RESUMEN
Microtubules regulate diverse cellular processes, including chromosome segregation, nuclear positioning, and cytokinesis. In many organisms, microtubule nucleation requires gamma-tubulin and associated proteins present at specific microtubule organizing centers (MTOCs). In fission yeast, interphase cytoplasmic microtubules originate from poorly characterized interphase MTOCs and spindle pole body (SPB), and during late anaphase from the equatorial MTOC (EMTOC). It has been previously shown that Mto1p (Mbo1p/Mod20p) function is important for the organization/nucleation of all cytoplasmic microtubules. Here, we show that Mto2p, a novel protein, interacts with Mto1p and is important for establishing a normal interphase cytoplasmic microtubule array. In addition, mto2Delta cells fail to establish a stable EMTOC and localize gamma-tubulin complex members to this medial structure. As predicted from these functions, Mto2p localizes to microtubules, the SPB, and the EMTOC in an Mto1p-dependent manner. mto2Delta cells fail to anchor the cytokinetic actin ring in the medial region of the cell and under conditions that mildly perturb actin structures, these rings unravel in mto2Delta cells. Our results suggest that the Mto2p and the EMTOC are critical for anchoring the cytokinetic actin ring to the medial region of the cell and for proper coordination of mitosis with cytokinesis.
Asunto(s)
Actinas/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Microtúbulos/metabolismo , Anafase , Anticuerpos Monoclonales/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Proteínas de Ciclo Celular/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Colorantes Fluorescentes , Proteínas Fúngicas/aislamiento & purificación , Proteínas de Unión al GTP/metabolismo , Eliminación de Gen , Proteínas Fluorescentes Verdes/metabolismo , Indoles , Microscopía Fluorescente , Microscopía por Video , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Centro Organizador de los Microtúbulos/metabolismo , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/crecimiento & desarrollo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Huso Acromático/metabolismo , Tiazoles/farmacología , Tiazolidinas , Tubulina (Proteína)/metabolismoRESUMEN
The Schizosaccharomyces pombe septation initiation network (SIN) triggers actomyosin ring constriction, septation, and cell division. It is organized at the spindle pole body (SPB) by the scaffold proteins Sid4p and Cdc11p. Here, we dissect the contributions of Sid4p and Cdc11p in anchoring SIN components and SIN regulators to the SPB. We find that Sid4p interacts with the SIN activator, Plo1p, in addition to Cdc11p and Dma1p. While the C terminus of Cdc11p is involved in binding Sid4p, its N-terminal half is involved in a wide variety of direct protein-protein interactions, including those with Spg1p, Sid2p, Cdc16p, and Cdk1p-Cdc13p. Given that the localizations of the remaining SIN components depend on Spg1p or Cdc16p, these data allow us to build a comprehensive model of SIN component organization at the SPB. FRAP experiments indicate that Sid4p and Cdc11p are stable SPB components, whereas signaling components of the SIN are dynamically associated with these structures. Our results suggest that the Sid4p-Cdc11p complex organizes a signaling hub on the SPB and that this hub coordinates cell and nuclear division.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Biológicos , Proteínas de Schizosaccharomyces pombe/metabolismo , Huso Acromático/metabolismo , Proteínas de Ciclo Celular/fisiología , División Celular/fisiología , Recuperación de Fluorescencia tras Fotoblanqueo , Immunoblotting , Proteínas Asociadas a Microtúbulos/fisiología , Mutagénesis Sitio-Dirigida , Plásmidos/genética , Pruebas de Precipitina , Unión Proteica , Schizosaccharomyces , Proteínas de Schizosaccharomyces pombe/fisiología , Huso Acromático/fisiología , Técnicas del Sistema de Dos HíbridosRESUMEN
The gamma-tubulin complex, via its ability to organize microtubules, is critical for accurate chromosome segregation and cytokinesis in the fission yeast, Schizosaccharomyces pombe. To better understand its roles, we have purified the S. pombe gamma-tubulin complex. Mass spectrometric analyses of the purified complex revealed known components and identified two novel proteins (i.e., Mbo1p and Gfh1p) with homology to gamma-tubulin-associated proteins from other organisms. We show that both Mbo1p and Gfh1p localize to microtubule organizing centers. Although cells deleted for either mbo1(+) or gfh1(+) are viable, they exhibit a number of defects associated with altered microtubule function such as defects in cell polarity, nuclear positioning, spindle orientation, and cleavage site specification. In addition, mbo1Delta and gfh1Delta cells exhibit defects in astral microtubule formation and anchoring, suggesting that these proteins have specific roles in astral microtubule function. This study expands the known roles of gamma-tubulin complex components in organizing different types of microtubule structures in S. pombe.
Asunto(s)
Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/fisiología , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/fisiología , Schizosaccharomyces/ultraestructura , Tubulina (Proteína)/metabolismo , Sitios de Unión , Proteínas de Unión a Calmodulina/análisis , Proteínas de Unión a Calmodulina/genética , Proteínas de Unión a Calmodulina/metabolismo , División Celular/genética , División Celular/fisiología , Polaridad Celular/genética , Eliminación de Gen , Proteínas Fluorescentes Verdes , Espacio Intranuclear/ultraestructura , Proteínas Asociadas a Microtúbulos/metabolismo , Centro Organizador de los Microtúbulos/metabolismo , Centro Organizador de los Microtúbulos/fisiología , Microtúbulos/metabolismo , Microtúbulos/fisiología , Modelos Biológicos , Datos de Secuencia Molecular , Mapeo de Interacción de Proteínas , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Alineación de Secuencia , Huso Acromático/genética , Huso Acromático/metabolismoRESUMEN
In the fission yeast Schizosaccharomyces pombe, the septation initiation network (SIN) triggers cytokinesis after mitosis. We investigated the relationship between Dma1p, a spindle checkpoint protein and cytokinesis inhibitor, and the SIN. Deletion of dma1 inactivates the spindle checkpoint and allows precocious SIN activation, while overexpressing Dma1p reduces SIN signaling. Dma1p seems to function by inhibiting the SIN activator, Plo1p kinase, since dma1 overexpression and deletion phenotypes suggest that Dma1p antagonizes Plo1p localization. Furthermore, failure to maintain high cyclin-dependent kinase (CDK) activity during spindle checkpoint activation in dma1 deletion cells requires Plo1p. Dma1p itself localizes to spindle pole bodies through interaction with Sid4p. Our observations suggest that Dma1p functions to prevent mitotic exit and cytokinesis during spindle checkpoint arrest by inhibiting SIN signaling.