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Molecular & Cellular Oncology 2021At the metaphase-to-anaphase transition, phosphatase activity feeds back to reverse early mitotic phosphorylation events. Our recent work indicates that the pseudokinase...
At the metaphase-to-anaphase transition, phosphatase activity feeds back to reverse early mitotic phosphorylation events. Our recent work indicates that the pseudokinase domain of the spindle checkpoint protein BUB1 (Budding Uninhibited by Benzimidazoles 1) mitotic checkpoint serine/threonine kinase B (BUB1B, BUBR1) maintains kinase-phosphatase balance at the outer kinetochore during mitotic exit.
PubMed: 33860079
DOI: 10.1080/23723556.2021.1876511 -
Methods in Molecular Biology (Clifton,... 2021A balanced progression through mitosis and cell division is largely dependent on orderly phosphorylation and ubiquitin-mediated proteolysis of regulatory and structural...
A balanced progression through mitosis and cell division is largely dependent on orderly phosphorylation and ubiquitin-mediated proteolysis of regulatory and structural proteins. These series of events ultimately secure genome stability and time-invariant cellular properties during cell proliferation. Two of the core enzymes regulating mitotic milestones in all eukaryotes are cyclin dependent kinase 1 (CDK1) with its coactivator cyclin B, and the E3 ubiquitin ligase anaphase promoting complex/cyclosome (APC/C). Discovering mechanisms and substrates for these enzymes is vital to understanding how cells move through mitosis and segregate chromosomes with high fidelity. However, the study of these enzymes has significant challenges. Purely in vitro studies discount the contributions of yet to be described regulators and misses the physiological context of cellular environment. In vivo studies are complicated by the fact that each of these enzymes, as well as many of their regulators and downstream targets, are essential. Moreover, long-term in vivo manipulations can result in cascading, indirect effects that can distort data analysis and interpretation. Many of these challenges can be circumvented using cell-free systems, which have historically played a critical role in identifying these enzymes and their contributions under quasicellular environments. Here, we describe the preparation of a newly developed human cell-free system that recapitulates an anaphase-like state of human cells. This new toolkit complements traditional cell-free systems from human cells and frog eggs and can be easily implemented in cell biology labs for direct and quantitative studies of mitotic signaling regulated by phosphorylation, APC/C-mediated proteolysis, and beyond.
Topics: Anaphase; Anaphase-Promoting Complex-Cyclosome; CDC2 Protein Kinase; Cell-Free System; Cyclin B1; HEK293 Cells; Humans; Mitosis; Mutation; Phosphorylation; Proteolysis; Ubiquitination
PubMed: 34085221
DOI: 10.1007/978-1-0716-1538-6_11 -
Current Biology : CB Mar 2023Error-free chromosome segregation in mitosis and meiosis relies on the assembly of a microtubule-based spindle that interacts with kinetochores to guide chromosomes to...
Error-free chromosome segregation in mitosis and meiosis relies on the assembly of a microtubule-based spindle that interacts with kinetochores to guide chromosomes to the cell equator before segregation in anaphase. Microtubules sprout from nucleation sites such as centrosomes, but kinetochores can also promote microtubule formation. It is unclear, however, how kinetochore-derived microtubules are generated and what their role is in chromosome segregation. Here, we show that the transient outer-kinetochore meshwork known as the fibrous corona serves as an autonomous microtubule nucleation platform. The fibrous corona is essential for the nucleation of kinetochore-derived microtubules, and when dissociated from the core kinetochore, it retains microtubule nucleation capacity. Nucleation relies on a fibrous-corona-bound pool of the LIC1 subunit of the dynein motor complex, which interacts with the γ-tubulin-tethering protein pericentrin (PCNT). PCNT is essential for microtubule nucleation from fibrous coronas, and in centrosome-depleted cells, where nearly all mitotic nucleation occurs at fibrous coronas, chromosome congression is fully dependent on PCNT. We further show that chromosomes in bovine oocytes, which naturally lack centrosomes, have highly expanded fibrous coronas that drive chromosome-derived microtubule nucleation. Preventing fibrous corona expansion in these cells impairs chromosome congression and causes spindle assembly defects. Our results show that fibrous coronas are autonomous microtubule-organizing centers that are important for spindle assembly, which may be especially relevant in acentrosomal cells such as oocytes.
Topics: Animals; Cattle; Chromosome Segregation; Microtubules; Kinetochores; Tubulin; Mitosis; Spindle Apparatus; Microtubule-Associated Proteins
PubMed: 36720222
DOI: 10.1016/j.cub.2023.01.010 -
Frontiers in Cell and Developmental... 2023Accurate chromosome segregation is vital for cell and organismal viability. The mitotic spindle, a bipolar macromolecular machine composed largely of dynamic... (Review)
Review
Accurate chromosome segregation is vital for cell and organismal viability. The mitotic spindle, a bipolar macromolecular machine composed largely of dynamic microtubules, is responsible for chromosome segregation during each cell replication cycle. Prior to anaphase, a bipolar metaphase spindle must be formed in which each pair of chromatids is attached to microtubules from opposite spindle poles. In this bipolar configuration pulling forces from the dynamic microtubules can generate tension across the sister kinetochores. The tension status acts as a signal that can destabilize aberrant kinetochore-microtubule attachments and reinforces correct, bipolar connections. Historically it has been challenging to isolate the specific role of tension in mitotic processes due to the interdependency of attachment and tension status at kinetochores. Recent technical and experimental advances have revealed new insights into how tension functions during mitosis. Here we summarize the evidence that tension serves as a biophysical signal that unifies multiple aspects of kinetochore and centromere function to ensure accurate chromosome segregation.
PubMed: 36755973
DOI: 10.3389/fcell.2023.1096333 -
PLoS Biology Jul 2022Precise spatiotemporal control of microtubule nucleation and organization is critical for faithful segregation of cytoplasmic and genetic material during cell division...
Precise spatiotemporal control of microtubule nucleation and organization is critical for faithful segregation of cytoplasmic and genetic material during cell division and signaling via the primary cilium in quiescent cells. Microtubule-associated proteins (MAPs) govern assembly, maintenance, and remodeling of diverse microtubule arrays. While a set of conserved MAPs are only active during cell division, an emerging group of MAPs acts as dual regulators in dividing and nondividing cells. Here, we elucidated the nonciliary functions and molecular mechanism of action of the ciliopathy-linked protein CCDC66, which we previously characterized as a regulator of ciliogenesis in quiescent cells. We showed that CCDC66 dynamically localizes to the centrosomes, the bipolar spindle, the spindle midzone, the central spindle, and the midbody in dividing cells and interacts with the core machinery of centrosome maturation and MAPs involved in cell division. Loss-of-function experiments revealed its functions during mitotic progression and cytokinesis. Specifically, CCDC66 depletion resulted in defective spindle assembly and orientation, kinetochore fiber stability, chromosome alignment in metaphase as well as central spindle and midbody assembly and organization in anaphase and cytokinesis. Notably, CCDC66 regulates mitotic microtubule nucleation via noncentrosomal and centrosomal pathways via recruitment of gamma-tubulin to the centrosomes and the spindle. Additionally, CCDC66 bundles microtubules in vitro and in cells by its C-terminal microtubule-binding domain. Phenotypic rescue experiments showed that the microtubule and centrosome-associated pools of CCDC66 individually or cooperatively mediate its mitotic and cytokinetic functions. Collectively, our findings identify CCDC66 as a multifaceted regulator of the nucleation and organization of the diverse mitotic and cytokinetic microtubule arrays and provide new insight into nonciliary defects that underlie ciliopathies.
Topics: Anaphase; Centrosome; Ciliopathies; Cytokinesis; Eye Proteins; Humans; Microtubule-Associated Proteins; Microtubules; Mitosis; Spindle Apparatus
PubMed: 35849559
DOI: 10.1371/journal.pbio.3001708 -
Cell Reports Jan 2021During mitotic chromosome segregation, the protease separase severs cohesin between sister chromatids. A probe for separase activity has shown that separase undergoes...
During mitotic chromosome segregation, the protease separase severs cohesin between sister chromatids. A probe for separase activity has shown that separase undergoes abrupt activation shortly before anaphase onset, after being suppressed throughout metaphase; however, the relevance of this control remains unclear. Here, we report that separase activates precociously, with respect to anaphase onset, during prolonged metaphase in multiple types of cancer cell lines. The artificial extension of metaphase in chromosomally stable diploid cells leads to precocious activation and, subsequently, to chromosomal bridges in anaphase, which seems to be attributable to incomplete cohesin removal. Conversely, shortening back of a prolonged metaphase restores the activation of separase and ameliorates anaphase bridge formation. These observations suggest that retarded metaphase progression affects the separase activation profile and its enzymatic proficiency. Our findings provide an unanticipated etiology for chromosomal instability in cancers and underscore the relevance of swift mitotic transitions for fail-safe chromosome segregation.
Topics: Animals; Chromosome Segregation; Humans; Mice; Mitosis; Rabbits; Separase
PubMed: 33472072
DOI: 10.1016/j.celrep.2020.108652 -
Open Biology Aug 2019The oncogenic transcription factor MYC modulates vast arrays of genes, thereby influencing numerous biological pathways including biogenesis, metabolism, proliferation,...
The oncogenic transcription factor MYC modulates vast arrays of genes, thereby influencing numerous biological pathways including biogenesis, metabolism, proliferation, apoptosis and pluripotency. When deregulated, MYC drives genomic instability via several mechanisms including aberrant proliferation, replication stress and ROS production. Deregulated MYC also promotes chromosome instability, but less is known about how MYC influences mitosis. Here, we show that deregulating MYC modulates multiple aspects of mitotic chromosome segregation. Cells overexpressing MYC have altered spindle morphology, take longer to align their chromosomes at metaphase and enter anaphase sooner. When challenged with a variety of anti-mitotic drugs, cells overexpressing MYC display more anomalies, the net effect of which is increased micronuclei, a hallmark of chromosome instability. Proteomic analysis showed that MYC modulates multiple networks predicted to influence mitosis, with the mitotic kinase PLK1 identified as a central hub. In turn, we show that MYC modulates several PLK1-dependent processes, namely mitotic entry, spindle assembly and SAC satisfaction. These observations thus underpin the pervasive nature of oncogenic MYC and provide a mechanistic rationale for MYC's ability to drive chromosome instability.
Topics: CRISPR-Cas Systems; Cell Line, Tumor; Cell Transformation, Neoplastic; Chromosomal Instability; Chromosome Segregation; Gene Amplification; Gene Expression Regulation; Genomic Instability; Humans; Mitosis; Mutagenesis; Proto-Oncogene Proteins c-myc
PubMed: 31455158
DOI: 10.1098/rsob.190136 -
Cell Cycle (Georgetown, Tex.) Oct 2021To ensure genome stability, chromosomes need to undergo proper condensation into two linked sister chromatids from prophase to prometaphase, followed by equal...
To ensure genome stability, chromosomes need to undergo proper condensation into two linked sister chromatids from prophase to prometaphase, followed by equal segregation at anaphase. Emerging evidence has shown that persistent DNA entanglements connecting the sister chromatids lead to the formation of ultrafine anaphase bridges (UFBs). If UFBs are not resolved soon after anaphase, they can induce chromosome missegregation. PICH (PLK1-interacting checkpoint helicase) is a DNA translocase that localizes on chromosome arms, centromeres and UFBs. It plays multiple essential roles in mitotic chromosome organization and segregation. PICH also recruits other associated proteins to UFBs, and together they mediate UFB resolution. Here, the proposed mechanism behind PICH's functions in chromosome organization and UFB resolution will be discussed. We summarize the regulation of PICH action at chromosome arms and centromeres, how PICH recognizes UFBs and recruits other UFB-associated factors, and finally how PICH promotes UFB resolution together with other DNA processing enzymes.
Topics: Anaphase; Centromere; Chromatids; Chromosome Segregation; Genomic Instability; Humans; Mitosis
PubMed: 34530686
DOI: 10.1080/15384101.2021.1970877 -
Frontiers in Plant Science 2021Most eukaryotic species propagate through sexual reproduction that requires male and female gametes. In flowering plants, it starts through a single round of DNA... (Review)
Review
Most eukaryotic species propagate through sexual reproduction that requires male and female gametes. In flowering plants, it starts through a single round of DNA replication (S phase) and two consecutive chromosome segregation (meiosis I and II). Subsequently, haploid mitotic divisions occur, which results in a male gametophyte (pollen grain) and a female gametophyte (embryo sac) formation. In order to obtain viable gametophytes, accurate chromosome segregation is crucial to ensure ploidy stability. A precise gametogenesis progression is tightly regulated in plants and is controlled by multiple mechanisms to guarantee a correct evolution through meiotic cell division and sexual differentiation. In the past years, research in the field has shown an important role of the conserved E3-ubiquitin ligase complex, Anaphase-Promoting Complex/Cyclosome (APC/C), in this process. The APC/C is a multi-subunit complex that targets proteins for degradation via proteasome 26S. The functional characterization of APC/C subunits in Arabidopsis, which is one of the main E3 ubiquitin ligase that controls cell cycle, has revealed that all subunits investigated so far are essential for gametophytic development and/or embryogenesis.
PubMed: 33719322
DOI: 10.3389/fpls.2021.642934 -
Molecular Cell Apr 2024The topological state of chromosomes determines their mechanical properties, dynamics, and function. Recent work indicated that interphase chromosomes are largely free...
The topological state of chromosomes determines their mechanical properties, dynamics, and function. Recent work indicated that interphase chromosomes are largely free of entanglements. Here, we use Hi-C, polymer simulations, and multi-contact 3C and find that, by contrast, mitotic chromosomes are self-entangled. We explore how a mitotic self-entangled state is converted into an unentangled interphase state during mitotic exit. Most mitotic entanglements are removed during anaphase/telophase, with remaining ones removed during early G1, in a topoisomerase-II-dependent process. Polymer models suggest a two-stage disentanglement pathway: first, decondensation of mitotic chromosomes with remaining condensin loops produces entropic forces that bias topoisomerase II activity toward decatenation. At the second stage, the loops are released, and the formation of new entanglements is prevented by lower topoisomerase II activity, allowing the establishment of unentangled and territorial G1 chromosomes. When mitotic entanglements are not removed in experiments and models, a normal interphase state cannot be acquired.
Topics: DNA Topoisomerases, Type II; Chromosomes; Mitosis; Interphase; Polymers
PubMed: 38521067
DOI: 10.1016/j.molcel.2024.02.025