Activity-driven chromatin organization during interphase: Compaction, segregation, and entanglement suppression.

In mammalian cells, the cohesin protein complex is believed to translocate along chromatin during interphase to form dynamic loops through a process called active loop extrusion. Chromosome conformation capture and imaging experiments have suggested that chromatin adopts a compact structure with limited interpenetration between chromosomes and between chromosomal sections. We developed a theory demonstrating that active loop extrusion causes the apparent fractal dimension of chromatin to cross-over between two and four at contour lengths on the order of 30 kilo-base pairs. The anomalously high fractal dimension [Formula: see text] is due to the inability of extruded loops to fully relax during active extrusion. Compaction on longer contour length scales extends within topologically associated domains (TADs), facilitating gene regulation by distal elements. Extrusion-induced compaction segregates TADs such that overlaps between TADs are reduced to less than 35% and increases the entanglement strand of chromatin by up to a factor of 50 to several Mega-base pairs. Furthermore, active loop extrusion couples cohesin motion to chromatin conformations formed by previously extruding cohesins and causes the mean square displacement of chromatin loci during lag times ([Formula: see text]) longer than tens of minutes to be proportional to [Formula: see text]. We validate our results with hybrid molecular dynamics-Monte Carlo simulations and show that our theory is consistent with experimental data. This work provides a theoretical basis for the compact organization of interphase chromatin, explaining the physical reason for TAD segregation and suppression of chromatin entanglements which contribute to efficient gene regulation.

Centromere pairing enables correct segregation of meiotic chromosomes.

Proper chromosome segregation in meiosis I relies on the formation of connections between homologous chromosomes. Crossovers between homologs provide a connection that allows them to attach correctly to the meiosis I spindle. Tension is transmitted across the crossover when the partners attach to microtubules from opposing poles of the spindle. Tension stabilizes microtubule attachments that will pull the partners toward opposite poles at anaphase. Paradoxically, in many organisms, non-crossover partners segregate correctly. The mechanism by which non-crossover partners become bioriented on the meiotic spindle is unknown. Both crossover and non-crossover partners pair their centromeres early in meiosis (prophase). In budding yeast, centromere pairing is correlated with subsequent correct segregation of the partners. The mechanism by which centromere pairing, in prophase, promotes later correct attachment of the partners to the metaphase spindle is unknown. We used live cell imaging to track the biorientation process of non-crossover chromosomes. We find that centromere pairing allows the establishment of connections between the partners that allows their later interdependent attachment to the meiotic spindle using tension-sensing biorientation machinery. Because all chromosome pairs experience centromere pairing, our findings suggest that crossover chromosomes also utilize this mechanism to achieve maximal segregation fidelity.

The mechanosensitive ion channel PIEZO1 promotes satellite cell function in muscle regeneration.

Muscle satellite cells (MuSCs), myogenic stem cells in skeletal muscles, play an essential role in muscle regeneration. After skeletal muscle injury, quiescent MuSCs are activated to enter the cell cycle and proliferate, thereby initiating regeneration; however, the mechanisms that ensure successful MuSC division, including chromosome segregation, remain unclear. Here, we show that PIEZO1, a calcium ion (Ca2+)-permeable cation channel activated by membrane tension, mediates spontaneous Ca2+ influx to control the regenerative function of MuSCs. Our genetic engineering approach in mice revealed that PIEZO1 is functionally expressed in MuSCs and that Piezo1 deletion in these cells delays myofibre regeneration after injury. These results are, at least in part, due to a mitotic defect in MuSCs. Mechanistically, this phenotype is caused by impaired PIEZO1-Rho signalling during myogenesis. Thus, we provide the first concrete evidence that PIEZO1, a bona fide mechanosensitive ion channel, promotes proliferation and regenerative functions of MuSCs through precise control of cell division.

PP2A-Cdc55 phosphatase regulates actomyosin ring contraction and septum formation during cytokinesis.

Eukaryotic cells divide and separate all their components after chromosome segregation by a process called cytokinesis to complete cell division. Cytokinesis is highly regulated by the recruitment of the components to the division site and through post-translational modifications such as phosphorylations. The budding yeast mitotic kinases Cdc28-Clb2, Cdc5, and Dbf2-Mob1 phosphorylate several cytokinetic proteins contributing to the regulation of cytokinesis. The PP2A-Cdc55 phosphatase regulates mitosis counteracting Cdk1- and Cdc5-dependent phosphorylation. This prompted us to propose that PP2A-Cdc55 could also be counteracting the mitotic kinases during cytokinesis. Here we show that in the absence of Cdc55, AMR contraction and the primary septum formation occur asymmetrically to one side of the bud neck supporting a role for PP2A-Cdc55 in cytokinesis regulation. In addition, by in vivo and in vitro assays, we show that PP2A-Cdc55 dephosphorylates the chitin synthase II (Chs2 in budding yeast) a component of the Ingression Progression Complexes (IPCs) involved in cytokinesis. Interestingly, the non-phosphorylable version of Chs2 rescues the asymmetric AMR contraction and the defective septa formation observed in cdc55∆ mutant cells. Therefore, timely dephosphorylation of the Chs2 by PP2A-Cdc55 is crucial for proper actomyosin ring contraction. These findings reveal a new mechanism of cytokinesis regulation by the PP2A-Cdc55 phosphatase and extend our knowledge of the involvement of multiple phosphatases during cytokinesis.

Machine learning classification of trajectories from molecular dynamics simulations of chromosome segregation.

In contrast to the well characterized mitotic machinery in eukaryotes it seems as if there is no universal mechanism organizing chromosome segregation in all bacteria. Apparently, some bacteria even use combinations of different segregation mechanisms such as protein machines or rely on physical forces. The identification of the relevant mechanisms is a difficult task. Here, we introduce a new machine learning approach to this problem. It is based on the analysis of trajectories of individual loci in the course of chromosomal segregation obtained by fluorescence microscopy. While machine learning approaches have already been applied successfully to trajectory classification in other areas, so far it has not been possible to use them to discriminate segregation mechanisms in bacteria. A main obstacle for this is the large number of trajectories required to train machine learning algorithms that we overcome here by using trajectories obtained from molecular dynamics simulations. We used these trajectories to train four different machine learning algorithms, two linear models and two tree-based classifiers, to discriminate segregation mechanisms and possible combinations of them. The classification was performed once using the complete trajectories as high-dimensional input vectors as well as on a set of features which were used to transform the trajectories into low-dimensional input vectors for the classifiers. Finally, we tested our classifiers on shorter trajectories with duration times comparable (or even shorter) than typical experimental trajectories and on trajectories measured with varying temporal resolutions. Our results demonstrate that machine learning algorithms are indeed capable of discriminating different segregation mechanisms in bacteria and to even resolve combinations of the mechanisms on rather short time scales.

Microtubule organizing centers regulate spindle positioning in mouse oocytes.

During female meiosis I (MI), spindle positioning must be tightly regulated to ensure the fidelity of the first asymmetric division and faithful chromosome segregation. Although the role of F-actin in regulating these critical processes has been studied extensively, little is known about whether microtubules (MTs) participate in regulating these processes. Using mouse oocytes as a model system, we characterize a subset of MT organizing centers that do not contribute directly to spindle assembly, termed mcMTOCs. Using laser ablation, STED super-resolution microscopy, and chemical manipulation, we show that mcMTOCs are required to regulate spindle positioning and faithful chromosome segregation during MI. We discuss how forces exerted by F-actin on the spindle are balanced by mcMTOC-nucleated MTs to anchor the spindle centrally and to regulate its timely migration. Our findings provide a model for asymmetric cell division, complementing the current F-actin-based models, and implicate mcMTOCs as a major player in regulating spindle positioning.

CENP-V is required for proper chromosome segregation through interaction with spindle microtubules in mouse oocytes.

Proper chromosome segregation is essential to avoid aneuploidy, yet this process fails with increasing age in mammalian oocytes. Here we report a role for the scarcely described protein CENP-V in oocyte spindle formation and chromosome segregation. We show that depending on the oocyte maturation state, CENP-V localizes to centromeres, to microtubule organizing centers, and to spindle microtubules. We find that Cenp-V-/- oocytes feature severe deficiencies, including metaphase I arrest, strongly reduced polar body extrusion, increased numbers of mis-aligned chromosomes and aneuploidy, multipolar spindles, unfocused spindle poles and loss of kinetochore spindle fibres. We also show that CENP-V protein binds, diffuses along, and bundles microtubules in vitro. The spindle assembly checkpoint arrests about half of metaphase I Cenp-V-/- oocytes from young adults only. This finding suggests checkpoint weakening in ageing oocytes, which mature despite carrying mis-aligned chromosomes. Thus, CENP-V is a microtubule bundling protein crucial to faithful oocyte meiosis, and Cenp-V-/- oocytes reveal age-dependent weakening of the spindle assembly checkpoint.

Kinetochore life histories reveal an Aurora-B-dependent error correction mechanism in anaphase.

Chromosome mis-segregation during mitosis leads to aneuploidy, which is a hallmark of cancer and linked to cancer genome evolution. Errors can manifest as "lagging chromosomes" in anaphase, although their mechanistic origins and likelihood of correction are incompletely understood. Here, we combine lattice light-sheet microscopy, endogenous protein labeling, and computational analysis to define the life history of >104 kinetochores. By defining the "laziness" of kinetochores in anaphase, we reveal that chromosomes are at a considerable risk of mis-segregation. We show that the majority of lazy kinetochores are corrected rapidly in anaphase by Aurora B; if uncorrected, they result in a higher rate of micronuclei formation. Quantitative analyses of the kinetochore life histories reveal a dynamic signature of metaphase kinetochore oscillations that forecasts their anaphase fate. We propose that in diploid human cells chromosome segregation is fundamentally error prone, with an additional layer of anaphase error correction required for stable karyotype propagation.

Bub1 and CENP-U redundantly recruit Plk1 to stabilize kinetochore-microtubule attachments and ensure accurate chromosome segregation.

Bub1 is required for the kinetochore/centromere localization of two essential mitotic kinases Plk1 and Aurora B. Surprisingly, stable depletion of Bub1 by ∼95% in human cells marginally affects whole chromosome segregation fidelity. We show that CENP-U, which is recruited to kinetochores by the CENP-P and CENP-Q subunits of the CENP-O complex, is required to prevent chromosome mis-segregation in Bub1-depleted cells. Mechanistically, Bub1 and CENP-U redundantly recruit Plk1 to kinetochores to stabilize kinetochore-microtubule attachments, thereby ensuring accurate chromosome segregation. Furthermore, unlike its budding yeast homolog, the CENP-O complex does not regulate centromeric localization of Aurora B. Consistently, depletion of Bub1 or CENP-U sensitizes cells to the inhibition of Plk1 but not Aurora B kinase activity. Taken together, our findings provide mechanistic insight into the regulation of kinetochore function, which may have implications for targeted treatment of cancer cells with mutations perturbing kinetochore recruitment of Plk1 by Bub1 or the CENP-O complex.

Nonrandom segregation of sister chromosomes by Escherichia coli MukBEF.

Structural maintenance of chromosomes (SMC) complexes contribute to chromosome organization in all domains of life. In Escherichia coli, MukBEF, the functional SMC homolog, promotes spatiotemporal chromosome organization and faithful chromosome segregation. Here, we address the relative contributions of MukBEF and the replication terminus (ter) binding protein, MatP, to chromosome organization-segregation. We show that MukBEF, but not MatP, is required for the normal localization of the origin of replication to midcell and for the establishment of translational symmetry between newly replicated sister chromosomes. Overall, chromosome orientation is normally maintained through division from one generation to the next. Analysis of loci flanking the replication termination region (ter), which demark the ends of the linearly organized portion of the nucleoid, demonstrates that MatP is required for maintenance of chromosome orientation. We show that DNA-bound ß2-processivity clamps, which mark the lagging strands at DNA replication forks, localize to the cell center, independent of replisome location but dependent on MukBEF action, and consistent with translational symmetry of sister chromosomes. Finally, we directly show that the older ("immortal") template DNA strand, propagated from previous generations, is preferentially inherited by the cell forming at the old pole, dependent on MukBEF and MatP. The work further implicates MukBEF and MatP as central players in chromosome organization, segregation, and nonrandom inheritance of genetic material and suggests a general framework for understanding how chromosome conformation and dynamics shape subcellular organization.

Cdc7-mediated phosphorylation of Cse4 regulates high-fidelity chromosome segregation in budding yeast.

Faithful chromosome segregation maintains chromosomal stability as errors in this process contribute to chromosomal instability (CIN), which has been observed in many diseases including cancer. Epigenetic regulation of kinetochore proteins such as Cse4 (CENP-A in humans) plays a critical role in high-fidelity chromosome segregation. Here we show that Cse4 is a substrate of evolutionarily conserved Cdc7 kinase, and that Cdc7-mediated phosphorylation of Cse4 prevents CIN. We determined that Cdc7 phosphorylates Cse4 in vitro and interacts with Cse4 in vivo in a cell cycle-dependent manner. Cdc7 is required for kinetochore integrity as reduced levels of CEN-associated Cse4, a faster exchange of Cse4 at the metaphase kinetochores, and defects in chromosome segregation, are observed in a cdc7-7 strain. Phosphorylation of Cse4 by Cdc7 is important for cell survival as constitutive association of a kinase-dead variant of Cdc7 (cdc7-kd) with Cse4 at the kinetochore leads to growth defects. Moreover, phospho-deficient mutations of Cse4 for consensus Cdc7 target sites contribute to CIN phenotype. In summary, our results have defined a role for Cdc7-mediated phosphorylation of Cse4 in faithful chromosome segregation.

A role for condensin in mediating transcriptional adaptation to environmental stimuli.

Nuclear organisation shapes gene regulation; however, the principles by which three-dimensional genome architecture influences gene transcription are incompletely understood. Condensin is a key architectural chromatin constituent, best known for its role in mitotic chromosome condensation. Yet at least a subset of condensin is bound to DNA throughout the cell cycle. Studies in various organisms have reported roles for condensin in transcriptional regulation, but no unifying mechanism has emerged. Here, we use rapid conditional condensin depletion in the budding yeast Saccharomyces cerevisiae to study its role in transcriptional regulation. We observe a large number of small gene expression changes, enriched at genes located close to condensin-binding sites, consistent with a possible local effect of condensin on gene expression. Furthermore, nascent RNA sequencing reveals that transcriptional down-regulation in response to environmental stimuli, in particular to heat shock, is subdued without condensin. Our results underscore the multitude by which an architectural chromosome constituent can affect gene regulation and suggest that condensin facilitates transcriptional reprogramming as part of adaptation to environmental changes.

Parental genome unification is highly error-prone in mammalian embryos.

Most human embryos are aneuploid. Aneuploidy frequently arises during the early mitotic divisions of the embryo, but its origin remains elusive. Human zygotes that cluster their nucleoli at the pronuclear interface are thought to be more likely to develop into healthy euploid embryos. Here, we show that the parental genomes cluster with nucleoli in each pronucleus within human and bovine zygotes, and clustering is required for the reliable unification of the parental genomes after fertilization. During migration of intact pronuclei, the parental genomes polarize toward each other in a process driven by centrosomes, dynein, microtubules, and nuclear pore complexes. The maternal and paternal chromosomes eventually cluster at the pronuclear interface, in direct proximity to each other, yet separated. Parental genome clustering ensures the rapid unification of the parental genomes on nuclear envelope breakdown. However, clustering often fails, leading to chromosome segregation errors and micronuclei, incompatible with healthy embryo development.

Permitted and restricted steps of human kinetochore assembly in mitotic cell extracts.

Mitotic kinetochores assemble via the hierarchical recruitment of numerous cytosolic components to the centromere region of each chromosome. However, how these orderly and localized interactions are achieved without spurious macromolecular assemblies forming from soluble kinetochore components in the cell cytosol remains poorly understood. We developed assembly assays to monitor the recruitment of green fluorescent protein-tagged recombinant proteins and native proteins from human cell extracts to inner kinetochore components immobilized on microbeads. In contrast to prior work in yeast and Xenopus egg extracts, we find that human mitotic cell extracts fail to support de novo assembly of microtubule-binding subcomplexes. A subset of interactions, such as those between CENP-A-containing nucleosomes and CENP-C, are permissive under these conditions. However, the subsequent phospho-dependent binding of the Mis12 complex is less efficient, whereas recruitment of the Ndc80 complex is blocked, leading to weak microtubule-binding activity of assembled particles. Using molecular variants of the Ndc80 complex, we show that auto-inhibition of native Ndc80 complex restricts its ability to bind to the CENP-T/W complex, whereas inhibition of the Ndc80 microtubule binding is driven by a different mechanism. Together, our work reveals regulatory mechanisms that guard against the spurious formation of cytosolic microtubule-binding kinetochore particles.

Inhibition of CDK4/6 kinases causes production of aneuploid oocytes by inactivating the spindle assembly checkpoint and accelerating first meiotic progression.

Cyclin D-CDK4/6 complex mediates the transition from the G1 to S phase in mammalian somatic cells. Meiotic oocytes pass through the G2/M transition and complete the first meiosis to reach maturation at the metaphase of meiosis II without intervening S phase, while Cyclin D-CDK4/6 complex is found to express during meiotic progression. Whether Cyclin D-CDK4/6 complex regulates meiotic cell cycle progression is not known. Here, we found its different role in oocyte meiosis: Cyclin D-CDK4/6 complex served as a regulator of spindle assembly checkpoint (SAC) to prevent aneuploidy in meiosis I. Inhibition of CDK4/6 kinases disrupted spindle assembly, chromosome alignment and kinetochore-microtubule attachments, but unexpectedly accelerated meiotic progression by inactivating SAC, consequently resulting in production of aneuploid oocytes. Further studies showed that the MPF activity decrease before first polar body extrusion was accelerated probably by inactivation of the SAC to promote ubiquitin-mediated cyclin B1 degradation. Taken together, these data reveal a novel role of Cyclin D-CDK4/6 complex in mediating control of the SAC in female meiosis I.

SET levels contribute to cohesion fatigue.

Chromosome instability (CIN) is a major hallmark of cancer cells and believed to drive tumor progression. Several cellular defects including weak centromeric cohesion are proposed to promote CIN, but the molecular mechanisms underlying these defects are poorly understood. In a screening for SET protein levels in various cancer cell lines, we found that most of the cancer cells exhibit higher SET protein levels than nontransformed cells, including RPE-1. Cancer cells with elevated SET often show weak centromeric cohesion, revealed by MG132-induced cohesion fatigue. Partial SET knockdown largely strengthens centromeric cohesion in cancer cells without increasing overall phosphatase 2A (PP2A) activity. Pharmacologically increased PP2A activity in these cancer cells barely ameliorates centromeric cohesion. These results suggest that compromised PP2A activity, a common phenomenon in cancer cells, may not be responsible for weak centromeric cohesion. Furthermore, centromeric cohesion in cancer cells can be strengthened by ectopic Sgo1 overexpression and weakened by SET WT, not by Sgo1-binding-deficient mutants. Altogether, these findings demonstrate that SET overexpression contributes to impaired centromeric cohesion in cancer cells and illustrate misregulated SET-Sgo1 pathway as an underlying mechanism.

Anaphase B: Long-standing models meet new concepts.

Mitotic cell divisions ensure stable transmission of genetic information from a mother to daughter cells in a series of generations. To ensure this crucial task is accomplished, the cell forms a bipolar structure called the mitotic spindle that divides sister chromatids to the opposite sides of the dividing mother cell. After successful establishment of stable attachments of microtubules to chromosomes and inspection of connections between them, at the heart of mitosis, the cell starts the process of segregation. This spectacular moment in the life of a cell is termed anaphase, and it involves two distinct processes: depolymerization of microtubules bound to chromosomes, which is also known as anaphase A, and elongation of the spindle or anaphase B. Both processes ensure physical separation of disjointed sister chromatids. In this chapter, we review the mechanisms of anaphase B spindle elongation primarily in mammalian systems, combining different pioneering ideas and concepts with more recent findings that shed new light on the force generation and regulation of biochemical modules operating during spindle elongation. Finally, we present a comprehensive model of spindle elongation that includes structural, biophysical, and molecular aspects of anaphase B.

Functional Coupling between DNA Replication and Sister Chromatid Cohesion Establishment.

Several lines of evidence suggest the existence in the eukaryotic cells of a tight, yet largely unexplored, connection between DNA replication and sister chromatid cohesion. Tethering of newly duplicated chromatids is mediated by cohesin, an evolutionarily conserved hetero-tetrameric protein complex that has a ring-like structure and is believed to encircle DNA. Cohesin is loaded onto chromatin in telophase/G1 and converted into a cohesive state during the subsequent S phase, a process known as cohesion establishment. Many studies have revealed that down-regulation of a number of DNA replication factors gives rise to chromosomal cohesion defects, suggesting that they play critical roles in cohesion establishment. Conversely, loss of cohesin subunits (and/or regulators) has been found to alter DNA replication fork dynamics. A critical step of the cohesion establishment process consists in cohesin acetylation, a modification accomplished by dedicated acetyltransferases that operate at the replication forks. Defects in cohesion establishment give rise to chromosome mis-segregation and aneuploidy, phenotypes frequently observed in pre-cancerous and cancerous cells. Herein, we will review our present knowledge of the molecular mechanisms underlying the functional link between DNA replication and cohesion establishment, a phenomenon that is unique to the eukaryotic organisms.

Back to the new beginning: Mitotic exit in space and time.

The ultimate goal of cell division is to generate two identical daughter cells that resemble the mother cell from which they derived. Once all the proper attachments to the spindle have occurred, the chromosomes have aligned at the metaphase plate and the spindle assembly checkpoint (a surveillance mechanism that halts cells form progressing in the cell cycle in case of spindle - microtubule attachment errors) has been satisfied, mitotic exit will occur. Mitotic exit has the purpose of completing the separation of the genomic material but also to rebuild the cellular structures necessary for the new cell cycle. This stage of mitosis received little attention until a decade ago, therefore our knowledge is much patchier than the molecular details we now have for the early stages of mitosis. However, it is emerging that mitotic exit is not just the simple reverse of mitotic entry and it is highly regulated in space and time. In this review I will discuss the main advances in the field that provided us with a better understanding on the key role of protein phosphorylation/de-phosphorylation in this transition together with the concept of their spatial regulation. As this field is much younger, I will highlight general consensus, contrasting views together with the outstanding questions awaiting for answers.