A fraction of barrier-to-autointegration factor (BAF) associates with centromeres and controls mitosis progression

AbstractBarrier-to-Autointegration Factor (BAF) is a conserved nuclear envelope (NE) component that binds chromatin and helps its anchoring to the NE. Cycles of phosphorylation and dephosphorylation control BAF function. Entering mitosis, phosphorylation releases BAF from chromatin and facilitates NE-disassembly. At mitotic exit, PP2A-mediated dephosphorylation restores chromatin binding and nucleates NE-reassembly. Here, we show that in Drosophila a small fraction of BAF (cenBAF) associates with centromeres. We also find that PP4 phosphatase, which is recruited to centromeres by CENP-C, prevents phosphorylation and release of cenBAF during mitosis. cenBAF is necessary for proper centromere assembly and accurate chromosome segregation, being critical for mitosis progression. Disrupting cenBAF localization prevents PP2A inactivation in mitosis compromising global BAF phosphorylation, which in turn leads to its persistent association with chromatin, delays anaphase onset and causes NE defects. These results suggest that, together with PP4 and CENP-C, cenBAF forms a centromere-based mechanism that controls chromosome segregation and mitosis progression.

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Morphology and behaviour of dinoflagellate chromosomes during the cell cycle and mitosis

Journal of Cell Science ◽

10.1242/jcs.113.7.1231 ◽

2000 ◽

Vol 113 (7) ◽

pp. 1231-1239 ◽

Cited By ~ 3

Author(s):

Y. Bhaud ◽

D. Guillebault ◽

J. Lennon ◽

H. Defacque ◽

M.O. Soyer-Gobillard ◽

...

Keyword(s):

Electron Microscopy ◽

Cell Cycle ◽

Confocal Microscopy ◽

Nuclear Envelope ◽

Chromosome Segregation ◽

Morphological Changes ◽

S Phase ◽

Chromosome Behaviour ◽

Double Number ◽

Do So

The morphology and behaviour of the chromosomes of dinoflagellates during the cell cycle appear to be unique among eukaryotes. We used synchronized and aphidicolin-blocked cultures of the dinoflagellate Crypthecodinium cohnii to describe the successive morphological changes that chromosomes undergo during the cell cycle. The chromosomes in early G(1) phase appeared to be loosely condensed with numerous structures protruding toward the nucleoplasm. They condensed in late G(1), before unwinding in S phase. The chromosomes in cells in G(2) phase were tightly condensed and had a double number of arches, as visualised by electron microscopy. During prophase, chromosomes elongated and split longitudinally, into characteristic V or Y shapes. We also used confocal microscopy to show a metaphase-like alignment of the chromosomes, which has never been described in dinoflagellates. The metaphase-like nucleus appeared flattened and enlarged, and continued to do so into anaphase. Chromosome segregation occurred via binding to the nuclear envelope surrounding the cytoplasmic channels and microtubule bundles. Our findings are summarized in a model of chromosome behaviour during the cell cycle.

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Differential requirement for Bub1 and Bub3 in regulation of meiotic versus mitotic chromosome segregation

The Journal of Cell Biology ◽

10.1083/jcb.201909136 ◽

2020 ◽

Vol 219 (4) ◽

Cited By ~ 2

Author(s):

Gisela Cairo ◽

Anne M. MacKenzie ◽

Soni Lacefield

Keyword(s):

Chromosome Segregation ◽

Protein Phosphatase ◽

Mitotic Chromosome ◽

Budding Yeast ◽

Meiotic Chromosome ◽

Protein Phosphatase 1 ◽

Aurora B ◽

Chromosome Missegregation ◽

Anaphase Onset ◽

Spindle Microtubules

Accurate chromosome segregation depends on the proper attachment of kinetochores to spindle microtubules before anaphase onset. The Ipl1/Aurora B kinase corrects improper attachments by phosphorylating kinetochore components and so releasing aberrant kinetochore–microtubule interactions. The localization of Ipl1 to kinetochores in budding yeast depends upon multiple pathways, including the Bub1–Bub3 pathway. We show here that in meiosis, Bub3 is crucial for correction of attachment errors. Depletion of Bub3 results in reduced levels of kinetochore-localized Ipl1 and concomitant massive chromosome missegregation caused by incorrect chromosome–spindle attachments. Depletion of Bub3 also results in shorter metaphase I and metaphase II due to premature localization of protein phosphatase 1 (PP1) to kinetochores, which antagonizes Ipl1-mediated phosphorylation. We propose a new role for the Bub1–Bub3 pathway in maintaining the balance between kinetochore localization of Ipl1 and PP1, a balance that is essential for accurate meiotic chromosome segregation and timely anaphase onset.

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The Ki-67 and RepoMan mitotic phosphatases assemble via an identical, yet novel mechanism

eLife ◽

10.7554/elife.16539 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 31

Author(s):

Ganesan Senthil Kumar ◽

Ezgi Gokhan ◽

Sofie De Munter ◽

Mathieu Bollen ◽

Paola Vagnarelli ◽

...

Keyword(s):

Protein Phosphatase ◽

Mitotic Chromosome ◽

Cancer Therapeutics ◽

Mitotic Exit ◽

Protein Phosphatase 1 ◽

Aurora B ◽

Aurora B Kinase ◽

Ki 67 ◽

Anaphase Onset

Ki-67 and RepoMan have key roles during mitotic exit. Previously, we showed that Ki-67 organizes the mitotic chromosome periphery and recruits protein phosphatase 1 (PP1) to chromatin at anaphase onset, in a similar manner as RepoMan (<xref ref-type="bibr" rid="bib2">Booth et al., 2014</xref>). Here we show how Ki-67 and RepoMan form mitotic exit phosphatases by recruiting PP1, how they distinguish between distinct PP1 isoforms and how the assembly of these two holoenzymes are dynamically regulated by Aurora B kinase during mitosis. Unexpectedly, our data also reveal that Ki-67 and RepoMan bind PP1 using an identical, yet novel mechanism, interacting with a PP1 pocket that is engaged only by these two PP1 regulators. These findings not only show how two distinct mitotic exit phosphatases are recruited to their substrates, but also provide immediate opportunities for the design of novel cancer therapeutics that selectively target the Ki-67:PP1 and RepoMan:PP1 holoenzymes.

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Phosphorylation-dependent mitotic SUMOylation drives nuclear envelope–chromatin interactions

The Journal of Cell Biology ◽

10.1083/jcb.202103036 ◽

2021 ◽

Vol 220 (12) ◽

Cited By ~ 1

Author(s):

Christopher Ptak ◽

Natasha O. Saik ◽

Ashwini Premashankar ◽

Diego L. Lapetina ◽

John D. Aitchison ◽

...

Keyword(s):

Gene Expression ◽

Nuclear Envelope ◽

Spatial Organization ◽

G1 Phase ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Chromatin Binding ◽

Chromatin Interactions ◽

Sumo Ligase ◽

Pore Complexes

In eukaryotes, chromatin binding to the inner nuclear membrane (INM) and nuclear pore complexes (NPCs) contributes to spatial organization of the genome and epigenetic programs important for gene expression. In mitosis, chromatin–nuclear envelope (NE) interactions are lost and then formed again as sister chromosomes segregate to postmitotic nuclei. Investigating these processes in S. cerevisiae, we identified temporally and spatially controlled phosphorylation-dependent SUMOylation events that positively regulate postmetaphase chromatin association with the NE. Our work establishes a phosphorylation-mediated targeting mechanism of the SUMO ligase Siz2 to the INM during mitosis, where Siz2 binds to and SUMOylates the VAP protein Scs2. The recruitment of Siz2 through Scs2 is further responsible for a wave of SUMOylation along the INM that supports the assembly and anchorage of subtelomeric chromatin at the INM and localization of an active gene (INO1) to NPCs during the later stages of mitosis and into G1-phase.

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CDK1 controls CHMP7-dependent nuclear envelope reformation

eLife ◽

10.7554/elife.59999 ◽

2021 ◽

Vol 10 ◽

Author(s):

Alberto T Gatta ◽

Yolanda Olmos ◽

Caroline L Stoten ◽

Qu Chen ◽

Peter B Rosenthal ◽

...

Keyword(s):

Nuclear Envelope ◽

Cell Cycle Control ◽

Nucleocytoplasmic Transport ◽

Control Programme ◽

Mitotic Exit ◽

Microtubule Network ◽

Endosomal Sorting ◽

Protein Biochemistry ◽

M Phase ◽

Membrane Sealing

Through membrane sealing and disassembly of spindle microtubules, the Endosomal Sorting Complex Required for Transport-III (ESCRT-III) machinery has emerged as a key player in the regeneration of a sealed nuclear envelope (NE) during mitotic exit, and in the repair of this organelle during interphase rupture. ESCRT-III assembly at the NE occurs transiently during mitotic exit and is initiated when CHMP7, an ER-localised ESCRT-II/ESCRT-III hybrid protein, interacts with the Inner Nuclear Membrane (INM) protein LEM2. Whilst classical nucleocytoplasmic transport mechanisms have been proposed to separate LEM2 and CHMP7 during interphase, it is unclear how CHMP7 assembly is suppressed in mitosis when NE and ER identities are mixed. Here, we use live cell imaging and protein biochemistry to examine the biology of these proteins during mitotic exit. Firstly, we show that CHMP7 plays an important role in the dissolution of LEM2 clusters that form at the NE during M-exit. Secondly, we show that CDK1 phosphorylates CHMP7 upon mitotic entry at Ser3 and Ser441 and that this phosphorylation reduces CHMP7's interaction with LEM2, limiting its assembly during M-phase. We show that spatiotemporal differences in the dephosphorylation of CHMP7 license its assembly at the NE during telophase, but restrict its assembly on the ER at this time. Without CDK1 phosphorylation, CHMP7 undergoes inappropriate assembly in the peripheral ER during M-exit, capturing LEM2 and downstream ESCRT-III components. Lastly, we establish that a microtubule network is dispensable for ESCRT-III assembly at the reforming nuclear envelope. These data identify a key cell-cycle control programme allowing ESCRT-III-dependent nuclear regeneration.

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Disruption of Centrosome Structure, Chromosome Segregation, and Cytokinesis by Misexpression of Human Cdc14A Phosphatase

Molecular Biology of the Cell ◽

10.1091/mbc.01-11-0535 ◽

2002 ◽

Vol 13 (7) ◽

pp. 2289-2300 ◽

Cited By ~ 92

Author(s):

Brett K. Kaiser ◽

Zachary A. Zimmerman ◽

Harry Charbonneau ◽

Peter K. Jackson

Keyword(s):

Cell Cycle ◽

Phosphatase Activity ◽

Chromosome Segregation ◽

Mitotic Spindle ◽

Genomic Stability ◽

Mitotic Exit ◽

Protein Abundance ◽

Cyclin Dependent Kinase ◽

Chromosome Partitioning

In budding yeast, the Cdc14p phosphatase activates mitotic exit by dephosphorylation of specific cyclin-dependent kinase (Cdk) substrates and seems to be regulated by sequestration in the nucleolus until its release in mitosis. Herein, we have analyzed the two human homologs of Cdc14p, hCdc14A and hCdc14B. We demonstrate that the human Cdc14A phosphatase is selective for Cdk substrates in vitro and that although the protein abundance and intrinsic phosphatase activity of hCdc14A and B vary modestly during the cell cycle, their localization is cell cycle regulated. hCdc14A dynamically localizes to interphase but not mitotic centrosomes, and hCdc14B localizes to the interphase nucleolus. These distinct patterns of localization suggest that each isoform of human Cdc14 likely regulates separate cell cycle events. In addition, hCdc14A overexpression induces the loss of the pericentriolar markers pericentrin and γ-tubulin from centrosomes. Overproduction of hCdc14A also causes mitotic spindle and chromosome segregation defects, defective karyokinesis, and a failure to complete cytokinesis. Thus, the hCdc14A phosphatase appears to play a role in the regulation of the centrosome cycle, mitosis, and cytokinesis, thereby influencing chromosome partitioning and genomic stability in human cells.

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Determinants of Drosophila zw10 protein localization and function

Journal of Cell Science ◽

10.1242/jcs.107.4.785 ◽

1994 ◽

Vol 107 (4) ◽

pp. 785-798 ◽

Cited By ~ 13

Author(s):

B.C. Williams ◽

M.L. Goldberg

Keyword(s):

Chromosome Segregation ◽

Sister Chromatid ◽

Protein Localization ◽

Metaphase Plate ◽

Obvious Effect ◽

Anaphase Onset ◽

Protein Functions ◽

Kinetochore Region ◽

And Function ◽

Feedback Pathway

We have examined several issues concerning how the Drosophila l(1)zw10 gene product functions to ensure proper chromosome segregation. (a) We have found that in zw10 mutant embryos and larval neuroblasts, absence of the zw10 protein has no obvious effect on either the congression of chromosomes to the metaphase plate or the morphology of the metaphase spindle, although many aberrations are observed subsequently in anaphase. This suggests that activity of the zw10 protein becomes essential at anaphase onset, a time at which the zw10 protein is redistributed to the kinetochore region of the chromosomes. (b) The zw10 protein appears to bind to kinetochores in mitotically arrested cells, eventually accumulating to high levels within the chromosome mass. Our results imply that zw10 may act as part of a novel feedback pathway that normally renders sister chromatid separation dependent upon spindle integrity. (c) The localization of zw10 protein is altered by two mitotic mutations, rough deal and abnormal anaphase resolution, that specifically disrupt anaphase. These findings indicate that the zw10 protein functions as part of a multicomponent mechanism ensuring proper chromosome segregation at the beginning of anaphase.

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The nucleolar phosphataseCdc14Bis dispensable for chromosome segregation and mitotic exit in human cells

Cell Cycle ◽

10.4161/cc.7.9.5792 ◽

2008 ◽

Vol 7 (9) ◽

pp. 1184-1190 ◽

Cited By ~ 47

Author(s):

Eli Berdougo ◽

Maxence V. Nachury ◽

Peter K. Jackson ◽

Prasad V. Jallepalli

Keyword(s):

Chromosome Segregation ◽

Mitotic Exit ◽

Human Cells

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The RAD51 recombinase protects mitotic chromatin in human cells

Nature Communications ◽

10.1038/s41467-021-25643-y ◽

2021 ◽

Vol 12 (1) ◽

Cited By ~ 1

Author(s):

Isabel E. Wassing ◽

Emily Graham ◽

Xanita Saayman ◽

Lucia Rampazzo ◽

Christine Ralf ◽

...

Keyword(s):

Chromosome Segregation ◽

Genome Stability ◽

Spindle Assembly Checkpoint ◽

De Novo ◽

Genomic Stability ◽

Human Cells ◽

Genome Integrity ◽

Chromosomal Replication ◽

Anaphase Onset ◽

Mitotic Chromatin

AbstractThe RAD51 recombinase plays critical roles in safeguarding genome integrity, which is fundamentally important for all living cells. While interphase functions of RAD51 in maintaining genome stability are well-characterised, its role in mitosis remains contentious. In this study, we show that RAD51 protects under-replicated DNA in mitotic human cells and, in this way, promotes mitotic DNA synthesis (MiDAS) and successful chromosome segregation. In cells experiencing mild replication stress, MiDAS was detected irrespective of mitotically generated DNA damage. MiDAS broadly required de novo RAD51 recruitment to single-stranded DNA, which was supported by the phosphorylation of RAD51 by the key mitotic regulator Polo-like kinase 1. Importantly, acute inhibition of MiDAS delayed anaphase onset and induced centromere fragility, suggesting a mechanism that prevents the satisfaction of the spindle assembly checkpoint while chromosomal replication remains incomplete. This study hence identifies an unexpected function of RAD51 in promoting genomic stability in mitosis.

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Role of spindle microtubules in the control of cell cycle timing.

The Journal of Cell Biology ◽

10.1083/jcb.80.3.674 ◽

1979 ◽

Vol 80 (3) ◽

pp. 674-691 ◽

Cited By ~ 66

Author(s):

G Sluder

Keyword(s):

Cell Cycle ◽

Nuclear Envelope ◽

Sea Urchin ◽

Control Cell ◽

Microtubule Assembly ◽

Nuclear Envelope Breakdown ◽

Anaphase Onset ◽

Spindle Cells ◽

Spindle Microtubules

Sea urchin eggs are used to investigate the involvement of spindle microtubules in the mechanisms that control the timing of cell cycle events. Eggs are treated for 4 min with Colcemid at prophase of the first mitosis. No microtubules are assembled for at least 3 h, and the eggs do not divide. These eggs show repeated cycles of nuclear envelope breakdown (NEB) and nuclear envelope reformation (NER). Mitosis (NEB to NER) is twice as long in Colcemid-treated eggs as in the untreated controls. Interphase (NER to NEB) is the same in both. Thus, each cycle is prolonged entirely in mitosis. The chromosomes of treated eggs condense and eventually split into separate chromatids which do not move apart. This "canaphase" splitting is substantially delayed relative to anaphase onset in the control eggs. Treated eggs are irradiated after NEB with 366-nm light to inactivate the Colcemid. This allows the eggs to assemble normal spindles and divide. Up to 14 min after NEB, delays in the start of microtubule assembly give equal delays in anaphase onset, cleavage, and the events of the following cell cycle. Regardless of the delay, anaphase follows irradiation by the normal prometaphase duration. The quantity of spindle microtubules also influences the timing of mitotic events. Short Colcemid treatments administered in prophase of second division cause eggs to assemble small spindles. One blastomere is irradiated after NEB to provide a control cell with a normal-sized spindle. Cells with diminished spindles always initiate anaphase later than their controls. Telophase events are correspondingly delayed. This work demonstrates that spindle microtubules are involved in the mechanisms that control the time when the cell will initiate anaphase, finish mitosis, and start the next cell cycle.

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