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1.
PLoS One ; 14(12): e0226327, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31877164

RESUMO

During mitosis, the structure of the Endoplasmic Reticulum (ER) displays a dramatic reorganization and remodeling, however, the mechanism driving these changes is poorly understood. Hairpin-containing ER transmembrane proteins that stabilize ER tubules have been identified as possible factors to promote these drastic changes in ER morphology. Recently, the Reticulon and REEP family of ER shaping proteins have been shown to heavily influence ER morphology by driving the formation of ER tubules, which are known for their close proximity with microtubules. Here, we examine the role of microtubules and other cytoskeletal factors in the dynamics of a Drosophila Reticulon, Reticulon-like 1 (Rtnl1), localization to spindle poles during mitosis in the early embryo. At prometaphase, Rtnl1 is enriched to spindle poles just prior to the ER retention motif KDEL, suggesting a possible recruitment role for Rtnl1 in the bulk localization of ER to spindle poles. Using image analysis-based methods and precise temporal injections of cytoskeletal inhibitors in the early syncytial Drosophila embryo, we show that microtubules are necessary for proper Rtnl1 localization to spindles during mitosis. Lastly, we show that astral microtubules, not microfilaments, are necessary for proper Rtnl1 localization to spindle poles, and is largely independent of the minus-end directed motor protein dynein. This work highlights the role of the microtubule cytoskeleton in Rtnl1 localization to spindles during mitosis and sheds light on a pathway towards inheritance of this major organelle.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Microtúbulos/metabolismo , Mitose , Animais , Drosophila melanogaster/metabolismo , Dineínas/metabolismo , Retículo Endoplasmático/metabolismo , Cinesina/metabolismo , Polos do Fuso/metabolismo
2.
Mol Med Rep ; 20(4): 3573-3582, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31485656

RESUMO

Microtubule­severing proteins (MTSPs) are a group of microtubule­associated proteins essential for multiple microtubule­related processes, including mitosis and meiosis. Katanin p60 ATPase­containing subunit A­like 1 (p60 katanin­like 1) is an MTSP that maintains the density of spindle microtubules at the poles in mitotic cells; however, to date, there have been no studies about its role in female meiosis. Using in vitro­matured (IVM) oocytes as a model, it was first revealed that p60 katanin­like 1 was predominant in the ovaries and oocytes, indicating its essential roles in oocyte meiosis. It was also revealed that p60 katanin­like 1 was concentrated at the spindle poles and co­localized and interacted with γ­tubulin, indicating that it may be involved in pole organization. Next, specific siRNA was used to deplete p60 katanin­like 1; the spindle organization was severely disrupted and characterized by an abnormal width:length ratio, multipolarity and extra aster microtubules out of the main spindles. Finally, it was determined that p60 katanin­like 1 knockdown retarded oocyte meiosis, reduced fertilization, and caused abnormal mitochondrial distribution. Collectively, these results indicated that p60 katanin­like 1 is essential for oocyte meiosis by ensuring the integrity of the spindle poles.


Assuntos
Katanina/metabolismo , Meiose , Microtúbulos/metabolismo , Oócitos/citologia , Polos do Fuso/metabolismo , Animais , Células Cultivadas , Feminino , Katanina/análise , Camundongos , Camundongos Endogâmicos ICR , Células NIH 3T3 , Oócitos/metabolismo , Oócitos/ultraestrutura , Polos do Fuso/ultraestrutura , Tubulina (Proteína)/análise , Tubulina (Proteína)/metabolismo
3.
Mol Biol Cell ; 30(19): 2503-2514, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31339442

RESUMO

Spindle microtubules, whose dynamics vary over time and at different locations, cooperatively drive chromosome segregation. Measurements of microtubule dynamics and spindle ultrastructure can provide insight into the behaviors of microtubules, helping elucidate the mechanism of chromosome segregation. Much work has focused on the dynamics and organization of kinetochore microtubules, that is, on the region between chromosomes and poles. In comparison, microtubules in the central-spindle region, between segregating chromosomes, have been less thoroughly characterized. Here, we report measurements of the movement of central-spindle microtubules during chromosome segregation in human mitotic spindles and Caenorhabditis elegans mitotic and female meiotic spindles. We found that these central-spindle microtubules slide apart at the same speed as chromosomes, even as chromosomes move toward spindle poles. In these systems, damaging central-spindle microtubules by laser ablation caused an immediate and complete cessation of chromosome motion, suggesting a strong coupling between central-spindle microtubules and chromosomes. Electron tomographic reconstruction revealed that the analyzed anaphase spindles all contain microtubules with both ends between segregating chromosomes. Our results provide new dynamical, functional, and ultrastructural characterizations of central-spindle microtubules during chromosome segregation in diverse spindles and suggest that central-spindle microtubules and chromosomes are strongly coupled in anaphase.


Assuntos
Segregação de Cromossomos/fisiologia , Fuso Acromático/metabolismo , Polos do Fuso/metabolismo , Anáfase/genética , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Linhagem Celular Tumoral , Segregação de Cromossomos/genética , Cromossomos/genética , Cromossomos/fisiologia , Humanos , Cinetocoros/metabolismo , Meiose/genética , Microtúbulos/metabolismo , Fuso Acromático/genética , Polos do Fuso/genética
4.
Mol Biol Cell ; 30(13): 1598-1609, 2019 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-31042116

RESUMO

The mitotic kinase, polo-like kinase 1 (PLK1), facilitates the assembly of the two mitotic spindle poles, which are required for the formation of the microtubule-based spindle that ensures appropriate chromosome distribution into the two forming daughter cells. Spindle poles are asymmetric in composition. One spindle pole contains the oldest mitotic centriole, the mother centriole, where the majority of cenexin, the mother centriole appendage protein and PLK1 binding partner, resides. We hypothesized that PLK1 activity is greater at the cenexin-positive older spindle pole. Our studies found that PLK1 asymmetrically localizes between spindle poles under conditions of chromosome misalignment, and chromosomes tend to misalign toward the oldest spindle pole in a cenexin- and PLK1-dependent manner. During chromosome misalignment, PLK1 activity is increased specifically at the oldest spindle pole, and this increase in activity is lost in cenexin-depleted cells. We propose a model where PLK1 activity elevates in response to misaligned chromosomes at the oldest spindle pole during metaphase.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Polos do Fuso/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Centríolos/metabolismo , Centrossomo/metabolismo , Cromossomos/metabolismo , Células HeLa , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/fisiologia , Humanos , Microtúbulos/metabolismo , Mitose/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Fuso Acromático/metabolismo , Polos do Fuso/enzimologia , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
5.
Cells ; 8(5)2019 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-31137886

RESUMO

The cell cycle is a series of events by which cellular components are accurately segregated into daughter cells, principally controlled by the oscillating activities of cyclin-dependent kinases (CDKs) and their co-activators. In eukaryotes, DNA replication is confined to a discrete synthesis phase while chromosome segregation occurs during mitosis. During mitosis, the chromosomes are pulled into each of the two daughter cells by the coordination of spindle microtubules, kinetochores, centromeres, and chromatin. These four functional units tie chromosomes to the microtubules, send signals to the cells when the attachment is completed and the division can proceed, and withstand the force generated by pulling the chromosomes to either daughter cell. Protein ubiquitination is a post-translational modification that plays a central role in cellular homeostasis. E3 ubiquitin ligases mediate the transfer of ubiquitin to substrate proteins determining their fate. One of the largest subfamilies of E3 ubiquitin ligases is the family of the tripartite motif (TRIM) proteins, whose dysregulation is associated with a variety of cellular processes and directly involved in human diseases and cancer. In this review we summarize the current knowledge and emerging concepts about TRIMs and their contribution to the correct regulation of cell cycle, describing how TRIMs control the cell cycle transition phases and their involvement in the different functional units of the mitotic process, along with implications in cancer progression.


Assuntos
Mitose/fisiologia , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Autofagia , Pontos de Checagem do Ciclo Celular , Centrossomo/metabolismo , Segregação de Cromossomos , Expressão Gênica , Humanos , Cinetocoros/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Polos do Fuso/metabolismo , Ubiquitina/metabolismo , Ubiquitinação
6.
Mol Biol Cell ; 30(14): 1645-1654, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31091161

RESUMO

Mitotic spindles are well known to be assembled from and dependent on microtubules. In contrast, whether actin filaments (F-actin) are required for or are even present in mitotic spindles has long been controversial. Here we have developed improved methods for simultaneously preserving F-actin and microtubules in fixed samples and exploited them to demonstrate that F-actin is indeed associated with mitotic spindles in intact Xenopus laevis embryonic epithelia. We also find that there is an "F-actin cycle," in which the distribution and organization of spindle F-actin changes over the course of the cell cycle. Live imaging using a probe for F-actin reveals that at least two pools of F-actin are associated with mitotic spindles: a relatively stable internal network of cables that moves in concert with and appears to be linked to spindles, and F-actin "fingers" that rapidly extend from the cell cortex toward the spindle and make transient contact with the spindle poles. We conclude that there is a robust endoplasmic F-actin network in normal vertebrate epithelial cells and that this network is also a component of mitotic spindles. More broadly, we conclude that there is far more internal F-actin in epithelial cells than is commonly believed.


Assuntos
Actinas/metabolismo , Epitélio/metabolismo , Fuso Acromático/metabolismo , Xenopus laevis/metabolismo , Animais , Sobrevivência Celular , Retículo Endoplasmático/metabolismo , Células Epiteliais/metabolismo , Forminas/metabolismo , Polos do Fuso/metabolismo
7.
Curr Genet ; 65(4): 851-855, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30788566

RESUMO

Saccharomyces cerevisiae has been widely used as a model system for the study of basic biological processes which are usually evolutionarily conserved from yeasts to multicellular eukaryotes. These studies are very important because they shed light on mechanisms that are altered in human diseases and help the development of new biomarkers and therapies. The mitotic spindle is a conserved apparatus that governs chromosome segregation during mitosis. Given its crucial role for genome stability and, therefore, for cell viability, its structure and function are strictly regulated. Recent findings reveal new levels of regulation in mitotic spindle dynamics and link spindle pole diversification with cell fate determination, health, disease and aging.


Assuntos
Envelhecimento/genética , Mitose/genética , Fuso Acromático/genética , Polos do Fuso/genética , Envelhecimento/metabolismo , Biomarcadores/metabolismo , Segregação de Cromossomos/genética , Humanos , Fuso Acromático/metabolismo
8.
Oncogene ; 38(22): 4199-4214, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30705408

RESUMO

The mitotic kinase Aurora A is crucial for various mitotic events. Its activation has been intensively investigated and is not yet completely understood. RITA, the RBP-J interacting and tubulin-associated protein, has been shown to modulate microtubule dynamics in mitosis. We asked if RITA could be related to the activation of Aurora A. We show here that RITA is colocalized with Aurora A and its activator TPX2 at spindle poles during mitosis. FLAG-RITA is precipitated with the complex of Aurora A, TPX2 and tubulin. Depletion of RITA increases exclusively active Aurora A and TPX2 at spindle poles in diverse cancer cell lines and in RITA knockout mouse embryonic fibroblasts. The enhanced active Aurora A, its substrate p-TACC3 and TPX2 are restored by adding back of RITA but not its Δtub mutant with an impaired tubulin-binding capability, indicating that RITA's role as Aurora A's modulator is mediated through its interaction with tubulin. Also, the mitotic failures in cells depleted of RITA are rescued by the inhibition of Aurora A. RITA itself does not directly interfere with the catalytic activity of Aurora A, instead, affects the microtubule binding of its activator TPX2. Moreover, Aurora A's activation correlates with microtubule stabilization induced by the microtubule stabilizer paclitaxel, implicating that stabilized microtubules caused by RITA depletion could also account for increased active Aurora A. Our data suggest a potential role for RITA in the activation of Aurora A at spindle poles by modulating the microtubule binding of TPX2 and the microtubule stability during mitosis.


Assuntos
Aurora Quinase A/metabolismo , Proteínas de Ligação a DNA/metabolismo , Mitose/fisiologia , Proteínas de Neoplasias/metabolismo , Polos do Fuso/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Células HeLa , Humanos , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo
9.
Essays Biochem ; 62(6): 803-813, 2018 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-30429281

RESUMO

The formation of a robust, bi-polar spindle apparatus, capable of accurate chromosome segregation, is a complex process requiring the co-ordinated nucleation, sorting, stabilization and organization of microtubules (MTs). Work over the last 25 years has identified protein complexes that act as functional modules to nucleate spindle MTs at distinct cellular sites such as centrosomes, kinetochores, chromatin and pre-existing MTs themselves. There is clear evidence that the extent to which these different MT nucleating pathways contribute to spindle mass both during mitosis and meiosis differs not only between organisms, but also in different cell types within an organism. This plasticity contributes the robustness of spindle formation; however, whether such plasticity is present in other aspects of spindle formation is less well understood. Here, we review the known roles of the protein complexes responsible for spindle pole focusing, investigating the evidence that these, too, act co-ordinately and differentially, depending on cellular context. We describe relationships between MT minus-end directed motors dynein and HSET/Ncd, depolymerases including katanin and MCAK, and direct minus-end binding proteins such as nuclear-mitotic apparatus protein, ASPM and Patronin/CAMSAP. We further explore the idea that the focused spindle pole acts as a non-membrane bound condensate and suggest that the metaphase spindle pole be treated as a transient organelle with context-dependent requirements for function.


Assuntos
Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitose , Proteínas Nucleares/metabolismo , Polos do Fuso/metabolismo , Animais , Humanos
10.
Curr Biol ; 28(19): R1131-R1135, 2018 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-30300593

RESUMO

During the cell cycle it is critical that the duplicated DNA faithfully segregates to give rise to two genetically identical daughter cells. An even distribution of the genome during mitosis is mediated by mitotic spindle microtubules, assisted by, among others, motor proteins of the kinesin superfamily. Chromokinesins are members of the kinesin superfamily that harbour a specific DNA-binding domain. The best characterized chromokinesins belong to the kinesin-4/Kif4 and kinesin-10/Kif22 families, respectively. Functional analysis of chromokinesins in several model systems revealed their involvement in chromosome arm orientation and oscillations. This is consistent with their originally proposed role in the generation of polar ejection forces that assist chromosome congression to the spindle equator. Kinesin-12/Kif15 members comprise a third family of chromokinesins, but their role remains less understood. Noteworthy, all chromokinesins exhibit chromosome-independent localization on spindle microtubules, and recent works have significantly extended the portfolio of mitotic processes in which chromokinesins play a role, from error correction and DNA compaction, to the regulation of spindle microtubule dynamics.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Cinesina/metabolismo , Cinesina/fisiologia , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiologia , Divisão Celular/fisiologia , Segregação de Cromossomos , Cromossomos/fisiologia , Dineínas/metabolismo , Humanos , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Mitose , Fuso Acromático/metabolismo , Polos do Fuso/fisiologia
11.
Sci Rep ; 8(1): 12791, 2018 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-30143724

RESUMO

Oocytes, including from mammals, lack centrioles, but neither the mechanism by which mature eggs lose their centrioles nor the exact stage at which centrioles are destroyed during oogenesis is known. To answer questions raised by centriole disappearance during oogenesis, using a transgenic mouse expressing GFP-centrin-2 (GFP CETN2), we traced their presence from e11.5 primordial germ cells (PGCs) through oogenesis and their ultimate dissolution in mature oocytes. We show tightly coupled CETN2 doublets in PGCs, oogonia, and pre-pubertal oocytes. Beginning with follicular recruitment of incompetent germinal vesicle (GV) oocytes, through full oocyte maturation, the CETN2 doublets separate within the pericentriolar material (PCM) and a rise in single CETN2 pairs is identified, mostly at meiotic metaphase-I and -II spindle poles. Partial CETN2 foci dissolution occurs even as other centriole markers, like Cep135, a protein necessary for centriole duplication, are maintained at the PCM. Furthermore, live imaging demonstrates that the link between the two centrioles breaks as meiosis resumes and that centriole association with the PCM is progressively lost. Microtubule inhibition shows that centriole dissolution is uncoupled from microtubule dynamics. Thus, centriole doublets, present in early G2-arrested meiotic prophase oocytes, begin partial reduction during follicular recruitment and meiotic resumption, later than previously thought.


Assuntos
Centríolos/metabolismo , Células Germinativas/metabolismo , Oócitos/metabolismo , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Centríolos/efeitos dos fármacos , Centrossomo/efeitos dos fármacos , Centrossomo/metabolismo , Feminino , Células Germinativas/citologia , Células Germinativas/efeitos dos fármacos , Proteínas de Fluorescência Verde/metabolismo , Metáfase/efeitos dos fármacos , Camundongos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Nocodazol/farmacologia , Oócitos/citologia , Oócitos/efeitos dos fármacos , Oogônios/citologia , Oogônios/efeitos dos fármacos , Oogônios/metabolismo , Ovário/embriologia , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/metabolismo , Polos do Fuso/efeitos dos fármacos , Polos do Fuso/metabolismo , Tubulina (Proteína)/metabolismo
12.
Science ; 361(6398): 189-193, 2018 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-30002254

RESUMO

At the beginning of mammalian life, the genetic material from each parent meets when the fertilized egg divides. It was previously thought that a single microtubule spindle is responsible for spatially combining the two genomes and then segregating them to create the two-cell embryo. We used light-sheet microscopy to show that two bipolar spindles form in the zygote and then independently congress the maternal and paternal genomes. These two spindles aligned their poles before anaphase but kept the parental genomes apart during the first cleavage. This spindle assembly mechanism provides a potential rationale for erroneous divisions into more than two blastomeric nuclei observed in mammalian zygotes and reveals the mechanism behind the observation that parental genomes occupy separate nuclear compartments in the two-cell embryo.


Assuntos
Segregação de Cromossomos , Embrião de Mamíferos/embriologia , Herança Materna/genética , Herança Paterna/genética , Polos do Fuso/metabolismo , Zigoto/metabolismo , Anáfase , Animais , Blastômeros/citologia , Núcleo Celular/metabolismo , Feminino , Genoma , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Microtúbulos/metabolismo
13.
EMBO Rep ; 19(8)2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29925526

RESUMO

Bipolar spindle assembly is necessary to ensure the proper progression of cell division. Loss of spindle pole integrity leads to multipolar spindles and aberrant chromosomal segregation. However, the mechanism underlying the maintenance of spindle pole integrity remains unclear. In this study, we show that the actin-binding protein adducin-1 (ADD1) is phosphorylated at S726 during mitosis. S726-phosphorylated ADD1 localizes to centrosomes, wherein it organizes into a rosette-like structure at the pericentriolar material. ADD1 depletion causes centriole splitting and therefore results in multipolar spindles during mitosis, which can be restored by re-expression of ADD1 and the phosphomimetic S726D mutant but not by the S726A mutant. Moreover, the phosphorylation of ADD1 at S726 is crucial for its interaction with TPX2, which is essential for spindle pole integrity. Together, our findings unveil a novel function of ADD1 in maintaining spindle pole integrity through its interaction with TPX2.


Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Polos do Fuso/metabolismo , Centríolos/metabolismo , Centrossomo/metabolismo , Deleção de Genes , Células HEK293 , Células HeLa , Humanos , Mitose , Fosforilação , Fosfosserina/metabolismo , Ligação Proteica
14.
Open Biol ; 8(6)2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29925632

RESUMO

Abnormal centrosome number and function have been implicated in tumour development. LIM kinase1 (LIMK1), a regulator of actin cytoskeleton dynamics, is found to localize at the mitotic centrosome. However, its role at the centrosome is not fully explored. Here, we report that LIMK1 depletion resulted in multi-polar spindles and defocusing of centrosomes, implicating its involvement in the regulation of mitotic centrosome integrity. LIMK1 could influence centrosome integrity by modulating centrosomal protein localization at the spindle pole. Interestingly, dynein light intermediate chains (LICs) are able to rescue the defects observed in LIMK1-depleted cells. We found that LICs are potential novel interacting partners and substrates of LIMK1 and that LIMK1 phosphorylation regulates cytoplasmic dynein function in centrosomal protein transport, which in turn impacts mitotic spindle pole integrity.


Assuntos
Centrossomo/metabolismo , Dineínas/metabolismo , Quinases Lim/genética , Quinases Lim/metabolismo , Citoplasma/metabolismo , Células HeLa , Humanos , Mitose , Fosforilação , Transporte Proteico , Fuso Acromático/metabolismo , Polos do Fuso/metabolismo
15.
Math Biosci ; 303: 46-51, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29792897

RESUMO

The mitotic spindle orientation and position is crucial for the fidelity of chromosome segregation during asymmetric cell division to generate daughter cells with different sizes or fates. This mechanism is best understood in the budding yeast Saccharomyces cerevisiae, named the spindle position checkpoint (SPOC). The SPOC inhibits cells from exiting mitosis until the mitotic spindle is properly oriented along the mother-daughter polarity axis. Despite many experimental studies, the mechanisms underlying SPOC regulation remains elusive and unexplored theoretically. Here, a minimal mathematical is developed to describe SPOC activation and silencing having autocatalytic feedback-loop. Numerical simulations of the nonlinear ordinary differential equations (ODEs) model accurately reproduce the phenotype of SPOC mechanism. Bifurcation analysis of the nonlinear ODEs reveals the orientation dependency on spindle pole bodies, and how this dependence is altered by parameter values. Partial differential equation (PDEs) model as well as linear stability analysis indicate that diffusion play no major role using experimental high diffusion values. These results provide for systems understanding on the molecular organization of spindle orientation system via mathematical modeling. The presented mathematical model is easy to understand and, within the above mentioned context, can be used as a base for further development of quantitative models in asymmetric cell-division.


Assuntos
Modelos Biológicos , Fuso Acromático/fisiologia , Fuso Acromático/ultraestrutura , Divisão Celular/fisiologia , Polaridade Celular/fisiologia , Simulação por Computador , Conceitos Matemáticos , Mitose/fisiologia , Dinâmica não Linear , Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/fisiologia , Polos do Fuso/fisiologia , Polos do Fuso/ultraestrutura
16.
Mol Biol Cell ; 29(13): 1664-1674, 2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29742018

RESUMO

CK1 protein kinases contribute to multiple biological processes, but how they are tailored to function in compartmentalized signaling events is largely unknown. Hhp1 and Hhp2 (Hhp1/2) are the soluble CK1 family members in Schizosaccharomyces pombe. One of their functions is to inhibit the septation initiation network (SIN) during a mitotic checkpoint arrest. The SIN is assembled by Sid4 at spindle pole bodies (SPBs), and though Hhp1/2 colocalize there, it is not known how they are targeted there or whether their SPB localization is required for SIN inhibition. Here, we establish that Hhp1/2 localize throughout the cell cycle to SPBs, as well as to the nucleus, cell tips, and division site. We find that their catalytic domains but not their enzymatic function are used for SPB targeting and that this targeting strategy is conserved in human CK1δ/ε localization to centrosomes. Further, we pinpoint amino acids in the Hhp1 catalytic domain required for SPB interaction; mutation of these residues disrupts Hhp1 association with the core SPB protein Ppc89, and the inhibition of cytokinesis in the setting of spindle stress. Taken together, these data have enabled us to define a molecular mechanism used by CK1 enzymes to target a specific cellular locale for compartmentalized signaling.


Assuntos
Compartimento Celular , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Transdução de Sinais , Polos do Fuso/metabolismo , Biocatálise , Domínio Catalítico , Ciclo Celular , Centrossomo/metabolismo , Modelos Moleculares , Transporte Proteico , Schizosaccharomyces/enzimologia , Corpos Polares do Fuso/metabolismo , Relação Estrutura-Atividade
17.
Biomolecules ; 9(1)2018 12 28.
Artigo em Inglês | MEDLINE | ID: mdl-30597919

RESUMO

For appropriate chromosome segregation, kinetochores on sister chromatids have to attach to microtubules from opposite spindle poles (bi-orientation). Chromosome alignment at the spindle equator, referred to as congression, can occur through the attachment of kinetochores to the lateral surface of spindle microtubules, facilitating bi-orientation establishment. However, the contribution of this phenomenon to mitotic fidelity has not been clarified yet. Here, we addressed whether delayed chromosome alignment to the spindle equator increases the rate of chromosome missegregation. Cancer cell lines depleted of Kid, a chromokinesin involved in chromosome congression, showed chromosome alignment with a slight delay, and increased frequency of lagging chromosomes. Delayed chromosome alignment concomitant with an increased rate of lagging chromosomes was also seen in cells depleted of kinesin family member 4A (KIF4A), another chromokinesin. Cells that underwent chromosome missegregation took relatively longer time to align chromosomes in both control and Kid/KIF4A-depleted cells. Tracking of late-aligning chromosomes showed that they exhibit a higher rate of lagging chromosomes. Intriguingly, the metaphase of cells that underwent chromosome missegregation was shortened, and delaying anaphase onset ameliorated the increased chromosome missegregation. These data suggest that late-aligning chromosomes do not have sufficient time to establish bi-orientation, leading to chromosome missegregation. Our data imply that delayed chromosome alignment is not only a consequence, but also a cause of defective bi-orientation establishment, which can lead to chromosomal instability in cells without severe mitotic defects.


Assuntos
Segregação de Cromossomos/fisiologia , Cinesina/metabolismo , Polos do Fuso/fisiologia , Linhagem Celular Tumoral , Instabilidade Cromossômica , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Cinesina/antagonistas & inibidores , Cinesina/genética , Microscopia de Fluorescência , Mitose , Prometáfase , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Estatísticas não Paramétricas
18.
Mol Cell Biol ; 38(2)2018 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-29061732

RESUMO

The migration of chromosomes during mitosis is mediated primarily by kinesins that bind to the chromosomes and move along the microtubules, exerting pulling and pushing forces on the centrosomes. We report that a DNA replication protein, Sld5, localizes to the centrosomes, resisting the microtubular pulling forces experienced during chromosome congression. In the absence of Sld5, centriolar satellites, which normally cluster around the centrosomes, are dissipated throughout the cytoplasm, resulting in the loss of their known function of recruiting the centrosomal protein, pericentrin. We observed that Sld5-deficient centrosomes lacking pericentrin were unable to endure the CENP-E- and Kid-mediated microtubular forces that converge on the centrosomes during chromosome congression, resulting in monocentriolar and acentriolar spindle poles. The minus-end-directed kinesin-14 motor protein, HSET, sustains the traction forces that mediate centrosomal fragmentation in Sld5-depleted cells. Thus, we report that a DNA replication protein has an as yet unknown function of ensuring spindle pole resistance to traction forces exerted during chromosome congression.


Assuntos
Centríolos/metabolismo , Centrossomo/fisiologia , Proteínas Cromossômicas não Histona/metabolismo , Membro 3 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/genética , Membro 3 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Centríolos/genética , Centrossomo/química , Proteínas Cromossômicas não Histona/genética , Cromossomos Humanos/metabolismo , Dano ao DNA , Células HeLa , Humanos , Interfase/fisiologia , Microtúbulos/química , Microtúbulos/fisiologia , Mitose , Polos do Fuso/fisiologia , Polos do Fuso/ultraestrutura , Imagem com Lapso de Tempo
19.
Mol Biol Cell ; 29(1): 10-28, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29142076

RESUMO

In many asymmetrically dividing cells, the microtubule-organizing centers (MTOCs; mammalian centrosome and yeast spindle pole body [SPB]) nucleate more astral microtubules on one of the two spindle poles than the other. This differential activity generally correlates with the age of MTOCs and contributes to orienting the mitotic spindle within the cell. The asymmetry might result from the two MTOCs being in distinctive maturation states. We investigated this model in budding yeast. Using fluorophores with different maturation kinetics to label the outer plaque components of the SPB, we found that the Cnm67 protein is mobile, whereas Spc72 is not. However, these two proteins were rapidly as abundant on both SPBs, indicating that SPBs mature more rapidly than anticipated. Superresolution microscopy confirmed this finding for Spc72 and for the γ-tubulin complex. Moreover, astral microtubule number and length correlated with the subcellular localization of SPBs rather than their age. Kar9-dependent orientation of the spindle drove the differential activity of the SPBs in astral microtubule organization rather than intrinsic differences between the spindle poles. Together, our data establish that Kar9 and spatial cues, rather than the kinetics of SPB maturation, control the asymmetry of astral microtubule organization between the preexisting and new SPBs.


Assuntos
Microtúbulos/metabolismo , Saccharomyces cerevisiae/metabolismo , Polos do Fuso/metabolismo , Cinética , Metáfase , Mitose , Modelos Biológicos , Complexos Multiproteicos/metabolismo , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/metabolismo
20.
J Cell Biochem ; 119(2): 2381-2395, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28885720

RESUMO

Stathmin/oncoprotein18 regulates microtubule dynamics and participates in mitotic entry and exit. We isolated stathmin as a physically interacting partner of KIFC1, a minus-end-directed kinesin functioning in bipolar spindle formation and maintenance. We found that stathmin depletion leads to multipolar spindle formation in IMR-90 normal human fibroblasts. Stathmin-depleted IMR-90 cells showed early mitotic delay but managed to undergo chromosome segregation by forming multiple poles or pseudo-bipoles. Consistent with these observations, lagging chromosomes, and micronuclei were elevated in stathmin-depleted IMR-90 cells, demonstrating that stathmin is essential for maintaining genomic stability during mitosis in human cells. Genomic instability induced by stathmin depletion led to premature senescence without any indication of cell death in normal IMR-90 cells. Double knock-down of both stathmin and p53 also did not induce cell death in IMR-90 cells, while the stathmin knock-down triggered apoptosis in p53-proficient human lung adenocarcinoma cells. Our results suggest that stathmin is essential in bipolar spindle formation to maintain genomic stability during mitosis, and the depletion of stathmin prevents the initiation of chromosome instability by inducing senescence in human normal fibroblasts.


Assuntos
Fibroblastos/citologia , Técnicas de Silenciamento de Genes , Instabilidade Genômica , Cinesina/metabolismo , Estatmina/genética , Estatmina/metabolismo , Células A549 , Linhagem Celular , Senescência Celular , Fibroblastos/metabolismo , Células HeLa , Humanos , Centro Organizador dos Microtúbulos/metabolismo , Mitose , Polos do Fuso/genética , Polos do Fuso/metabolismo
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