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
Nuclear formation in Xenopus egg extracts requires cytosol and is inhibited by GTP gamma S, indicating a requirement for GTPase activity. Nuclear envelope (NE) vesicle fusion is extensively inhibited by GTP gamma S and two mutant forms of the Ran GTPase, Q69L and T24N. Depletion of either Ran or RCC1, the exchange factor for Ran, from the assembly reaction also inhibits this step of NE formation. Ran depletion can be complemented by the addition of Ran loaded with either GTP or GDP but not with GTP gamma S. RCC1 depletion is only complemented by RCC1 itself or by RanGTP. Thus, generation of RanGTP by RCC1 and GTP hydrolysis by Ran are both required for the extensive membrane fusion events that lead to NE formation.
Assuntos
Proteínas de Ciclo Celular , Fatores de Troca do Nucleotídeo Guanina , Guanosina Trifosfato/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares , Xenopus laevis , Proteína ran de Ligação ao GTP/metabolismo , Substituição de Aminoácidos , Animais , Cromatina/química , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Feminino , Imunofluorescência , Corantes Fluorescentes , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/farmacologia , Hidrólise/efeitos dos fármacos , Masculino , Fusão de Membrana/efeitos dos fármacos , Mutação , Membrana Nuclear/efeitos dos fármacos , Oócitos/citologia , Oócitos/metabolismo , Solubilidade , Cabeça do Espermatozoide/metabolismo , Proteínas de Xenopus , Proteína ran de Ligação ao GTP/genéticaRESUMO
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.
Assuntos
Proteínas de Ciclo Celular , Cromatina/fisiologia , Proteínas de Ligação a DNA/fisiologia , Fatores de Troca do Nucleotídeo Guanina , Guanosina Trifosfato/fisiologia , Mitose/fisiologia , Proteínas Nucleares/fisiologia , Fuso Acromático/fisiologia , Animais , Extratos Celulares , Guanosina Trifosfato/metabolismo , Humanos , Técnicas In Vitro , Microtúbulos/fisiologia , Modelos Biológicos , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Recombinantes , Schizosaccharomyces , Transdução de Sinais , Proteínas de Xenopus , Xenopus laevis , Proteína ran de Ligação ao GTPRESUMO
In many eukaryotic cells, protein secretion is regulated by extracellular signalling molecules giving rise to increased intracellular Ca2+ and activation of kinases and phosphatases. To test whether components involved in the first step of secretion, the translocation of proteins across the endoplasmic reticulum (ER) membrane, are regulated by Ca2+-dependent phosphorylation and dephosphorylation, we have investigated the effect of Ca2+ on kinases associated with the rough ER. Using purified rough microsomes from dog pancreas we found that Ca2+-dependent isoforms of protein kinase C (PKC) are associated with the rough ER and phosphorylate essential components of the protein translocation machinery. Phosphorylation of microsomal proteins by PKCs increased protein translocation efficiency in vitro. We also found that proteins of the translocation machinery became phosphorylated in intact cells. This suggests a further level of regulation of protein translocation across the ER membrane.
