RESUMEN
Stathmin is an important regulator of microtubule polymerization and dynamics. When unphosphorylated it destabilizes microtubules in two ways, by reducing the microtubule polymer mass through sequestration of soluble tubulin into an assembly-incompetent T2S complex (two alpha:beta tubulin dimers per molecule of stathmin), and by increasing the switching frequency (catastrophe frequency) from growth to shortening at plus and minus ends by binding directly to the microtubules. Phosphorylation of stathmin on one or more of its four serine residues (Ser(16), Ser(25), Ser(38), and Ser(63)) reduces its microtubule-destabilizing activity. However, the effects of phosphorylation of the individual serine residues of stathmin on microtubule dynamic instability have not been investigated systematically. Here we analyzed the effects of stathmin singly phosphorylated at Ser(16) or Ser(63), and doubly phosphorylated at Ser(25) and Ser(38), on its ability to modulate microtubule dynamic instability at steady-state in vitro. Phosphorylation at either Ser(16) or Ser(63) strongly reduced or abolished the ability of stathmin to bind to and sequester soluble tubulin and its ability to act as a catastrophe factor by directly binding to the microtubules. In contrast, double phosphorylation of Ser(25) and Ser(38) did not affect the binding of stathmin to tubulin or microtubules or its catastrophe-promoting activity. Our results indicate that the effects of stathmin on dynamic instability are strongly but differently attenuated by phosphorylation at Ser(16) and Ser(63) and support the hypothesis that selective targeting by Ser(16)-specific or Ser(63)-specific kinases provides complimentary mechanisms for regulating microtubule function.
Asunto(s)
Microtúbulos/fisiología , Estatmina/farmacología , Alanina/metabolismo , Animales , Axonema/efectos de los fármacos , Axonema/fisiología , Cinética , Microscopía por Video , Microtúbulos/efectos de los fármacos , Microtúbulos/ultraestructura , Fosforilación , Fosfoserina/metabolismo , Desnaturalización Proteica , Renaturación de Proteína , Erizos de Mar , Estatmina/metabolismo , Tubulina (Proteína)/efectos de los fármacos , Tubulina (Proteína)/metabolismoRESUMEN
Dicentric chromosomes undergo breakage during mitosis as a result of the attachment of two centromeres on one sister chromatid to opposite spindle poles. Studies utilizing a conditional dicentric chromosome III in Saccharomyces cerevisiae have shown that dicentric chromosome repair occurs primarily by deletion of one centromere via a RAD52-dependent recombination pathway. We report that dicentric chromosome resolution requires RAD1, a gene involved in the single-strand annealing DNA repair pathway. We additionally show that single-strand annealing repair of a dicentric chromosome can occur in the absence of RAD52. RAD52-independent repair requires the adaptation-defective cdc5-ad allele of the yeast polo kinase and the DNA damage checkpoint gene RAD9. Dicentric chromosome breakage in cdc5-ad rad52 mutant cells is associated with a prolonged mitotic arrest, during which nuclei undergo microtubule-dependent oscillations, accompanied by dynamic changes in nuclear morphology. We further demonstrate that the frequency of spontaneous direct repeat recombination is suppressed in yeast cells treated with benomyl, a drug that perturbs microtubules. Our findings indicate that microtubule-dependent processes facilitate recombination.
Asunto(s)
Relojes Biológicos/genética , Estructuras del Núcleo Celular/genética , Cromosomas/genética , Reparación del ADN/genética , Proteínas de Drosophila , Endonucleasas/genética , Mitosis/genética , Saccharomyces cerevisiae/genética , Benomilo/farmacología , Relojes Biológicos/efectos de los fármacos , Proteínas de Ciclo Celular/genética , Estructuras del Núcleo Celular/efectos de los fármacos , Estructuras del Núcleo Celular/metabolismo , Centrómero/genética , Cromosomas/efectos de los fármacos , Cromosomas/metabolismo , Reparación del ADN/efectos de los fármacos , Enzimas Reparadoras del ADN , ADN de Cadena Simple/genética , Proteínas de Unión al ADN/genética , Relación Dosis-Respuesta a Droga , Fungicidas Industriales/farmacología , Microtúbulos/efectos de los fármacos , Microtúbulos/genética , Proteínas Serina-Treonina Quinasas/genética , Proteína Recombinante y Reparadora de ADN Rad52 , Recombinación Genética/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiaeRESUMEN
Microtubule dynamics are influenced by interactions of microtubules with cellular factors and by changes in the primary sequence of the tubulin molecule. Mutations of yeast beta-tubulin C354, which is located near the binding site of some antimitotic compounds, reduce microtubule dynamicity greater than 90% in vivo and in vitro. The resulting intrinsically stable microtubules allowed us to determine which, if any, cellular processes are dependent on dynamic microtubules. The average number of cytoplasmic microtubules decreased from 3 in wild-type to 1 in mutant cells. The single microtubule effectively located the bud site before bud emergence. Although spindles were positioned near the bud neck at the onset of anaphase, the mutant cells were deficient in preanaphase spindle alignment along the mother-bud axis. Spindle microtubule dynamics and spindle elongation rates were also severely depressed in the mutants. The pattern and extent of cytoplasmic microtubule dynamics modulation through the cell cycle may reveal the minimum dynamic properties required to support growth. The ability to alter intrinsic microtubule dynamics and determine the in vivo phenotype of cells expressing the mutant tubulin provides a critical advance in assessing the dynamic requirements of an essential gene function.