RESUMO
Spindle assembly checkpoint proteins have been thought to reside in the peripheral corona region of the kinetochore, distal to microtubule attachment sites at the outer plate. However, recent biochemical evidence indicates that checkpoint proteins are closely linked to the core kinetochore microtubule attachment site comprised of the Knl1-Mis12-Ndc80 (KMN) complexes/KMN network. In this paper, we show that the Knl1-Zwint1 complex is required to recruit the Rod-Zwilch-Zw10 (RZZ) and Mad1-Mad2 complexes to the outer kinetochore. Consistent with this, nanometer-scale mapping indicates that RZZ, Mad1-Mad2, and the C terminus of the dynein recruitment factor Spindly are closely juxtaposed with the KMN network in metaphase cells when their dissociation is blocked and the checkpoint is active. In contrast, the N terminus of Spindly is â¼75 nm outside the calponin homology domain of the Ndc80 complex. These results reveal how checkpoint proteins are integrated within the substructure of the kinetochore and will aid in understanding the coordination of microtubule attachment and checkpoint signaling during chromosome segregation.
Assuntos
Cinetocoros/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular , Microtúbulos/metabolismo , Motivos de Aminoácidos , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/metabolismo , Dineínas/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Cinetocoros/efeitos dos fármacos , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Metáfase/efeitos dos fármacos , Proteínas dos Microtúbulos/química , Proteínas dos Microtúbulos/metabolismo , Microtúbulos/efeitos dos fármacos , Proteínas Mutantes/metabolismo , Nocodazol/farmacologia , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Subunidades Proteicas/metabolismoRESUMO
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
Kinetochores mediate chromosome segregation at mitosis. They are thought to contain both active, force-producing and passive, frictional interfaces with microtubules whose relative locations have been unclear. We inferred mechanical deformation within single kinetochores during metaphase oscillations by measuring average separations between fluorescently labeled kinetochore subunits in living cells undergoing mitosis. Inter-subunit distances were shorter in kinetochores moving toward poles than in those moving away. Inter-subunit separation decreased abruptly when kinetochores switched to poleward movement and decreased further when pulling force increased, suggesting that active force generation during poleward movement compresses kinetochores. The data revealed an active force-generating interface within kinetochores and a separate passive frictional interface located at least 20 nanometers away poleward. Together, these interfaces allow persistent attachment with intermittent active force generation.
Assuntos
Segregação de Cromossomos/fisiologia , Cinetocoros/fisiologia , Metáfase , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos/genética , Cinetocoros/química , Fenômenos Mecânicos , Movimento (Física) , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , PotoroidaeRESUMO
Cdt1, a protein critical for replication origin licensing in G1 phase, is degraded during S phase but re-accumulates in G2 phase. We now demonstrate that human Cdt1 has a separable essential mitotic function. Cdt1 localizes to kinetochores during mitosis through interaction with the Hec1 component of the Ndc80 complex. G2-specific depletion of Cdt1 arrests cells in late prometaphase owing to abnormally unstable kinetochore-microtubule (kMT) attachments and Mad1-dependent spindle-assembly-checkpoint activity. Cdt1 binds a unique loop extending from the rod domain of Hec1 that we show is also required for kMT attachment. Mutation of the loop domain prevents Cdt1 kinetochore localization and arrests cells in prometaphase. Super-resolution fluorescence microscopy indicates that Cdt1 binding to the Hec1 loop domain promotes a microtubule-dependent conformational change in the Ndc80 complex in vivo. These results support the conclusion that Cdt1 binding to Hec1 is essential for an extended Ndc80 configuration and stable kMT attachment.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Sequência de Aminoácidos , Pontos de Checagem do Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas do Citoesqueleto , Células HeLa , Humanos , Cinetocoros/química , Microtúbulos/química , Mitose , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genéticaRESUMO
Kinetochores bound to kinetochore microtubules (kMTs) exhibit directional instability in mammalian and other mitotic vertebrate cells, oscillating between poleward (P) and away-from-the-pole (AP) movements. These oscillations are coupled to changes in length of kMTs in a way that maintains a net stretch of the centromere. To understand how sister kinetochore directional instability and kMT plus-end dynamic instability are coupled to oscillations in centromere stretch, we tracked at high resolution the positions of fluorescent kinetochores and their poles for oscillating chromosomes within spindles of metaphase PtK1 cells. We found that the kinetics of P and AP movement are nonlinear and different. By subtracting contributions from the poleward flux of kMTs, we found that maximum centromere stretch occurred when the leading kinetochore switched from depolymerization to polymerization, whereas minimum centromere stretch occurred on average 7 s after the initially trailing kinetochore switched from polymerization to depolymerization. These differences produce oscillations in centromere stretch at about twice the frequency of kinetochore directional instability and at about twice the frequency of centromere oscillations back and forth across the spindle equator.
Assuntos
Centrômero/metabolismo , Cinetocoros/metabolismo , Metáfase , Animais , Linhagem Celular , Cromossomos/metabolismo , Dipodomys , Fuso AcromáticoRESUMO
Successful completion of mitosis requires that sister kinetochores become attached end-on to the plus ends of spindle microtubules (MTs) in prometaphase, thereby forming kinetochore microtubules (kMTs) that tether one sister to one spindle pole and the other sister to the opposite pole. Sites for kMT attachment provide at least four key functions: robust and dynamic kMT anchorage; force generation that can be coupled to kMT plus-end dynamics; correction of errors in kMT attachment; and control of the spindle assembly checkpoint (SAC). The SAC typically delays anaphase until chromosomes achieve metaphase alignment with each sister kinetochore acquiring a full complement of kMTs. Although it has been known for over 30 years that MT motor proteins reside at kinetochores, a highly conserved network of protein complexes, called the KMN network, has emerged in recent years as the primary interface between the kinetochore and kMTs. This Commentary will summarize recent advances in our understanding of the role of the KMN network for the key kinetochore functions, with a focus on human cells.
Assuntos
Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Proteínas do Citoesqueleto , Humanos , Proteínas Nucleares/metabolismoRESUMO
Cse4 is the budding yeast homologue of CENP-A, a modified histone H3 that specifies the base of kinetochores in all eukaryotes. Budding yeast is unique in having only one kinetochore microtubule attachment site per centromere. The centromere is specified by CEN DNA, a sequence-specific binding complex (CBF3), and a Cse4-containing nucleosome. Here we compare the ratio of kinetochore proximal Cse4-GFP fluorescence at anaphase to several standards including purified EGFP molecules in vitro to generate a calibration curve for the copy number of GFP-fusion proteins. Our results yield a mean of ~5 Cse4s, ~3 inner kinetochore CBF3 complexes, and ~20 outer kinetochore Ndc80 complexes. Our calibrated measurements increase 2.5-3-fold protein copy numbers at eukaryotic kinetochores based on previous ratio measurements assuming two Cse4s per budding yeast kinetochore. All approximately five Cse4s may be associated with the CEN nucleosome, but we show that a mean of three Cse4s could be located within flanking nucleosomes at random sites that differ between chromosomes.
Assuntos
Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Nucleossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Cinetocoros/metabolismoRESUMO
Fluorescent speckle microscopy (FSM) is a method for measuring the movements and dynamic assembly of macromolecular assemblies such as cytoskeletal filaments (e.g., microtubules and actin) or focal adhesions within large arrays in living cells or in preparations in vitro. The discovery of the method depended on recognizing the importance of unexpected fluorescence images of microtubules obtained by time-lapse recording of vertebrate epithelial cells in culture. In cells that were injected with fluorescent tubulin at ~10% of the cytosol pool, microtubules typically appeared as smooth threads with a nearly constant fluorescence intensity. One day, when an unusually low concentration of fluorescent tubulin was injected into cells, the images from a sensitive cooled charge-coupled detector camera showed microtubules with an unusual "speckled" appearance-there were fluorescent dots with variable intensity and spacing along the microtubules. A first thought was that the speckles were an artifact. With further thought, we surmised that the speckles could be telling us something about stochastic association of tubulin dimers with the growing end of a microtubule. Numerous experiments confirmed the latter hypothesis. Subsequently the method we call FSM has proven to be very valuable. The speckles turned out not to be a meaningless artifact, but rather a serendipitous find.
Assuntos
Microscopia de Fluorescência/métodos , Microtúbulos/metabolismo , Animais , Células Cultivadas , Humanos , Análise de Célula Única/métodos , Imagem com Lapso de Tempo , Tubulina (Proteína)/metabolismoRESUMO
To define the molecular architecture of the kinetochore in vertebrate cells, we measured the copy number of eight kinetochore proteins that link kinetochore microtubules (MTs [kMTs]) to centromeric DNA. We used a fluorescence ratio method and chicken DT40 cell lines in which endogenous loci encoding the analyzed proteins were deleted and complemented using integrated green fluorescent protein fusion transgenes. For a mean of 4.3 kMTs at metaphase, the protein copy number per kMT is between seven and nine for members of the MT-binding KNL-1/Mis12 complex/Ndc80 complex network. It was between six and nine for four members of the constitutive centromere-associated network: centromere protein C (CENP-C), CENP-H, CENP-I, and CENP-T. The similarity in copy number per kMT for all of these proteins suggests that each MT end is linked to DNA by six to nine fibrous unit attachment modules in vertebrate cells, a conclusion that indicates architectural conservation between multiple MT-binding vertebrate and single MT-binding budding yeast kinetochores.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Animais , Linhagem Celular , Galinhas , Proteínas Cromossômicas não Histona/genética , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Digital fluorescence microscopy is commonly used to track individual proteins and their dynamics in living cells. However, extracting molecule-specific information from fluorescence images is often limited by the noise and blur intrinsic to the cell and the imaging system. Here we discuss a method called "model-convolution," which uses experimentally measured noise and blur to simulate the process of imaging fluorescent proteins whose spatial distribution cannot be resolved. We then compare model-convolution to the more standard approach of experimental deconvolution. In some circumstances, standard experimental deconvolution approaches fail to yield the correct underlying fluorophore distribution. In these situations, model-convolution removes the uncertainty associated with deconvolution and therefore allows direct statistical comparison of experimental and theoretical data. Thus, if there are structural constraints on molecular organization, the model-convolution method better utilizes information gathered via fluorescence microscopy, and naturally integrates experiment and theory.
RESUMO
Recent high-resolution studies of kinetochore structure have transformed the way researchers think about this crucial macro-molecular complex, which is essential for ensuring chromosome segregation occurs faithfully during cell division. Kinetochores mediate the interaction between chromosomes and the plus-ends of dynamic spindle microtubules and control the timing of anaphase onset by regulating the spindle assembly checkpoint (SAC). There is much debate in the SAC research community as to whether mitotic cells sense only microtubule attachment at the kinetochore, or both attachment and tension, before committing to anaphase. In this Commentary, we present a brief history of the tension-versus-attachment debate, summarize recent advances in our understanding of kinetochore structure and focus on the implications of a phenomenon known as intrakinetochore stretch for SAC regulation. We also hypothesize how intrakinetochore stretch might impact SAC function by regulating both microtubule attachment stability and the localization and activity of checkpoint components at the kinetochore.
Assuntos
Anáfase , Cinetocoros/metabolismo , Transdução de Sinais , Animais , Aurora Quinases , Fenômenos Biomecânicos , Humanos , Cinetocoros/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Fuso Acromático/metabolismoRESUMO
Generation of motile force is one of the main functions of the eukaryotic kinetochore during cell division. In recent years, the KMN network of proteins (Ndc80 complex, Mis12 complex, and KNL-1 complex) has emerged as a highly conserved core microtubule-binding complex at the kinetochore. It plays a major role in coupling force generation to microtubule plus-end polymerization and depolymerization. In this review, we discuss current theoretical mechanisms of force generation, and then focus on emerging information about mechanistic contributions from the Ndc80 complex in eukaryotes and the microtubule-binding Dam1/DASH complex from fungi. New information has also become available from super-resolution light microscopy on the protein architecture of the kinetochore-microtubule attachment site in both budding yeast and humans, which provides further insight into the mechanism of force generation. We briefly discuss potential contributions of motors, other microtubule-associated proteins, and microtubule depolymerases. Using the above evidence, we present speculative models of force generation at the kinetochore.
Assuntos
Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Estresse Mecânico , Células HeLa , Humanos , Cinetocoros/ultraestrutura , Microtúbulos/ultraestrutura , Modelos Teóricos , Proteínas Motores Moleculares/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismoRESUMO
The mitotic spindle is essential for chromosome segregation and must be large enough to accommodate all of the chromatin in the dividing cell. In this issue, Dinarina et al. (2009) grow "fields" of spindles on coverslips to investigate the relationship between chromatin and spindle size as well as intrinsic mechanisms of spindle assembly.
Assuntos
Cromatina , Fuso Acromático , Animais , Extratos Celulares , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMO
Chromosome segregation requires assembly of kinetochores on centromeric chromatin to mediate interactions with spindle microtubules and control cell-cycle progression. To elucidate the protein architecture of human kinetochores, we developed a two-color fluorescence light microscopy method that measures average label separation, Delta, at <5 nm accuracy. Delta analysis of 16 proteins representing core structural complexes spanning the centromeric chromatin-microtubule interface, when correlated with mechanical states of spindle-attached kinetochores, provided a nanometer-scale map of protein position and mechanical properties of protein linkages. Treatment with taxol, which suppresses microtubule dynamics and activates the spindle checkpoint, revealed a specific switch in kinetochore architecture. Cumulatively, Delta analysis revealed that compliant linkages are restricted to the proximity of chromatin, suggested a model for how the KMN (KNL1/Mis12 complex/Ndc80 complex) network provides microtubule attachment and generates pulling forces from depolymerization, and identified an intrakinetochore molecular switch that may function in controlling checkpoint activity.
Assuntos
Cinetocoros/química , Cinetocoros/metabolismo , Microtúbulos/química , Microtúbulos/metabolismo , Proteínas do Citoesqueleto , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , Metáfase , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas NuclearesRESUMO
The kinetochore is a macromolecular protein machine [1] that links centromeric chromatin to the plus ends of one or more microtubules (MTs) and segregates chromosomes during cell division. Its core structure consists of eight multicomponent protein complexes, most of which are conserved in all eukaryotes. We use an in vivo two-color fluorescence microscopy technique to determine, for the first time, the location of these proteins along the budding yeast kinetochore axis at nanometer resolution. Together with kinetochore protein counts [2, 3], these localizations predict the 3D protein architecture of a metaphase kinetochore-microtubule attachment and provide new functional insights. We also find that the kinetochore becomes much shorter in anaphase as metaphase tension is lost. Shortening is due mainly to a decrease in the length of the Ndc80 complex, which may result either from intramolecular bending of the Ndc80 complex at the kink within the stalk region of the Ndc80-Nuf2 dimer [4, 5] or from a change in its orientation relative to the microtubule axis. Conformational changes within the Ndc80 and Mtw1 complexes may serve as mechanical cues for tension-dependent regulation of MT attachment and the spindle-assembly checkpoint. The geometry of the core structure of the budding yeast kinetochore reported here is remarkably similar to that found in mammalian kinetochores, indicating that kinetochore structure is conserved in eukaryotes with either point or regional centromeres.
Assuntos
Cinetocoros/ultraestrutura , Microscopia de Fluorescência/métodos , Microtúbulos/metabolismo , Fuso Acromático/ultraestrutura , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cinetocoros/metabolismo , Microtúbulos/ultraestrutura , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/ultraestrutura , Conformação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/ultraestrutura , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Fuso Acromático/metabolismoRESUMO
BACKGROUND: Bipolar spindle assembly is critical for achieving accurate segregation of chromosomes. In the absence of centrosomes, meiotic spindles achieve bipolarity by a combination of chromosome-initiated microtubule nucleation and stabilization and motor-driven organization of microtubules. Once assembled, the spindle structure is maintained on a relatively long time scale despite the high turnover of the microtubules that comprise it. To study the underlying mechanisms responsible for spindle assembly and steady-state maintenance, we used microneedle manipulation of preassembled spindles in Xenopus egg extracts. RESULTS: When two meiotic spindles were brought close enough together, they interacted, creating an interconnected microtubule structure with supernumerary poles. Without exception, the perturbed system eventually re-established bipolarity, forming a single spindle of normal shape and size. Bipolar spindle fusion was blocked when cytoplasmic dynein function was perturbed, suggesting a critical role for the motor in this process. The fusion of Eg5-inhibited monopoles also required dynein function but only occurred if the initial interpolar separation was less than twice the microtubule radius of a typical monopole. CONCLUSIONS: Our experiments uniquely illustrate the architectural plasticity of the spindle and reveal a robust ability of the system to attain a bipolar morphology. We hypothesize that a major mechanism driving spindle fusion is dynein-mediated sliding of oppositely oriented microtubules, a novel function for the motor, and posit that this same mechanism might also be involved in normal spindle assembly and homeostasis.
Assuntos
Dineínas/metabolismo , Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Animais , Polaridade Celular , Segregação de Cromossomos , Cinesinas/genética , Cinesinas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Oócitos/citologia , Oócitos/fisiologia , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMO
Bub1 is a serine/threonine kinase originally described as a core component of the spindle assembly checkpoint (SAC) mechanism in yeast. Bub1 binding at kinetochores has been reported to be required for SAC function and localization of other SAC components. A proper SAC is believed to be essential for murine embryonic development, as all previously described null mutations in SAC components in mice cause embryonic lethality. We produced mice harboring a Bub1 mutant allele lacking exons 2 and 3, resulting in a hypomorphic mutant expressed at <5% of wild-type levels. Despite this significant reduction, homozygous mutant animals are viable on a mixed 129P2/B6 or FVB background but display increased tumorigenesis with aging, whereas mice with a C57Bl/6J background die perinatally. Bub1 mutant murine embryonic fibroblasts (MEFs) display defects in chromosome congression to the metaphase plate, severe chromosome missegregation, and aneuploidy accompanied by high levels of premature senescence. Mutant MEFs have a robust SAC in response to nocodazole treatment but an impaired response to Taxol. Mutant MEFs also show reduced kinetochore localization of BubR1, but not of Mad2. The significant reduction in SAC response to Taxol, but not nocodazole, coupled with the reduced binding of BubR1, but not Mad2, indicates that Bub1 is particularly critical for the SAC response to a lack of tension on kinetochores. Thus, Bub1 is essential for proper chromosome segregation, a defect that can lead to severe phenotypes, including perinatal lethality and a predisposition to cancer.
Assuntos
Transformação Celular Neoplásica/genética , Proteínas Serina-Treonina Quinases/genética , Alelos , Aneuploidia , Animais , Proteínas de Ciclo Celular , Transformação Celular Neoplásica/metabolismo , Segregação de Cromossomos , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Fibroblastos/fisiologia , Cinetocoros/efeitos dos fármacos , Cinetocoros/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Nocodazol/farmacologia , Paclitaxel/farmacologia , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/genética , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/enzimologia , Fuso Acromático/genéticaRESUMO
Aurora-A is a conserved kinase implicated in mitotic regulation and carcinogenesis. Aurora-A was previously implicated in mitotic entry and spindle assembly, although contradictory results prevented a clear understanding of the roles of Aurora-A in mammals. We developed a conditional null mutation in the mouse Aurora-A gene to investigate Aurora-A functions in primary cells ex vivo and in vivo. We show here that conditional Aurora-A ablation in cultured embryonic fibroblasts causes impaired mitotic entry and mitotic arrest with a profound defect in bipolar spindle formation. Germ line Aurora-A deficiency causes embryonic death at the blastocyst stage with pronounced cell proliferation failure, mitotic arrest, and monopolar spindle formation. Aurora-A deletion in mid-gestation embryos causes an increase in mitotic and apoptotic cells. These results indicate that murine Aurora-A facilitates, but is not absolutely required for, mitotic entry in murine embryonic fibroblasts and is essential for centrosome separation and bipolar spindle formation in vitro and in vivo. Aurora-A deletion increases apoptosis, suggesting that molecular therapies targeting Aurora-A may be effective in inducing tumor cell apoptosis. Aurora-A conditional mutant mice provide a valuable system for further defining Aurora-A functions and for predicting effects of Aurora-A therapeutic intervention.
Assuntos
Desenvolvimento Embrionário , Proteínas Serina-Treonina Quinases/metabolismo , Fuso Acromático/enzimologia , Alelos , Animais , Apoptose , Aurora Quinase A , Aurora Quinases , Blastocisto/citologia , Blastocisto/enzimologia , Proliferação de Células , Perda do Embrião , Embrião de Mamíferos/citologia , Embrião de Mamíferos/enzimologia , Feminino , Fibroblastos/citologia , Fibroblastos/enzimologia , Deleção de Genes , Marcação de Genes , Camundongos , Mitose , Mutação/genética , Ploidias , Gravidez , Proteínas Serina-Treonina Quinases/deficiênciaRESUMO
Point and regional centromeres specify a unique site on each chromosome for kinetochore assembly. The point centromere in budding yeast is a unique 150-bp DNA sequence, which supports a kinetochore with only one microtubule attachment. In contrast, regional centromeres are complex in architecture, can be up to 5 Mb in length, and typically support many kinetochore-microtubule attachments. We used quantitative fluorescence microscopy to count the number of core structural kinetochore protein complexes at the regional centromeres in fission yeast and Candida albicans. We find that the number of CENP-A nucleosomes at these centromeres reflects the number of kinetochore-microtubule attachments instead of their length. The numbers of kinetochore protein complexes per microtubule attachment are nearly identical to the numbers in a budding yeast kinetochore. These findings reveal that kinetochores with multiple microtubule attachments are mainly built by repeating a conserved structural subunit that is equivalent to a single microtubule attachment site.
Assuntos
Candida albicans/citologia , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Schizosaccharomyces/citologia , Autoantígenos/metabolismo , Proteína Centromérica A , Proteínas Cromossômicas não Histona/metabolismo , DNA Fúngico/metabolismo , Fluorescência , Fase G2 , Metáfase , Saccharomyces cerevisiae/citologia , Proteínas de Schizosaccharomyces pombeRESUMO
In budding yeast, the mitotic spindle is comprised of 32 kinetochore microtubules (kMTs) and approximately 8 interpolar MTs (ipMTs). Upon anaphase onset, kMTs shorten to the pole, whereas ipMTs increase in length. Overlapping MTs are responsible for the maintenance of spindle integrity during anaphase. To dissect the requirements for anaphase spindle stability, we introduced a conditionally functional dicentric chromosome into yeast. When centromeres from the same sister chromatid attach to opposite poles, anaphase spindle elongation is delayed and a DNA breakage-fusion-bridge cycle ensues that is dependent on DNA repair proteins. We find that cell survival after dicentric chromosome activation requires the MT-binding proteins Kar3p, Bim1p, and Ase1p. In their absence, anaphase spindles are prone to collapse and buckle in the presence of a dicentric chromosome. Our analysis reveals the importance of Bim1p in maintaining a stable ipMT overlap zone by promoting polymerization of ipMTs during anaphase, whereas Kar3p contributes to spindle stability by cross-linking spindle MTs.