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1.
Nat Rev Mol Cell Biol ; 21(3): 151-166, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32034394

RESUMO

During division, eukaryotic cells undergo a dramatic, complex and coordinated remodelling of their cytoskeleton and membranes. For cell division to occur, chromosomes must be segregated and new cellular structures, such as the spindle apparatus, must be assembled. Pre-existing organelles, such as the nuclear envelope, endoplasmic reticulum and Golgi apparatus, must be disassembled or remodelled, distributed and reformed. Smaller organelles such as mitochondria as well as cytoplasmic content must also be properly distributed between daughter cells. This mixture of organelles and cytoplasm is bound by a plasma membrane that is itself subject to remodelling as division progresses. The lipids resident in these different membrane compartments play important roles in facilitating the division process. In recent years, we have begun to understand how membrane remodelling is coordinated during division; however, there is still much to learn. In this Review, we discuss recent insights into how these important cellular events are performed and regulated.


Assuntos
Divisão Celular/fisiologia , Membranas/metabolismo , Organelas/fisiologia , Animais , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Células Eucarióticas/citologia , Complexo de Golgi/metabolismo , Humanos , Membranas/fisiologia , Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Organelas/metabolismo , Fuso Acromático/metabolismo
2.
Nat Commun ; 11(1): 24, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31911655

RESUMO

The spindle assembly checkpoint (SAC) prevents premature chromosome segregation by inactivating the anaphase promoting complex/cyclosome (APC/C) until all chromosomes are properly attached to mitotic spindles. Here we identify a role for Cullin-RING ubiquitin ligase complex 4 (CRL4), known for modulating DNA replication, as a crucial mitotic regulator that triggers the termination of the SAC and enables chromosome segregation. CRL4 is recruited to chromatin by the replication origin binding protein RepID/DCAF14/PHIP. During mitosis, CRL4 dissociates from RepID and replaces it with RB Binding Protein 7 (RBBP7), which ubiquitinates the SAC mediator BUB3 to enable mitotic exit. During interphase, BUB3 is protected from CRL4-mediated degradation by associating with promyelocytic leukemia (PML) nuclear bodies, ensuring its availability upon mitotic onset. Deficiencies in RepID, CRL4 or RBBP7 delay mitotic exit, increase genomic instability and enhance sensitivity to paclitaxel, a microtubule stabilizer and anti-tumor drug.


Assuntos
Anáfase , Proteínas de Ciclo Celular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Metáfase , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mitose , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteína da Leucemia Promielocítica/genética , Proteína da Leucemia Promielocítica/metabolismo , Ligação Proteica , Proteólise , Proteína 7 de Ligação ao Retinoblastoma/genética , Proteína 7 de Ligação ao Retinoblastoma/metabolismo , Fuso Acromático/metabolismo , Ubiquitina-Proteína Ligases/genética
3.
Nat Commun ; 11(1): 277, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31937754

RESUMO

Migration of meiosis-I (MI) spindle from the cell center to a sub-cortical location is a critical step for mouse oocytes to undergo asymmetric meiotic cell division. In this study, we investigate the mechanism by which formin-2 (FMN2) orchestrates the initial movement of MI spindle. By defining protein domains responsible for targeting FMN2, we show that spindle-periphery localized FMN2 is required for spindle migration. The spindle-peripheral FMN2 nucleates short actin bundles from vesicles derived likely from the endoplasmic reticulum (ER) and concentrated in a layer outside the spindle. This layer is in turn surrounded by mitochondria. A model based on polymerizing actin filaments pushing against mitochondria, thus generating a counter force on the spindle, demonstrated an inherent ability of this system to break symmetry and evolve directional spindle motion. The model is further supported through experiments involving spatially biasing actin nucleation via optogenetics and disruption of mitochondrial distribution and dynamics.


Assuntos
Actinas/metabolismo , Meiose , Oócitos/citologia , Organelas/metabolismo , Fuso Acromático/metabolismo , Animais , Divisão Celular Assimétrica , Vesículas Citoplasmáticas/metabolismo , Retículo Endoplasmático/metabolismo , Feminino , Forminas/química , Forminas/genética , Forminas/metabolismo , Camundongos , Mitocôndrias/metabolismo , Modelos Biológicos , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Oócitos/metabolismo , Domínios Proteicos , Deleção de Sequência
4.
Nat Commun ; 11(1): 270, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31937751

RESUMO

Phase separation of substrates and effectors is proposed to enhance biological reaction rates and efficiency. Targeting protein for Xklp2 (TPX2) is an effector of branching microtubule nucleation in spindles and functions with the substrate tubulin by an unknown mechanism. Here we show that TPX2 phase separates into a co-condensate with tubulin, which mediates microtubule nucleation in vitro and in isolated cytosol. TPX2-tubulin co-condensation preferentially occurs on pre-existing microtubules, the site of branching microtubule nucleation, at the endogenous and physiologically relevant concentration of TPX2. Truncation and chimera versions of TPX2 suggest that TPX2-tubulin co-condensation enhances the efficiency of TPX2-mediated branching microtubule nucleation. Finally, the known inhibitor of TPX2, the importin-α/ß heterodimer, regulates TPX2 condensation in vitro and, consequently, branching microtubule nucleation activity in isolated cytosol. Our study demonstrates how regulated phase separation can simultaneously enhance reaction efficiency and spatially coordinate microtubule nucleation, which may facilitate rapid and accurate spindle formation.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Proteínas de Ciclo Celular/química , Citosol/metabolismo , Carioferinas/metabolismo , Meiose , Proteínas Associadas aos Microtúbulos/química , Centro Organizador dos Microtúbulos/metabolismo , Óvulo/citologia , Óvulo/metabolismo , Fuso Acromático/metabolismo , Proteínas de Xenopus/química , Xenopus laevis
5.
Biochim Biophys Acta Mol Cell Res ; 1867(4): 118636, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31884069

RESUMO

Kinesin-6 KIF20A is essential for microtubule organization and central spindle assembly during cytokinesis. However, the functions of KIF20A in meiotic division and spermatogenesis remain elusive. Here, we report that kinesin-6 KIF20A locates at the microtubules in mouse spermatogenic cells and co-localizes with the spindle midzone and midbody. We demonstrate that central spindle organization and chromosomal stability are regulated by KIF20A in male meiotic division. KIF20A inhibition leads to the defects in central spindle assembly and cytokinetic abscission, and finally results in the increase of aneuploid cells and the alteration of cell populations in the spermatogenic cells. Furthermore, we have revealed that kinesin-6 KIF20A is associated with the formation and maturation of the acrosomes during spermatogenesis. Our findings have identified the specific roles of KIF20A in central spindle organization in meiotic division.


Assuntos
Acrossomo/metabolismo , Cinesina/metabolismo , Espermatogênese , Fuso Acromático/metabolismo , Animais , Células Cultivadas , Células HeLa , Humanos , Cinesina/genética , Masculino , Camundongos , Camundongos Endogâmicos ICR
6.
BMC Mol Cell Biol ; 20(1): 58, 2019 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-31823729

RESUMO

BACKGROUND: Astral microtubules emanating from the mitotic centrosomes play pivotal roles in defining cell division axis and tissue morphogenesis. Previous studies have demonstrated that human transforming acidic coiled-coil 3 (TACC3), the most conserved TACC family protein, regulates formation of astral microtubules at centrosomes in vertebrate cells by affecting γ-tubulin ring complex (γ-TuRC) assembly. However, the molecular mechanisms underlying such function were not completely understood. RESULTS: Here, we show that Aurora A site-specific phosphorylation in TACC3 regulates formation of astral microtubules by stabilizing γ-TuRC assembly in human cells. Mutation of the most conserved Aurora A targeting site, Ser 558 to alanine (S558A) in TACC3 results in robust loss of astral microtubules and disrupts localization of the γ-tubulin ring complex (γ-TuRC) proteins at the spindle poles. Under similar condition, phospho-mimicking S558D mutation retains astral microtubules and the γ-TuRC proteins in a manner similar to control cells expressed with wild type TACC3. Time-lapse imaging reveals that S558A mutation leads to defects in positioning of the spindle-poles and thereby causes delay in metaphase to anaphase transition. Biochemical results determine that the Ser 558- phosphorylated TACC3 interacts with the γ-TuRC proteins and further, S558A mutation impairs the interaction. We further reveal that the mutation affects the assembly of γ-TuRC from the small complex components. CONCLUSIONS: The results demonstrate that TACC3 phosphorylation stabilizes γ- tubulin ring complex assembly and thereby regulates formation of centrosomal asters. They also implicate a potential role of TACC3 phosphorylation in the functional integrity of centrosomes/spindle poles.


Assuntos
Aurora Quinase A/metabolismo , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Motivos de Aminoácidos , Aurora Quinase A/genética , Centrossomo/metabolismo , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/genética , Fosforilação , Fuso Acromático/genética , Fuso Acromático/metabolismo , Tubulina (Proteína)/genética
7.
Elife ; 82019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31794381

RESUMO

Occludin (OCLN) mutations cause human microcephaly and cortical malformation. A tight junction component thought absent in neuroepithelium after neural tube closure, OCLN isoform-specific expression extends into corticogenesis. Full-length and truncated isoforms localize to neuroprogenitor centrosomes, but full-length OCLN transiently localizes to plasma membranes while only truncated OCLN continues at centrosomes throughout neurogenesis. Mimicking human mutations, full-length OCLN depletion in mouse and in human CRISPR/Cas9-edited organoids produce early neuronal differentiation, reduced progenitor self-renewal and increased apoptosis. Human neural progenitors were more severely affected, especially outer radial glial cells, which mouse embryonic cortex lacks. Rodent and human mutant progenitors displayed reduced proliferation and prolonged M-phase. OCLN interacted with mitotic spindle regulators, NuMA and RAN, while full-length OCLN loss impaired spindle pole morphology, astral and mitotic microtubule integrity. Thus, early corticogenesis requires full-length OCLN to regulate centrosome organization and dynamics, revealing a novel role for this tight junction protein in early brain development.


Assuntos
Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/metabolismo , Ocludina/metabolismo , Junções Íntimas/metabolismo , Aneuploidia , Animais , Apoptose , Sistemas CRISPR-Cas , Diferenciação Celular , Proliferação de Células , Centrossomo/metabolismo , Córtex Cerebral/patologia , Modelos Animais de Doenças , Edição de Genes , Humanos , Camundongos , Camundongos Knockout , Microcefalia/genética , Microcefalia/patologia , Microtúbulos/metabolismo , Mutagênese , Mutação , Neurogênese/genética , Neurogênese/fisiologia , Ocludina/genética , Fuso Acromático/metabolismo , Junções Íntimas/genética
8.
PLoS Biol ; 17(11): e3000531, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31682603

RESUMO

Recycling endosomes regulate plasma membrane recycling. Recently, recycling endosome-associated proteins have been implicated in the positioning and orientation of the mitotic spindle and cytokinesis. Loss of MYO5B, encoding the recycling endosome-associated myosin Vb, is associated with tumor development and tissue architecture defects in the gastrointestinal tract. Whether loss of MYO5B expression affects mitosis is not known. Here, we demonstrate that loss of MYO5B expression delayed cytokinesis, perturbed mitotic spindle orientation, led to the misorientation of the plane of cell division during the course of mitosis, and resulted in the delamination of epithelial cells. Remarkably, the effects on spindle orientation, but not cytokinesis, were a direct consequence of physical hindrance by giant late endosomes, which were formed in a chloride channel-sensitive manner concomitant with a redistribution of chloride channels from the cell periphery to late endosomes upon loss of MYO5B. Rab7 availability was identified as a limiting factor for the development of giant late endosomes. In accordance, increasing rab7 availability corrected mitotic spindle misorientation and cell delamination in cells lacking MYO5B expression. In conclusion, we identified a novel role for MYO5B in the regulation of late endosome size control and identify the inability to control late endosome size as an unexpected novel mechanism underlying defects in cell division orientation and epithelial architecture.


Assuntos
Endossomos/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , Fuso Acromático/metabolismo , Animais , Células CACO-2 , Adesão Celular/fisiologia , Divisão Celular/fisiologia , Linhagem Celular , Membrana Celular/metabolismo , Citocinese/genética , Citocinese/fisiologia , Endossomos/genética , Células Epiteliais/metabolismo , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitose/fisiologia , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/genética , Proteínas rab de Ligação ao GTP/metabolismo
9.
Mol Cells ; 42(12): 840-849, 2019 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-31722512

RESUMO

The spatiotemporal mitotic processes are controlled qualitatively by phosphorylation and qualitatively by ubiquitination. Although the SKP1-CUL1-F-box protein (SCF) complex and the anaphase-promoting complex/cyclosome (APC/C) mainly mediate ubiquitin-dependent proteolysis of mitotic regulators, the E3 ligase for a large portion of mitotic proteins has yet to be identified. Here, we report c-Cbl as an E3 ligase that degrades DDA3, a protein involved in spindle dynamics. Depletion of c-Cbl led to increased DDA3 protein levels, resulting in increased recruitment of Kif2a to the mitotic spindle, a concomitant reduction in spindle formation, and chromosome alignment defects. Furthermore, c-Cbl depletion induced centrosome over-duplication and centriole amplification. Therefore, we concluded that c-Cbl controls spindle dynamics and centriole duplication through its E3 ligase activity against DDA3.


Assuntos
Centríolos/metabolismo , Mitose , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Fuso Acromático/metabolismo , Ciclo Celular , Centrossomo/metabolismo , Células HeLa , Humanos , Cinesina/metabolismo , Microtúbulos/metabolismo , Proteínas Proto-Oncogênicas c-cbl/genética , RNA Interferente Pequeno , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
10.
Nat Commun ; 10(1): 4513, 2019 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-31586073

RESUMO

The midbody is an organelle assembled at the intercellular bridge between the two daughter cells at the end of mitosis. It controls the final separation of the daughter cells and has been involved in cell fate, polarity, tissue organization, and cilium and lumen formation. Here, we report the characterization of the intricate midbody protein-protein interaction network (interactome), which identifies many previously unknown interactions and provides an extremely valuable resource for dissecting the multiple roles of the midbody. Initial analysis of this interactome revealed that PP1ß-MYPT1 phosphatase regulates microtubule dynamics in late cytokinesis and de-phosphorylates the kinesin component MKLP1/KIF23 of the centralspindlin complex. This de-phosphorylation antagonizes Aurora B kinase to modify the functions and interactions of centralspindlin in late cytokinesis. Our findings expand the repertoire of PP1 functions during mitosis and indicate that spatiotemporal changes in the distribution of kinases and counteracting phosphatases finely tune the activity of cytokinesis proteins.


Assuntos
Citocinese/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Mapas de Interação de Proteínas/fisiologia , Proteína Fosfatase 1/metabolismo , Aurora Quinase B/metabolismo , Sítios de Ligação/genética , Células HeLa , Humanos , Microscopia Intravital , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/metabolismo , Mitose/fisiologia , Mutagênese Sítio-Dirigida , Fosforilação/fisiologia , Proteína Fosfatase 1/genética , RNA Interferente Pequeno/metabolismo , Fuso Acromático/metabolismo , Imagem com Lapso de Tempo
11.
Dev Cell ; 51(3): 313-325.e10, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31588029

RESUMO

In the eukaryotic cell cycle, a threshold level of cyclin B accumulation triggers the G2-to-M transition, and subsequent cyclin B destruction triggers mitotic exit. The anaphase-promoting complex/cyclosome (APC/C) is the E3 ubiquitin ligase that, together with its co-activator Cdc20, targets cyclin B for destruction during mitotic exit. Here, we show that two pathways act in concert to protect cyclin B from Cdc20-activated APC/C in G2, in order to enable cyclin B accumulation and the G2-to-M transition. The first pathway involves the Mad1-Mad2 spindle checkpoint complex, acting in a distinct manner from checkpoint signaling after mitotic entry but employing a common molecular mechanism-the promotion of Mad2-Cdc20 complex formation. The second pathway involves cyclin-dependent kinase phosphorylation of Cdc20, which is known to reduce Cdc20's affinity for the APC/C. Cooperation of these two mechanisms, which target distinct APC/C binding interfaces of Cdc20, enables cyclin B accumulation and the G2-to-M transition.


Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas Cdc20/metabolismo , Ciclina B/metabolismo , Fase G2 , Mitose , Proteólise , Animais , Proteínas de Caenorhabditis elegans/metabolismo , Linhagem Celular Tumoral , Quinases Ciclina-Dependentes/metabolismo , Fertilidade , Humanos , Modelos Biológicos , Fosforilação , Ligação Proteica , Fuso Acromático/metabolismo
12.
Int J Mol Sci ; 20(20)2019 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-31640125

RESUMO

Gestational diabetes mellitus (GDM) is a type of unbalanced glucose tolerance that occurs during pregnancy, which affects approximately 10% of pregnancies worldwide. Fetuin-A is associated with insulin resistance, and the concentration of circulating fetuin-A increases in women with GDM, however, the role of fetuin-A in the placenta remains unclear. In this study, we enrolled placental samples from twenty pregnant women with GDM and twenty non-GDM pregnant women and found that the abundance of fetuin-A was upregulated in terms of mRNA and protein levels. Fetuin-A inhibited placental cell growth by inducing apoptosis and inhibiting S phase entry. Irregular alignment of mitotic chromosomes and aberrant mitotic spindle poles were observed. In addition, centrosome amplification was induced by fetuin-A treatment, and these amplified centrosomes nucleated microtubules with disorganized microtubule arrays in placental cells. Furthermore, fetuin-A inhibited autophagy, and thus blocked the growth of the primary cilium, a cellular antenna that regulates placenta development and differentiation. Thus, our study uncovered the novel function of fetuin-A in regulating placental cell growth and ciliogenesis.


Assuntos
Diabetes Gestacional/metabolismo , Placenta/metabolismo , Placentação , alfa-2-Glicoproteína-HS/genética , alfa-2-Glicoproteína-HS/metabolismo , Adulto , Apoptose , Estudos de Casos e Controles , Diferenciação Celular , Células Cultivadas , Centrossomo/metabolismo , Diabetes Gestacional/genética , Feminino , Humanos , Idade Materna , Placenta/citologia , Gravidez , Fuso Acromático/metabolismo , Regulação para Cima
13.
Nat Commun ; 10(1): 4651, 2019 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-31604948

RESUMO

Mammalian oocytes assemble a bipolar acentriolar microtubule spindle to segregate chromosomes during asymmetric division. There is increasing evidence that actin in the spindle interior not only participates in spindle migration and positioning but also protects oocytes from chromosome segregation errors leading to aneuploidy. Here we show that actin is an integral component of the meiotic machinery that closely interacts with microtubules during all major events of human oocyte maturation from the time point of spindle assembly till polar body extrusion and metaphase arrest. With the aid of drugs selectively affecting cytoskeleton dynamics and transiently disturbing the integrity of the two cytoskeleton systems, we identify interdependent structural rearrangements indicative of a close communication between actin and microtubules as fundamental feature of human oocytes. Our data support a model of actin-microtubule interplay that is essential for bipolar spindle assembly and correct partitioning of the nuclear genome in human oocyte meiosis.


Assuntos
Actinas/fisiologia , Segregação de Cromossomos/fisiologia , Oócitos/citologia , Fuso Acromático/metabolismo , Feminino , Humanos , Meiose , Microtúbulos/fisiologia , Oócitos/ultraestrutura , Corpos Polares/citologia , Corpos Polares/metabolismo , Corpos Polares/ultraestrutura , Fuso Acromático/ultraestrutura , Tubulina (Proteína)/metabolismo
14.
Int J Mol Sci ; 20(20)2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31618856

RESUMO

Proper bipolar spindle assembly underlies accurate chromosome segregation. A cohort of microtubule-associated proteins orchestrates spindle microtubule formation in a spatiotemporally coordinated manner. Among them, the conserved XMAP215/TOG family of microtubule polymerase plays a central role in spindle assembly. In fission yeast, two XMAP215/TOG members, Alp14 and Dis1, share essential roles in cell viability; however how these two proteins functionally collaborate remains undetermined. Here we show the functional interplay and specification of Alp14 and Dis1. Creation of new mutant alleles of alp14, which display temperature sensitivity in the absence of Dis1, enabled us to conduct detailed analyses of a double mutant. We have found that simultaneous inactivation of Alp14 and Dis1 results in early mitotic arrest with very short, fragile spindles. Intriguingly, these cells often undergo spindle collapse, leading to a lethal "cut" phenotype. By implementing an artificial targeting system, we have shown that Alp14 and Dis1 are not functionally exchangeable and as such are not merely redundant paralogues. Interestingly, while Alp14 promotes microtubule nucleation, Dis1 does not. Our results uncover that the intrinsic specification, not the spatial regulation, between Alp14 and Dis1 underlies the collaborative actions of these two XMAP215/TOG members in mitotic progression, spindle integrity and genome stability.


Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/fisiologia , Cinetocoros/metabolismo , Mitose , Modelos Moleculares , Fenótipo , Fuso Acromático/metabolismo
15.
Crit Rev Biochem Mol Biol ; 54(4): 352-370, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31573359

RESUMO

Biophysical studies of the yeast centromere have shown that the organization of the centromeric chromatin plays a crucial role in maintaining proper tension between sister kinetochores during mitosis. While centromeric chromatin has traditionally been considered a simple spring, recent work reveals the centromere as a multifaceted, tunable shock absorber. Centromeres can differ from other regions of the genome in their heterochromatin state, supercoiling state, and enrichment of structural maintenance of chromosomes (SMC) protein complexes. Each of these differences can be utilized to alter the effective stiffness of centromeric chromatin. In budding yeast, the SMC protein complexes condensin and cohesin stiffen chromatin by forming and cross-linking chromatin loops, respectively, into a fibrous structure resembling a bottlebrush. The high density of the loops compacts chromatin while spatially isolating the tension from spindle pulling forces to a subset of the chromatin. Paradoxically, the molecular crowding of chromatin via cohesin and condensin also causes an outward/poleward force. The structure allows the centromere to act as a shock absorber that buffers the variable forces generated by dynamic spindle microtubules. Based on the distribution of SMCs from bacteria to human and the conserved distance between sister kinetochores in a wide variety of organisms (0.4 to 1 micron), we propose that the bottlebrush mechanism is the foundational principle for centromere function in eukaryotes.


Assuntos
Segregação de Cromossomos/fisiologia , Cinetocoros/fisiologia , Saccharomyces cerevisiae/fisiologia , Adenosina Trifosfatases/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Heterocromatina/metabolismo , Humanos , Microtúbulos/metabolismo , Mitose/fisiologia , Complexos Multiproteicos/metabolismo , Neoplasias/metabolismo , Filogenia , Fuso Acromático/metabolismo
16.
BMC Biol ; 17(1): 73, 2019 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-31521166

RESUMO

BACKGROUND: Spindle microtubule organization, regulated by microtubule-associated proteins, is critical for cell division. Proper organization of kinetochore fiber (K-fiber), connecting spindle poles and kinetochores, is a prerequisite for precise chromosomal alignment and faithful genetic material transmission. However, the mechanisms of K-fiber organization and dynamic maintenance are still not fully understood. RESULTS: We reveal that two previously uncharacterized coiled-coil domain proteins CCDC74A and CCDC74B (CCDC74A/B) are spindle-localized proteins in mammalian cells. They bind directly to microtubules through two separate domains and bundle microtubules both in vivo and in vitro. These functions are required for K-fiber organization, bipolar spindle formation, and chromosomal alignment. Moreover, CCDC74A/B form homodimers in vivo, and their self-association activity is necessary for microtubule bundling and K-fiber formation. CONCLUSIONS: We characterize CCDC74A and CCDC74B as microtubule-associated proteins that localize to spindles and are important K-fiber crosslinkers required for bipolar spindle formation and chromosome alignment.


Assuntos
Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Animais , Células COS , Chlorocebus aethiops , Células HEK293 , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/genética , Mitose
18.
Nat Cell Biol ; 21(9): 1138-1151, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31481795

RESUMO

One of the first steps in mitotic spindle assembly is the dissolution of the centrosome linker followed by centrosome separation driven by EG5, a tetrameric plus-end-directed member of the kinesin-5 family. However, even in the absence of the centrosome linker, the two centrosomes are kept together by an ill-defined microtubule-dependent mechanism. Here we show that KIFC3, a minus-end-directed kinesin-14, provides microtubule-based centrosome cohesion. KIFC3 forms a homotetramer that pulls the two centrosomes together via a specific microtubule network. At mitotic onset, KIFC3 activity becomes the main driving force of centrosome cohesion to prevent premature spindle formation after linker dissolution as it counteracts the increasing EG5-driven pushing forces. KIFC3 is eventually inactivated by NEver in mitosis-related Kinase 2 (NEK2) to enable EG5-driven bipolar spindle assembly. We further show that persistent centrosome cohesion in mitosis leads to chromosome mis-segregation. Our findings reveal a mechanism of spindle assembly that is evolutionary conserved from yeast to humans.


Assuntos
Centrossomo/metabolismo , Cinesina/metabolismo , Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Segregação de Cromossomos/fisiologia , Células HeLa , Humanos , Cinesina/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mitose , Quinases Relacionadas a NIMA/metabolismo
19.
Cells ; 8(8)2019 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-31370271

RESUMO

The ubiquitin-like protein SUMO is a regulator involved in most cellular mechanisms. Recent studies have discovered new modes of function for this protein. Of particular interest is the ability of SUMO to organize proteins in larger assemblies, as well as the role of SUMO-dependent ubiquitylation in their disassembly. These mechanisms have been largely described in the context of DNA repair, transcriptional regulation, or signaling, while much less is known on how SUMO facilitates organization of microtubule-dependent processes during mitosis. Remarkably however, SUMO has been known for a long time to modify kinetochore proteins, while more recently, extensive proteomic screens have identified a large number of microtubule- and spindle-associated proteins that are SUMOylated. The aim of this review is to focus on the possible role of SUMOylation in organization of the spindle and kinetochore complexes. We summarize mitotic and microtubule/spindle-associated proteins that have been identified as SUMO conjugates and present examples regarding their regulation by SUMO. Moreover, we discuss the possible contribution of SUMOylation in organization of larger protein assemblies on the spindle, as well as the role of SUMO-targeted ubiquitylation in control of kinetochore assembly and function. Finally, we propose future directions regarding the study of SUMOylation in regulation of spindle organization and examine the potential of SUMO and SUMO-mediated degradation as target for antimitotic-based therapies.


Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Fuso Acromático/metabolismo , Animais , Humanos , Proteômica , Ubiquitinação
20.
Curr Opin Cell Biol ; 60: 139-144, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31377657

RESUMO

Cells need to regulate the size and shape of their organelles for proper function. For example, the mitotic spindle adapts its size to changes in cell size over several orders of magnitude, but we lack a mechanistic understanding of how this is achieved. Here, we review our current knowledge of how small and large spindles assemble and ask which microtubule-based biophysical processes (nucleation, polymerization dynamics, transport) may be responsible for spindle size regulation. Finally, we review possible cell-scale mechanisms that put spindle size under the regulation of cell size.


Assuntos
Tamanho Celular , Fuso Acromático/metabolismo , Fenômenos Biofísicos , Microtúbulos/metabolismo , Modelos Biológicos , Polimerização
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