RESUMO
It is considered that sister chromatids are held together immediately after replication by special protein complex--cohesin that consists of Smc1--Smc3 core dimer and two additional subunits, Scc1 and Scc3. This process is called cohesion. We have characterized binding of cohesin complex to early- and late-replicated chromatin at different stages of the cell cycle in human cells HeLa and HT1080 using superresolution microscopy (based on Structural ilumination microscopy--SIM) and immunoelectron microscopy. It has been shown that cohesins do not play important role in cohesion of heterochromatic domains, but they provide cohesion and organization of subdomains in euchromatic regions.
Assuntos
Proteínas de Ciclo Celular/química , Proteoglicanas de Sulfatos de Condroitina/química , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/química , Eucromatina/metabolismo , Heterocromatina/metabolismo , Proteínas Nucleares/química , Fosfoproteínas/química , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proteoglicanas de Sulfatos de Condroitina/genética , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Cromátides/ultraestrutura , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA , Eucromatina/ultraestrutura , Expressão Gênica , Células HeLa , Heterocromatina/ultraestrutura , Humanos , Microscopia Imunoeletrônica , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ligação Proteica , Multimerização ProteicaRESUMO
A method of nuclear matrix and chromosomal scaffold preparation from cultured animal cells was developed. After the high-salt extraction, interphase and mitotic cells were not detached from the coverslips that enabled us to analyse the nuclear matrix and chromosomal scaffold in cells at all mitotic phases. Morphological methods (phase contrast microscopy and electron microscopy of ultrathin sections) did not reveal any structures that could be identified as a chromosomal scaffold. However, after staining with antibodies to XCAP-E and topoisomerase IIalpha some structures were revealed in metaphase cells having both localization and morphology of a chromosomal scaffold. The cell residuals were not stained with antibodies to XCAP-E and topoisomerase IIalpha, if the nuclear matrix and chromosomal scaffold were destabilized by addition of beta-mercaptoethanol.
Assuntos
Cromossomos/metabolismo , Matriz Nuclear/metabolismo , Fixação de Tecidos/métodos , Animais , Antígenos de Neoplasias/isolamento & purificação , Antígenos de Neoplasias/metabolismo , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Cromossomos/ultraestrutura , DNA Topoisomerases Tipo II/isolamento & purificação , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Humanos , Regiões de Interação com a Matriz , Camundongos , Microscopia Eletrônica , Matriz Nuclear/ultraestrutura , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , Cloreto de Sódio/química , Suínos , Proteínas de Xenopus/isolamento & purificação , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMO
In the present work we have studied the distribution of some proteins participating in the nuclear envelope assembly (lamins A/C, B and LAP2 alpha) in mitotic cells and after hypotonic treatment with 15% Hank's solution. In untreated cells, these proteins are localized in the nuclei of interphase cells migrate to the cytoplasm during mitosis. Hypotonic treatment of interphase, prophase and telophase cells does not lead to considerable relocalization of lamins A/C and B. However, unlike normal mitosis, in prometaphase and metaphase cells their chromosomes acquire affinity to lamins and LAP2 alpha. Comparative analysis of lamins and LAP2 alpha distribution have revealed that chromosomes have special sites for binding with different proteins.
Assuntos
Núcleo Celular/metabolismo , Cromossomos/ultraestrutura , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Linhagem Celular , Núcleo Celular/ultraestrutura , Proteínas de Ligação a DNA/metabolismo , Imunofluorescência , Células HeLa , Humanos , Soluções Hipotônicas , Interfase , Lamina Tipo A/metabolismo , Lamina Tipo B/metabolismo , Proteínas de Membrana/metabolismo , Microscopia Eletrônica , Mitose , Membrana Nuclear/ultraestrutura , Concentração OsmolarRESUMO
Chromatin associated with the nuclear envelope appears in the interphase nuclei as a layer of anchorosomes, granules 20-25 nm in diameter. The fraction of chromatin directly associated with the nuclear envelope is resistant to decondensation, shows a low level of DNA methylation, and contains specific acid-soluble proteins. However, mechanisms underlying the interaction of chromatin with the nuclear envelope are not fully understood. Specifically, it is not known whether anchorosomes are permanent structures or if they undergo reversible disassembly during mitosis, when contacts between chromatin and the nuclear envelope are destroyed. We obtained immune serum recognizing a 68 kDa protein from the nuclear envelopes fraction and studied the localization of this protein in interphase and mitotic cells. We show that this protein present in the NE/anchorosomal fraction does not remain bound with chromosomes during mitosis. It dissociates from chromosomes at the beginning of the prophase and then can be identified again at the periphery of the newly forming nuclei in the telophase.
