RESUMO
Mitotic centromere-associated kinesin (MCAK) is an ATP-dependent microtubule (MT) depolymerase regulated by Aurora kinase (AURK) phosphorylation and implicated in resolution of improper MT attachments in mitosis. Distribution of MCAK was studied in oocyte maturation by anti-MCAK antibody, anti-tubulin antibody, anti-AURKB antibody and anti-centromere antibody (ACA) and by the expression of MCAK-enhanced green fluorescent protein fusion protein in maturing mouse oocytes. Function was assessed by knockdown of MCAK and Mad2, by inhibiting AURK or the proteasome, by live imaging with polarization microscope and by chromosomal analysis. The results show that MCAK is transiently recruited to the nucleus and transits to spindle poles, ACA-positive domains and chiasmata at prometaphase I. At metaphase I and II, it is present at centrosomes and centromeres next to AURKB and checkpoint proteins Mad2 and BubR1. It is retained at centromeres at telophase I and also at the midbody. Knockdown of MCAK causes a delay in chromosome congression but does not prevent bipolar spindle assembly. MCAK knockdown also induces a meiosis I arrest, which is overcome by knockdown of Mad2 resulting in chiasma resolution, chromosome separation, formation of aberrant meiosis II spindles and increased hypoploidy. In conclusion, MCAK appears to possess a unique distribution and function in oocyte maturation. It is required for meiotic progression from meiosis I to meiosis II associated with silencing of the spindle assembly checkpoint. Alterations in abundance and activity of MCAK, as implicated in aged oocytes, may therefore contribute to the loss of control of cell cycle and chromosome behaviour, thus increasing risk for errors in chromosome segregation and aneuploidy.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Centrômero/enzimologia , Cinesinas/metabolismo , Meiose , Mitose , Oócitos/enzimologia , Fuso Acromático/enzimologia , Animais , Aurora Quinase B , Aurora Quinases , Proteínas de Ciclo Celular/genética , Nucléolo Celular/enzimologia , Células Cultivadas , Centrômero/efeitos dos fármacos , Segregação de Cromossomos , Inibidores de Cisteína Proteinase/farmacologia , Feminino , Cinesinas/genética , Proteínas Mad2 , Camundongos , Microinjeções , Oócitos/efeitos dos fármacos , Fosforilação , Ploidias , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico , Interferência de RNA , Proteínas Recombinantes de Fusão/metabolismo , Fuso Acromático/efeitos dos fármacos , Fatores de TempoRESUMO
Using antipeptide antibodies to conserved regions of the kinesin motor domain, we cloned a kinesin-related protein that associates with the centromere region of mitotic chromosomes. We call the protein MCAK, for mitotic centromere-associated kinesin. MCAK appears concentrated on centromeres at prophase and persists until telophase, after which time the localization disperses. It is found throughout the centromere region and between the kinetochore plates of isolated mitotic CHO chromosomes, in contrast to two other kinetochore-associated microtubule motors: cytoplasmic dynein and CENP-E (Yen et al., 1992), which are closer to the outer surface of the kinetochore plates. Sequence analysis shows MCAK to be a kinesin-related protein with the motor domain located in the center of the protein. It is 60-70% similar to kif2, a kinesin-related protein originally cloned from mouse brain with a centrally located motor domain (Aizawa et al., 1992). MCAK protein is present in interphase and mitotic CHO cells and is transcribed as a single 3.4-kb message.
Assuntos
Centrômero/fisiologia , Centrômero/ultraestrutura , Cinesinas/fisiologia , Cinetocoros/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Células CHO , Proteínas Cromossômicas não Histona/fisiologia , Clonagem Molecular , Cricetinae , Dineínas/fisiologia , Cinesinas/química , Cinesinas/isolamento & purificação , Cinetocoros/ultraestrutura , Mitose , Dados de Sequência Molecular , Plasmídeos , Reação em Cadeia da Polimerase , Estrutura Secundária de Proteína , Fases de Leitura , Fuso Acromático/ultraestrutura , Frações Subcelulares/metabolismoRESUMO
The cell cycle of the marine centric diatom Stephanopyxis turris consists of a series of spatially and temporally well-ordered events. We have used immunofluorescence microscopy to examine the role of cytoplasmic microtubules in these events. At interphase, microtubules radiate out from the microtubule-organizing center, forming a network around the nucleus and extending much of the length and breadth of the cell. As the cell enters mitosis, this network breaks down and a highly ordered mitotic spindle is formed. Peripheral microtubule bundles radiate out from each spindle pole and swing out and away from the central spindle during anaphase. Treatment of synchronized cells with 2.5 X 10(-8) M Nocodazole reversibly inhibited nuclear migration concurrent with the disappearance of the extensive cytoplasmic microtubule arrays associated with migrating nuclei. Microtubule arrays and mitotic spindles that reformed after the drug was washed out appeared normal. In contrast, cells treated with 5.0 X 10(-8) M Nocodazole were not able to complete nuclear migration after the drug was washed out and the mitotic spindles that formed were multipolar. Normal and multipolar spindles that were displaced toward one end of the cell by the drug treatment had no effect on the plane of division during cytokinesis. The cleavage furrow always bisected the cell regardless of the position of the mitotic spindle, resulting in binucleate/anucleate daughter cells. This suggests that in S. turris, unlike animal cells, the location of the plane of division is cortically determined before mitosis.
Assuntos
Ciclo Celular , Divisão Celular , Eucariotos/citologia , Microtúbulos/fisiologia , Benzimidazóis/farmacologia , Compartimento Celular/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Núcleo Celular/fisiologia , Citoplasma/fisiologia , Citoplasma/ultraestrutura , Imunofluorescência , Microscopia Eletrônica , Microtúbulos/efeitos dos fármacos , Microtúbulos/ultraestrutura , Movimento , Nocodazol , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/fisiologia , Fuso Acromático/ultraestruturaRESUMO
We have used indirect immunofluorescence in combination with correlative EM to subdivide the mammalian kinetochore into two domains based on the localization of specific antigens. We demonstrate here that the fibrous corona on the distal face of the kinetochore plate contains tubulin (previously shown by Mitchison, T. J., and M. W. Kirschner. 1985. J. Cell Biol. 101:755-765) and the minus end-directed, ATP-dependent microtubule motor protein, dynein; whereas a 50-kD CREST antigen is located internal to these components in the kinetochore. Tubulin and dynein can be extracted from the kinetochore by 150 mM KI, leaving other, as yet uncharacterized, components of the kinetochore corona intact. Microtubules and tubulin subunits will associate with kinetochores in vitro after extraction with 150 mM KI, suggesting that other functionally significant, corona-associated molecules remain unextracted. Our results suggest that the corona region of the kinetochore contains the machinery for chromosome translocation along microtubules.
Assuntos
Cromossomos/ultraestrutura , Fuso Acromático/ultraestrutura , Animais , Centrômero/química , Centrômero/fisiologia , Centrômero/ultraestrutura , Cromossomos/química , Cromossomos/fisiologia , Cricetinae , Cricetulus , Dineínas/análise , Dineínas/imunologia , Dineínas/fisiologia , Eletroforese em Gel de Poliacrilamida , Feminino , Imunofluorescência , Soros Imunes/imunologia , Immunoblotting , Microscopia Imunoeletrônica , Microtúbulos/química , Microtúbulos/fisiologia , Microtúbulos/ultraestrutura , Ovário/química , Ovário/citologia , Ovário/ultraestrutura , Fuso Acromático/química , Fuso Acromático/fisiologia , Tubulina (Proteína)/análise , Tubulina (Proteína)/imunologia , Tubulina (Proteína)/fisiologiaRESUMO
Mitotic centromere-associated kinesin (MCAK) is recruited to the centromere at prophase and remains centromere associated until after telophase. MCAK is a homodimer that is encoded by a single gene and has no associated subunits. A motorless version of MCAK that binds centromeres but not microtubules disrupts chromosome segregation during anaphase. Antisense-induced depletion of MCAK results in the same defect. MCAK overexpression induces centromere-independent bundling and eventual loss of spindle microtubule polymer suggesting that centromere-associated bundling and/or depolymerization activity is required for anaphase. Live cell imaging indicates that MCAK may be required to coordinate the onset of sister centromere separation.
Assuntos
Anáfase/fisiologia , Cinesinas/fisiologia , Mitose/genética , Animais , Células CHO , Centrômero/metabolismo , Cromossomos/genética , Cricetinae , Cinesinas/genética , Microtúbulos/metabolismo , Fuso Acromático/metabolismoRESUMO
Using pan-kinesin antibodies to screen a differentiating C2C12 cell library, we identified the kinesin proteins KIF3A, KIF3B, and conventional kinesin heavy chain to be present in differentiating skeletal muscle. We compared the expression and subcellular localization characteristics of these kinesins in myogenic cells to others previously identified in muscle, neuronal, and mitotic systems (KIF1C, KIF3C, and mitotic-centromere-associated kinesin). Because members of the KIF3 subfamily of kinesin-related proteins showed altered subcellular fractionation characteristics in differentiating cells, we focused our study of kinesins in muscle on the function of kinesin-II. Kinesin-II is a motor complex comprised of dimerized KIF3A and KIF3B proteins and a tail-associated protein, KAP. The Xenopus homologue of KIF3B, Xklp3, is predominantly localized to the region of the Golgi apparatus, and overexpression of motorless-Xklp3 in Xenopus A6 cells causes mislocalization of Golgi components (). In C2C12 myoblasts and myotubes, KIF3B is diffuse and punctate, and not primarily associated with the Golgi. Overexpression of motorless-KIF3B does not perturb localization of Golgi components in myogenic cells, and myofibrillogenesis is normal. In adult skeletal muscle, KIF3B colocalizes with the excitation-contraction-coupling membranes. We propose that these membranes, consisting of the transverse-tubules and sarcoplasmic reticulum, are dynamic structures in which kinesin-II may function to actively assemble and maintain in myogenic cells.
Assuntos
Proteínas de Ligação ao Cálcio/fisiologia , Cinesinas/metabolismo , Proteínas Musculares/fisiologia , Músculo Esquelético/metabolismo , Animais , Anticorpos/imunologia , Diferenciação Celular , Divisão Celular , Linhagem Celular , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Cinesinas/genética , Cinesinas/imunologia , Proteínas Motores Moleculares/metabolismo , Músculo Esquelético/citologia , Miofibrilas/metabolismo , Retículo Sarcoplasmático/metabolismo , Frações Subcelulares/metabolismo , Vesículas Transportadoras/metabolismo , Xenopus , Proteínas de XenopusRESUMO
This review summarizes results concerning the molecular nature of the higher eucaryotic kinetochore. The first major section of this review includes kinetochore proteins whose general functions remain to be determined, precluding their entry into a discrete functional category. Many of the proteins in this section, however, are likely to be involved in kinetochore formation or structure. The second major section is concerned with how microtubule motor proteins function to cause chromosome movement. The microtubule motors dynein, CENP-E, and MCAK have all been observed at the kinetochore. While their precise functions are not well understood, all three are implicated in chromosome movement during mitosis. Finally, the last section deals with kinetochore components that play a role in the spindle checkpoint; a checkpoint that delays mitosis until all kinetochores have attached to the mitotic spindle. Brief reviews of kinetochore morphology and of an important technical breakthrough that enabled the molecular dissection of the kinetochore are also included.
Assuntos
Cinetocoros/fisiologia , Animais , Síndrome CREST/etiologia , Proteínas Cromossômicas não Histona/fisiologia , Células Eucarióticas , Humanos , Cinetocoros/ultraestrutura , Microscopia Eletrônica , Modelos Biológicos , Proteínas Motores Moleculares/fisiologia , Transdução de Sinais , Fuso Acromático/fisiologiaRESUMO
We have found that nocodazole reversibly inhibits nuclear migration and can be used to induce karyokinesis before the completion of nuclear migration, resulting in spindles that are displaced toward the hypothecal end of the cell. Surprisingly, displacement of mitotic nuclei results in complete spatial uncoupling of karyokinesis from cytokinesis. Nocodazole-induced displacement of mitotic nuclei will neither alter the position of the original furrow nor induce additional furrows. This demonstrates that in S. turris the location of the presumptive cleavage furrow is not determined by the position of the spindle but is cortically determined before mitosis. Therefore, although cell division in S. turris resembles certain mechanochemical aspects of cleavage in animal cells, our evidence suggests that the spatial regulation of the cytokinetic apparatus relies on a mechanism of cortical determination that is characteristic of plant cells.
Assuntos
Divisão Celular/fisiologia , Eucariotos/citologia , Fuso Acromático/fisiologia , Núcleo Celular/fisiologia , Citoesqueleto/fisiologia , Nocodazol , Fuso Acromático/efeitos dos fármacos , Tubulina (Proteína)/fisiologiaAssuntos
Tubulina (Proteína)/análogos & derivados , Animais , Biotina , Encéfalo/metabolismo , Soluções Tampão , Bovinos , Centrifugação com Gradiente de Concentração/métodos , Etilmaleimida , Corantes Fluorescentes , Guanosina Trifosfato/análogos & derivados , Indicadores e Reagentes , Ligação Proteica , Tubulina (Proteína)/isolamento & purificaçãoAssuntos
Clonagem Molecular/métodos , Regulação da Expressão Gênica no Desenvolvimento , Cinesinas/imunologia , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Bacteriófago lambda/genética , Southern Blotting/métodos , Western Blotting/métodos , Células CHO , Cricetinae , Cricetulus , DNA Complementar/genética , Técnica Indireta de Fluorescência para Anticorpo , Biblioteca Gênica , Soros Imunes , Cinesinas/biossíntese , Cinesinas/genética , Dados de Sequência Molecular , Desenvolvimento Muscular , Proteínas Musculares/biossíntese , Proteínas Musculares/genética , Proteínas Musculares/imunologia , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/metabolismo , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/imunologia , Estrutura Terciária de Proteína , Coelhos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Ensaio de Placa ViralRESUMO
Despite over 100 year of research, the mechanisms that cells use to ensure the proper segregation of chromosomes during mitosis are still surprisingly obscure. However, recent high resolution video light microscopic studies of dividing cells are telling us new and important information about chromosome behavior. Molecular genetics is enabling us to build a more complete list of the components involved in chromosome segregation. And in vitro assays for chromosome segregation are providing information about the signals that control the equipartitioning of sister chromatids during cell division.
Assuntos
Autoantígenos , Cromossomos/fisiologia , Mitose , Animais , Proteína Centromérica A , Proteínas Cromossômicas não Histona/fisiologia , Células HeLa , HumanosRESUMO
Mitotic spindles isolated from the diatom Stephanopyxis turris consist of two half-spindles of closely interdigitating microtubules that slide relative to one another in the presence of ATP, reinitiating spindle elongation (anaphase B) in vitro. Purified spindles that have been exposed to ATP-gamma-S undergo ATP-dependent reactivation more readily than do control spindles. Thiophosphorylated proteins in such spindles are located in the spindle midzone, kinetochores, and a portion of the pole complex. One major thiophosphorylated peptide of 205 kd is detected in extracts prepared from spindles labeled with [35S]ATP-gamma-S, and is also localized in the spindle midzone by using an antibody that recognizes thiophosphorylated proteins. It is likely that this 205 kd peptide is either a positive regulator or mechanochemical transducer of microtubule sliding when it is in a phosphorylated state.
Assuntos
Anáfase , Eucariotos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Eucariotos/ultraestrutura , Imunofluorescência , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , FosforilaçãoRESUMO
Mitotic centromere-associated kinesin (MCAK) is a microtubule depolymerizer that is consistent with its role in promoting chromosome segregation during mitosis. Here we show that the conserved motor domain of MCAK is necessary but not sufficient for microtubule depolymerization in cells or in vitro. The addition of only 30 amino acids N-terminal to the motor restores depolymerization activity. Furthermore, dimerization studies revealed that the smallest functional MCAK deletion constructs are monomers. These results define a highly conserved domain within MCAK and related (KIN I) kinesins that is critical for depolymerization activity and show that this depolymerization is not dependent on MCAK dimerization.
Assuntos
Cinesinas/química , Microtúbulos/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Dimerização , Cinesinas/fisiologia , Dados de Sequência MolecularRESUMO
The interplay between microtubules and microtubule-based motors is fundamental to basic aspects of cellular function, such as the intracellular transport of organelles and alterations in cellular morphology during cell locomotion and division. Motor proteins are unique in that they couple nucleotide hydrolysis to force production that can do work. The force transduction by proteins belonging to the kinesin and dynein superfamilies has been thought only to power movement of these motors along the surface of microtubules; however, a growing body of evidence, both genetic and biochemical, suggests that motors can also directly influence the polymerization dynamics of microtubules. For example, at the vertebrate kinetochore, motors interact directly with microtubule ends and modulate polymerization dynamics to orchestrate chromosome movements during mitosis. Although a role for motors in regulating microtubule length has been established, the mechanisms used by motors to promote microtubule growth or shrinkage are unclear, as is an understanding of why cells might choose motors to control dynamics rather than a variety of non-motor proteins known to affect microtubule stability. Elucidation of the exact mechanisms by which motors alter the exchange of tubulin subunits at microtubule ends in vitro may shed light on how microtubule stability is regulated to produce the array of dynamic behavior seen in cells.
Assuntos
Proteínas dos Microtúbulos , Proteínas Associadas aos Microtúbulos/fisiologia , Microtúbulos/fisiologia , Sequência de Aminoácidos , Animais , Cinesinas/metabolismo , Cinetocoros/metabolismo , Cinetocoros/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas Motores Moleculares , Dados de Sequência Molecular , Fosfoproteínas/metabolismo , Saccharomyces cerevisiae/genética , EstatminaRESUMO
Oocytes of the echiuroid, Urechis caupo, contain an abundant maternal mRNA that encodes a protein very similar to LBP/p40, originally identified as a non-integrin 67 kDa laminin binding protein. We have sequenced the Urechis caupo mRNA for LBP/p40, and a similar mRNA from the Hawaiian sea urchin, Tripneustes gratilla. Both of the encoded proteins, as well as LBP/p40 proteins from other sources, share significant homology in the amino 2/3 of the proteins, but diverge extensively at the carboxyl ends. LBP/p40 protein is present in growing and in full-grown U. caupo oocytes. The protein concentration remains constant for the first 48 hours of embryogenesis and then begins to decline. In sucrose gradients run with homogenates from coelomocytes, oocytes, and early embryos, all of the LBP/p40 protein appears to be associated with either polysomes or free 40 S ribosomal subunits. In later embryos, an increasing proportion of the protein is found in the soluble fraction. Immunohistochemistry indicates that LBP/p40 is uniformly distributed in early U. caupo embryos, with no localization at the cell surface. In later embryos LBP/p40 is localized in specific parts of the embryo which may correspond to neural tissue.
Assuntos
Anelídeos/metabolismo , Embrião não Mamífero/metabolismo , Oócitos/metabolismo , Peptídeos/metabolismo , Biossíntese de Proteínas , Precursores de Proteínas , Receptores de Laminina , Sequência de Aminoácidos , Animais , Anelídeos/embriologia , Sequência de Bases , Feminino , Variação Genética , Humanos , Laminina/metabolismo , Dados de Sequência Molecular , Peso Molecular , Biossíntese Peptídica , Peptídeos/química , Ouriços-do-Mar , Homologia de Sequência de AminoácidosRESUMO
Mitotic centromere-associated kinesin (MCAK) is important for anaphase chromosome segregation. MCAK is diffusely localized to both the cytoplasm and the nucleus during interphase. At prophase MCAK is recruited to mitotic centromeres. It is associated with centromeres throughout mitosis and then returns to exhibiting a diffuse nuclear and cytoplasmic localization during interphase. MCAK has several predicted nuclear localization sequences. The subcellular distribution of expressed deletion constructs of GFP-MCAK suggest that the nucleocytoplasmic ratio of MCAK protein is dependent on a balance between several predicted nuclear localization sequences (NLS) and a putative nuclear exclusion sequence (NES) in the amino-terminal region of MCAK. Amino acid substitutions in the ATP-binding domain of the MCAK motor affect nuclear localization, which, in turn, influences the degree of centromere binding.
Assuntos
Trifosfato de Adenosina/metabolismo , Cinesinas/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Anáfase/fisiologia , Animais , Sítios de Ligação , Células CHO/química , Núcleo Celular/metabolismo , Células Cultivadas , Centrômero/fisiologia , Cricetinae , Citoplasma/metabolismo , Proteínas de Fluorescência Verde , Humanos , Interfase/fisiologia , Proteínas Luminescentes/metabolismo , Camundongos , Pessoa de Meia-Idade , Mitose , Dados de Sequência Molecular , Mutação , Ratos , Proteínas Recombinantes/metabolismo , Alinhamento de SequênciaRESUMO
To identify kinesin-related proteins that may be important for mitotic function in embryonic and tissue culture cells we have generated polyclonal antibodies to two synthetic peptides corresponding to conserved regions of the kinesin motor domain. In Xenopus eggs we have identified a family of microtubule-binding proteins, recognized by one or both affinity-purified peptide antibodies but not by monoclonal antibodies that recognize conventional kinesin heavy chain. Like kinesin, most of these proteins bind to microtubules only upon addition of AMP-PNP or nucleotide depletion and are released upon subsequent addition of ATP. At least one protein, however, exhibits markedly distinct properties, binding readily to microtubules in the absence of AMP-PNP and/or nucleotide depletion. We also report that, unlike antibodies to conventional kinesin, the peptide antibodies to the kinesin motor domain immunofluorescently label spindles and kinetochores in mitotic tissue culture cells, suggesting that kinesin-like proteins may have important roles in chromosome movement and mitosis.
Assuntos
Cinesinas/imunologia , Mitose/fisiologia , Oligopeptídeos/imunologia , Fuso Acromático/química , Trifosfato de Adenosina/farmacologia , Adenilil Imidodifosfato/farmacologia , Sequência de Aminoácidos , Animais , Formação de Anticorpos , Células CHO/química , Centrômero/química , Centrômero/imunologia , Cricetinae , Imunofluorescência , Proteínas Associadas aos Microtúbulos/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/isolamento & purificação , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Mitose/imunologia , Dados de Sequência Molecular , Óvulo/química , Fuso Acromático/imunologia , XenopusRESUMO
Injection of purified autoantibodies against human centromeric proteins into HeLa cells during interphase disrupts the organization of the kinetochore and interferes with chromosomal movements during the subsequent mitosis even though the chromosomes retain the ability to bind microtubules. We have investigated the hypothesis that this phenotype arises from effects on cytoplasmic dynein, the microtubule motor protein. In previous experiments we found that introduction of anticentromere antibodies into cell nuclei during the G1- or S-phases causes a prometaphase-like arrest, while injections during G2-phase cause a metaphase arrest. We show here that, in both cases, the level of detectable cytoplasmic dynein at kinetochores is significantly decreased. In contrast, when injected cells were permitted to enter mitosis in the absence of microtubules (conditions where trilaminar kinetochores could be detected by electron microscopy), the intensity of dynein labeling on the kinetochores was identical to that seen in uninjected control cells exposed to colcemid. Therefore, the loss of dynein label on mitotic kinetochores was correlated both with the injection of anticentromere antibodies and with the presence of intact spindle microtubules. We suggest that the injection of anticentromere antibodies somehow weakens the association of dynein with the kinetochore, so that when microtubules are present, these motor molecules are pulled away from the kinetochores as they generate force. This model offers an explanation for the failure of chromosomes of injected cells to move normally in mitosis even though they have attached microtubules.
Assuntos
Proteínas Cromossômicas não Histona/imunologia , Dineínas/genética , Cinetocoros/fisiologia , Mitose/efeitos dos fármacos , Anticorpos/farmacologia , Autoantígenos/genética , Autoantígenos/imunologia , Centrômero/genética , Centrômero/imunologia , Proteínas Cromossômicas não Histona/genética , Citoplasma/química , Demecolcina/farmacologia , Dineínas/química , Dineínas/efeitos dos fármacos , Células HeLa , Humanos , Imunoglobulina G/farmacologia , Cinetocoros/química , Cinetocoros/efeitos dos fármacos , Metáfase/efeitos dos fármacos , Microinjeções , Microtúbulos/metabolismo , Mitose/imunologia , Modelos Genéticos , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/genética , Coloração e RotulagemRESUMO
What is the origin of the forces generating chromosome and spindle movements in mitosis? Both microtubule dynamics and microtubule-dependent motors have been proposed as the source of these motor forces. Cytoplasmic dynein and kinesin are two soluble proteins that power membranous organelle movements on microtubules. Kinesin directs movement of organelles to the 'plus' end of microtubules, and is found at the mitotic spindle in sea urchin embryos, but not in mammalian cells. Cytoplasmic dynein translocates organelles to the 'minus' end of microtubules, and is composed of two heavy chains and several light chains. We report here that monoclonal antibodies to two of these subunits and to another polypeptide that associates with dynein localize the protein to the mitotic spindle and to the kinetochores of isolated chromosomes, suggesting that cytoplasmic dynein is important in powering movements of the spindle and chromosomes in dividing cells.
Assuntos
Adenosina Trifosfatases/metabolismo , Cromossomos/ultraestrutura , Dineínas/metabolismo , Microtúbulos/ultraestrutura , Fuso Acromático/ultraestrutura , Animais , Linhagem Celular , Células Cultivadas , Embrião de Galinha , Cromossomos/enzimologia , Dineínas/análise , Dineínas/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Fibroblastos/enzimologia , Fibroblastos/ultraestrutura , Immunoblotting , Microtúbulos/enzimologia , Peso Molecular , Fuso Acromático/enzimologiaRESUMO
The spindle in dividing cells of the diatom Stephanopyxis turris contains three distinct classes of microtubules: central spindle microtubules, which slide over each other and grow during anaphase spindle elongation; kinetochore-attached microtubules, which are located on the outer surface of the central spindle; and peripheral microtubules, which fan out from the spindle poles in astral-like arrays. The poles are multilayered structures, which remain attached to the spindle after isolation. In vitro, after addition of ATP, central spindles elongate and the two half-spindles slide completely apart with a concurrent decrease in the extent and magnitude of the zone of microtubule overlap. Spindle elongation takes place in spindles whose chromatin has been removed by enzymic digestion and the extent of elongation in vitro is increased by the addition of neurotubulin. After ATP addition the arrays of interdigitating microtubules in the zone of overlap become disordered and selectively depolymerize from the overlap zone polewards. In some reactivated spindles an unusual structure, a striated fibre, can be seen running from the pole plates part of the way towards the spindle midzone. The fibre has no precedent in mitotic ultrastructure and its function is unclear. These results demonstrate that we can duplicate the essential elements of anaphase B in vitro and that this system will be useful for further studies of the molecular basis of spindle elongation.