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Molecular Biology of the Cell Jun 2019The endoplasmic reticulum (ER) is extensively remodeled during metazoan open mitosis. However, whether the ER becomes more tubular or more cisternal during mitosis is...
The endoplasmic reticulum (ER) is extensively remodeled during metazoan open mitosis. However, whether the ER becomes more tubular or more cisternal during mitosis is controversial, and dedicated factors governing the morphology of the mitotic ER have remained elusive. Here, we describe the ER membrane proteins REEP3 and REEP4 as major determinants of ER morphology in metaphase cells. REEP3/4 are specifically required for generating the high-curvature morphology of mitotic ER and promote ER tubulation through their reticulon homology domains (RHDs). This ER-shaping activity of REEP3/4 is distinct from their previously described function to clear ER from metaphase chromatin. We further show that related REEP proteins do not contribute to mitotic ER shaping and provide evidence that the REEP3/4 carboxyterminus mediates regulation of the proteins. These findings confirm that ER converts to higher curvature during mitosis, identify REEP3/4 as specific and crucial morphogenic factors mediating ER tubulation during mitosis, and define the first cell cycle-specific role for RHD proteins.
Topics: Amino Acid Sequence; Chromatin; Endoplasmic Reticulum; HeLa Cells; Humans; Membrane Transport Proteins; Metaphase; Mitosis; Protein Domains
PubMed: 30995177
DOI: 10.1091/mbc.E18-11-0698 -
Cell Cycle (Georgetown, Tex.) Jun 2021ATP metabolism during mitosis needs to be coordinated with numerous energy-demanding activities, especially in cancer cells whose metabolic pathways are reprogramed to...
ATP metabolism during mitosis needs to be coordinated with numerous energy-demanding activities, especially in cancer cells whose metabolic pathways are reprogramed to sustain rapid proliferation in a nutrient-deficient environment. Although strategies targeting the energy metabolic pathways have shown therapeutic efficacy in preclinical cancer models, how normal cells and cancer cells differentially respond to energy shortage is unclear. In this study, using time-lapse microscopy, we found that cancer cells displayed unique mitotic phenotypes in a dose-dependent manner upon decreasing ATP (i.e. energy) supply. When reduction in ATP concentration was moderate, chromosome movements in mitosis were barely affected, while the metaphase-anaphase transition was significantly prolonged due to reduced tension between the sister-kinetochores, which delayed the satisfaction of the spindle assembly checkpoint. Further reduction in ATP concentration led to a decreased level of Aurora-B at the centromere, resulting in increased chromosome mis-segregation after metaphase delay. In contrast to cancer cells, ATP restriction in non-transformed cells induced cell cycle arrest in interphase, rather than causing mitotic defects. In addition, data mining of cancer patient database showed a correlation between signatures of energy production and chromosomal instability possibly resulted from mitotic defects. Together, these results reveal that energy restriction induces differential responses in normal and cancer cells, with chromosome mis-segregation only observed in cancer cells. This points to targeting energy metabolism as a potentially cancer-selective therapeutic strategy.
Topics: Adenosine Triphosphate; Anaphase; Aurora Kinase B; Chromosome Segregation; Energy Metabolism; Female; HeLa Cells; Humans; Interphase; Kinetochores; Metaphase; Microscopy; NAD; Signal Transduction; Spindle Apparatus; Time-Lapse Imaging; Uterine Cervical Neoplasms
PubMed: 34048314
DOI: 10.1080/15384101.2021.1930679 -
Cell Reports Aug 2022Chromosome alignment at the spindle equator promotes proper chromosome segregation and depends on pulling forces exerted at kinetochore fiber tips together with polar...
Chromosome alignment at the spindle equator promotes proper chromosome segregation and depends on pulling forces exerted at kinetochore fiber tips together with polar ejection forces. However, kinetochore fibers are also subjected to forces driving their poleward flux. Here we introduce a flux-driven centering model that relies on flux generated by forces within the overlaps of bridging and kinetochore fibers. This centering mechanism works so that the longer kinetochore fiber fluxes faster than the shorter one, moving the kinetochores toward the center. We develop speckle microscopy in human spindles and confirm the key prediction that kinetochore fiber flux is length dependent. Kinetochores are better centered when overlaps are shorter and the kinetochore fiber flux slower than the bridging fiber flux. We identify Kif18A and Kif4A as overlap and flux regulators and NuMA as a fiber coupler. Thus, length-dependent sliding forces exerted by the bridging fiber onto kinetochore fibers support chromosome alignment.
Topics: Anaphase; Cell Cycle Proteins; Chromosome Segregation; Chromosomes; Humans; Kinesins; Kinetochores; Metaphase; Microtubules; Spindle Apparatus
PubMed: 35926461
DOI: 10.1016/j.celrep.2022.111169 -
PloS One 2014Proper spindle positioning and orientation are essential for accurate mitosis which requires dynamic interactions between microtubule and actin filament (F-actin)....
Proper spindle positioning and orientation are essential for accurate mitosis which requires dynamic interactions between microtubule and actin filament (F-actin). Although mounting evidence demonstrates the role of F-actin in cortical cytoskeleton dynamics, it remains elusive as to the structure and function of F-actin-based networks in spindle geometry. Here we showed a ring-like F-actin structure surrounding the mitotic spindle which forms since metaphase and maintains in MG132-arrested metaphase HeLa cells. This cytoplasmic F-actin structure is relatively isotropic and less dynamic. Our computational modeling of spindle position process suggests a possible mechanism by which the ring-like F-actin structure can regulate astral microtubule dynamics and thus mitotic spindle orientation. We further demonstrated that inhibiting Plk1, Mps1 or Myosin, and disruption of microtubules or F-actin polymerization perturbs the formation of the ring-like F-actin structure and alters spindle position and symmetric division. These findings reveal a previously unrecognized but important link between mitotic spindle and ring-like F-actin network in accurate mitosis and enables the development of a method to theoretically illustrate the relationship between mitotic spindle and cytoplasmic F-actin.
Topics: Actin Cytoskeleton; Actins; Cell Line, Tumor; Cytoskeleton; HeLa Cells; Humans; Metaphase; Microtubules; Mitosis; Myosins; Spindle Apparatus
PubMed: 25299690
DOI: 10.1371/journal.pone.0102547 -
The Journal of Cell Biology Dec 2019During mitosis, the centrosome expands its capacity to nucleate microtubules. Understanding the mechanisms of centrosomal microtubule nucleation is, however, constrained...
During mitosis, the centrosome expands its capacity to nucleate microtubules. Understanding the mechanisms of centrosomal microtubule nucleation is, however, constrained by a lack of knowledge of the amount of soluble and polymeric tubulin at mitotic centrosomes. Here we combined light microscopy and serial-section electron tomography to measure the amount of dimeric and polymeric tubulin at mitotic centrosomes in early embryos. We show that a one-cell stage centrosome at metaphase contains >10,000 microtubules with a total polymer concentration of 230 µM. Centrosomes concentrate soluble α/β tubulin by about 10-fold over the cytoplasm, reaching peak values of 470 µM, giving a combined total monomer and polymer tubulin concentration at centrosomes of up to 660 µM. These findings support in vitro data suggesting that microtubule nucleation in centrosomes is driven in part by concentrating soluble tubulin.
Topics: Animals; Caenorhabditis elegans; Centrosome; Cytoplasm; Dimerization; Image Processing, Computer-Assisted; Imaging, Three-Dimensional; Metaphase; Microscopy, Electron; Microtubules; Mitosis; Nocodazole; Polymers; RNA Interference; Solubility; Tubulin
PubMed: 31636117
DOI: 10.1083/jcb.201902069 -
Chromosome Research : An International... Sep 2016We have found that reagents that reduce oxidized cysteines lead to destabilization of metaphase chromosome folding, suggesting that chemically linked cysteine residues...
We have found that reagents that reduce oxidized cysteines lead to destabilization of metaphase chromosome folding, suggesting that chemically linked cysteine residues may play a structural role in mitotic chromosome organization, in accord with classical studies by Dounce et al. (J Theor Biol 42:275-285, 1973) and Sumner (J Cell Sci 70:177-188, 1984a). Human chromosomes isolated into buffer unfold when exposed to dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP). In micromanipulation experiments which allow us to examine the mechanics of individual metaphase chromosomes, we have found that the gel-like elastic stiffness of native metaphase chromosomes is dramatically suppressed by DTT and TCEP, even before the chromosomes become appreciably unfolded. We also report protein labeling experiments on human metaphase chromosomes which allow us to tag oxidized and reduction-sensitive cysteine residues. PAGE analysis using fluorescent labels shows a small number of labeled bands. Mass spectrometry analysis of similarly labeled proteins provides a list of candidates for proteins with oxidized cysteines involved in chromosome organization, notably including components of condensin I, cohesin, the nucleosome-interacting proteins RCC1 and RCC2, as well as the RNA/DNA-binding protein NONO/p54NRB.
Topics: Adenosine Triphosphatases; Animals; Cell Cycle Proteins; Cell Line; Chromosomal Proteins, Non-Histone; Chromosomes, Human; Cysteine; DNA-Binding Proteins; Dithiothreitol; Electrophoresis, Gel, Two-Dimensional; Guanine Nucleotide Exchange Factors; HEK293 Cells; Humans; Karyotype; Mass Spectrometry; Metaphase; Micromanipulation; Multiprotein Complexes; Notophthalmus viridescens; Nuclear Matrix-Associated Proteins; Nuclear Proteins; Octamer Transcription Factors; Oxidation-Reduction; Phosphines; RNA-Binding Proteins; Cohesins
PubMed: 27145786
DOI: 10.1007/s10577-016-9528-6 -
The Journal of Cell Biology Jan 2001Duo1p and Dam1p were previously identified as spindle proteins in the budding yeast, Saccharomyces cerevisiae. Here, analyses of a diverse collection of duo1 and dam1...
Duo1p and Dam1p were previously identified as spindle proteins in the budding yeast, Saccharomyces cerevisiae. Here, analyses of a diverse collection of duo1 and dam1 alleles were used to develop a deeper understanding of the functions and interactions of Duo1p and Dam1p. Based on the similarity of mutant phenotypes, genetic interactions between duo1 and dam1 alleles, interdependent localization to the mitotic spindle, and Duo1p/Dam1p coimmunoprecipitation from yeast protein extracts, these analyses indicated that Duo1p and Dam1p perform a shared function in vivo as components of a protein complex. Duo1p and Dam1p are not required to assemble bipolar spindles, but they are required to maintain metaphase and anaphase spindle integrity. Immunofluorescence and electron microscopy of duo1 and dam1 mutant spindles revealed a diverse variety of spindle defects. Our results also indicate a second, previously unidentified, role for the Duo1p/Dam1p complex. duo1 and dam1 mutants show high rates of chromosome missegregation, premature anaphase events while arrested in metaphase, and genetic interactions with a subset of kinetochore components consistent with a role in kinetochore function. In addition, Duo1p and Dam1p localize to kinetochores in chromosome spreads, suggesting that this complex may serve as a link between the kinetochore and the mitotic spindle.
Topics: Amino Acid Sequence; Anaphase; Cell Cycle Proteins; Chromosomes, Fungal; Cytoskeletal Proteins; Fungal Proteins; Kinetochores; Metaphase; Microscopy, Electron; Microtubule-Associated Proteins; Mitosis; Molecular Sequence Data; Mutagenesis; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Spindle Apparatus
PubMed: 11149931
DOI: 10.1083/jcb.152.1.197 -
Cytoskeleton (Hoboken, N.J.) Nov 2019Chromosome segregation is mediated by spindle microtubules that attach to the kinetochore via dynamic protein complexes, such as Ndc80, Ska, Cdt1 and ch-TOG during...
Chromosome segregation is mediated by spindle microtubules that attach to the kinetochore via dynamic protein complexes, such as Ndc80, Ska, Cdt1 and ch-TOG during mitotic metaphase. While experimental studies have previously shown that these proteins and protein complexes are all essential for maintaining a stable kinetochore-microtubule (kMT) interface, their exact roles in the mitotic metaphase remains elusive. In this study, we employed experimental and computational methods in order to characterize how these proteins can strengthen kMT attachments in both nonload-bearing and load-bearing conditions, typical of prometaphase and metaphase, respectively. Immunofluorescence staining of HeLa cells showed that the levels of Ska and Cdt1 significantly increased from prometaphase to metaphase, while levels of the Ndc80 complex remained unchanged. Our new computational model showed that by incorporating binding and unbinding of each protein complex coupled with a biased diffusion mechanism, the displacement of a possible complex formed by Ndc80-Ska-Cdt1 is significantly higher than that of Ndc80 alone or Ndc80-Ska. In addition, when we incorporate Ndc80/ch-TOG in the model, rupture force and time of attachment of the kMT interface increases. These results support the hypothesis that Ndc80-associated proteins strengthen kMT attachments, and that the interplay between kMT protein complexes in metaphase ensures stable attachments.
Topics: Cell Cycle Proteins; Chromosomal Proteins, Non-Histone; Computer Simulation; Cytoskeletal Proteins; HeLa Cells; Humans; Kinetochores; Metaphase; Microtubules; Mitosis; Protein Binding
PubMed: 31525284
DOI: 10.1002/cm.21562 -
Science (New York, N.Y.) May 2016The position and orientation of the mitotic spindle is precisely regulated to ensure the accurate partition of the cytoplasm between daughter cells and the correct...
The position and orientation of the mitotic spindle is precisely regulated to ensure the accurate partition of the cytoplasm between daughter cells and the correct localization of the daughters within growing tissue. Using magnetic tweezers to perturb the position of the spindle in intact cells, we discovered a force-generating machinery that maintains the spindle at the cell center during metaphase and anaphase in one- and two-cell Caenorhabditis elegans embryos. The forces increase with the number of microtubules and are larger in smaller cells. The machinery is rigid enough to suppress thermal fluctuations to ensure precise localization of the mitotic spindle, yet compliant enough to allow molecular force generators to fine-tune the position of the mitotic spindle to facilitate asymmetric division.
Topics: Anaphase; Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Centrosome; Elasticity; Embryo, Nonmammalian; Kinesins; Metaphase; Mitosis; RNA Interference; Spindle Apparatus; Viscosity
PubMed: 27230381
DOI: 10.1126/science.aad9745 -
The Journal of Cell Biology Feb 1994We argue that hypotheses for how chromosomes achieve a metaphase alignment, that are based solely on a tug-of-war between poleward pulling forces produced along the... (Review)
Review
We argue that hypotheses for how chromosomes achieve a metaphase alignment, that are based solely on a tug-of-war between poleward pulling forces produced along the length of opposing kinetochore fibers, are no longer tenable for vertebrates. Instead, kinetochores move themselves and their attached chromosomes, poleward and away from the pole, on the ends of relatively stationary but shortening/elongating kinetochore fiber microtubules. Kinetochores are also "smart" in that they switch between persistent constant-velocity phases of poleward and away from the pole motion, both autonomously and in response to information within the spindle. Several molecular mechanisms may contribute to this directional instability including kinetochore-associated microtubule motors and kinetochore microtubule dynamic instability. The control of kinetochore directional instability, to allow for congression and anaphase, is likely mediated by a vectorial mechanism whose magnitude and orientation depend on the density and orientation or growth of polar microtubules. Polar microtubule arrays have been shown to resist chromosome poleward motion and to push chromosomes away from the pole. These "polar ejection forces" appear to play a key role in regulating kinetochore directional instability, and hence, positions achieved by chromosomes on the spindle.
Topics: Anaphase; Animals; Centromere; Chromosomes; Metaphase; Microtubules; Mitosis; Models, Biological; Spindle Apparatus
PubMed: 8294508
DOI: 10.1083/jcb.124.3.223