RESUMO
The first centromeric protein identified in any species was CENP-A, a divergent member of the histone H3 family that was recognised by autoantibodies from patients with scleroderma-spectrum disease. It has recently been suggested to rename this protein CenH3. Here, we argue that the original name should be maintained both because it is the basis of a long established nomenclature for centromere proteins and because it avoids confusion due to the presence of canonical histone H3 at centromeres.
Assuntos
Autoantígenos/genética , Proteínas Cromossômicas não Histona/genética , Histonas/genética , Autoantígenos/metabolismo , Centrômero , Proteína Centromérica A , Proteínas Cromossômicas não Histona/metabolismo , Histonas/metabolismo , Humanos , Cinetocoros , Escleroderma Sistêmico/genética , Terminologia como AssuntoRESUMO
Specific proteinases of the Ced-3/interleukin-1 beta converting enzyme family trigger a multi-enzyme cascade that drives apoptotic events. Studies with newly developed cell-free systems have begun to identify the substrates of these and other proteinases in apoptosis. Studies with intact cells have revealed that cleavage of the genome into domain-sized fragments precedes the activation of a second nuclease that fragments the DNA into nucleosome-sized pieces.
Assuntos
Apoptose/fisiologia , Caspases , Núcleo Celular/metabolismo , Animais , Proteínas de Caenorhabditis elegans , Caspase 1 , Cisteína Endopeptidases/metabolismo , Endopeptidases/metabolismo , Esterases/metabolismo , Proteínas de Helminto/metabolismo , Humanos , Mitose/fisiologiaRESUMO
Recent studies have begun to yield some insight into the structural and regulatory components of centromeres, and new assays have been developed that promise to be of use in advancing our understanding of centromere structure and function. In the budding yeast Saccharomyces cerevisiae new proteins that are required for centromere function have been identified and an in vitro microtubule-binding assay that should assist in dissecting the process of centromere microtubule attachment has been developed. The centromere-specific DNA sequences in the fission yeast Schizosaccharomyces pombe have been identified and partially characterized. In addition, several mammalian centromere proteins have been further characterized, and localization and inhibition studies suggest roles for these proteins in the regulation and assembly of a functional kinetochore.
Assuntos
Centrômero/fisiologia , Fuso Acromático/fisiologia , Animais , Autoanticorpos , Centrômero/ultraestrutura , Proteínas Cromossômicas não Histona/fisiologia , DNA Fúngico/genética , Humanos , Microtúbulos/fisiologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Schizosaccharomyces/genética , Schizosaccharomyces/fisiologia , Fuso Acromático/ultraestrutura , Relação Estrutura-AtividadeRESUMO
Apaf-1(-/-) or caspase-3(-/-) cells treated with a variety of apoptosis inducers manifest apoptosis-associated alterations including the translocation of apoptosis-inducing factor (AIF) from mitochondria to nuclei, large scale DNA fragmentation, and initial chromatin condensation (stage I). However, when compared with normal control cells, Apaf-1(-/-) or caspase-3(-/-) cells fail to exhibit oligonucleosomal chromatin digestion and a more advanced pattern of chromatin condensation (stage II). Microinjection of such cells with recombinant AIF only causes peripheral chromatin condensation (stage I), whereas microinjection with activated caspase-3 or its downstream target caspase-activated DNAse (CAD) causes a more pronounced type of chromatin condensation (stage II). Similarly, when added to purified HeLa nuclei, AIF causes stage I chromatin condensation and large-scale DNA fragmentation, whereas CAD induces stage II chromatin condensation and oligonucleosomal DNA degradation. Furthermore, in a cell-free system, concomitant neutralization of AIF and CAD is required to suppress the nuclear DNA loss caused by cytoplasmic extracts from apoptotic wild-type cells. In contrast, AIF depletion alone suffices to suppress the nuclear DNA loss contained in extracts from apoptotic Apaf-1(-/-) or caspase-3(-/-) cells. As a result, at least two redundant parallel pathways may lead to chromatin processing during apoptosis. One of these pathways involves Apaf-1 and caspases, as well as CAD, and leads to oligonucleosomal DNA fragmentation and advanced chromatin condensation. The other pathway, which is caspase-independent, involves AIF and leads to large-scale DNA fragmentation and peripheral chromatin condensation.
Assuntos
Apoptose/fisiologia , Caspases/metabolismo , Núcleo Celular/metabolismo , Flavoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas/metabolismo , Animais , Antineoplásicos/farmacologia , Fator de Indução de Apoptose , Fator Apoptótico 1 Ativador de Proteases , Arsenitos/farmacologia , Caspase 3 , Caspases/genética , Células Cultivadas , Cisplatino/farmacologia , Grupo dos Citocromos c/genética , Grupo dos Citocromos c/metabolismo , Fragmentação do DNA , Desoxirribonucleases/genética , Desoxirribonucleases/metabolismo , Etoposídeo/farmacologia , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Flavoproteínas/genética , Corantes Fluorescentes/metabolismo , Células HeLa , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Microinjeções , Proteínas/genética , Proteínas Recombinantes/metabolismoRESUMO
Proteolytic cleavage of the cohesin subunit Scc1 is a consistent feature of anaphase onset, although temporal differences exist between eukaryotes in cohesin loss from chromosome arms, as distinct from centromeres. We describe the effects of genetic deletion of Scc1 in chicken DT40 cells. Scc1 loss caused premature sister chromatid separation but did not disrupt chromosome condensation. Scc1 mutants showed defective repair of spontaneous and induced DNA damage. Scc1-deficient cells frequently failed to complete metaphase chromosome alignment and showed chromosome segregation defects, suggesting aberrant kinetochore function. Notably, the chromosome passenger INCENP did not localize normally to centromeres, while the constitutive kinetochore proteins CENP-C and CENP-H behaved normally. These results suggest a role for Scc1 in mitotic regulation, along with cohesion.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiologia , Cromátides/metabolismo , Cinetocoros/metabolismo , Animais , Antibacterianos/farmacologia , Proteínas de Ciclo Celular/genética , Linhagem Celular , Núcleo Celular/metabolismo , Galinhas , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Reparo do DNA , Doxiciclina/farmacologia , Citometria de Fluxo , Proteínas Fúngicas , Humanos , Hibridização in Situ Fluorescente , Substâncias Macromoleculares , Microscopia de Força Atômica , Microscopia de Fluorescência , Proteínas Nucleares/metabolismo , Fenótipo , Fosfoproteínas , Subunidades Proteicas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Saccharomyces cerevisiae , CoesinasRESUMO
Although apoptosis is a major factor in metazoan morphogenesis and tissue homeostasis, its underlying biochemical mechanisms are poorly understood. This is now beginning to change as cell-free systems for the study of apoptosis start to reveal some of the activities involved. As suggested by earlier genetic analyses, a proteinase with properties resembling those of the interleukin-1-beta-converting enzyme (ICE) has been shown to initiate the apoptotic cascade in vitro. Curiously, results obtained with the cell-free systems suggest that essential downstream effectors of the apoptotic response may be intrinsic components of healthy nuclei.
RESUMO
Chromosomal passengers are proteins that move from centromeres to the spindle midzone during mitosis. Recent experiments show that the passengers inner centromere protein (INCENP) and aurora-B kinase are in a macromolecular complex that might also contain a third passenger, survivin. The chromosomal passenger complex functions throughout mitosis in chromosome condensation and segregation, and at the end of mitosis, in the completion of cytokinesis.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Proteínas Associadas aos Microtúbulos , Microtúbulos/metabolismo , Mitose/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas/metabolismo , Aurora Quinase B , Aurora Quinases , Morte Celular/fisiologia , Proteínas Inibidoras de Apoptose , Proteínas de Neoplasias , SurvivinaRESUMO
In the preceding article we described a polyclonal antibody that recognizes cSc-1, a major polypeptide component of the chicken mitotic chromosome scaffold. This polypeptide was shown to be chicken topoisomerase II. In the experiments described in the present article we use indirect immunofluorescence and immunoelectron microscopy to examine the distribution of topoisomerase II within intact chromosomes. We also describe a simple experimental protocol that differentiates antigens that are interspersed along the chromatin fiber from those that occupy restricted domains within the chromosome. These experiments indicate that the distribution of the enzyme appears to be independent of the bulk chromatin. Our data suggest that topoisomerase II is bound to the bases of the radial loop domains of mitotic chromosomes.
Assuntos
Cromossomos/ultraestrutura , DNA Topoisomerases Tipo I/metabolismo , Mitose , Animais , Galinhas , Cromatina/enzimologia , Cromatina/ultraestrutura , Cromossomos/enzimologia , Microscopia EletrônicaRESUMO
We report the development of a new method for producing mitotic extracts from tissue culture cells. These extracts reproducibly promote the condensation of chromatin in vitro when incubated with purified interphase nuclei. This condensation reaction is not species specific, since nuclei from chicken, human, and hamster cell lines all undergo chromatin condensation upon incubation with the extract. We have used this extract to investigate the role of DNA topoisomerase II (topo II) in the chromosome condensation process. Chromatin condensation does not require the presence of soluble topo II in the mitotic extract. However, the extent of formation of discrete chromosome-like structures correlates with the level of endogenous topo II present in the interphase nuclei. Our results further suggest that chromatin condensation in this extract may involve two processes: chromatin compaction and resolution into discrete chromosomes.
Assuntos
Cromatina/fisiologia , Cromossomos/fisiologia , DNA Topoisomerases Tipo II/fisiologia , Mitose/fisiologia , Animais , Núcleo Celular/fisiologia , Sistema Livre de Células , Embrião de Galinha , Drosophila , Eritrócitos/fisiologia , Células HeLa , Humanos , Interfase , Membrana Nuclear/metabolismoRESUMO
We have developed procedures for depositing intact mitotic chromosomes and isolated residual scaffolds on electron microscope grids at controlled and reproducible levels of compaction. The chromosomes were isolated using a recently developed aqueous method. Our study has addressed two different aspects of chromosome structure. First, we present a method for improved visualization of radial chromatin loops in undisrupted mitotic chromosomes. Second, we have visualized a nonhistone protein residual scaffold isolated from nuclease-digested chromosomes under conditions of low salt protein extraction. These scaffolds, which have an extremely simple protein composition, are the size of chromosomes, are fibrous in nature, and are found to retain differentiated regions that appear to derive from the kinetochores and the chromatid axis. When our standard preparation conditions were used, the scaffold appearance was found to be very reproducible. If the ionic conditions were varied, however, the scaffold appearance underwent dramatic changes. In the presence of millimolar concentrations of Mg++ or high concentrations of NaCl, the fibrous scaffold protein network was observed to undergo a lateral aggregation or assembly into a coarse meshlike structure. The alteration of scaffold structure was apparently reversible. This observation is consistent with a model in which the scaffolding network plays a dynamic role in chromosome condensation at mitosis.
Assuntos
Cromossomos/ultraestrutura , Metáfase , Cromatina/ultraestrutura , Proteínas Cromossômicas não Histona/análise , Cromossomos/análise , Células HeLa , Humanos , Magnésio/farmacologia , Microscopia Eletrônica , Conformação Proteica , Cloreto de Sódio/farmacologiaRESUMO
Serum of patients with systemic lupus erythematosus (SLE) contains crossreacting autoantibodies which recognize histones in nucleosomes or when they are induced to form octamers in solution in the presence of 2 M NaCl, but not when they are dissociated free in solution at physiological ionic strength. We have found that histones stored in eggs of Xenopus laevis for use in rapid nuclear synthesis during early development react with this antibody. This reaction has been observed by radioimmunoassay, inhibition of chromatin assembly by the extracts in the presence of antibody, and, in a preliminary result, by identification of a histone-antibody complex bound to protein A-sepharose. Further evidence that the extract antigen corresponds to the stored histone pool comes from sedimentation and charge fractionation experiments where the chromatin assembly activity and antigen (measured by radioimmunoassay) were found to cofractionate. BEcause the extract histones are not bound to DNA, our results suggest that they are stored as a soluble complex in a conformation similar or identical to the octameric core of the nucleosome. Our data suggest that the histones in this complex are bound to an anionic factor or factors which presumably replaces the DNA in shielding the positive charges on the histones.
Assuntos
Histonas , Óvulo/análise , Animais , Anticorpos Antinucleares , Complexo Antígeno-Anticorpo , Centrifugação com Gradiente de Concentração , Cromatina/metabolismo , Feminino , Histonas/imunologia , Histonas/metabolismo , Nucleossomos , Conformação Proteica , Radioimunoensaio , Xenopus laevisRESUMO
We have used an antibody probe to measure the levels of topoisomerase II in several transformed and developmentally regulated normal cell types. Transformed cells contain roughly 1 X 10(6) copies of the enzyme. During erythropoiesis in chicken embryos the enzyme level drops from 7.8 X 10(4) copies per erythroblast to less than 300 copies per erythrocyte concomitant with the cessation of mitosis in the blood. Cultured myoblasts also lose topoisomerase II upon fusion into nonproliferating myotubes. When peripheral blood lymphocytes (which lack detectable topoisomerase II) commence proliferation, they express topoisomerase II de novo. Appearance of the enzyme exactly parallels the onset of DNA replication. These results suggest that topoisomerase II is not required for transcription in higher eukaryotes, but that it may function during DNA replication. Furthermore, topoisomerase II is a sensitive and specific marker for proliferating cells.
Assuntos
Divisão Celular , DNA Topoisomerases Tipo II/metabolismo , Animais , Linhagem Celular , Embrião de Galinha , Replicação do DNA , DNA Topoisomerases Tipo II/imunologia , Eritrócitos/enzimologia , Eritropoese , Técnicas Imunológicas , Ativação Linfocitária , Músculos/citologia , Músculos/enzimologia , Transcrição GênicaRESUMO
We used antisera from patients with the CREST syndrome of scleroderma (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) to show that an antigenic component of the kinetochore present in metaphase chromosomes is also present in nonhistone chromosome scaffolds isolated following extensive digestion of the DNA and extraction of the bulk of chromosomal protein. All sera from 12 scleroderma CREST patients previously shown by immunofluorescence microscopy to have circulating antikinetochore antibodies recognise a protein of Mr 77,000 (CREST-77) in an immunoblotting assay. 9 of the 12 sera also recognise an antigen of Mr 110,000 (CREST-110). These proteins are present in isolated chromosomes and nonhistone scaffolds derived from them by two different procedures. Sera of five scleroderma CREST patients who are antikinetochore negative (by immunofluorescence) bind to neither protein in immunoblots. These data suggest that CREST-77 (and possibly CREST-110) is a component of the human kinetochore, and that the kinetochore is an integral part of the mitotic chromosome scaffolding.
Assuntos
Cromossomos/ultraestrutura , Metáfase , Fuso Acromático/ultraestrutura , Autoanticorpos/imunologia , Proteínas Cromossômicas não Histona/análise , Proteínas Cromossômicas não Histona/imunologia , Humanos , Peso Molecular , Escleroderma Sistêmico/imunologiaRESUMO
We have combined in vivo and in vitro approaches to investigate the function of CENP-B, a major protein of human centromeric heterochromatin. Expression of epitope-tagged deletion derivatives of CENP-B in HeLa cells revealed that a single domain less than 158 residues from the amino terminus of the protein is sufficient to localize CENP-B to centromeres. Centromere localization was abolished if as few as 28 amino acids were removed from the amino terminus of CENP-B. The centromere localization signal of CENP-B can function in an autonomous fashion, relocating a fused bacterial enzyme to centromeres. The centromere localization domain of CENP-B specifically binds in vitro to a subset of alpha-satellite DNA monomers. These results suggest that the primary mechanism for localization of CENP-B to centromeres involves the recognition of a DNA sequence found at centromeres. Analysis of the distribution of this sequence in alpha-satellite DNA suggests that CENP-B binding may have profound effects on chromatin structure at centromeres.
Assuntos
Autoantígenos , Centrômero/química , Cromatina/química , Proteínas Cromossômicas não Histona/análise , Proteínas de Ligação a DNA/análise , Sequência de Bases , Proteína B de Centrômero , Regulação da Expressão Gênica , Células HeLa , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação ProteicaRESUMO
We describe a novel set of polypeptide antigens that shows a dramatic change in structural localization during mitosis. Through metaphase these antigens define a new chromosomal substructure that is located between the sister chromatids. Because the antigens are concentrated in the pericentromeric region, we have provisionally termed them the INCENPs (inner centromere proteins). The INCENPs (two polypeptides of 155 and 135 kD) were identified with a monoclonal antibody that was raised against the bulk proteins of the mitotic chromosome scaffold fraction. These two polypeptides are the most tightly bound chromosomal proteins known. When scaffolds are prepared, 100% of the detectable INCENPs remain scaffold associated. We were therefore unprepared for the fate of the INCENPs at anaphase. As the sister chromatids separate, the INCENPs dissociate fully from them, remaining behind at the metaphase plate as the chromatids migrate to the spindle poles. During anaphase the INCENPs are found on coarse fibers in the central spindle, and also in close apposition to the cell membrane in the region of the forming contractile ring. During telophase, the INCENPs gradually become focused onto the forming midbody, together with which they are ultimately discarded. Several possible in vivo roles for the INCENPs are suggested by these data: regulation of sister chromatid pairing, stabilization of the plane of cleavage, and separation of spindle poles at anaphase.
Assuntos
Centrômero/ultraestrutura , Cromossomos/ultraestrutura , Proteínas Nucleares/fisiologia , Anáfase , Anticorpos Monoclonais , Antígenos Nucleares , Linhagem Celular , Imunofluorescência , Metáfase , Mitose , Proteínas Nucleares/imunologiaRESUMO
Antibodies to a set of structurally related autoantigens (p23-25) bind to a previously uncharacterized, large structural domain in the nucleus of a variety of human cell types. This subnuclear domain is visible by phase contrast alone as a region of decreased density after several different fixation protocols. The morphology of this region changes dramatically during the cell cycle and we have given it the name PIKA (for polymorphic interphase karyosomal association) based on preliminary evidence that the PIKA proteins may be associated with chromatin. The function of the PIKA is not yet known, but our immunolocalization data indicate that it is unlikely to be associated with regions of ongoing DNA replication, heterogeneous nuclear RNA storage, or mRNA processing. The discovery of the PIKA provides evidence supporting an emerging model of nuclear structure. It now appears that the nucleus is organized into distinct domains which include not only the nucleolus, but also previously unidentified regions such as the PIKAs. Furthermore, structural rearrangements undergone by the nucleolus and the PIKAs may be indicative of a broad tendency for nuclear organization to change in a cell cycle-specific fashion.
Assuntos
Compartimento Celular , Núcleo Celular/ultraestrutura , Animais , Autoantígenos/imunologia , Ciclo Celular , Núcleo Celular/imunologia , Células Cultivadas , Clonagem Molecular , DNA/análise , Imunofluorescência , Células HeLa , Humanos , Soros Imunes , Microscopia Imunoeletrônica , Polimorfismo Genético , CoelhosRESUMO
The family of three structurally related autoantigens CENP-A (17 kD), CENP-B (80 kD), and CENP-C (140 kD) are the best characterized components of the human centromere, and they have been widely assumed to be components of the kinetochore. Kinetochore components are currently of great interest since this structure, which has long been known to be the site of microtubule attachment to the chromosome, is now believed to be a site of force production for anaphase chromosome movement. In the present study we have mapped the distribution of CENP-B in mitotic chromosomes by immunoelectron microscopy using two monospecific polyclonal antibodies together with a newly developed series of ultra-small 1-nm colloidal gold probes. We were surprised to find that greater than 95% of CENP-B is distributed throughout the centromeric heterochromatin beneath the kinetochore. This strongly supports other emerging evidence that CENP-B is specifically associated with alpha-satellite heterochromatin. Although in certain instances CENP-B can be seen to be concentrated immediately adjacent to the lower surface of the kinetochore, the outer plate remains virtually unlabeled. Similar analysis with a human autoimmune serum that recognizes all three CENP antigens reveals an additional unsuspected feature of kinetochore structure. In addition to recognizing antigens in the centromeric heterochromatin, the autoantiserum recognizes a concentration of antigens lateral to the kinetochore. This difference in staining pattern may reflect the presence of a "collar" of chromatin rich in CENP-C and/or CENP-A encircling the kinetochore plates.
Assuntos
Autoantígenos , Centrômero/análise , Proteínas Cromossômicas não Histona/análise , Cromossomos/análise , Proteínas de Ligação a DNA , Autoanticorpos , Centrômero/imunologia , Centrômero/ultraestrutura , Proteína B de Centrômero , Células HeLa , Heterocromatina/análise , Humanos , Imuno-Histoquímica , Interfase , Microscopia Eletrônica , Mitose/fisiologia , PermeabilidadeRESUMO
We have performed a biochemical and double-stranded RNA-mediated interference (RNAi) analysis of the role of two chromosomal passenger proteins, inner centromere protein (INCENP) and aurora B kinase, in cultured cells of Drosophila melanogaster. INCENP and aurora B function is tightly interlinked. The two proteins bind to each other in vitro, and DmINCENP is required for DmAurora B to localize properly in mitosis and function as a histone H3 kinase. DmAurora B is required for DmINCENP accumulation at centromeres and transfer to the spindle at anaphase. RNAi for either protein dramatically inhibited the ability of cells to achieve a normal metaphase chromosome alignment. Cells were not blocked in mitosis, however, and entered an aberrant anaphase characterized by defects in sister kinetochore disjunction and the presence of large amounts of amorphous lagging chromatin. Anaphase A chromosome movement appeared to be normal, however cytokinesis often failed. DmINCENP and DmAurora B are not required for the correct localization of the kinesin-like protein Pavarotti (ZEN-4/CHO1/MKLP1) to the midbody at telophase. These experiments reveal that INCENP is required for aurora B kinase function and confirm that the chromosomal passengers have essential roles in mitosis.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos/fisiologia , Proteínas de Drosophila , Histonas/metabolismo , Cinetocoros/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Anáfase/fisiologia , Animais , Aurora Quinase B , Aurora Quinases , Divisão Celular/fisiologia , Células Cultivadas , Proteínas Cromossômicas não Histona/genética , Cromossomos/metabolismo , Drosophila , Metáfase/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/genética , Microtúbulos/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Telófase/fisiologiaRESUMO
Here, we describe the cloning and characterization of ScII, the second most abundant protein after topoisomerase II, of the chromosome scaffold fraction to be identified. ScII is structurally related to a protein, Smc1p, previously found to be required for accurate chromosome segregation in Saccharomyces cerevisiae. ScII and the other members of the emerging family of SMC1-like proteins are likely to be novel ATPases, with NTP-binding A and B sites separated by two lengthy regions predicted to form an alpha-helical coiled-coil. Analysis of the ScII B site predicted that ScII might use ATP by a mechanism similar to the bacterial recN DNA repair and recombination enzyme. ScII is a mitosis-specific scaffold protein that colocalizes with topoisomerase II in mitotic chromosomes. However, ScII appears not to be associated with the interphase nuclear matrix. ScII might thus play a role in mitotic processes such as chromosome condensation or sister chromatid disjunction, both of which have been previously shown to involve topoisomerase II.
Assuntos
Adenosina Trifosfatases/química , Proteínas Aviárias , Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Cromossomos/química , Proteínas de Ligação a DNA/química , Mitose , Proteínas Nucleares/química , Adenosina Trifosfatases/análise , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Núcleo Celular/metabolismo , Células Cultivadas , Embrião de Galinha , Galinhas , Cromátides/química , Clonagem Molecular , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/química , Interfase , Modelos Moleculares , Dados de Sequência Molecular , Matriz Nuclear/química , Proteínas Nucleares/análise , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleotídeos/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/químicaRESUMO
NuMA is a large nuclear protein whose relocation to the spindle poles is required for bipolar mitotic spindle assembly. We show here that this process depends on directed NuMA transport toward microtubule minus ends powered by cytoplasmic dynein and its activator dynactin. Upon nuclear envelope breakdown, large cytoplasmic aggregates of green fluorescent protein (GFP)-tagged NuMA stream poleward along spindle fibers in association with the actin-related protein 1 (Arp1) protein of the dynactin complex and cytoplasmic dynein. Immunoprecipitations and gel filtration demonstrate the assembly of a reversible, mitosis-specific complex of NuMA with dynein and dynactin. NuMA transport is required for spindle pole assembly and maintenance, since disruption of the dynactin complex (by increasing the amount of the dynamitin subunit) or dynein function (with an antibody) strongly inhibits NuMA translocation and accumulation and disrupts spindle pole assembly.