RESUMO
The nuclear envelope (NE) of eukaryotic cells separates nucleoplasm from cytoplasm, mediates nucleo-cytoplasmic transport, and contributes to the control of gene expression. The NE consists of three major components: the nuclear membranes, the nuclear pore complexes (NPCs), and the nuclear lamina. The list of identified NE proteins has increased considerably during recent years but is most likely not complete. In most eukaryotes, the NE breaks down and is then reassembled during mitosis. The assembly of NPCs and the association and fusion of nuclear membranes around decondensing chromosomes are tightly coordinated processes. Here, we report the identification and characterization of MEL-28, a large protein essential for the assembly of a functional NE in C. elegans embryos. RNAi depletion or genetic mutation of mel-28 severely impairs nuclear morphology and leads to abnormal distribution of both integral NE proteins and NPCs. The structural defects of the NE were associated with functional defects and lack of nuclear exclusion of soluble proteins. MEL-28 localizes to NPCs during interphase, to kinetochores in early to middle mitosis then is widely distributed on chromatin late in mitosis. We show that MEL-28 is an early-assembling, stable NE component required for all aspects of NE assembly.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Cinetocoros/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Zigoto/crescimento & desenvolvimento , Animais , Sequência Conservada , Proteínas de Ligação a DNA , Mitose/fisiologia , Membrana Nuclear/fisiologia , Membrana Nuclear/ultraestrutura , Poro Nuclear , Interferência de RNARESUMO
The metazoan nuclear envelope (NE) breaks down and re-forms during each cell cycle. Nuclear pore complexes (NPCs), which allow nucleocytoplasmic transport during interphase, assemble into the re-forming NE at the end of mitosis. Using in vitro NE assembly, we show that the vertebrate homologue of MEL-28 (maternal effect lethal), a recently discovered NE component in Caenorhabditis elegans, functions in postmitotic NPC assembly. MEL-28 interacts with the Nup107-160 complex (Nup for nucleoporin), an important building block of the NPC, and is essential for the recruitment of the Nup107-160 complex to chromatin. We suggest that MEL-28 acts as a seeding point for NPC assembly.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Ciclo Celular/fisiologia , Cromatina/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Animais , Caenorhabditis elegans , Proteínas de Ligação a DNA , Escherichia coli , Imunofluorescência , Humanos , Interferência de RNA , RNA Interferente Pequeno/genética , XenopusRESUMO
The metazoan nuclear envelope (NE) breaks down and reforms at each mitosis. Nuclear pore complexes (NPCs), which allow nucleocytoplasmic transport during interphase, assemble into the reforming NE at the end of mitosis. Using in vitro NE assembly assays, we show that one of the two transmembrane nucleoporins, pom121, is essential for NE formation, whereas the second, gp210, is dispensable. Depletion of either pom121-containing membrane vesicles or the protein alone does not affect vesicle binding to chromatin but prevents their fusion to form a closed NE. When the Nup107-160 complex, which is essential for integration of NPCs into the NE, is also depleted, pom121 becomes dispensable for NE formation, suggesting a close functional link between NPC and NE formation and the existence of a checkpoint that monitors NPC assembly state.
Assuntos
Proteínas de Membrana/fisiologia , Membrana Nuclear/fisiologia , Complexo de Proteínas Formadoras de Poros Nucleares/fisiologia , Poro Nuclear/fisiologia , Proteínas Nucleares/fisiologia , Animais , Sequência de Bases , Células Cultivadas , DNA Complementar/genética , Feminino , Técnicas In Vitro , Fusão de Membrana/fisiologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiologia , Proteínas de Membrana/genética , Modelos Biológicos , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas Nucleares/genética , RNA Interferente Pequeno/genética , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfecção , Proteínas de Xenopus , Xenopus laevisRESUMO
Nuclear envelope (NE) formation during cell division in multicellular organisms is a central yet poorly understood biological process. We report that the conserved nucleoporin Nup155 has an essential function in NE formation in Caenorhabditis elegans embryos and in Xenopus laevis egg extracts. In vivo depletion of Nup155 led to failure of nuclear lamina formation and defects in chromosome segregation at anaphase. Nup155 depletion inhibited accumulation of nucleoporins at the nuclear periphery, including those recruited to chromatin early in NE formation. Electron microscopy analysis revealed that Nup155 is also required for the formation of a continuous nuclear membrane in vivo and in vitro. Time-course experiments indicated that Nup155 is recruited to chromatin at the time of NE sealing, suggesting that nuclear pore complex assembly has to progress to a relatively late stage before NE membrane assembly occurs.