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Scientific Reports May 2017EG5 (KIF11) is a member of the kinesin-like protein family involved in centrosome separation and bipolar spindle formation. When a cell enters mitosis, CDK1...
EG5 (KIF11) is a member of the kinesin-like protein family involved in centrosome separation and bipolar spindle formation. When a cell enters mitosis, CDK1 phosphorylates EG5 at Thr926 and promotes EG5 localization on the mitotic spindle which drives bipolar spindle formation. EG5 provides power for spindle movement and thus controls the dynamics of spindle assembly. However, little is known about EG5 regulation or how EG5 detaches from the spindle upon mitotic exit. In this study we identify EG5 as a novel substrate of PP2A phosphatase, and we show that the PP2A/B55α complex plays an important role in mitotic exit by a mechanism involving EG5. The PP2A/B55α complex physically associates with the EG5 C-terminal tail domain and dephosphorylates EG5 at Thr926 that enables mitotic exit. Conversely PP2A knockdown cells show a high level of phospho-EG5 in late metaphase, which is associated with a delay in mitotic exit. These phenotypic features are similar to those induced by EG5/T926D transfection that mimics phosphorylated EG5 status. Our results argue that PP2A controls mitotic exit through EG5 dephosphorylation. Lack of PP2A leads to abnormal EG5 activation, resulting in delay of mitotic exit.
Topics: Anaphase; Chromosome Segregation; Gene Knockdown Techniques; HeLa Cells; Humans; Kinesins; Metaphase; Mitosis; Phosphorylation; Phosphothreonine; Protein Binding; Protein Phosphatase 2
PubMed: 28487562
DOI: 10.1038/s41598-017-01915-w -
Cell Reports Nov 2016To achieve chromosome segregation during mitosis, sister chromatids must undergo a dramatic change in their behavior to switch from balanced oscillations at the...
To achieve chromosome segregation during mitosis, sister chromatids must undergo a dramatic change in their behavior to switch from balanced oscillations at the metaphase plate to directed poleward motion during anaphase. However, the factors that alter chromosome behavior at the metaphase-to-anaphase transition remain incompletely understood. Here, we perform time-lapse imaging to analyze anaphase chromosome dynamics in human cells. Using multiple directed biochemical, genetic, and physical perturbations, our results demonstrate that differences in the global phosphorylation states between metaphase and anaphase are the major determinant of chromosome motion dynamics. Indeed, causing a mitotic phosphorylation state to persist into anaphase produces dramatic metaphase-like oscillations. These induced oscillations depend on both kinetochore-derived and polar ejection forces that oppose poleward motion. Thus, our analysis of anaphase chromosome motion reveals that dephosphorylation of multiple mitotic substrates is required to suppress metaphase chromosome oscillatory motions and achieve directed poleward motion for successful chromosome segregation.
Topics: Anaphase; Chromatids; Chromosomes, Human; HeLa Cells; Humans; Kinetochores; Metaphase; Models, Biological; Movement; Okadaic Acid; Phosphorylation
PubMed: 27829144
DOI: 10.1016/j.celrep.2016.10.046 -
Cell Cycle (Georgetown, Tex.) Oct 2019Aneuploidy caused by abnormal chromosome segregation during early embryo development leads to embryonic death or congenital malformation. Centromere protein F (CENPF) is...
Aneuploidy caused by abnormal chromosome segregation during early embryo development leads to embryonic death or congenital malformation. Centromere protein F (CENPF) is a member of centromere protein family that regulates chromosome segregation during mitosis. However, its necessity in early embryo development has not been fully investigated. In this study, expression and function of CENPF was investigated in mouse early embryogenesis. Detection of CENPF expression and localization revealed a cytoplasm, spindle and nuclear membrane related dynamic pattern throughout mitotic progression. Farnesyltransferase inhibitor (FTI) was employed to inhibit CENPF farnesylation in zygotes. The results showed that CENPF degradation was inhibited and its specific localization on nuclear membranes in morula and blastocyst vanished after FTI treatment. Also, CAAX motif mutation leads to failure of CENPF-C630 localization in morula and blastocyst. These results indicate that farnesylation plays a key role during CENPF degradation and localization in early embryos. To further assess CENPF function in parthenogenetic or fertilized embryos development, morpholino (MO) and Trim-Away were used to disturb CENPF function. CENPF knockdown in Metaphase II (MII) oocytes, zygotes or embryos with MO approach resulted in failure to develop into morulae and blastocysts, revealing its indispensable role in both parthenogenetic and fertilized embryos. Disturbing of CENPF with Trim-Away approach in zygotes resulted in impaired development of 2-cell and 4-cell, but did not affect the morula and blastocyst formation because of the recovered expression of CENPF. Taken together, our data suggest CENPF plays an important role during early embryonic development in mice. : CENPF: centromere protein F; MO: morpholino; FTI: Farnesyltransferase inhibitor; CENPE: centromere protein E; IVF: fertilization; MII: metaphase II; SAC: spindle assembly checkpoint; Mad1: mitotic arrest deficient 1; BUB1: budding uninhibited by benzimidazole 1; BUBR1: BUB1 mitotic checkpoint serine/threonine kinase B; Cdc20: cell division cycle 20.
Topics: Animals; Blastocyst; Centromere; Chromosomal Proteins, Non-Histone; Embryo, Mammalian; Embryonic Development; Farnesyltranstransferase; Female; Gene Knockdown Techniques; Metaphase; Mice; Mice, Inbred ICR; Microfilament Proteins; Morpholinos; Morula; Oocytes; Parthenogenesis; Piperidines; Pregnancy; Prenylation; Pyridines; Zygote
PubMed: 31478449
DOI: 10.1080/15384101.2019.1661173 -
Nature Communications Nov 2017To divide, most animal cells drastically change shape and round up against extracellular confinement. Mitotic cells facilitate this process by generating intracellular...
To divide, most animal cells drastically change shape and round up against extracellular confinement. Mitotic cells facilitate this process by generating intracellular pressure, which the contractile actomyosin cortex directs into shape. Here, we introduce a genome-scale microcantilever- and RNAi-based approach to phenotype the contribution of > 1000 genes to the rounding of single mitotic cells against confinement. Our screen analyzes the rounding force, pressure and volume of mitotic cells and localizes selected proteins. We identify 49 genes relevant for mitotic rounding, a large portion of which have not previously been linked to mitosis or cell mechanics. Among these, depleting the endoplasmic reticulum-localized protein FAM134A impairs mitotic progression by affecting metaphase plate alignment and pressure generation by delocalizing cortical myosin II. Furthermore, silencing the DJ-1 gene uncovers a link between mitochondria-associated Parkinson's disease and mitotic pressure. We conclude that mechanical phenotyping is a powerful approach to study the mechanisms governing cell shape.
Topics: Actin Cytoskeleton; Actomyosin; Animals; Biomechanical Phenomena; Cell Shape; HeLa Cells; High-Throughput Screening Assays; Humans; Membrane Proteins; Metaphase; Mice; Microscopy, Atomic Force; Mitosis; Myosin Type II; Parkinson Disease; Phenotype; Pressure; Protein Deglycase DJ-1; Single-Cell Analysis; Spindle Apparatus; Surface Tension; Transgenes
PubMed: 29097687
DOI: 10.1038/s41467-017-01147-6 -
The Journal of Cell Biology Jan 2018Precise regulation of kinetochore-microtubule attachments is essential for successful chromosome segregation. Central to this regulation is Aurora B kinase, which...
Precise regulation of kinetochore-microtubule attachments is essential for successful chromosome segregation. Central to this regulation is Aurora B kinase, which phosphorylates kinetochore substrates to promote microtubule turnover. A critical target of Aurora B is the N-terminal "tail" domain of Hec1, which is a component of the NDC80 complex, a force-transducing link between kinetochores and microtubules. Although Aurora B is regarded as the "master regulator" of kinetochore-microtubule attachment, other mitotic kinases likely contribute to Hec1 phosphorylation. In this study, we demonstrate that Aurora A kinase regulates kinetochore-microtubule dynamics of metaphase chromosomes, and we identify Hec1 S69, a previously uncharacterized phosphorylation target site in the Hec1 tail, as a critical Aurora A substrate for this regulation. Additionally, we demonstrate that Aurora A kinase associates with inner centromere protein (INCENP) during mitosis and that INCENP is competent to drive accumulation of the kinase to the centromere region of mitotic chromosomes. These findings reveal that both Aurora A and B contribute to kinetochore-microtubule attachment dynamics, and they uncover an unexpected role for Aurora A in late mitosis.
Topics: Animals; Aurora Kinase A; Aurora Kinase B; Cell Line, Tumor; Centromere; Chromosomal Proteins, Non-Histone; Chromosome Segregation; Cytoskeletal Proteins; HeLa Cells; Humans; Kinetochores; Metaphase; Microtubules; Nuclear Proteins; Phosphorylation; Potoroidae; Protein Binding; Spindle Apparatus
PubMed: 29187526
DOI: 10.1083/jcb.201707160 -
Proceedings of the National Academy of... Jul 2020The metaphase spindle is a dynamic structure orchestrating chromosome segregation during cell division. Recently, soft matter approaches have shown that the spindle...
The metaphase spindle is a dynamic structure orchestrating chromosome segregation during cell division. Recently, soft matter approaches have shown that the spindle behaves as an active liquid crystal. Still, it remains unclear how active force generation contributes to its characteristic spindle-like shape. Here we combine theory and experiments to show that molecular motor-driven forces shape the structure through a barreling-type instability. We test our physical model by titrating dynein activity in egg extract spindles and quantifying the shape and microtubule orientation. We conclude that spindles are shaped by the interplay between surface tension, nematic elasticity, and motor-driven active forces. Our study reveals how motor proteins can mold liquid crystalline droplets and has implications for the design of active soft materials.
Topics: Animals; Biomechanical Phenomena; Dyneins; Elasticity; Liquid Crystals; Metaphase; Microtubules; Mitosis; Spindle Apparatus; Surface Tension; Xenopus Proteins; Xenopus laevis
PubMed: 32601228
DOI: 10.1073/pnas.2002446117 -
Molecular Biology of the Cell Feb 2019The nuclear envelope (NE) aids in organizing the interphase genome by tethering chromatin to the nuclear periphery. During mitotic entry, NE-chromatin contacts are...
The nuclear envelope (NE) aids in organizing the interphase genome by tethering chromatin to the nuclear periphery. During mitotic entry, NE-chromatin contacts are broken. Here, we report on the consequences of impaired NE removal from chromatin for cell division of human cells. Using a membrane-chromatin tether that cannot be dissociated when cells enter mitosis, we show that a failure in breaking membrane-chromatin interactions impairs mitotic chromatin organization, chromosome segregation and cytokinesis, and induces an aberrant NE morphology in postmitotic cells. In contrast, chromosome segregation and cell division proceed successfully when membrane attachment to chromatin is induced during metaphase, after chromosomes have been singularized and aligned at the metaphase plate. These results indicate that the separation of membranes and chromatin is critical during prometaphase to allow for proper chromosome compaction and segregation. We propose that one cause of these defects is the multivalency of membrane-chromatin interactions.
Topics: Cell Nucleus Shape; Chromatin; Chromosome Segregation; Endoplasmic Reticulum; HeLa Cells; Humans; Intracellular Membranes; M Phase Cell Cycle Checkpoints; Membrane Proteins; Metaphase; Mitosis; Nuclear Envelope; Protein Binding; Solubility
PubMed: 30586323
DOI: 10.1091/mbc.E18-10-0609 -
International Journal of Cancer Aug 2011km23-1 is a dynein light chain that was identified as a TGFβ receptor-interacting protein. To investigate whether km23-1 controls human ovarian carcinoma cell (HOCC)...
km23-1 is a dynein light chain that was identified as a TGFβ receptor-interacting protein. To investigate whether km23-1 controls human ovarian carcinoma cell (HOCC) growth, we established a tet-off inducible expression system in SKOV-3 cells in which the expression of km23-1 is induced upon doxycycline removal. We found that forced expression of km23-1 inhibited both anchorage-dependent and anchorage-independent growth of SKOV-3 cells. More importantly, induction of km23-1 expression substantially reduced the tumorigenicity of SKOV-3 cells in a xenograft model in vivo. Fluorescence-activated cell sorting analysis of SKOV-3 and IGROV-1 HOCCs demonstrated that the cells were accumulating at G2/M. Phospho-MEK, phospho-ERK and cyclin B1 were elevated, as was the mitotic index, suggesting that km23-1 suppresses HOCCs growth by inducing a mitotic delay. Immunofluorescence analyses demonstrated that the cells were accumulating at prometaphase/metaphase with increases in multipolar and multinucleated cells. Further, although the mitotic spindle assembly checkpoint protein BubR1 was present at the prometaphase kinetochore in Dox+/- cells, it was inappropriately retained at the metaphase kinetochore in Dox- cells. Thus, the mechanism by which high levels of km23-1 suppress ovarian carcinoma growth in vitro and inhibit ovary tumor formation in vivo appears to involve a BubR1-related mitotic delay.
Topics: Animals; Cell Division; Cell Line, Tumor; Cytoplasmic Dyneins; Dyneins; Female; Humans; Metaphase; Mice; Mice, Nude; Mitosis; Neoplasm Transplantation; Ovarian Neoplasms; Prometaphase; Transplantation, Heterologous; Up-Regulation
PubMed: 21469138
DOI: 10.1002/ijc.25954 -
The Journal of Biological Chemistry Sep 2016During cell division, accurate chromosome segregation is tightly regulated by Polo-like kinase 1 (PLK1) and opposing activities of Aurora B kinase and protein...
During cell division, accurate chromosome segregation is tightly regulated by Polo-like kinase 1 (PLK1) and opposing activities of Aurora B kinase and protein phosphatase 1 (PP1). However, the regulatory mechanisms underlying the aforementioned hierarchical signaling cascade during mitotic chromosome segregation have remained elusive. Sds22 is a conserved regulator of PP1 activity, but how it regulates PP1 activity in space and time during mitosis remains elusive. Here we show that Sds22 is a novel and cognate substrate of PLK1 in mitosis, and the phosphorylation of Sds22 by PLK1 elicited an inhibition of PP1-mediated dephosphorylation of Aurora B at threonine 232 (Thr) in a dose-dependent manner. Overexpression of a phosphomimetic mutant of Sds22 causes a dramatic increase in mitotic delay, whereas overexpression of a non-phosphorylatable mutant of Sds22 results in mitotic arrest. Mechanistically, the phosphorylation of Sds22 by PLK1 strengthens the binding of Sds22 to PP1 and inhibits the dephosphorylation of Thr of Aurora B to ensure a robust, error-free metaphase-anaphase transition. These findings delineate a conserved signaling hierarchy that orchestrates dynamic protein phosphorylation and dephosphorylation of critical mitotic regulators during chromosome segregation to guard chromosome stability.
Topics: Anaphase; Aurora Kinase B; Cell Cycle Proteins; Chromosomal Instability; Chromosome Segregation; Chromosomes, Human; HEK293 Cells; HeLa Cells; Humans; Metaphase; Phosphorylation; Protein Phosphatase 1; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Polo-Like Kinase 1
PubMed: 27557660
DOI: 10.1074/jbc.M116.745372 -
Current Biology : CB May 2002Anaphase, mitotic exit, and cytokinesis proceed in rapid succession, and while mitotic exit is a requirement for cytokinesis in yeast, it may not be a direct requirement...
Anaphase, mitotic exit, and cytokinesis proceed in rapid succession, and while mitotic exit is a requirement for cytokinesis in yeast, it may not be a direct requirement for furrow initiation in animal cells. In this report, we physically manipulated the proximity of the mitotic apparatus (MA) to the cell cortex in combination with microinjection of effectors of the spindle checkpoint and CDK1 activity to determine how the initiation of cytokinesis is coupled to the onset of anaphase and mitotic exit. Whereas precocious contact between the MA and the cell surface advanced the onset of cytokinesis into early anaphase A, furrowing could not be advanced prior to the metaphase-anaphase transition. Additionally, while cells arrested in anaphase could be induced to initiate cleavage furrows, cells arrested in metaphase could not. Finally, activation of the mitotic checkpoint in one spindle of a binucleate cell failed to arrest cytokinesis induced by the control spindle but did inhibit the formation of furrows between the arrested MA and the control, nonarrested MA. Our experiments suggest that the competence of the mitotic apparatus to initiate cytokinesis is not dependent on cyclin degradation but does require anaphase-promoting complex (APC) activity and, thus, inactivation of the mitotic checkpoint.
Topics: Anaphase; Anaphase-Promoting Complex-Cyclosome; Animals; Blastomeres; Calcium-Binding Proteins; Carrier Proteins; Cell Cycle Proteins; Cell Division; Cyclin B; Embryo, Nonmammalian; Fungal Proteins; Ligases; Metaphase; Mitosis; Nuclear Proteins; Sea Urchins; Spindle Apparatus; Time Factors; Ubiquitin-Protein Ligase Complexes
PubMed: 12015124
DOI: 10.1016/s0960-9822(02)00838-2