-
The Journal of Biological Chemistry Jun 2023Mitotic kinetochores are initially captured by dynamic microtubules via a "search-and-capture" mechanism. The microtubule motor, dynein, is critical for kinetochore...
Mitotic kinetochores are initially captured by dynamic microtubules via a "search-and-capture" mechanism. The microtubule motor, dynein, is critical for kinetochore capture as it has been shown to transport microtubule-attached chromosomes toward the spindle pole during prometaphase. The microtubule-binding nuclear division cycle 80 (Ndc80) complex that is recruited to kinetochores in prophase is known to play a central role in forming kinetochore-microtubule (kMT) attachments in metaphase. It is not yet clear, however, how Ndc80 contributes to initial kMT capture during prometaphase. Here, by combining CRISPR/Cas9-mediated knockout and RNAi technology with assays specific to study kMT capture, we show that mitotic cells lacking Ndc80 exhibit substantial defects in this function during prometaphase. Rescue experiments show that Ndc80 mutants deficient in microtubule-binding are unable to execute proper kMT capture. While cells inhibited of dynein alone are predominantly able to make initial kMT attachments, cells co-depleted of Ndc80 and dynein show severe defects in kMT capture. Further, we use an in vitro total internal reflection fluorescence microscopy assay to reconstitute microtubule capture events, which suggest that Ndc80 and dynein coordinate with each other for microtubule plus-end capture and that the phosphorylation status of Ndc80 is critical for productive kMT capture. A novel interaction between Ndc80 and dynein that we identify in prometaphase extracts might be critical for efficient plus-end capture. Thus, our studies, for the first time, identify a distinct event in the formation of initial kMT attachments, which is directly mediated by Ndc80 and in coordination with dynein is required for efficient kMT capture and chromosome alignment.
Topics: Dyneins; Kinetochores; Nuclear Proteins; Microtubules; Mitosis; Spindle Apparatus; Microtubule-Associated Proteins; Cell Cycle Proteins
PubMed: 37060995
DOI: 10.1016/j.jbc.2023.104711 -
Cell Reports Apr 2023We create a computational framework that utilizes loop extrusion (LE) by multiple condensin I/II motors to predict changes in chromosome organization during mitosis. The...
We create a computational framework that utilizes loop extrusion (LE) by multiple condensin I/II motors to predict changes in chromosome organization during mitosis. The theory accurately reproduces the experimental contact probability profiles for the mitotic chromosomes in HeLa and DT40 cells. The LE rate is smaller at the start of mitosis and increases as the cells approach metaphase. Condensin II-mediated mean loop size is about six times larger than loops because of condensin I. The loops, which overlap each other, are stapled to a central dynamically changing helical scaffold formed by the motors during the LE process. A polymer physics-based data-driven method that uses the Hi-C contact map as the only input shows that the helix is characterized as random helix perversions (RHPs) in which the handedness changes randomly along the scaffold. The theoretical predictions, which are testable using imaging experiments, do not contain any parameters.
Topics: Humans; Chromosomes; Adenosine Triphosphatases; DNA-Binding Proteins; Mitosis
PubMed: 37027299
DOI: 10.1016/j.celrep.2023.112348 -
Frontiers in Bioinformatics 2023The invariant cell lineage of allows unambiguous assignment of the identity for each cell, which offers a unique opportunity to study developmental dynamics such as the...
The invariant cell lineage of allows unambiguous assignment of the identity for each cell, which offers a unique opportunity to study developmental dynamics such as the timing of cell division, dynamics of gene expression, and cell fate decisions at single-cell resolution. However, little is known about cell morphodynamics, including the extent to which they are variable between individuals, mainly due to the lack of sufficient amount and quality of quantified data. In this study, we systematically quantified the cell morphodynamics in 52 embryos from the two-cell stage to mid-gastrulation at the high spatiotemporal resolution, 0.5 μm thickness of optical sections, and 30-second intervals of recordings. Our data allowed systematic analyses of the morphological features. We analyzed sphericity dynamics and found a significant increase at the end of metaphase in every cell, indicating the universality of the mitotic cell rounding. Concomitant with the rounding, the volume also increased in most but not all cells, suggesting less universality of the mitotic swelling. Combining all features showed that cell morphodynamics was unique for each cell type. The cells before the onset of gastrulation could be distinguished from all the other cell types. Quantification of reproducibility in cell-cell contact revealed that variability in division timings and cell arrangements produced variability in contacts between the embryos. However, the area of such contacts occupied less than 5% of the total area, suggesting the high reproducibility of spatial occupancies and adjacency relationships of the cells. By comparing the morphodynamics of identical cells between the embryos, we observed diversity in the variability between cells and found it was determined by multiple factors, including cell lineage, cell generation, and cell-cell contact. We compared the variabilities of cell morphodynamics and cell-cell contacts with those in ascidian embryos. The variabilities were larger in , despite smaller differences in embryo size and number of cells at each developmental stage.
PubMed: 37026092
DOI: 10.3389/fbinf.2023.1082531 -
Frontiers in Cell and Developmental... 2023Cell division events require regulatory systems to ensure that events happen in a distinct order. The classic view of temporal control of the cell cycle posits that... (Review)
Review
Cell division events require regulatory systems to ensure that events happen in a distinct order. The classic view of temporal control of the cell cycle posits that cells order events by linking them to changes in Cyclin Dependent Kinase (CDK) activities. However, a new paradigm is emerging from studies of anaphase where chromatids separate at the central metaphase plate and then move to opposite poles of the cell. These studies suggest that distinct events are ordered depending upon the location of each chromosome along its journey from the central metaphase plate to the elongated spindle poles. This system is dependent upon a gradient of Aurora B kinase activity that emerges during anaphase and acts as a spatial beacon to control numerous anaphase/telophase events and cytokinesis. Recent studies also suggest that Aurora A kinase activity specifies proximity of chromosomes or proteins to spindle poles during prometaphase. Together these studies argue that a key role for Aurora kinases is to provide spatial information that controls events depending upon the location of chromosomes or proteins along the mitotic spindle.
PubMed: 36994100
DOI: 10.3389/fcell.2023.1139367 -
Cells Mar 2023The () gene was discovered about 40 years ago and shown to be required for both mitotic and meiotic cell division. Subsequent studies showed that is highly conserved... (Review)
Review
The () gene was discovered about 40 years ago and shown to be required for both mitotic and meiotic cell division. Subsequent studies showed that is highly conserved and that mutations in its human ortholog (; or ) are the most common cause of autosomal recessive primary microcephaly. This finding greatly stimulated research on and its fly and mouse () orthologs. The three Asp orthologous proteins bind the microtubules (MTs) minus ends during cell division and also function in interphase nuclei. Investigations on different cell types showed that Asp/Aspm/ASPM depletion disrupts one or more of the following mitotic processes: aster formation, spindle pole focusing, centrosome-spindle coupling, spindle orientation, metaphase-to-anaphase progression, chromosome segregation, and cytokinesis. In addition, ASPM physically interacts with components of the DNA repair and replication machineries and is required for the maintenance of chromosomal DNA stability. We propose the working hypothesis that the // genes play the same conserved functions in , mouse, and human cells. Human microcephaly is a genetically heterogeneous disorder caused by mutations in 30 different genes that play a variety of functions required for cell division and chromosomal DNA integrity. Our hypothesis postulates that recapitulates the functions of most human microcephaly genes and provides a justification for why is the most frequently mutated gene in autosomal recessive primary microcephaly.
Topics: Animals; Humans; Mice; DNA; Drosophila; Microcephaly; Mitosis; Nerve Tissue Proteins; Microtubule-Associated Proteins
PubMed: 36980263
DOI: 10.3390/cells12060922 -
Nature Communications Mar 2023Spindle formation in male meiosis relies on the canonical centrosome system, which is distinct from acentrosomal oocyte meiosis, but its specific regulatory mechanisms...
Spindle formation in male meiosis relies on the canonical centrosome system, which is distinct from acentrosomal oocyte meiosis, but its specific regulatory mechanisms remain unknown. Herein, we report that DYNLRB2 (Dynein light chain roadblock-type-2) is a male meiosis-upregulated dynein light chain that is indispensable for spindle formation in meiosis I. In Dynlrb2 KO mouse testes, meiosis progression is arrested in metaphase I due to the formation of multipolar spindles with fragmented pericentriolar material (PCM). DYNLRB2 inhibits PCM fragmentation through two distinct pathways; suppressing premature centriole disengagement and targeting NuMA (nuclear mitotic apparatus) to spindle poles. The ubiquitously expressed mitotic counterpart, DYNLRB1, has similar roles in mitotic cells and maintains spindle bipolarity by targeting NuMA and suppressing centriole overduplication. Our work demonstrates that two distinct dynein complexes containing DYNLRB1 or DYNLRB2 are separately used in mitotic and meiotic spindle formations, respectively, and that both have NuMA as a common target.
Topics: Mice; Animals; Male; Dyneins; Spindle Apparatus; Centrosome; Meiosis; Metaphase
PubMed: 36973253
DOI: 10.1038/s41467-023-37370-7 -
Scientific Reports Mar 2023Gloriosine, the predominant metabolite of Gloriosa superba L., shares chemical properties with colchicine. We analyze the microtubule-binding affinity of gloriosine at...
Gloriosine, the predominant metabolite of Gloriosa superba L., shares chemical properties with colchicine. We analyze the microtubule-binding affinity of gloriosine at the colchicine binding site (CBS) using an in silico-in vivo approach. The In silico docking of gloriosine showed a binding score of (-) 7.5 kcal/Mol towards β-tubulin at CBS and was validated by overlapping the coupling pose of the docked ligand with co-crystallized colchicine. 2D plots (Ligplot +) showed > 85% overlap between gloriosine and colchicine. The ADMET profile of gloriosine was in accordance with Lipinski's rule of five. Gloriosine belongs to class II toxicity with anLD value of 6 mg/kg. In vivo and transmission electron microscopy studies revealed that gloriosine induces abnormalities in cell division such as condensed chromosomes in C-metaphase and enlarged nucleus with increased nuclear material. Gloriosine treated cells exhibited mitotic index of about 14% compared to control of 24% and high anti-proliferative activity i.e. 63.94% cell viability at a low concentration (0.0004 mg/ml). We conclude that gloriosine has a strong affinity for β-tubulin at CBS and thus can be used as a colchicine alternative in cytology and other clinical conditions.
Topics: Colchicine; Tubulin; Microtubules; Binding Sites; Protein Binding
PubMed: 36964265
DOI: 10.1038/s41598-023-31187-6 -
Proceedings of the National Academy of... Mar 2023DNA compaction is required for the condensation and resolution of chromosomes during mitosis, but the relative contribution of individual chromatin factors to this...
DNA compaction is required for the condensation and resolution of chromosomes during mitosis, but the relative contribution of individual chromatin factors to this process is poorly understood. We developed a physiological, cell-free system using high-speed egg extracts and optical tweezers to investigate real-time mitotic chromatin fiber formation and force-induced disassembly on single DNA molecules. Compared to interphase extract, which compacted DNA by ~60%, metaphase extract reduced DNA length by over 90%, reflecting differences in whole-chromosome morphology under these two conditions. Depletion of the core histone chaperone ASF1, which inhibits nucleosome assembly, decreased the final degree of metaphase fiber compaction by 29%, while depletion of linker histone H1 had a greater effect, reducing total compaction by 40%. Compared to controls, both depletions reduced the rate of compaction, led to more short periods of decompaction, and increased the speed of force-induced fiber disassembly. In contrast, depletion of condensin from metaphase extract strongly inhibited fiber assembly, resulting in transient compaction events that were rapidly reversed under high force. Altogether, these findings support a speculative model in which condensin plays the predominant role in mitotic DNA compaction, while core and linker histones act to reduce slippage during loop extrusion and modulate the degree of DNA compaction.
Topics: Animals; Xenopus laevis; Chromatin; Chromosomes; DNA; Mitosis
PubMed: 36917660
DOI: 10.1073/pnas.2221309120 -
Molecular Biology of the Cell May 2023Microtubules are ubiquitous cytoskeletal polymers with essential functions in chromosome segregation, intracellular transport, and cellular morphogenesis. End-binding...
Microtubules are ubiquitous cytoskeletal polymers with essential functions in chromosome segregation, intracellular transport, and cellular morphogenesis. End-binding proteins (EBs) form the nodes of intricate microtubule plus-end interaction networks. Which EB binding partners are most critical for cell division and how cells organize a microtubule cytoskeleton in the absence of an EB protein are open questions. Here, we perform a detailed analysis of deletion and point mutants of the budding yeast EB protein Bim1. We demonstrate that Bim1 executes its key mitotic functions as part of two cargo complexes-Bim1-Kar9 in the cytoplasm and Bim1-Bik1-Cik1-Kar3 in the nucleus. The latter complex acts during initial metaphase spindle assembly and supports tension establishment and sister chromatid biorientation. We demonstrate that engineered plus-end targeting of Cik1-Kar3 and overexpression of the microtubule crosslinker Ase1 restore distinct aspects of the spindle phenotype. In addition to defining key Bim1-cargo complexes our study also characterizes redundant mechanisms that allow cells to proliferate in the absence of Bim1.
Topics: Microtubule-Associated Proteins; Microtubule Proteins; Saccharomyces cerevisiae Proteins; Saccharomyces cerevisiae; Saccharomycetales; Cell Cycle Proteins; Microtubules; Mitosis; Chromosome Segregation
PubMed: 36884292
DOI: 10.1091/mbc.E23-02-0054 -
Frontiers in Cell and Developmental... 2023was originally identified as a part of an aberrant fusion gene in Ewing sarcoma, the second most common pediatric bone cancer. Due to formation of the fusion gene in...
was originally identified as a part of an aberrant fusion gene in Ewing sarcoma, the second most common pediatric bone cancer. Due to formation of the fusion gene in the tumor genome, the cell loses one wild type allele. Our previous study demonstrated that the loss of (homologue of human ) in zebrafish leads to the high incidence of mitotic dysfunction, of aneuploidy, and of tumorigenesis in the mutant background. To dissect the molecular function of EWSR1, we successfully established a stable DLD-1 cell line that enables a conditional knockdown of EWSR1 using an Auxin Inducible Degron (AID) system. When both genes of DLD-1 cell were tagged with at its 5'-end using a CRISPR/Cas9 system, treatment of the () DLD-1 cells with a plant-based Auxin (AUX) led to the significant levels of degradation of AID-EWSR1 proteins. During anaphase, the knockdown (AUX+) cells displayed higher incidence of lagging chromosomes compared to the control (AUX-) cells. This defect was proceeded by a lower incidence of the localization of Aurora B at inner centromeres, and by a higher incidence of the protein at Kinetochore proximal centromere compared to the control cells during pro/metaphase. Despite these defects, the EWSR1 knockdown cells did not undergo mitotic arrest, suggesting that the cell lacks the error correction mechanism. Significantly, the EWSR1 knockdown (AUX+) cells induced higher incidence of aneuploidy compared to the control (AUX-) cells. Since our previous study demonstrated that EWSR1 interacts with the key mitotic kinase, Aurora B, we generated replacement lines of and (a mutant that has low affinity for Aurora B) in the () DLD-1 cells. The EWSR1-mCherry rescued the high incidence of aneuploidy of EWSR1 knockdown cells, whereas EWSR1-mCherry:R565A failed to rescue the phenotype. Together, we demonstrate that EWSR1 prevents the induction of lagging chromosomes, and of aneuploidy through the interaction with Aurora B.
PubMed: 36875767
DOI: 10.3389/fcell.2023.987153