisms by which NE stress contributes to disease pathogenesis are introduced.

The Chromosomal Passenger Complex (CPC) regulates a plethora of processes during multiple stages of nuclear and cytoplasmic division. Early during mitosis, the CPC is recruited to centromeres and kinetochores, and ensures that the duplicated chromosomes become properly connected to microtubules from opposite poles of the mitotic spindle. Progression into anaphase is accompanied by a striking relocation of the CPC from centromeres to the antiparallel microtubule overlaps of the anaphase spindle and to the equatorial cortex. This translocation requires direct interactions of the CPC with the kinesin-6 family member MKLP2/KIF20A, and the inactivation of cyclin B-cyclin-dependent kinase-1 (CDK1). Here, we review recent progress in the regulation of this relocation event. Furthermore, we discuss why the CPC must be relocated during early anaphase in light of recent advances in the functions of the CPC post metaphase.

Scaffold attachment factor A (SAF-A) or hnRNP U is a nuclear RNA-binding protein with a well-documented role in processing newly transcribed RNA. Recent studies also indicate that SAF-A can oligomerise in an ATP-dependent manner and interact with RNA to form a dynamic nuclear mesh. This mesh is thought to regulate nuclear and chromatin architecture, yet a mechanistic understanding is lacking. Here, we review developments in the field to understand how the SAF-A/RNA mesh affects chromatin organisation in interphase and mitosis. As SAF-A has an intrinsically disordered domain we discuss how the chromatin mesh is related to nuclear phase-separated condensates, which in other situations have been shown to regulate transcription and cell functions. Finally, we infer possible links between diseases emerging from SAF-A mutations and its role in chromatin organisation and regulation.

Mitotic rate is an important prognostic predictor in invasive breast carcinoma. Current guidelines recommend counting mitoses from 10 contiguous high power fields (HPFs) in the core biopsy. We propose a method to score mitotic activity in 1 HPF at the most mitotically active area of the tumour edge, or the interface between invasive tumour and benign breast tissue. We propose a score of 1, 2, or 3, corresponding to ≤1, 2, or ≥3 mitoses in 1 HPF, respectively. A total of 141 breast core biopsies with corresponding surgical excisions were blindly examined. We counted the number of mitotic figures in 1 HPF and in 10 contiguous HPFs in the core biopsy and compared with the mitotic count from 10 contiguous HPFs in the excision which is considered the gold standard. Concordance rates and interobserver agreement rates were calculated. The concordance rate was 82.3%, 78.7% and 82.3% between 1 HPF versus 10 HPFs in the core biopsy, 1 HPF in the core biopsy versus 10 HPFs in the excision and 10 HPFs in the core biopsy vs 10 HPFs in the excision, respectively. In the core biopsy, all three investigators agreed in 73.8% and 83.7% of the cases using the 1 HPF method and the 10 HPFs method, respectively; in the excision specimen, agreement was reached in 82.3% of the cases. The 1 HPF method showed similar concordance rate and interobserver agreement compared to the conventional method in the prediction of the mitotic score in the excision in all score groups. When stratified by mitotic score, the 1 HPF method predicted superior correlation with excision in the score 1 group than the 10 HPFs method, but not in the score 2 or 3 groups. From these findings we conclude that the proposed 1 HPF method can be used in clinical practice to grade invasive breast carcinomas in core biopsies, with the possibility of being utilised in small biopsies with less than 10 HPFs of invasive carcinoma.

Approximately half of purified mammalian RNA polymerase II (Pol II) is associated with a tightly interacting sub-stoichiometric subunit, Gdown1. Previous studies have established that Gdown1 inhibits transcription initiation through competitive interactions with general transcription factors and blocks the Pol II termination activity of transcription termination factor 2 (TTF2). However, the biological functions of Gdown1 remain poorly understood. Here, we utilized genetic, microscopic, and multi-omics approaches to functionally characterize Gdown1 in three human cell lines. Acute depletion of Gdown1 caused minimal direct effects on transcription. We show that Gdown1 resides predominantly in the cytoplasm of interphase cells, shuttles between the cytoplasm and nucleus, and is regulated by nuclear export. Gdown1 enters the nucleus at the onset of mitosis. Consistently, genetic ablation of Gdown1 is associated with partial de-repression of mitotic transcription, and Gdown1 KO cells present with evidence of aberrant mitoses coupled to p53 pathway activation. Evidence is presented demonstrating that Gdown1 modulates the combined functions of purified productive elongation factors PAF1C, RTF1, SPT6, DSIF and P-TEFb in vitro. Collectively, our findings support a model wherein the Pol II-regulatory function of Gdown1 occurs during mitosis and is required for genome integrity.

The kinetochore is essential for faithful chromosome segregation during mitosis and is assembled through dynamic processes involving numerous kinetochore proteins. Various experimental strategies have been used to understand kinetochore assembly processes. Fluorescence recovery after photobleaching (FRAP) analysis is also a useful strategy for revealing the dynamics of kinetochore assembly. In this study, we introduced fluorescence protein-tagged kinetochore protein cDNAs into each endogenous locus and performed FRAP analyses in chicken DT40 cells. Centromeric protein (CENP)-C was highly mobile in interphase, but immobile during mitosis. CENP-C mutants lacking the CENP-A-binding domain became mobile during mitosis. In contrast to CENP-C, CENP-T and CENP-H were immobile during both interphase and mitosis. The mobility of Dsn1, which is a component of the Mis12 complex and directly binds to CENP-C, depended on CENP-C mobility during mitosis. Thus, our FRAP assays provide dynamic aspects of how the kinetochore is assembled.

La escolarización debería alfabetizar científicamente a la ciudadanía para tomar decisiones con criterio sobre cuestiones socio-científicas, como el uso de pseudoterapias para tratar del cáncer. Por ello, se necesita conocer la forma de evaluar lo aprendido en todas las etapas educativas. Sin embargo, apenas hay estudios que consideren cómo se evalúa el aprendizaje de Biología adquirido durante el Bachillerato en las pruebas de acceso a la universidad (EvAU). De manera que, en este trabajo, se analiza la demanda cognitiva requerida para responder las 188 preguntas sobre un proceso biológico tan relevante como la división celular, que han sido formuladas en los exámenes EvAU de Biología de la Comunidad de Madrid desde 2002 hasta 2019; distinguiendo entre preguntas de producto, de relato y de proceso. Los resultados muestran un predominio de las preguntas de relato y de producto, con menor demanda cognitiva, viéndose una clara tendencia a aumentar las segundas hasta 2016. Además, algunos enunciados se repiten, centrándose principalmente en: mitosis, células animales, dinámica cromosómica o etapas como la anafase; obviando estructuras y momentos del proceso importantes, como el huso acromático o la prometafase. Se discuten posibles mejoras como la inclusión de preguntas de proceso que demanden tomar decisiones, Schooling should make citizens scientifically literate in order to make informed decisions on socio-scientific issues, such as theuse of pseudo-therapies to treat cancer. Therefore, it is necessary to know how learning is being assessed at all educational stages. However, there are hardly any studies that consider how the learning of Biology acquired during the Baccalaureate is evaluated inthe university entrance exams (EvAU). us, in this paper, we analyse the cognitive demand required to answer the 188 questionson a biological process as relevant as cell division, which have been asked in the EvAU Biology exams of the Community of Madridfrom 2002 to 2019; distinguishing among product, narrative and process questions. e results show a predominance of narrativeand product questions, with lower cognitive demand, with a clear trend towards an increase in product questions until 2016. Inaddition, some statements are repeated and questions tend to focus on: mitosis, animal cells, chromosome dynamics or stages suchas anaphase; ignoring other important structures and moments of the process such as the achromatic spindle or the prometaphase.Possible improvements are discussed, such as the inclusion of process questions that require decision making, Proyecto del Ministerio de Ciencia, Innovación y Universidades [PGC2018-096581-B-C22; T.E.M. y B.B.T], Proyecto del Ministerio de Economía, Industria y Competitividad [EDU2017-82688-P; J.M.P.M.], Cátedra UNESCO de Educación para la Justicia Social [T.E.M., B.B.T y J.M.P.M.]

Breast cancer is prone to relapse and metastasize to many vital organs, contributing to most of the breast cancer-related death and accentuating the importance of systematic identification of key factors regulating the metastasis of breast cancer. In this study, we performed a genome-wide CRISPR/Cas9 knock out screen in an orthotopic murine model of breast cancer for essential genes monitoring the progression and metastasis of breast cancer. We found one member of the zinc finger protein (ZNF) family, i.e., ZNF319, was among the top candidate genes. We further confirmed the lower expression of ZNF319 in the tumor tissue of breast cancer patients by analyzing tissue sections with IHC staining and TCGA database. Consistently, higher expression of ZNF319 correlates with better clinical outcome in almost all subtypes of breast cancer. Moreover, knocking down or overexpressing ZNF319 in breast cancer cells dramatically affects the breast cancer growth and metastasis capacity both in vitro and in vivo, suggesting ZNF319 functions as a strong suppressor of breast cancer progression. Lastly, the transcriptome analysis on ZNF319-silenced breast cancer cells shows that ZNF319 is involved in multiple crucial signaling pathways and biological processes, especially in cell cycle and proliferation. GO and KEGG analyses of our RNA-seq results reveal the up-regulation of E2F and G2/M related genes in ZNF319-silenced cells, suggesting that ZNF319 monitors the cell cycle during the breast cancer progression through the regulation of the E2F target genes and G2/M checkpoint. In summary, our study identifies ZNF319 as a novel metastasis suppressor gene arresting tumor cell cycle in breast cancer and thus presents a novel potential therapeutic target for breast cancer treatment.

Splicing precursor messenger RNA (pre-mRNA) is a critical step to produce physiologically functional protein. Splicing failure not only gives rise to dysfunctional proteins but also generates abnormal protein function, which causes several diseases. Several pre-mRNA splicing factors are reported to regulate mitosis directly at mitotic structures and/or indirectly through controlling the pre-mRNA splicing for mitotic proteins. In this study, we described the mitotic functions of SF3B14, a component of the spliceosomal U2 small nuclear ribonucleoprotein (snRNP), which we identified as a candidate involved in mitosis based on the large-scale RNA interference (RNAi) screen of the nucleolar proteome database. We observed that SF3B14 depletion caused prolonged mitosis and several mitotic defects, such as monopolar spindle and chromosome misalignment during metaphase. Although SF3B14 was found in the nucleolar proteome database, our immunofluorescent stainings demonstrated that SF3B14 was predominantly localized in the nucleoplasm and excluded from the nucleolus during interphase. In addition, SF3B14 did not colocalize with specific mitotic structures during mitosis, which is not in line with its direct mitotic function. Notably, we found that the SF3B14 depletion reduced protein levels of TUBGCP6, required for centrosome regulation, and increased the unspliced/spliced ratio of its mRNA. Taken together, we propose that the pre-mRNA of TUBGCP6 is one of the targets for SF3B14 splicing through which SF3B14 controls mitotic chromosome behavior.

Bi-oriented attachment of microtubules to the centromere is a pre-requisite for faithful chromosome segregation during mitosis. Budding yeast have point centromeres containing the cis-element proteins CDE-I, -II, and -III, which interact with trans-acting factors such as Cbf1, Cse4, and Ndc10. Our previous genetic screens, using a comprehensive library of histone point mutants, revealed that the TBS-I, -II, and -III regions of nucleosomes are required for faithful chromosome segregation. In TBS-III deficient cells, peri-centromeric nucleosomes containing the H2A.Z homolog Htz1 are lacking, however, it is unclear why chromosome segregation is defective in these cells. Here, we show that, in cells lacking TBS-III, both chromatin binding at the centromere and the total amount of some of the centromere proteins are reduced, and transcription through the centromere is up-regulated during M-phase. Moreover, the chromatin binding of Cse4, Mif2, Cbf1, Ndc10, and Scm3 was reduced upon ectopic transcription through the centromere in wild-type cells. These results suggest that transcription through the centromere displaces key centromere proteins and, consequently, destabilizes the interaction between centromeres and microtubules, leading to defective chromosome segregation. The identification of new roles for histone binding residues in TBS-III will shed new light on nucleosome function during chromosome segregation.

Purpose Tau/MAPT (microtubule associated protein tau) protein is actively studied for the pathologic consequences of its aberrant proteostasis in central nervous system leading to neurodegenerative diseases. Besides its ability to generate insoluble toxic oligomers, Tau homeostasis has attracted attention for its involvement in the formation of the mitotic spindle. This evidence, in association with the description of Tau expression in extra-neuronal tissues, and mainly in cancer tissues, constitutes the rationale for a more in-depth investigation of Tau role also in neoplastic diseases. Methods In our study, we investigated the expression of phosphorylated Tau in prostate cancer cell lines with particular focus on the residue Thr231 present in microtubule binding domain. Results The analysis of prostate cancer cells synchronized with nocodazole demonstrated that the expression of Tau protein phosphorylated at residue Thr231 is restricted to G2/M cell cycle phase. The phosphorylated form was unable to bind tubulin and it does not localize on mitotic spindle. As demonstrated by the use of specific inhibitors, the phosphorylation status of Tau is under the direct control of cdk5 and PP2A, while cdk1 activation was able to exert an indirect control. These mechanisms were also active in cells treated with docetaxel, where counteracting the expression of the dephosphorylated form, by kinase inhibition or protein silencing, determined resistance to drug toxicity. Conclusions We hypothesize that phosphorylation status of Tau is a key marker for G2/M phase in prostate cancer cells and that the forced modulation of Tau phosphorylation can interfere with the capacity of cell to efficiently progress through G2/M phase.

Scripts for NE assembly analysis in mitosis. The files contain the core scripts and template notebooks that were used for analyzing the image data presented in the accepted publication. Demo data for running the analysis notebook is uploaded. Other raw microscopy data are not uploaded owing to their large size and complexity (generated from various number of experiments performed in different conditions), but are available upon request (see associated publication for details). Other detailed instruction for how to use the scripts and analyze the data is available upon request(see associated publication for details). The scripts do not report original algorithms or methods. They are built on the collections of algorithms including scikit-image and scipy. For other details of the scripts (or future update), please refer to: https://github.com/shiwei23/AnalysisTool_Mitosis.

Funder: Boehringer Ingelheim Fonds, Funder: Bonn University, Funder: Max Planck Society, Funder: Carnegie Mellon University, The key to ensuring proper chromosome segregation during mitosis is the kinetochore (KT), a tightly regulated multiprotein complex that links the centromeric chromatin to the spindle microtubules and as such leads the segregation process. Understanding its architecture, function, and regulation is therefore essential. However, due to its complexity and dynamics, only its individual subcomplexes could be studied in structural detail so far. In this study, we construct a nanometer-precise in situ map of the human-like regional KT of Schizosaccharomyces pombe using multi-color single-molecule localization microscopy. We measure each protein of interest (POI) in conjunction with two references, cnp1CENP-A at the centromere and sad1 at the spindle pole. This allows us to determine cell cycle and mitotic plane, and to visualize individual centromere regions separately. We determine protein distances within the complex using Bayesian inference, establish the stoichiometry of each POI and, consequently, build an in situ KT model with unprecedented precision, providing new insights into the architecture.

The ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome) is essential for the control of mitosis, and its activity is subject to tight regulation. In early mitosis, APC/C is inhibited by the mitotic checkpoint system, but subsequently it regains activity and promotes metaphase-anaphase transition by targeting cyclin B and securin for degradation. The phosphorylation of APC/C by the mitotic protein kinase Cdk1-cyclin B facilitates its interaction with its coactivator Cdc20, while the phosphorylation of Cdc20 inhibits its binding to APC/C. This raises the question of how Cdc20 binds to APC/C under conditions of high Cdk1 activity. It seemed possible that the opposing action of protein phosphatases produces a fraction of unphosphorylated Cdc20 that binds to APC/C. We found, however, that while inhibitors of protein phosphatases PP2A and PP1 increased the overall phosphorylation of Cdc20 in anaphase extracts from Xenopus eggs, they did not decrease the levels of Cdc20 bound to APC/C. Searching for alternative mechanisms, we found that following the binding of Cdc20 to APC/C, it became significantly protected against phosphorylation by Cdk1. Protection was mainly at threonine sites at the N-terminal region of Cdc20, known to affect its interaction with APC/C. A model is proposed according to which a pool of unphosphorylated Cdc20, originating from initial stages of mitosis or from phosphatase action, combines with phosphorylated APC/C and thus becomes stabilized against further phosphorylation, possibly by steric hindrance of Cdk1 action. This pool of APC Cdc20 appears to be required for the regulation of APC/C activity at different stages of mitosis.