CDK1 controls CHMP7-dependent nuclear envelope reformation

AbstractThrough the process of annular fusion 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 during mitotic exit, and in the repair of this organelle during interphase rupture. ESCRT-III polymerisation at the nuclear envelope occurs transiently during mitotic exit and CHMP7, an ER-localised ESCRT-II/ESCRT-III hybrid protein, initiates assembly in a manner dependent upon the 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. We show that CHMP7 plays an important role in disaggregating LEM2 clusters at the reforming nuclear envelope during mitotic exit to allow proper nuclear regeneration. Further, we show that CDK1 phosphorylates CHMP7 upon mitotic entry and suppresses its polymerisation, preventing its inappropriate capture of LEM2 in the peripheral ER during mitotic exit. 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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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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Membrane binding by CHMP7 coordinates ESCRT-III dependent nuclear envelope reformation

10.1101/049221 ◽

2016 ◽

Cited By ~ 1

Author(s):

Yolanda Olmos ◽

Anna Perdrix ◽

Jeremy G Carlton

Keyword(s):

Nuclear Envelope ◽

Membrane Binding ◽

Point Mutations ◽

Binding Activity ◽

Mitotic Exit ◽

Exit Point ◽

Cellular Functions ◽

Endosomal Sorting ◽

N Terminus ◽

And Function

AbstractAmongst other cellular functions, the Endosomal Sorting Complex Required for Transport-III (ESCRT-III) machinery controls nuclear envelope (NE) reformation during mitotic exit by sealing holes in the reforming NE. ESCRT-III also acts to repair this organelle upon migration-induced rupture. The ESCRT-III component CHMP7 is responsible for recruitment of ESCRT-III to the NE. Here, we show that the N-terminus of CHMP7, comprising tandem Winged Helix (WH)-domains, is a membrane-binding module. This activity allows CHMP7 to bind to the Endoplasmic Reticulum (ER), an organelle continuous with the NE, and provides a platform to direct NE-recruitment of ESCRT-III during mitotic exit. Point mutations that disrupt membrane-binding prevent CHMP7 localising to the ER and its subsequent enrichment at the reforming NE. These mutations prevent both assembly of downstream ESCRT-III components at the reforming NE and proper establishment of post-mitotic nucleo-cytoplasmic compartmentalisation. These data identify a novel membrane-binding activity within an ESCRT-III subunit that is essential for post-mitotic nuclear regeneration.One Sentence SummaryCHMP7’s atypical N-terminus is a membrane-binding module that allows assembly and function of ESCRT-III at the nuclear envelope during mitotic exit.

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Mutational analyses of fs(1)Ya, an essential, developmentally regulated, nuclear envelope protein in Drosophila.

Genetics ◽

10.1093/genetics/141.4.1473 ◽

1995 ◽

Vol 141 (4) ◽

pp. 1473-1481 ◽

Cited By ~ 1

Author(s):

J Liu ◽

K Song ◽

M F Wolfner

Keyword(s):

Cell Cycle ◽

Embryonic Development ◽

Nuclear Envelope ◽

Envelope Protein ◽

Chromatin Condensation ◽

Cell Cycle Control ◽

Nuclear Lamina ◽

Mitotic Division ◽

Developmentally Regulated ◽

Egg Maturation

Abstract The fs(1)Ya protein (YA) is an essential, maternally encoded, nuclear lamina protein that is under both developmental and cell cycle control. A strong Ya mutation results in early arrest of embryos. To define the function of YA in the nuclear envelope during early embryonic development, we characterized the phenotypes of four Ya mutants alleles and determined their molecular lesions. Ya mutant embryos arrest with abnormal nuclear envelopes prior to the first mitotic division; a proportion of embryos from two leaky Ya mutants proceed beyond this but arrest after several abnormal divisions. Ya unfertilized eggs contain nuclei of different sizes and condensation states, apparently due to abnormal fusion of the meiotic products immediately after meiosis. Lamin is localized at the periphery of the uncondensed nuclei in these eggs. These results suggest that YA function is required during and after egg maturation to facilitate proper chromatin condensation, rather than to allow a lamin-containing nuclear envelope to form. Two leaky Ya alleles that partially complement have lesions at opposite ends of the YA protein, suggesting that the N- and C-termini are important for YA function and that YA might interact with itself either directly or indirectly.

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Regulating chromosomal movement by the cochaperone FKB-6 ensures timely pairing and synapsis

The Journal of Cell Biology ◽

10.1083/jcb.201606126 ◽

2017 ◽

Vol 216 (2) ◽

pp. 393-408 ◽

Cited By ~ 7

Author(s):

Benjamin Alleva ◽

Nathan Balukoff ◽

Amy Peiper ◽

Sarit Smolikove

Keyword(s):

Nuclear Envelope ◽

Chromosome Pairing ◽

Meiotic Prophase ◽

Homologous Chromosome ◽

Strand Break ◽

Crossing Over ◽

Chromosome Movement ◽

Microtubule Network ◽

Homologous Chromosome Pairing ◽

Proper Movement

In meiotic prophase I, homologous chromosome pairing is promoted through chromosome movement mediated by nuclear envelope proteins, microtubules, and dynein. After proper homologue pairing has been established, the synaptonemal complex (SC) assembles along the paired homologues, stabilizing their interaction and allowing for crossing over to occur. Previous studies have shown that perturbing chromosome movement leads to pairing defects and SC polycomplex formation. We show that FKB-6 plays a role in SC assembly and is required for timely pairing and proper double-strand break repair kinetics. FKB-6 localizes outside the nucleus, and in its absence, the microtubule network is altered. FKB-6 is required for proper movement of dynein, increasing resting time between movements. Attenuating chromosomal movement in fkb-6 mutants partially restores the defects in synapsis, in agreement with FKB-6 acting by decreasing chromosomal movement. Therefore, we suggest that FKB-6 plays a role in regulating dynein movement by preventing excess chromosome movement, which is essential for proper SC assembly and homologous chromosome pairing.

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Computational modelling of mitotic exit in budding yeast: the role of separase and Cdc14 endocycles

Journal of The Royal Society Interface ◽

10.1098/rsif.2010.0649 ◽

2011 ◽

Vol 8 (61) ◽

pp. 1128-1141 ◽

Cited By ~ 20

Author(s):

P. K. Vinod ◽

Paula Freire ◽

Ahmed Rattani ◽

Andrea Ciliberto ◽

Frank Uhlmann ◽

...

Keyword(s):

Regulatory Networks ◽

Budding Yeast ◽

Cell Cycle Control ◽

Computational Modelling ◽

Eukaryotic Cell ◽

Mitotic Exit ◽

Intuitive Reasoning ◽

Systematic Analysis ◽

Systems View

The operating principles of complex regulatory networks are best understood with the help of mathematical modelling rather than by intuitive reasoning. Hereby, we study the dynamics of the mitotic exit (ME) control system in budding yeast by further developing the Queralt's model. A comprehensive systems view of the network regulating ME is provided based on classical experiments in the literature. In this picture, Cdc20–APC is a critical node controlling both cyclin (Clb2 and Clb5) and phosphatase (Cdc14) branches of the regulatory network. On the basis of experimental situations ranging from single to quintuple mutants, the kinetic parameters of the network are estimated. Numerical analysis of the model quantifies the dependence of ME control on the proteolytic and non-proteolytic functions of separase. We show that the requirement of the non-proteolytic function of separase for ME depends on cyclin-dependent kinase activity. The model is also used for the systematic analysis of the recently discovered Cdc14 endocycles. The significance of Cdc14 endocycles in eukaryotic cell cycle control is discussed as well.

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In Vivo Dynamics of Nuclear Pore Complexes in Yeast

The Journal of Cell Biology ◽

10.1083/jcb.136.6.1185 ◽

1997 ◽

Vol 136 (6) ◽

pp. 1185-1199 ◽

Cited By ~ 82

Author(s):

Mirella Bucci ◽

Susan R. Wente

Keyword(s):

Nuclear Envelope ◽

Recipient Cell ◽

Nucleocytoplasmic Transport ◽

Yeast Cells ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Central Location ◽

Nuclear Membranes ◽

Mutant Strains ◽

Pore Complexes

While much is known about the role of nuclear pore complexes (NPCs) in nucleocytoplasmic transport, the mechanism of NPC assembly into pores formed through the double lipid bilayer of the nuclear envelope is not well defined. To investigate the dynamics of NPCs, we developed a live-cell assay in the yeast Saccharomyces cerevisiae. The nucleoporin Nup49p was fused to the green fluorescent protein (GFP) of Aequorea victoria and expressed in nup49 null haploid yeast cells. When the GFP–Nup49p donor cell was mated with a recipient cell harboring only unlabeled Nup49p, the nuclei fused as a consequence of the normal mating process. By monitoring the distribution of the GFP–Nup49p, we could assess whether NPCs were able to move from the donor section of the nuclear envelope to that of the recipient nucleus. We observed that fluorescent NPCs moved and encircled the entire nucleus within 25 min after fusion. When assays were done in mutant kar1-1 strains, where nuclear fusion does not occur, GFP–Nup49p appearance in the recipient nucleus occurred at a very slow rate, presumably due to new NPC biogenesis or to exchange of GFP– Nup49p into existing recipient NPCs. Interestingly, in a number of existing mutant strains, NPCs are clustered together at permissive growth temperatures. This has been explained with two different hypotheses: by movement of NPCs through the double nuclear membranes with subsequent clustering at a central location; or, alternatively, by assembly of all NPCs at a central location (such as the spindle pole body) with NPCs in mutant cells unable to move away from this point. Using the GFP–Nup49p system with a mutant in the NPCassociated factor Gle2p that exhibits formation of NPC clusters only at 37°C, it was possible to distinguish between these two models for NPC dynamics. GFP– Nup49p-labeled NPCs, assembled at 23°C, moved into clusters when the cells were shifted to growth at 37°C. These results indicate that NPCs can move through the double nuclear membranes and, moreover, can do so to form NPC clusters in mutant strains. Such clusters may result by releasing NPCs from a nuclear tether, or by disappearance of a protein that normally prevents pore aggregation. This system represents a novel approach for identifying regulators of NPC assembly and movement in the future.

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Characterization of Cep135, a novel coiled-coil centrosomal protein involved in microtubule organization in mammalian cells

The Journal of Cell Biology ◽

10.1083/jcb.200108088 ◽

2002 ◽

Vol 156 (1) ◽

pp. 87-100 ◽

Cited By ~ 94

Author(s):

Toshiro Ohta ◽

Russell Essner ◽

Jung-Hwa Ryu ◽

Robert E. Palazzo ◽

Yumi Uetake ◽

...

Keyword(s):

Mammalian Cells ◽

Coiled Coil ◽

Amino Acid Residues ◽

Protein Overexpression ◽

Microtubule Organization ◽

Microtubule Network ◽

Centrosomal Protein ◽

Wide Range ◽

Spindle Microtubules

By using monoclonal antibodies raised against isolated clam centrosomes, we have identified a novel 135-kD centrosomal protein (Cep135), present in a wide range of organisms. Cep135 is located at the centrosome throughout the cell cycle, and localization is independent of the microtubule network. It distributes throughout the centrosomal area in association with the electron-dense material surrounding centrioles. Sequence analysis of cDNA isolated from CHO cells predicted a protein of 1,145–amino acid residues with extensive α-helical domains. Expression of a series of deletion constructs revealed the presence of three independent centrosome-targeting domains. Overexpression of Cep135 resulted in the accumulation of unique whorl-like particles in both the centrosome and the cytoplasm. Although their size, shape, and number varied according to the level of protein expression, these whorls were composed of parallel dense lines arranged in a 6-nm space. Altered levels of Cep135 by protein overexpression and/or suppression of endogenous Cep135 by RNA interference caused disorganization of interphase and mitotic spindle microtubules. Thus, Cep135 may play an important role in the centrosomal function of organizing microtubules in mammalian cells.

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Monoclonal antibodies identify a group of nuclear pore complex glycoproteins.

The Journal of Cell Biology ◽

10.1083/jcb.104.5.1143 ◽

1987 ◽

Vol 104 (5) ◽

pp. 1143-1156 ◽

Cited By ~ 314

Author(s):

C M Snow ◽

A Senior ◽

L Gerace

Keyword(s):

Monoclonal Antibodies ◽

Nuclear Envelope ◽

Nuclear Pore Complex ◽

Peptide Mapping ◽

Polyclonal Antibodies ◽

Integral Membrane Protein ◽

Nucleocytoplasmic Transport ◽

Nuclear Pore ◽

Nuclear Surface ◽

Punctate Pattern

Using monoclonal antibodies we identified a group of eight polypeptides of rat liver nuclear envelopes that have common epitopes. Most or all of these proteins are structurally distinct, as shown by tryptic peptide mapping and analysis with polyclonal antibodies. While these polypeptides are relatively tightly bound to nuclear membranes, only one is an integral membrane protein. The eight antigens cofractionate with the nuclear pore complex under various conditions of ionic strength and detergent. It can be seen by immunofluorescence microscopy that the monoclonal antibodies reacting with these antigens stain the nuclear surface of interphase cells in a finely punctate pattern. When the nuclear envelope is disassembled and subsequently reformed during mitosis, the proteins are reversibly dispersed throughout the cytoplasm in the form of minute foci. By EM immunogold localization on isolated nuclear envelopes, the monoclonal antibodies label exclusively the nuclear pore complex, at both its nucleoplasmic and cytoplasmic margins. Considered together, our biochemical and localization data indicate that the eight nuclear envelope polypeptides are pore complex components. As shown in the accompanying paper (Holt, G. D., C. M. Snow, A. Senior, R. S. Haltiwanger, L. Gerace, and G. W. Hart, J. Cell Biol., 104:1157-1164) these eight polypeptides contain a novel form of glycosylation, O-linked N-acetylglucosamine. The relative abundance and disposition of these O-linked glycoproteins in the pore complex are consistent with their having a role in nucleocytoplasmic transport.

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Nuclear Channels in Hela Cells: Nucleocytoplasmic Transport or Reassembly of the Nuclear Envelope?

Microscopy and Microanalysis ◽

10.1017/s1431927600029275 ◽

2001 ◽

Vol 7 (S2) ◽

pp. 640-641

Author(s):

J.R. Palisano ◽

E.K. Traister

Keyword(s):

Nuclear Envelope ◽

Hela Cells ◽

Fluorescent Microscopy ◽

Calf Serum ◽

Cancer Cell Line ◽

Nucleocytoplasmic Transport ◽

Culture Collection ◽

Cervical Cancer Cell Line ◽

Electron Microscopic ◽

And Function

Although nuclear channels (NC) have been described in a variety of cells, their function has not been fully investigated. This investigation used immunofluorescent techniques to examine the formation and function of NC in HeLa cells, a human cervical cancer cell line. NC are defined as invaginations of the nuclear envelope (NE) that traverse the nucleoplasm, thus forming a ring-like nucleus. The resulting tunnel is devoid of nucleic acid and may contain cytoplasmic elements. to date, all the reports of the presence of NC are from electron microscopic investigations, which clearly demonstrate the occurrence of NC in the presence of what appear to be interphase nuclei. This investigation was initiated because the NC that we observed in HeLa cells could be seen with fluorescent microscopy and only appeared during telophase and early interphase.HeLa cells, obtained from American Type Culture Collection, were cultured on glass coverslips in minimum essential medium (MEM) containing 5% calf serum (CS) and a mixture of 5% CO2 in air at 37°C.

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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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