RESUMEN
The network structure of densely packed chromatin within the nucleus of eukaryotic cells acts in concert with nonequilibrium processes. Using statistical physics simulations, we explore the control provided by transient crosslinking of the chromatin network by structural-maintenance-of-chromosome (SMC) proteins over (i) the physical properties of the chromatin network and (ii) condensate formation of embedded molecular species. We find that the density and lifetime of transient SMC crosslinks regulate structural relaxation modes and tune the sol-vs-gel state of the chromatin network, which imparts control over the kinetic pathway to condensate formation. Specifically, lower density, shorter-lived crosslinks induce sollike networks and a droplet-fusion pathway, whereas higher density, longer-lived crosslinks induce gellike networks and an Ostwald-ripening pathway.
Asunto(s)
Cromatina , Cromatina/metabolismo , Cinética , Condensados Biomoleculares/metabolismo , Modelos Moleculares , Reactivos de Enlaces Cruzados/químicaRESUMEN
Centromere (CEN) identity is specified epigenetically by specialized nucleosomes containing evolutionarily conserved CEN-specific histone H3 variant CENP-A (Cse4 in Saccharomyces cerevisiae, CENP-A in humans), which is essential for faithful chromosome segregation. However, the epigenetic mechanisms that regulate Cse4 function have not been fully defined. In this study, we show that cell cycle-dependent methylation of Cse4-R37 regulates kinetochore function and high-fidelity chromosome segregation. We generated a custom antibody that specifically recognizes methylated Cse4-R37 and showed that methylation of Cse4 is cell cycle regulated with maximum levels of methylated Cse4-R37 and its enrichment at the CEN chromatin occur in the mitotic cells. Methyl-mimic cse4-R37F mutant exhibits synthetic lethality with kinetochore mutants, reduced levels of CEN-associated kinetochore proteins and chromosome instability (CIN), suggesting that mimicking the methylation of Cse4-R37 throughout the cell cycle is detrimental to faithful chromosome segregation. Our results showed that SPOUT methyltransferase Upa1 contributes to methylation of Cse4-R37 and overexpression of UPA1 leads to CIN phenotype. In summary, our studies have defined a role for cell cycle-regulated methylation of Cse4 in high-fidelity chromosome segregation and highlight an important role of epigenetic modifications such as methylation of kinetochore proteins in preventing CIN, an important hallmark of human cancers.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomycetales , Humanos , Ciclo Celular , Centrómero/metabolismo , Proteína A Centromérica/metabolismo , Inestabilidad Cromosómica , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Metilación , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismoRESUMEN
Chromatin motions depend on and may regulate genome functions, in particular the DNA damage response. In yeast, DNA double-strand breaks (DSBs) globally increase chromatin diffusion, whereas in higher eukaryotes the impact of DSBs on chromatin dynamics is more nuanced. We mapped the motions of chromatin microdomains in mammalian cells using diffractive optics and photoactivatable chromatin probes and found a high level of spatial heterogeneity. DNA damage reduces heterogeneity and imposes spatially defined shifts in motions: Distal to DNA breaks, chromatin motions are globally reduced, whereas chromatin retains higher mobility at break sites. These effects are driven by context-dependent changes in chromatin compaction. Photoactivated lattices of chromatin microdomains are ideal to quantify microscale coupling of chromatin motion. We measured correlation distances up to 2 µm in the cell nucleus, spanning chromosome territories, and speculate that this correlation distance between chromatin microdomains corresponds to the physical separation of A and B compartments identified in chromosome conformation capture experiments. After DNA damage, chromatin motions become less correlated, a phenomenon driven by phase separation at DSBs. Our data indicate tight spatial control of chromatin motions after genomic insults, which may facilitate repair at the break sites and prevent deleterious contacts of DSBs, thereby reducing the risk of genomic rearrangements.
Asunto(s)
Ensamble y Desensamble de Cromatina , Cromatina , Roturas del ADN de Doble Cadena , Reparación del ADN , Animales , Cromatina/química , Cromosomas , Saccharomyces cerevisiae/genéticaRESUMEN
Faithful chromosome segregation maintains chromosomal stability as errors in this process contribute to chromosomal instability (CIN), which has been observed in many diseases including cancer. Epigenetic regulation of kinetochore proteins such as Cse4 (CENP-A in humans) plays a critical role in high-fidelity chromosome segregation. Here we show that Cse4 is a substrate of evolutionarily conserved Cdc7 kinase, and that Cdc7-mediated phosphorylation of Cse4 prevents CIN. We determined that Cdc7 phosphorylates Cse4 in vitro and interacts with Cse4 in vivo in a cell cycle-dependent manner. Cdc7 is required for kinetochore integrity as reduced levels of CEN-associated Cse4, a faster exchange of Cse4 at the metaphase kinetochores, and defects in chromosome segregation, are observed in a cdc7-7 strain. Phosphorylation of Cse4 by Cdc7 is important for cell survival as constitutive association of a kinase-dead variant of Cdc7 (cdc7-kd) with Cse4 at the kinetochore leads to growth defects. Moreover, phospho-deficient mutations of Cse4 for consensus Cdc7 target sites contribute to CIN phenotype. In summary, our results have defined a role for Cdc7-mediated phosphorylation of Cse4 in faithful chromosome segregation.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica/fisiología , Proteínas de Unión al ADN/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/fisiología , Centrómero/metabolismo , Proteína A Centromérica/metabolismo , Cromatina/metabolismo , Inestabilidad Cromosómica , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/fisiología , Cromosomas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Epigénesis Genética , Histonas/metabolismo , Cinetocoros/metabolismo , Proteínas Nucleares/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/fisiología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologíaRESUMEN
R-loops, the byproduct of DNA-RNA hybridization and the displaced single-stranded DNA (ssDNA), have been identified in bacteria, yeasts, and other eukaryotic organisms. The persistent presence of R-loops contributes to defects in DNA replication and repair, gene expression, and genomic integrity. R-loops have not been detected at centromeric (CEN) chromatin in wild-type budding yeast. Here we used an hpr1∆ strain that accumulates R-loops to investigate the consequences of R-loops at CEN chromatin and chromosome segregation. We show that Hpr1 interacts with the CEN-histone H3 variant, Cse4, and prevents the accumulation of R-loops at CEN chromatin for chromosomal stability. DNA-RNA immunoprecipitation (DRIP) analysis showed an accumulation of R-loops at CEN chromatin that was reduced by overexpression of RNH1 in hpr1∆ strains. Increased levels of ssDNA, reduced levels of Cse4 and its assembly factor Scm3, and mislocalization of histone H3 at CEN chromatin were observed in hpr1∆ strains. We determined that accumulation of R-loops at CEN chromatin contributes to defects in kinetochore biorientation and chromosomal instability (CIN) and these phenotypes are suppressed by RNH1 overexpression in hpr1∆ strains. In summary, our studies provide mechanistic insights into how accumulation of R-loops at CEN contributes to defects in kinetochore integrity and CIN.
Asunto(s)
Centrómero/metabolismo , Cromatina/química , Inestabilidad Cromosómica , Cinetocoros/metabolismo , Estructuras R-Loop , Saccharomycetales/metabolismo , Ciclo Celular , ADN de Hongos/metabolismo , Genoma Fúngico , Histonas/metabolismo , Modelos Biológicos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/citología , Saccharomycetales/genéticaRESUMEN
Anaphase onset is an irreversible cell cycle transition that is triggered by the activation of the protease Separase. Separase cleaves the Mcd1 (also known as Scc1) subunit of Cohesin, a complex of proteins that physically links sister chromatids, triggering sister chromatid separation. Separase is regulated by the degradation of the anaphase inhibitor Securin which liberates Separase from inhibitory Securin/Separase complexes. In many organisms, Securin is not essential suggesting that Separase is regulated by additional mechanisms. In this work, we show that in budding yeast Cdk1 activates Separase (Esp1 in yeast) through phosphorylation to trigger anaphase onset. Esp1 activation is opposed by protein phosphatase 2A associated with its regulatory subunit Cdc55 (PP2ACdc55) and the spindle protein Slk19. Premature anaphase spindle elongation occurs when Securin (Pds1 in yeast) is inducibly degraded in cells that also contain phospho-mimetic mutations in ESP1, or deletion of CDC55 or SLK19. This striking phenotype is accompanied by advanced degradation of Mcd1, disruption of pericentric Cohesin organization and chromosome mis-segregation. Our findings suggest that PP2ACdc55 and Slk19 function redundantly with Pds1 to inhibit Esp1 within pericentric chromatin, and both Pds1 degradation and Cdk1-dependent phosphorylation of Esp1 act together to trigger anaphase onset.
Asunto(s)
Anafase/fisiología , Proteína Quinasa CDC2/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteína Fosfatasa 2/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Separasa/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Mutación , Fosforilación , Proteína Fosfatasa 2/genética , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Securina/genética , Securina/metabolismo , Separasa/genética , Huso Acromático/genética , CohesinasRESUMEN
Centromeres are specialized domains of heterochromatin that provide the foundation for the kinetochore. Centromeric heterochromatin is characterized by specific histone modifications, a centromere-specific histone H3 variant (CENP-A), and the enrichment of cohesin, condensin, and topoisomerase II. Centromere DNA varies orders of magnitude in size from 125 bp (budding yeast) to several megabases (human). In metaphase, sister kinetochores on the surface of replicated chromosomes face away from each other, where they establish microtubule attachment and bi-orientation. Despite the disparity in centromere size, the distance between separated sister kinetochores is remarkably conserved (approximately 1 µm) throughout phylogeny. The centromere functions as a molecular spring that resists microtubule-based extensional forces in mitosis. This review explores the physical properties of DNA in order to understand how the molecular spring is built and how it contributes to the fidelity of chromosome segregation.
Asunto(s)
Centrómero/genética , Segregación Cromosómica/genética , Heterocromatina/genética , Meiosis/genética , Autoantígenos/genética , Proteína A Centromérica , Proteínas Cromosómicas no Histona/genética , Humanos , Cinetocoros/metabolismo , Microtúbulos/genética , SchizosaccharomycesRESUMEN
Lipid droplet (LD) utilization is an important cellular activity that regulates energy balance and release of lipid second messengers. Because fatty acids exhibit both beneficial and toxic properties, their release from LDs must be controlled. Here we demonstrate that yeast Sfh3, an unusual Sec14-like phosphatidylinositol transfer protein, is an LD-associated protein that inhibits lipid mobilization from these particles. We further document a complex biochemical diversification of LDs during sporulation in which Sfh3 and select other LD proteins redistribute into discrete LD subpopulations. The data show that Sfh3 modulates the efficiency with which a neutral lipid hydrolase-rich LD subclass is consumed during biogenesis of specialized membrane envelopes that package replicated haploid meiotic genomes. These results present novel insights into the interface between phosphoinositide signaling and developmental regulation of LD metabolism and unveil meiosis-specific aspects of Sfh3 (and phosphoinositide) biology that are invisible to contemporary haploid-centric cell biological, proteomic, and functional genomics approaches.
Asunto(s)
Metabolismo de los Lípidos , Proteínas de Transferencia de Fosfolípidos/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/metabolismo , Homeostasis , Membranas Intracelulares/metabolismo , Modelos Moleculares , Fosfolipasas/metabolismo , Proteínas de Transferencia de Fosfolípidos/química , Proteínas de Transferencia de Fosfolípidos/metabolismo , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Esporas Fúngicas/metabolismoRESUMEN
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.
Asunto(s)
Centrómero/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Nucleosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Fluorescencia , Proteínas Fluorescentes Verdes/metabolismo , Cinetocoros/metabolismoRESUMEN
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.
Asunto(s)
Cinetocoros/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Estrés Mecánico , Células HeLa , Humanos , Cinetocoros/ultraestructura , Microtúbulos/ultraestructura , Modelos Teóricos , Proteínas Motoras Moleculares/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismoRESUMEN
Accurate segregation of duplicated chromosomes ensures that daughter cells get one and only one copy of each chromosome. Errors in chromosome segregation result in aneuploidy and have severe consequences on human health. Incorrect chromosome number and chromosomal instability are hallmarks of tumor cells. Hence, segregation errors are thought to be a major cause of tumorigenesis. A study of the physical mechanical basis of chromosome segregation is essential to understand the processes that can lead to errors. Tremendous progress has been made in recent years in identifying the proteins necessary for chromosome movement and segregation, but the mechanism and structure of critical force generating components and the molecular basis of centromere stiffness remain poorly understood.
Asunto(s)
Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Huso Acromático/fisiología , Biofisica , Cromatina/genética , Cromatina/fisiología , ADN de Hongos/química , ADN de Hongos/genética , Cinetocoros/fisiología , Proteínas de Microtúbulos/genética , Proteínas de Microtúbulos/fisiología , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/fisiología , Microtúbulos/fisiología , Mitosis/genética , Mitosis/fisiología , Proteínas Motoras Moleculares/genética , Proteínas Motoras Moleculares/fisiología , Saccharomyces cerevisiae/ultraestructura , Huso Acromático/genéticaRESUMEN
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.
Asunto(s)
Candida albicans/citología , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Schizosaccharomyces/citología , Autoantígenos/metabolismo , Proteína A Centromérica , Proteínas Cromosómicas no Histona/metabolismo , ADN de Hongos/metabolismo , Fluorescencia , Fase G2 , Metafase , Saccharomyces cerevisiae/citología , Proteínas de Schizosaccharomyces pombeRESUMEN
BACKGROUND: Cohesin proteins link sister chromatids and provide the basis for tension between bioriented sister chomatids in mitosis. Cohesin is concentrated at the centromere region of the chromosome despite the fact that sister centromeres can be separated by 800 nm in vivo. The function of cohesin at sites of separated DNA is unknown. RESULTS: We provide evidence that the kinetochore promotes the organization of pericentric chromatin into a cruciform in mitosis such that centromere-flanking DNA adopts an intramolecular loop, whereas sister-chromatid arms are paired intermolecularly. Visualization of cohesin subunits by fluorescence microscopy revealed a cylindrical structure that encircles the central spindle and spans the distance between sister kinetochores. Kinetochore assembly at the apex of the loop initiates intrastrand loop formation that extends approximately 25 kb (12.5 kb on either side of the centromere). Two centromere loops (one from each sister chromatid) are stretched between the ends of sister-kinetochore microtubules along the spindle axis. At the base of the loop there is a transition to intermolecular sister-chromatid pairing. CONCLUSIONS: The C loop conformation reveals the structural basis for sister-kinetochore clustering in budding yeast and for kinetochore biorientation and thus resolves the paradox of maximal interstrand separation in regions of highest cohesin concentration.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Mitosis/fisiología , Proteínas Nucleares/metabolismo , Fenómenos Biomecánicos , Conformación Molecular , Saccharomyces cerevisiae , Huso Acromático/metabolismo , CohesinasRESUMEN
Genetically encoded fluorescent proteins are an essential tool in cell biology, widely used for investigating cellular processes with molecular specificity. Direct uses of fluorescent proteins include studies of the in vivo cellular localization and dynamics of a protein, as well as measurement of its in vivo concentration. In this chapter, we focus on the use of genetically encoded fluorescent protein as an accurate reporter of in vivo protein numbers. Using the challenge of counting the number of copies of kinetochore proteins in budding yeast as a case study, we discuss the basic considerations in developing a technique for the accurate evaluation of intracellular fluorescence signal. This discussion includes criteria for the selection of a fluorescent protein with optimal characteristics, selection of microscope and image acquisition system components, the design of a fluorescence signal quantification technique, and possible sources of measurement errors. We also include a brief survey of available calibration standards for converting the fluorescence measurements into a number of molecules, since the availability of such a standard usually determines the design of the signal measurement technique as well as the accuracy of final measurements. Finally, we show that, as in the case of budding yeast kinetochore proteins, the in vivo intracellular protein numbers determined from fluorescence measurements can also be employed to elucidate details of cellular structures.
Asunto(s)
Proteínas Fluorescentes Verdes , Cinetocoros/química , Proteínas Luminiscentes , Microscopía Fluorescente/métodos , Proteínas de Saccharomyces cerevisiae/análisis , Saccharomyces cerevisiae/química , Ciclo Celular , Proteínas Fluorescentes Verdes/genética , Cinetocoros/ultraestructura , Proteínas Luminiscentes/genética , Microscopía Fluorescente/instrumentación , Técnicas de Sonda Molecular , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/química , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/química , Huso Acromático/química , Huso Acromático/ultraestructuraRESUMEN
Fluorescence microscopy provides a powerful method for localization of structures in biological specimens. However, aspects of the image formation process such as noise and blur from the microscope's point-spread function combine to produce an unintuitive image transformation on the true structure of the fluorescing molecules in the specimen, hindering qualitative and quantitative analysis of even simple structures in unprocessed images. We introduce FluoroSim, an interactive fluorescence microscope simulator that can be used to train scientists who use fluorescence microscopy to understand the artifacts that arise from the image formation process, to determine the appropriateness of fluorescence microscopy as an imaging modality in an experiment, and to test and refine hypotheses of model specimens by comparing the output of the simulator to experimental data. FluoroSim renders synthetic fluorescence images from arbitrary geometric models represented as triangle meshes. We describe three rendering algorithms on graphics processing units for computing the convolution of the specimen model with a microscope's point-spread function and report on their performance. We also discuss several cases where the microscope simulator has been used to solve real problems in biology.
RESUMEN
Kinetochore attachment to spindle microtubule plus-ends is necessary for accurate chromosome segregation during cell division in all eukaryotes. The centromeric DNA of each chromosome is linked to microtubule plus-ends by eight structural-protein complexes. Knowing the copy number of each of these complexes at one kinetochore-microtubule attachment site is necessary to understand the molecular architecture of the complex, and to elucidate the mechanisms underlying kinetochore function. We have counted, with molecular accuracy, the number of structural protein complexes in a single kinetochore-microtubule attachment using quantitative fluorescence microscopy of GFP-tagged kinetochore proteins in the budding yeast Saccharomyces cerevisiae. We find that relative to the two Cse4p molecules in the centromeric histone, the copy number ranges from one or two for inner kinetochore proteins such as Mif2p, to 16 for the DAM-DASH complex at the kinetochore-microtubule interface. These counts allow us to visualize the overall arrangement of a kinetochore-microtubule attachment. As most of the budding yeast kinetochore proteins have homologues in higher eukaryotes, including humans, this molecular arrangement is likely to be replicated in more complex kinetochores that have multiple microtubule attachments.
Asunto(s)
Cinetocoros/química , Microtúbulos/química , Complejos Multiproteicos/química , Sitios de Unión , Proteínas Cromosómicas no Histona/análisis , Proteínas Cromosómicas no Histona/química , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/química , Proteínas Fluorescentes Verdes , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Complejos Multiproteicos/análisis , Proteínas de Saccharomyces cerevisiae/análisis , Proteínas de Saccharomyces cerevisiae/química , Huso AcromáticoRESUMEN
The budding yeast kinetochore is comprised of >60 proteins and associates with 120 bp of centromeric (CEN) DNA. Kinetochore proteins are highly dynamic and exhibit programmed cell cycle changes in localization. The CEN-specific histone, Cse4p, is one of a few stable kinetochore components and remains associated with CEN DNA throughout mitosis. In contrast, several other kinetochore proteins have been observed along interpolar microtubules and at the midzone during anaphase. The inner kinetochore protein, Ndc10p, is enriched at the spindle midzone in late anaphase. We show that Ndc10p is transported to the plus-ends of interpolar microtubules at the midzone during anaphase, a process that requires survivin (Bir1p), a member of the aurora kinase (Ipl1p) complex, and Cdc14p phosphatase. In addition, Ndc10p is required for essential non-kinetochore processes during mitosis. Cells lacking functional Ndc10p show defects in spindle stability during anaphase and failure to split the septin ring during cytokinesis. This latter phenotype leads to a cell separation defect in ndc10-1 cells. We propose that Ndc10p plays a direct role in maintaining spindle stability during anaphase and coordinates the completion of cell division after chromosome segregation.
Asunto(s)
Citocinesis , Proteínas de Unión al ADN/metabolismo , Cinetocoros/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Huso Acromático/química , Huso Acromático/metabolismo , Anafase , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Proteínas Fúngicas/metabolismo , Cinetocoros/metabolismo , Microtúbulos/química , Microtúbulos/metabolismo , Transporte de Proteínas , Proteínas Tirosina Fosfatasas/metabolismoRESUMEN
It has been hypothesized that spatial gradients in kMT dynamic instability facilitate mitotic spindle formation and chromosome movement. To test this hypothesis requires the analysis of kMT dynamics, which have not been resolved at the single kMT level in living cells. The budding yeast spindle offers an attractive system in which to study kMT dynamics because, in contrast to animal cells, there is only one kMT per kinetochore. To visualize metaphase kMT plus-end dynamics in yeast, a strain containing a green fluorescent protein fusion to the kinetochore protein, Cse4, was imaged by fluorescence microscopy. Although individual kinetochores were not resolvable, we found that models of kMT dynamics could be evaluated by simulating the stochastic kMT dynamics and then simulating the fluorescence imaging of kMT plus-end-associated kinetochores. Statistical comparison of model-predicted images to experimentally observed images demonstrated that a pure dynamic instability model for kMT dynamics in the yeast metaphase spindle was unacceptable. However, when a temporally stable spatial gradient in the catastrophe or rescue frequency was added to the model, there was reasonable agreement between the model and the experiment. These results provide the first evidence of temporally stable spatial gradients of kMT catastrophe and/or rescue frequency in living cells.