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1.
Nat Rev Mol Cell Biol ; 12(5): 320-32, 2011 May.
Artículo en Inglés | MEDLINE | ID: mdl-21508988

RESUMEN

Fidelity during chromosome segregation is essential to prevent aneuploidy. The proteins and chromatin at the centromere form a unique site for kinetochore attachment and allow the cell to sense and correct errors during chromosome segregation. Centromeric chromatin is characterized by distinct chromatin organization, epigenetics, centromere-associated proteins and histone variants. These include the histone H3 variant centromeric protein A (CENPA), the composition and deposition of which have been widely investigated. Studies have examined the structural and biophysical properties of the centromere and have suggested that the centromere is not simply a 'landing pad' for kinetochore formation, but has an essential role in mitosis by assembling and directing the organization of the kinetochore.


Asunto(s)
Centrómero/metabolismo , Cromatina/metabolismo , Segregación Cromosómica , Cinetocoros/metabolismo , Animales , Histonas/metabolismo , Humanos , Modelos Genéticos , Huso Acromático/metabolismo
2.
Mol Cell ; 52(6): 819-31, 2013 Dec 26.
Artículo en Inglés | MEDLINE | ID: mdl-24268574

RESUMEN

The organization of chromosomes into territories plays an important role in a wide range of cellular processes, including gene expression, transcription, and DNA repair. Current understanding has largely excluded the spatiotemporal dynamic fluctuations of the chromatin polymer. We combine in vivo chromatin motion analysis with mathematical modeling to elucidate the physical properties that underlie the formation and fluctuations of territories. Chromosome motion varies in predicted ways along the length of the chromosome, dependent on tethering at the centromere. Detachment of a tether upon inactivation of the centromere results in increased spatial mobility. A confined bead-spring chain tethered at both ends provides a mechanism to generate observed variations in local mobility as a function of distance from the tether. These predictions are realized in experimentally determined higher effective spring constants closer to the centromere. The dynamic fluctuations and territorial organization of chromosomes are, in part, dictated by tethering at the centromere.


Asunto(s)
Centrómero/metabolismo , Cromatina/metabolismo , Cromosomas Fúngicos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/química , Cromatina/genética , Ensamble y Desensamble de Cromatina , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas Fúngicos/química , Elasticidad , Genotipo , Modelos Genéticos , Movimiento (Física) , Conformación de Ácido Nucleico , Nucleosomas/metabolismo , Fenotipo , Conformación Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Tiempo , Imagen de Lapso de Tiempo
3.
J Cell Physiol ; 229(2): 132-8, 2014 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23893718

RESUMEN

One of the most fundamental concepts of microscopy is that of resolution-the ability to clearly distinguish two objects as separate. Recent advances such as structured illumination microscopy (SIM) and point localization techniques including photoactivated localization microscopy (PALM), and stochastic optical reconstruction microscopy (STORM) strive to overcome the inherent limits of resolution of the modern light microscope. These techniques, however, are not always feasible or optimal for live cell imaging. Thus, in this review, we explore three techniques for extracting high resolution data from images acquired on a widefield microscope-deconvolution, model convolution, and Gaussian fitting. Deconvolution is a powerful tool for restoring a blurred image using knowledge of the point spread function (PSF) describing the blurring of light by the microscope, although care must be taken to ensure accuracy of subsequent quantitative analysis. The process of model convolution also requires knowledge of the PSF to blur a simulated image which can then be compared to the experimentally acquired data to reach conclusions regarding its geometry and fluorophore distribution. Gaussian fitting is the basis for point localization microscopy, and can also be applied to tracking spot motion over time or measuring spot shape and size. All together, these three methods serve as powerful tools for high-resolution imaging using widefield microscopy.


Asunto(s)
Microscopía/métodos , Fenómenos Ópticos , Dispersión de Radiación
4.
Elife ; 52016 05 11.
Artículo en Inglés | MEDLINE | ID: mdl-27166637

RESUMEN

In budding yeast, if the spindle becomes mispositioned, cells prevent exit from mitosis by inhibiting the mitotic exit network (MEN). The MEN is a signaling cascade that localizes to spindle pole bodies (SPBs) and activates the phosphatase Cdc14. There are two competing models that explain MEN regulation by spindle position. In the 'zone model', exit from mitosis occurs when a MEN-bearing SPB enters the bud. The 'cMT-bud neck model' posits that cytoplasmic microtubule (cMT)-bud neck interactions prevent MEN activity. Here we find that 1) eliminating cMT- bud neck interactions does not trigger exit from mitosis and 2) loss of these interactions does not precede Cdc14 activation. Furthermore, using binucleate cells, we show that exit from mitosis occurs when one SPB enters the bud despite the presence of a mispositioned spindle. We conclude that exit from mitosis is triggered by a correctly positioned spindle rather than inhibited by improper spindle position.


Asunto(s)
Microtúbulos/metabolismo , Mitosis , Saccharomyces cerevisiae/fisiología , Cuerpos Polares del Huso/metabolismo , Proteínas de Ciclo Celular/metabolismo , Modelos Biológicos , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
5.
Methods Cell Biol ; 123: 347-65, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24974037

RESUMEN

Biological questions are increasingly being addressed using a wide range of quantitative analytical tools to examine protein complex composition. Knowledge of the absolute number of proteins present provides insights into organization, function, and maintenance and is used in mathematical modeling of complex cellular dynamics. In this chapter, we outline and describe three microscopy-based methods for determining absolute protein numbers--fluorescence correlation spectroscopy, stepwise photobleaching, and ratiometric comparison of fluorescence intensity to known standards. In addition, we discuss the various fluorescently labeled proteins that have been used as standards for both stepwise photobleaching and ratiometric comparison analysis. A detailed procedure for determining absolute protein number by ratiometric comparison is outlined in the second half of this chapter. Counting proteins by quantitative microscopy is a relatively simple yet very powerful analytical tool that will increase our understanding of protein complex composition.


Asunto(s)
Procesamiento de Imagen Asistido por Computador/métodos , Proteínas del Citoesqueleto/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo/métodos , Recuperación de Fluorescencia tras Fotoblanqueo/normas , Proteínas Fluorescentes Verdes/metabolismo , Cinetocoros/metabolismo , Microscopía Fluorescente/métodos , Microscopía Fluorescente/normas , Fotoblanqueo , Estándares de Referencia , Saccharomycetales/metabolismo , Relación Señal-Ruido
6.
Mol Biol Cell ; 25(5): 712-27, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24403601

RESUMEN

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/metabolismo
7.
J Cell Biol ; 200(6): 757-72, 2013 Mar 18.
Artículo en Inglés | MEDLINE | ID: mdl-23509068

RESUMEN

The mechanisms by which sister chromatids maintain biorientation on the metaphase spindle are critical to the fidelity of chromosome segregation. Active force interplay exists between predominantly extensional microtubule-based spindle forces and restoring forces from chromatin. These forces regulate tension at the kinetochore that silences the spindle assembly checkpoint to ensure faithful chromosome segregation. Depletion of pericentric cohesin or condensin has been shown to increase the mean and variance of spindle length, which have been attributed to a softening of the linear chromatin spring. Models of the spindle apparatus with linear chromatin springs that match spindle dynamics fail to predict the behavior of pericentromeric chromatin in wild-type and mutant spindles. We demonstrate that a nonlinear spring with a threshold extension to switch between spring states predicts asymmetric chromatin stretching observed in vivo. The addition of cross-links between adjacent springs recapitulates coordination between pericentromeres of neighboring chromosomes.


Asunto(s)
Cromatina/metabolismo , Segregación Cromosómica/fisiología , Cromosomas Fúngicos/metabolismo , Modelos Biológicos , Saccharomyces cerevisiae/metabolismo , Huso Acromático/metabolismo
8.
Curr Biol ; 22(6): 471-81, 2012 Mar 20.
Artículo en Inglés | MEDLINE | ID: mdl-22365852

RESUMEN

BACKGROUND: Tension sensing of bioriented chromosomes is essential for the fidelity of chromosome segregation. The spindle assembly checkpoint (SAC) conveys lack of tension or attachment to the anaphase promoting complex. Components of the SAC (Bub1) phosphorylate histone H2A (S121) and recruit the protector of cohesin, Shugoshin (Sgo1), to the inner centromere. How the chromatin structural modifications of the inner centromere are integrated into the tension sensing mechanisms and the checkpoint are not known. RESULTS: We have identified a Bub1/Sgo1-dependent structural change in the geometry and dynamics of kinetochores and the pericentric chromatin upon reduction of microtubule dynamics. The cluster of inner kinetochores contract, whereas the pericentric chromatin and cohesin that encircle spindle microtubules undergo a radial expansion. Despite its increased spatial distribution, the pericentric chromatin is less dynamic. The change in dynamics is due to histone H2A phosphorylation and Sgo1 recruitment to the pericentric chromatin, rather than microtubule dynamics. CONCLUSIONS: Bub1 and Sgo1 act as a rheostat to regulate the chromatin spring and maintain force balance. Through histone H2A S121 phosphorylation and recruitment of Sgo1, Bub1 kinase softens the chromatin spring in response to changes in microtubule dynamics. The geometric alteration of all 16 kinetochores and pericentric chromatin reflect global changes in the pericentromeric region and provide mechanisms for mechanically amplifying damage at a single kinetochore microtubule.


Asunto(s)
Cromatina/metabolismo , Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Benomilo/farmacología , Centrómero/metabolismo , ADN de Hongos/química , ADN de Hongos/metabolismo , Histonas/metabolismo , Cinetocoros/metabolismo , Modelos Biológicos , Proteínas Nucleares/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Estrés Mecánico
9.
Mol Biol Cell ; 23(13): 2560-70, 2012 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-22593210

RESUMEN

Nucleosome positioning is important for the structural integrity of chromosomes. During metaphase the mitotic spindle exerts physical force on pericentromeric chromatin. The cell must adjust the pericentromeric chromatin to accommodate the changing tension resulting from microtubule dynamics to maintain a stable metaphase spindle. Here we examine the effects of spindle-based tension on nucleosome dynamics by measuring the histone turnover of the chromosome arm and the pericentromere during metaphase in the budding yeast Saccharomyces cerevisiae. We find that both histones H2B and H4 exhibit greater turnover in the pericentromere during metaphase. Loss of spindle-based tension by treatment with the microtubule-depolymerizing drug nocodazole or compromising kinetochore function results in reduced histone turnover in the pericentromere. Pericentromeric histone dynamics are influenced by the chromatin-remodeling activities of STH1/NPS1 and ISW2. Sth1p is the ATPase component of the Remodels the Structure of Chromatin (RSC) complex, and Isw2p is an ATP-dependent DNA translocase member of the Imitation Switch (ISWI) subfamily of chromatin-remodeling factors. The balance between displacement and insertion of pericentromeric histones provides a mechanism to accommodate spindle-based tension while maintaining proper chromatin packaging during mitosis.


Asunto(s)
Cinetocoros/metabolismo , Metafase , Nucleosomas/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas/metabolismo , Fenómenos Biomecánicos , Proteínas de Ciclo Celular/metabolismo , Ensamble y Desensamble de Cromatina , Cromosomas Fúngicos/metabolismo , Semivida , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Huso Acromático/metabolismo , Factores de Transcripción/metabolismo
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