Interrelated effects of chromosome size, mechanics, number, location-orientation and polar ejection force on the spindle accuracy: a 3D computational study.

The search-and-capture model of spindle assembly has been a guiding principle for understanding prometaphase for decades. The computational model presented allows one to address two questions: how rapidly the microtubule-kinetochore connections are made, and how accurate these connections are. In most previous numerical simulations, the model geometry was drastically simplified. Using the CellDynaMo computational platform, we previously introduced a geometrically and mechanically realistic 3D model of the prometaphase mitotic spindle, and used it to evaluate thermal noise and microtubule kinetics effects on the capture of a single chromosome. Here, we systematically investigate how geometry and mechanics affect a spindle assembly's speed and accuracy, including nuanced distinctions between merotelic, mero-amphitelic, and mero-syntelic chromosomes. We find that softening of the centromere spring improves accuracy for short chromosome arms, but accuracy disappears for long chromosome arms. Initial proximity of chromosomes to one spindle pole makes assembly accuracy worse, while initial chromosome orientation matters less. Chromokinesins, added onto flexible chromosome arms, allow modeling of the polar ejection force, improving a spindle assembly's accuracy for a single chromosome. However, spindle space crowding by multiple chromosomes worsens assembly accuracy. Our simulations suggest that the complex microtubule network of the early spindle is key to rapid and accurate assembly.

Subjective health status: an easily available, independent, robust and significant predictive factor at the prometaphase of vaccination programs for the vaccination behavior of Chinese adults.

BACKGROUND: The World Health Organization (WHO) proposed COVID-19 vaccination as an emergent and important method to end the COVID-19 pandemic. Since China started vaccination programs in December 2020, vaccination has spread to provinces and municipalities nationwide. Previous research has focused on people's vaccination willingness and its influencing factors but has not examined vaccination behavior. We examine the effectiveness of psychosocial factors in predicting vaccination behavior. METHODS: A cross-sectional online survey was performed among Chinese adults on 8 May and 4 June 2021. The statistical analysis of the data included univariate analysis, receiver operator characteristics (ROC) analysis and ordinal multiclassification logistic regression model analysis. RESULTS: Of the 1300 respondents, 761 (58.5%) were vaccinated. Univariate analysis showed that a high education level and good subjective health status were protective factors for vaccination behavior, while suffering from chronic diseases was a risk factor. ROC analysis showed that subjective health status (AUC = 0.625, 95% CI: 0.594-0.656, P < 0.001) was the best predictor of vaccination behavior. Logistic regression analysis with subjective health status as a dependent variable indicated that older age, female sex, depression, neurasthenia, obsession, hypochondriasis and chronic disease were significant risk factors, while positive coping tendencies were a significant protective factor. CONCLUSION: Our study found a simple and effective marker, subjective health status, that can predict vaccination behavior. This finding can guide future epidemic prevention work.

Synchronizing Mammalian Cells for Mitotic Analysis of the Localization of Survivin.

This paper describes an easy method to enrich the harvest of adherent mammalian cells at each stage of mitosis (from prometaphase to cytokinesis) by combining Eg5 inhibition using dimethylenastron (DMA) with mitotic shake-off, followed by timed release from the drug.

A prometaphase mechanism of securin destruction is essential for meiotic progression in mouse oocytes.

Successful cell division relies on the timely removal of key cell cycle proteins such as securin. Securin inhibits separase, which cleaves the cohesin rings holding chromosomes together. Securin must be depleted before anaphase to ensure chromosome segregation occurs with anaphase. Here we find that in meiosis I, mouse oocytes contain an excess of securin over separase. We reveal a mechanism that promotes excess securin destruction in prometaphase I. Importantly, this mechanism relies on two phenylalanine residues within the separase-interacting segment (SIS) of securin that are only exposed when securin is not bound to separase. We suggest that these residues facilitate the removal of non-separase-bound securin ahead of metaphase, as inhibiting this period of destruction by mutating both residues causes the majority of oocytes to arrest in meiosis I. We further propose that cellular securin levels exceed the amount an oocyte is capable of removing in metaphase alone, such that the prometaphase destruction mechanism identified here is essential for correct meiotic progression in mouse oocytes.

Prometaphase.

The establishment of a metaphase plate in which all chromosomes are attached to mitotic spindle microtubules and aligned at the cell equator is required for faithful chromosome segregation in metazoans. The achievement of this configuration relies on the precise coordination between several concurrent mechanisms that start upon nuclear envelope breakdown, mediate chromosome capture at their kinetochores during mitotic spindle assembly and culminate with the congression of all chromosomes to the spindle equator. This period is called 'prometaphase'. Because the nature of chromosome capture by mitotic spindle microtubules is error prone, the cell is provided of error correction mechanisms that sense and correct most erroneous kinetochore-microtubule attachments before committing to separate sister chromatids in anaphase. In this review, aimed for newcomers in the field, more than providing an exhaustive mechanistic coverage of each and every concurrent mechanism taking place during prometaphase, we provide an integrative overview of these processes that ultimately promote the subsequent faithful segregation of chromosomes during mitosis.

Mps1 promotes poleward chromosome movements in meiotic prometaphase.

In prophase of meiosis I, homologous chromosomes pair and become connected by cross-overs. Chiasmata, the connections formed by cross-overs, enable the chromosome pair, called a bivalent, to attach as a single unit to the spindle. When the meiotic spindle forms in prometaphase, most bivalents are associated with one spindle pole and then go through a series of oscillations on the spindle, attaching to and detaching from microtubules until the partners of the bivalent become bioriented-attached to microtubules from opposite sides of the spindle. The conserved kinase, Mps1, is essential for the bivalents to be pulled by microtubules across the spindle in prometaphase. Here we show that MPS1 is needed for efficient triggering of the migration of microtubule-attached kinetochores toward the poles and promotes microtubule depolymerization. Our data support the model Mps1 acts at the kinetochore to coordinate the successful attachment of a microtubule and the triggering of microtubule depolymerization to then move the chromosome.

Microtubule-Targeted Self-Assembly Triggers Prometaphase-Metaphase Oscillations Suppressing Tumor Growth.

Microtubules are highly strategic targets of cancer therapies. Small molecule antimitotic agents are so far the best chemotherapeutic medication in cancer treatment. However, the high rate of neuropathy and drug resistance limit their clinical usage. Inspired by the multicomponent-targeting feature of molecular self-assembly (MSA) overcoming drug resistance, we synthesized peptide-based rotor molecules that self-assemble in response to the surrounding environment to target the microtubule array. The MSAs self-adjust morphologically in response to the pH change and viscosity variations during Golgi-endosome trafficking, escape trafficking cargos, and eventually bind to the microtubule array physically in a nonspecific manner. Such unrefined nano-bio interactions suppress regional tubulin polymerization triggering atypical prometaphase--metaphase oscillations to inhibit various cancer cells proliferating without inducing obvious neurotoxicity. The MSA also exerts potent antiproliferative effects in the subcutaneous cervix cancer xenograft tumor model equivalent to Cisplatin, better than the classic antimitotic drug Taxol.

Temperature-Induced uncoupling of cell cycle regulators.

The early stages of development involve complex sequences of morphological changes that are both reproducible from embryo to embryo and often robust to environmental variability. To investigate the relationship between reproducibility and robustness we examined cell cycle progression in early Drosophila embryos at different temperatures. Our experiments show that while the subdivision of cell cycle steps is conserved across a wide range of temperatures (5-35 â€‹°C), the relative duration of individual steps varies with temperature. We find that the transition into prometaphase is delayed at lower temperatures relative to other cell cycle events, arguing that it has a different mechanism of regulation. Using an in vivo biosensor, we quantified the ratio of activities of the major mitotic kinase, Cdk1 and one of the major mitotic phosphatases PP1. Comparing activation profile with cell cycle transition times at different temperatures indicates that in early fly embryos activation of Cdk1 drives entry into prometaphase but is not required for earlier cell cycle events. In fact, chromosome condensation can still occur when Cdk1 activity is inhibited pharmacologically. These results demonstrate that different kinases are rate-limiting for different steps of mitosis, arguing that robust inter-regulation may be needed for rapid and ordered mitosis.

Destabilization of Long Astral Microtubules via Cdk1-Dependent Removal of GTSE1 from Their Plus Ends Facilitates Prometaphase Spindle Orientation.

The precise regulation of microtubule dynamics over time and space in dividing cells is critical for several mitotic mechanisms that ultimately enable cell proliferation, tissue organization, and development. Astral microtubules, which extend from the centrosome toward the cell cortex, must be present for the mitotic spindle to properly orient, as well as for the faithful execution of anaphase and cytokinesis. However, little is understood about how the dynamic properties of astral microtubules are regulated spatiotemporally, or the contribution of astral microtubule dynamics to spindle positioning. The mitotic regulator Cdk1-CyclinB promotes destabilization of centrosomal microtubules and increased microtubule dynamics as cells enter mitosis, but how Cdk1 activity modulates astral microtubule stability, and whether it impacts spindle positioning, is unknown. Here, we uncover a mechanism revealing that Cdk1 destabilizes astral microtubules in prometaphase and thereby influences spindle reorientation. Phosphorylation of the EB1-dependent microtubule plus-end tracking protein GTSE1 by Cdk1 in early mitosis abolishes its interaction with EB1 and recruitment to microtubule plus ends. Loss of Cdk1 activity, or mutation of phosphorylation sites in GTSE1, induces recruitment of GTSE1 to growing microtubule plus ends in mitosis. This decreases the catastrophe frequency of astral microtubules and causes an increase in the number of long astral microtubules reaching the cell cortex, which restrains the ability of cells to reorient spindles along the long cellular axis in early mitosis. Astral microtubules thus must not only be present but also dynamic to allow the spindle to reorient, a state assisted by selective destabilization of long astral microtubules via Cdk1.

MDA-MB-157 Cell Line Presents High Levels of MAD2L2 and Dysregulated Mitosis.

BACKGROUND/AIM: Accurate regulation of the spindle assembly checkpoint (SAC) and anaphase promoting complex/cyclosome (APC/C) are essential for the correct execution of mitosis. In this work, we focused on MAD2L2 (REV7), a central translesion (TLS) protein, which also functions as a mitotic regulator by inhibiting APC/C in prometaphase. MATERIALS AND METHODS: Using bioinformatics analysis, live cell imaging and APC/C protein binding and degradation assays, we explored the influence of MAD2L2 over-expression in breast cancer. RESULTS: A significant over-expression of MAD2L2 was found in triple negative breast cancers (TNBC), compared to other breast cancers, correlating to poor patient prognosis. We also identified significant over-expression of MAD2L2 in the MDA-MB-157 triple negative (TN) cell line. A high percentage of MDA-MB-157 cells failed to complete mitosis and died during mitosis or shortly after. In addition, these cells completed mitosis at a significantly slower rate than control cells. MDA-MB-157 cells present high levels of mitotic slippage upon nocodazole treatment and acute dysregulation in APC/C function and substrate degradation. Moreover, silencing of MAD2L2 in the MDA-MB-157 cell line improved mitotic phenotypes. CONCLUSION: MAD2L2 over-expression supports the carcinogenic phenotype of MDA-MB-157 cells by promoting uncontrolled mitosis.

Synchronization of human retinal pigment epithelial-1 cells in mitosis.

Human retinal pigment epithelial-1 (RPE-1) cells are increasingly being used as a model to study mitosis because they represent a non-transformed alternative to cancer cell lines, such as HeLa cervical adenocarcinoma cells. However, the lack of an efficient method to synchronize RPE-1 cells in mitosis precludes their application for large-scale biochemical and proteomics assays. Here, we report a protocol to synchronize RPE-1 cells based on sequential treatments with the Cdk4 and Cdk6 inhibitor PD 0332991 (palbociclib) and the microtubule-depolymerizing drug nocodazole. With this method, the vast majority (80-90%) of RPE-1 cells arrested at prometaphase and exited mitosis synchronously after release from nocodazole. Moreover, the cells fully recovered and re-entered the cell cycle after the palbociclib-nocodazole block. Finally, we show that this protocol could be successfully employed for the characterization of the protein-protein interaction network of the kinetochore protein Ndc80 by immunoprecipitation coupled with mass spectrometry. This synchronization method significantly expands the versatility and applicability of RPE-1 cells to the study of cell division and might be applied to other cell lines that do not respond to treatments with DNA synthesis inhibitors.

Escape from the checkpoint: Nek2A binds a unique conformation of the APC/C-MCC complex.

Cell division depends on the timely degradation of numerous proteins by the anaphase-promoting complex/cyclosome (APC/C). The APC/C is a large E3 ubiquitin ligase that in complex with Cdc20 recognises degrons in its substrates. The ability of APC/C-Cdc20 to bind degrons is prevented by the binding of the mitotic checkpoint complex (MCC) which constitutes the "wait anaphase" signal. Curiously, the mitotic kinase Nek2A is insensitive to the presence of the MCC. How Nek2A avoids MCC inhibition has been unclear but now work from Alfieri and colleagues published in this issue of EMBO reports provides an explanation [1]. It shows that Nek2A is able to bind a specific open conformation of the APC/C-MCC complex that allows Nek2A ubiquitination. A dimer of Nek2A binds two distinct binding pockets on the APC/C through C-terminal MR motifs and thus independently of degrons. One of the MR binding pockets is only available for interaction in the open form of APC/C-MCC explaining Nek2A selectivity for this conformation. Whether other substrates bind the APC/C directly without using canonical degrons will be important to determine.

A unique binding mode of Nek2A to the APC/C allows its ubiquitination during prometaphase.

The anaphase-promoting complex (APC/C) is the key E3 ubiquitin ligase which directs mitotic progression and exit by catalysing the sequential ubiquitination of specific substrates. The activity of the APC/C in mitosis is restrained by the spindle assembly checkpoint (SAC), which coordinates chromosome segregation with the assembly of the mitotic spindle. The SAC effector is the mitotic checkpoint complex (MCC), which binds and inhibits the APC/C. It is incompletely understood how the APC/C switches substrate specificity in a cell cycle-specific manner. For instance, it is unclear how in prometaphase, when APC/C activity towards cyclin B and securin is repressed by the MCC, the kinase Nek2A is ubiquitinated. Here, we combine biochemical and structural analysis with functional studies in cells to show that Nek2A is a conformational-specific binder of the APC/C-MCC complex (APC/CMCC ) and that, in contrast to cyclin A, Nek2A can be ubiquitinated efficiently by the APC/C in conjunction with both the E2 enzymes UbcH10 and UbcH5. We propose that these special features of Nek2A allow its prometaphase-specific ubiquitination.

TRIM8 interacts with KIF11 and KIFC1 and controls bipolar spindle formation and chromosomal stability.

The faithful inheritance of chromosomes is essential for the propagation of organisms. In eukaryotes, central to this process is the mitotic spindle. Recently, we have identified TRIM8 as a gene aberrantly expressed in gliomas whose expression reduces the clonogenic potential in the patients' glioma cells. TRIM8 encodes an E3 ubiquitin ligase involved in various pathological processes, including hypertrophy, antiviral defense, encephalopathy, and cancer development. To gain insights into the TRIM8 functions, we characterized the TRIM8 interactome in primary mouse embryonic neural stem cells using proteomics. We found that TRIM8 interacts with KIFC1, and KIF11/Eg5, two master regulators of mitotic spindle assembly and cytoskeleton reorganization. By exploring the TRIM8 role in the mitotic spindle machinery, we showed that TRIM8 localizes at the mitotic spindle during mitosis and plays a role in centrosome separation at the beginning of mitosis with a subsequent delay of the mitotic progression and impact on chromosomal stability.

Pentagamavunon-1 (PGV-1) inhibits ROS metabolic enzymes and suppresses tumor cell growth by inducing M phase (prometaphase) arrest and cell senescence.

We previously showed that curcumin, a phytopolyphenol found in turmeric (Curcuma longa), targets a series of enzymes in the ROS metabolic pathway, induces irreversible growth arrest, and causes apoptosis. In this study, we tested Pentagamavunon-1 (PGV-1), a molecule related to curcumin, for its inhibitory activity on tumor cells in vitro and in vivo. PGV-1 exhibited 60 times lower GI50 compared to that of curcumin in K562 cells, and inhibited the proliferation of cell lines derived from leukemia, breast adenocarcinoma, cervical cancer, uterine cancer, and pancreatic cancer. The inhibition of growth by PGV-1 remained after its removal from the medium, which suggests that PGV-1 irreversibly prevents proliferation. PGV-1 specifically induced prometaphase arrest in the M phase of the cell cycle, and efficiently induced cell senescence and cell death by increasing intracellular ROS levels through inhibition of ROS-metabolic enzymes. In a xenograft mouse model, PGV-1 had marked anti-tumor activity with little side effects by oral administration, whereas curcumin rarely inhibited tumor formation by this administration. Therefore, PGV-1 is a potential therapeutic to induce tumor cell apoptosis with few side effects and low risk of relapse.

CTCF sites display cell cycle-dependent dynamics in factor binding and nucleosome positioning.

CCCTC-binding factor (CTCF) plays a key role in the formation of topologically associating domains (TADs) and loops in interphase. During mitosis TADs are absent, but how TAD formation is dynamically controlled during the cell cycle is not known. Several contradicting observations have been made regarding CTCF binding to mitotic chromatin using both genomics- and microscopy-based techniques. Here, we have used four different assays to address this debate. First, using 5C, we confirmed that TADs and CTCF loops are readily detected in interphase, but absent during prometaphase. Second, ATAC-seq analysis showed that CTCF sites display greatly reduced accessibility and lose the CTCF footprint in prometaphase, suggesting loss of CTCF binding and rearrangement of the nucleosomal array around the binding motif. In contrast, transcription start sites remain accessible in prometaphase, although adjacent nucleosomes can also become repositioned and occupy at least a subset of start sites during mitosis. Third, loss of site-specific CTCF binding was directly demonstrated using CUT&RUN. Histone modifications and histone variants are maintained in mitosis, suggesting a role in bookmarking of active CTCF sites. Finally, live-cell imaging, fluorescence recovery after photobleaching, and single molecule tracking showed that almost all CTCF chromatin binding is lost in prometaphase. Combined, our results demonstrate loss of CTCF binding to CTCF sites during prometaphase and rearrangement of the chromatin landscape around CTCF motifs. This, combined with loss of cohesin, would contribute to the observed loss of TADs and CTCF loops during mitosis and reveals that CTCF sites, key architectural cis-elements, display cell cycle stage-dependent dynamics in factor binding and nucleosome positioning.

Synthesis and biological evaluation of N-biphenyl-nicotinic based moiety compounds: A new class of antimitotic agents for the treatment of Hodgkin Lymphoma.

We previously demonstrated that some N-biphenylanilides caused cell-cycle arrest at G2/M transition in breast cancer cells. Among them we choose three derivatives, namely PTA34, PTA73 and RS35 for experimentation in solid tumor cell lines, classical Hodgkin Lymphoma (cHL) cell lines and bona fide normal cell lines. Almost all tumor cells were sensitive to compounds in the nanomolar range whereas, they were not cytotoxic to normal ones. Interestingly the compounds caused a strong G2/M phase arrest in cHL cell lines, thus, here we investigated whether they affected the integrity of microtubules in such cells. We found that they induced a long prometaphase arrest, followed by induction of apoptosis which involved mitochondria. PTA73 and RS35 induced the mitotic arrest through the fragmentation of microtubules which prevented the kinethocore-mitotic spindle interaction and the exit from mitosis. PTA34 is instead a tubulin-targeting agent because it inhibited the tubulin polymerization as vinblastine. As such, PTA34 maintained the Cyclin B1-CDK1 regulatory complex activated during the G2/M arrest while inducing the inactivation of Bcl-2 through phosphorylation in Ser70, the degradation of Mcl-1 and a strong activation of BIML and BIMS proapoptotic isoforms. In addition PTA34 exerted an antiangiogenic effect by suppressing microvascular formation.

CHROMOSOMAL INSTABILITY AS A CYTOGENETIC MARKER IN CHILDREN WITH VARYING DEGREES OF CONTROL OF ASTHMA.

The aim of investigation was to learn cytogenetic karyotype characteristics of children with asthma, with varying degrees of control in order to find markers of disease severity. The study involved 82 children aged 6 to 18 years, patients with asthma who were treated in Allergic Department of Regional Pediatric Hospital of Ivano-Frankivsk. Regarding the level of controlled asthma as a result of the application of test control asthma (GINA, 2011) children were distributed as follows: - 22 (30.6%) with controlled (CBA), 24 (33.3%) - is partly controlled (PCBA) 26 (36.1%) - with uncontrolled asthma (NCBA). The control group children were selected by random sampling, living in different parts of the Ivano-Frankivsk region (10 persons). Research of cytogenetic features of the karyotype in children with bronchial asthma was conducted by studying specimens of prometaphase chromosomes of peripheral blood lymphocytes. We analyzed associations of acrocentral number of chromosomes, karyotype features. The study involved 82 children aged 6 to 18 years with asthma of varying degrees of its control over the outcome of asthma control test. Children with uncontrolled asthma received significantly higher rate of chromosome acrocentral associations 13-15 (DD), 21-22 (GG) and 13-15 - 21-22 (DG). In patients with asthma we observed a lower mitotic activity compared with that in healthy ones (PN <0.05), and with a lower degree of controllability it decreased. Recognition of the genetic mechanisms of asthma leads to a new understanding of the genesis of the disease and can move forward in developing methods of treatment and prevention.

Activation of the apoptotic pathway during prolonged prometaphase blocks daughter cell proliferation.

When untransformed human cells spend >1.5 h in prometaphase under standard culture conditions, all daughters arrest in G1 despite normal division of their mothers. We investigate what happens during prolonged prometaphase that leads to daughter cell arrest in the absence of DNA damage. We find that progressive loss of anti-apoptotic MCL-1 activity and oxidative stress act in concert to partially activate the apoptosis pathway, resulting in the delayed death of some daughters and senescence for the rest. At physiological oxygen levels, longer prometaphase durations are needed for all daughters to arrest. Partial activation of apoptosis during prolonged prometaphase leads to persistent caspase activity, which activates the kinase cascade mediating the post-mitotic activation of p38. This in turn activates p53, and the consequent expression of p21stops the cell cycle. This mechanism can prevent cells suffering intractable mitotic defects, which modestly prolong mitosis but allow its completion without DNA damage, from producing future cell generations that are susceptible to the evolution of a transformed phenotype.

The microtubule-associated protein HURP recruits the centrosomal protein TACC3 to regulate K-fiber formation and support chromosome congression.

Kinetochore fibers (K-fibers) are microtubule bundles attached to chromosomes. Efficient K-fiber formation is required for chromosome congression, crucial for faithful chromosome segregation in cells. However, the mechanisms underlying K-fiber formation before chromosome biorientation remain unclear. Depletion of hepatoma up-regulated protein (HURP), a RanGTP-dependent microtubule-associated protein localized on K-fibers, has been shown to result in low-efficiency K-fiber formation. Therefore, here we sought to identify critical interaction partners of HURP that may modulate this function. Using co-immunoprecipitation and bimolecular fluorescence complementation assays, we determined that HURP interacts directly with the centrosomal protein transforming acidic coiled coil-containing protein 3 (TACC3), a centrosomal protein, both in vivo and in vitro through the HURP1-625 region. We found that HURP is important for TACC3 function during kinetochore microtubule assembly at the chromosome region in prometaphase. Moreover, HURP regulates stable lateral kinetochore attachment and chromosome congression in early mitosis by modulation of TACC3. These findings provide new insight into the coordinated regulation of K-fiber formation and chromosome congression in prometaphase by microtubule-associated proteins.