RESUMO
It was recently reported that GTP-bound Ran induces microtubule and pseudo-spindle assembly in mitotic egg extracts in the absence of chromosomes and centrosomes, and that chromosomes induce the assembly of spindle microtubules in these extracts through generation of Ran-GTP. Here we examine the effects of Ran-GTP on microtubule nucleation and dynamics and show that Ran-GTP has independent effects on both the nucleation activity of centrosomes and the stability of centrosomal microtubules. We also show that inhibition of Ran-GTP production, even in the presence of duplicated centrosomes and kinetochores, prevents assembly of a bipolar spindle in M-phase extracts.
Assuntos
Microtúbulos/metabolismo , Fuso Acromático/fisiologia , Proteína ran de Ligação ao GTP/metabolismo , Animais , Centrossomo/metabolismo , Cromossomos/metabolismo , Replicação do DNA , Humanos , Masculino , Microscopia de Fluorescência , Oócitos/química , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Proteínas Proto-Oncogênicas c-mos/farmacologia , Espermatozoides/química , Espermatozoides/citologia , Xenopus laevis , Proteína ran de Ligação ao GTP/genéticaRESUMO
The small GTPase Ran, bound to GTP, is required for the induction of spindle formation by chromosomes in M phase. High concentrations of Ran.GTP are proposed to surround M phase chromatin. We show that the action of Ran.GTP in spindle formation requires TPX2, a microtubule-associated protein previously known to target a motor protein, Xklp2, to microtubules. TPX2 is normally inactivated by binding to the nuclear import factor, importin alpha, and is displaced from importin alpha by the action of Ran.GTP. TPX2 is required for Ran.GTP and chromatin-induced microtubule assembly in M phase extracts and mediates spontaneous microtubule assembly when present in excess over free importin alpha. Thus, components of the nuclear transport machinery serve to regulate spindle formation in M phase.
Assuntos
Proteínas de Ciclo Celular , Proteínas Associadas aos Microtúbulos/metabolismo , Mitose/fisiologia , Proteínas de Neoplasias , Proteínas Nucleares/metabolismo , Fosfoproteínas , Fuso Acromático/metabolismo , Proteínas de Xenopus , Proteína ran de Ligação ao GTP/metabolismo , Animais , Cromatina/metabolismo , Clonagem Molecular , Feminino , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/isolamento & purificação , GTP Fosfo-Hidrolases/metabolismo , Expressão Gênica/fisiologia , Células HeLa , Humanos , Carioferinas , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/isolamento & purificação , Microtúbulos/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Xenopus laevis , Proteína ran de Ligação ao GTP/genética , Proteína ran de Ligação ao GTP/isolamento & purificaçãoRESUMO
Chromosomes are segregated by two antiparallel arrays of microtubules arranged to form the spindle apparatus. During cell division, the nucleation of cytosolic microtubules is prevented and spindle microtubules nucleate from centrosomes (in mitotic animal cells) or around chromosomes (in plants and some meiotic cells). The molecular mechanism by which chromosomes induce local microtubule nucleation in the absence of centrosomes is unknown, but it can be studied by adding chromatin beads to Xenopus egg extracts. The beads nucleate microtubules that eventually reorganize into a bipolar spindle. RCC1, the guanine-nucleotide-exchange factor for the GTPase protein Ran, is a component of chromatin. Using the chromatin bead assay, we show here that the activity of chromosome-associated RCC1 protein is required for spindle formation. Ran itself, when in the GTP-bound state (Ran-GTP), induces microtubule nucleation and spindle-like structures in M-phase extract. We propose that RCC1 generates a high local concentration of Ran-GTP around chromatin which in turn induces the local nucleation of microtubules.
