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Nature Communications Jan 2019Faithful chromosome segregation requires proper chromosome congression at prometaphase and dynamic maintenance of the aligned chromosomes at metaphase. Chromosome...
Faithful chromosome segregation requires proper chromosome congression at prometaphase and dynamic maintenance of the aligned chromosomes at metaphase. Chromosome missegregation can result in aneuploidy, birth defects and cancer. The kinetochore-bound KMN network and the kinesin motor CENP-E are critical for kinetochore-microtubule attachment and chromosome stability. The linear ubiquitin chain assembly complex (LUBAC) attaches linear ubiquitin chains to substrates, with well-established roles in immune response. Here, we identify LUBAC as a key player of chromosome alignment during mitosis. LUBAC catalyzes linear ubiquitination of the kinetochore motor CENP-E, which is specifically required for the localization of CENP-E at attached kinetochores, but not unattached ones. KNL1 acts as a receptor of linear ubiquitin chains to anchor CENP-E at attached kinetochores in prometaphase and metaphase. Thus, linear ubiquitination promotes chromosome congression and dynamic chromosome alignment by coupling the dynamic kinetochore microtubule receptor CENP-E to the static one, the KMN network.
Topics: Animals; Carrier Proteins; Chromosomal Proteins, Non-Histone; Chromosome Segregation; Fibroblasts; Gene Knockdown Techniques; HEK293 Cells; HeLa Cells; Humans; Intracellular Signaling Peptides and Proteins; Kinetochores; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microtubule-Associated Proteins; Mitosis; Primary Cell Culture; RNA, Small Interfering; Ubiquitin; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 30655516
DOI: 10.1038/s41467-018-08043-7 -
The EMBO Journal Dec 2010Eukaryotic cells segregate their chromosomes accurately to opposite poles during mitosis, which is necessary for maintenance of their genetic integrity. This process... (Review)
Review
Eukaryotic cells segregate their chromosomes accurately to opposite poles during mitosis, which is necessary for maintenance of their genetic integrity. This process mainly relies on the forces generated by kinetochore-microtubule (KT-MT) attachment. During prometaphase, the KT initially interacts with a single MT extending from a spindle pole and then moves towards a spindle pole. Subsequently, MTs from the other spindle pole also interact with the KT. Eventually, one sister KT becomes attached to MTs from one pole while the other sister to those from the other pole (sister KT bi-orientation). If sister KTs interact with MTs with aberrant orientation, this must be corrected to attain proper bi-orientation (error correction) before the anaphase is initiated. Here, I discuss how KTs initially interact with MTs and how this interaction develops into bi-orientation; both processes are fundamentally crucial for proper chromosome segregation in the subsequent anaphase.
Topics: Chromosome Segregation; Kinetochores; Microtubules; Mitosis; Models, Biological
PubMed: 21102558
DOI: 10.1038/emboj.2010.294 -
Mechanisms of Development Jul 2008The Drosophila Myb protein, DMyb, is a transcription factor important for cell proliferation and development. Unlike the mRNAs produced by mammalian myb genes,...
The Drosophila Myb protein, DMyb, is a transcription factor important for cell proliferation and development. Unlike the mRNAs produced by mammalian myb genes, Drosophila myb transcripts do not fluctuate substantially during the cell cycle. A comprehensive analysis of the localization and degradation of the DMyb protein has now revealed that DMyb is present in nuclei during S phase of all mitotically active tissues throughout embryogenesis and larval development. However, DMyb and Mip130, another member of the Myb complex, are not uniformly distributed throughout the nucleus. Instead, both proteins, which colocalize, appear to be specifically excluded from heterochromatic regions of chromosomes. Furthermore, DMyb and Mip130 are unstable proteins that are degraded during prometaphase of mitosis. The timing of their degradation is reminiscent of Cyclin A, but at least for DMyb, the mechanism differs; although DMyb degradation is dependent on core APC/C components, it does not depend on the Fizzy or Fizzy-related adaptor proteins. DMyb levels are also high in actively endoreplicating polyploid cells, but there is no indication of cyclical degradation. We conclude that cell cycle specific degradation of DMyb and Mip130 is likely to be utilized as a key regulatory mechanism in down-regulating their levels and the activity of the Myb complex.
Topics: Animals; Carrier Proteins; Cell Cycle Proteins; Drosophila Proteins; Drosophila melanogaster; Euchromatin; Gene Expression Regulation, Developmental; Prometaphase; Proto-Oncogene Proteins c-myb
PubMed: 18424081
DOI: 10.1016/j.mod.2008.02.005 -
Bioinformatics (Oxford, England) Jun 2022Lattice light-sheet microscopy (LLSM) is revolutionizing cell biology since it enables fast, high-resolution extended imaging in three dimensions combined with a drastic...
MOTIVATION
Lattice light-sheet microscopy (LLSM) is revolutionizing cell biology since it enables fast, high-resolution extended imaging in three dimensions combined with a drastic reduction in photo-toxicity and bleaching. However, analysis of such datasets still remains a major challenge.
RESULTS
Automated tracking of kinetochores, the protein complex facilitating and controlling microtubule attachment of the chromosomes within the mitotic spindle, provides quantitative assessment of chromosome dynamics in mitosis. Here, we extend existing open-source kinetochore tracking software (KiT) to track (and pair) kinetochores throughout prometaphase to anaphase in LLSM data. One of the key improvements is a regularization term in the objective function to enforce biological information about the number of kinetochores in a human mitotic cell, as well as improved diagnostic tools. This software provides quantitative insights into how kinetochores robustly ensure congression and segregation of chromosomes during mitosis.
AVAILABILITY AND IMPLEMENTATION
KiT is free, open-source software implemented in MATLAB and can be downloaded as a package from https://github.com/cmcb-warwick/KiT. The source repository is available at https://bitbucket.org/jarmond/kit (tag v2.4.0) and under continuing development.
SUPPLEMENTARY INFORMATION
Supplementary data are available at Bioinformatics online.
Topics: Humans; Kinetochores; Spindle Apparatus; Anaphase; Microtubules; Software; Chromosome Segregation
PubMed: 35579370
DOI: 10.1093/bioinformatics/btac330 -
Journal of Cell Science Dec 2012Successful completion of mitosis requires that sister kinetochores become attached end-on to the plus ends of spindle microtubules (MTs) in prometaphase, thereby forming... (Review)
Review
Successful completion of mitosis requires that sister kinetochores become attached end-on to the plus ends of spindle microtubules (MTs) in prometaphase, thereby forming kinetochore microtubules (kMTs) that tether one sister to one spindle pole and the other sister to the opposite pole. Sites for kMT attachment provide at least four key functions: robust and dynamic kMT anchorage; force generation that can be coupled to kMT plus-end dynamics; correction of errors in kMT attachment; and control of the spindle assembly checkpoint (SAC). The SAC typically delays anaphase until chromosomes achieve metaphase alignment with each sister kinetochore acquiring a full complement of kMTs. Although it has been known for over 30 years that MT motor proteins reside at kinetochores, a highly conserved network of protein complexes, called the KMN network, has emerged in recent years as the primary interface between the kinetochore and kMTs. This Commentary will summarize recent advances in our understanding of the role of the KMN network for the key kinetochore functions, with a focus on human cells.
Topics: Cytoskeletal Proteins; Humans; Kinetochores; Microtubule-Associated Proteins; Microtubules; Nuclear Proteins; Spindle Apparatus
PubMed: 23418356
DOI: 10.1242/jcs.093724 -
The Plant Journal : For Cell and... Jul 2013In plants, as in all eukaryotic organisms, microtubule- and actin-filament based structures play fundamental roles during cell division. In addition to the mitotic... (Review)
Review
In plants, as in all eukaryotic organisms, microtubule- and actin-filament based structures play fundamental roles during cell division. In addition to the mitotic spindle, plant cells have evolved a unique cytoskeletal structure that designates a specific division plane before the onset of mitosis via formation of a cortical band of microtubules and actin filaments called the preprophase band. During cytokinesis, a second plant-specific microtubule and actin filament structure called the phragmoplast directs vesicles to create the new cell wall. In response to intrinsic and extrinsic cues, many plant cells form a preprophase band in G2 , then the preprophase band recruits specific proteins to populate the cortical division site prior to disassembly of the preprophase band in prometaphase. These proteins are thought to act as a spatial reminder that actively guides the phragmoplast towards the cortical division site during cytokinesis. A number of proteins involved in determination and maintenance of the plane of cell division have been identified. Our current understanding of the molecular interactions of these proteins and their regulation of microtubules is incomplete, but advanced imaging techniques and computer simulations have validated some early concepts of division site determination.
Topics: Actin Cytoskeleton; Arabidopsis; Bryopsida; Cell Division; Cell Wall; Cytokinesis; Cytoskeleton; Microtubules; Mitosis; Plant Cells; Plant Proteins; Prophase; Tradescantia; Zea mays
PubMed: 23496276
DOI: 10.1111/tpj.12177 -
BioRxiv : the Preprint Server For... Aug 2023The mammalian RAD52 protein is a DNA repair factor that has both strand annealing and recombination mediator activities, yet is dispensable for cell viability. To...
The mammalian RAD52 protein is a DNA repair factor that has both strand annealing and recombination mediator activities, yet is dispensable for cell viability. To characterize genetic contexts that reveal dependence on RAD52 to sustain cell viability (i.e., synthetic lethal relationships), we performed genome-wide CRISPR knock-out screens. Subsequent secondary screening found that depletion of ERCC6L in RAD52-deficient cells causes reduced viability and elevated genome instability, measured as accumulation of 53BP1 into nuclear foci. Furthermore, loss of RAD52 causes elevated levels of anaphase ultrafine bridges marked by ERCC6L, and conversely depletion of ERCC6L causes elevated RAD52 foci both in prometaphase and interphase cells. These effects were enhanced with combination treatments using hydroxyurea and the topoisomerase IIα inhibitor ICRF-193, and the timing of these treatments are consistent with defects in addressing such stress in mitosis. Thus, loss of RAD52 appears to cause an increased reliance on ERCC6L in mitosis, and vice versa. Consistent with this notion, combined depletion of ERCC6L and disrupting G2/M progression via CDK1 inhibition causes a marked loss of viability in RAD52-deficient cells. We suggest that RAD52 and ERCC6L play compensatory roles in protecting genome stability in mitosis.
PubMed: 37662271
DOI: 10.1101/2023.08.23.554522 -
Proceedings of the National Academy of... Jun 2020Topoisomerase IIα (TOP2A) is a core component of mitotic chromosomes and important for establishing mitotic chromosome condensation. The primary roles of TOP2A in...
Topoisomerase IIα (TOP2A) is a core component of mitotic chromosomes and important for establishing mitotic chromosome condensation. The primary roles of TOP2A in mitosis have been difficult to decipher due to its multiple functions across the cell cycle. To more precisely understand the role of TOP2A in mitosis, we used the auxin-inducible degron (AID) system to rapidly degrade the protein at different stages of the human cell cycle. Removal of TOP2A prior to mitosis does not affect prophase timing or the initiation of chromosome condensation. Instead, it prevents chromatin condensation in prometaphase, extends the length of prometaphase, and ultimately causes cells to exit mitosis without chromosome segregation occurring. Surprisingly, we find that removal of TOP2A from cells arrested in prometaphase or metaphase cause dramatic loss of compacted mitotic chromosome structure and conclude that TOP2A is crucial for maintenance of mitotic chromosomes. Treatments with drugs used to poison/inhibit TOP2A function, such as etoposide and ICRF-193, do not phenocopy the effects on chromosome structure of TOP2A degradation by AID. Our data point to a role for TOP2A as a structural chromosome maintenance enzyme locking in condensation states once sufficient compaction is achieved.
Topics: Chromosome Segregation; Chromosome Structures; Chromosomes, Human; Cytokinesis; DNA Topoisomerases, Type II; HCT116 Cells; Heterochromatin; Humans; Metaphase; Mitosis
PubMed: 32414923
DOI: 10.1073/pnas.2001760117 -
Scientific Reports Mar 2018Faithful chromosome segregation is ensured by the establishment of bi-orientation; the attachment of sister kinetochores to the end of microtubules extending from...
Faithful chromosome segregation is ensured by the establishment of bi-orientation; the attachment of sister kinetochores to the end of microtubules extending from opposite spindle poles. In addition, kinetochores can also attach to lateral surfaces of microtubules; called lateral attachment, which plays a role in chromosome capture and transport. However, molecular basis and biological significance of lateral attachment are not fully understood. We have addressed these questions by focusing on the prometaphase rosette, a typical chromosome configuration in early prometaphase. We found that kinetochores form uniform lateral attachments in the prometaphase rosette. Many transient kinetochore components are maximally enriched, in an Aurora B activity-dependent manner, when the prometaphase rosette is formed. We revealed that rosette formation is driven by rapid poleward motion of dynein, but can occur even in its absence, through slow kinetochore movements caused by microtubule depolymerization that is supposedly dependent on kinetochore tethering at microtubule ends by CENP-E. We also found that chromosome connection to microtubules is extensively lost when lateral attachment is perturbed in cells defective in end-on attachment. Our findings demonstrate that lateral attachment is an important intermediate in bi-orientation establishment and chromosome alignment, playing a crucial role in incorporating chromosomes into the nascent spindle.
Topics: Chromosome Segregation; Dyneins; HeLa Cells; Humans; Kinetochores; Microtubules; Prometaphase; Rosette Formation; Spindle Apparatus
PubMed: 29497093
DOI: 10.1038/s41598-018-22164-5 -
The Journal of Cell Biology Mar 2018The mitotic spindle checkpoint delays anaphase onset in the presence of unattached kinetochores, and efficient checkpoint signaling requires kinetochore localization of...
The mitotic spindle checkpoint delays anaphase onset in the presence of unattached kinetochores, and efficient checkpoint signaling requires kinetochore localization of the Rod-ZW10-Zwilch (RZZ) complex. In the present study, we show that human Chmp4c, a protein involved in membrane remodeling, localizes to kinetochores in prometaphase but is reduced in chromosomes aligned at the metaphase plate. Chmp4c promotes stable kinetochore-microtubule attachments and is required for proper mitotic progression, faithful chromosome alignment, and segregation. Depletion of Chmp4c diminishes localization of RZZ and Mad1-Mad2 checkpoint proteins to prometaphase kinetochores and impairs mitotic arrest when microtubules are depolymerized by nocodazole. Furthermore, Chmp4c binds to ZW10 through a small C-terminal region, and constitutive Chmp4c kinetochore targeting causes a ZW10-dependent checkpoint metaphase arrest. In addition, Chmp4c spindle functions do not require endosomal sorting complex required for transport-dependent membrane remodeling. These results show that Chmp4c regulates the mitotic spindle checkpoint by promoting localization of the RZZ complex to unattached kinetochores.
Topics: Cell Cycle Checkpoints; Cell Cycle Proteins; Endosomal Sorting Complexes Required for Transport; HeLa Cells; Humans; Kinetochores; Mad2 Proteins; Nuclear Proteins; Signal Transduction; Spindle Apparatus
PubMed: 29362225
DOI: 10.1083/jcb.201709005