ABSTRACT
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.
Subject(s)
Cell Cycle Proteins/chemistry , Chondroitin Sulfate Proteoglycans/chemistry , Chromatids/metabolism , Chromosomal Proteins, Non-Histone/chemistry , Euchromatin/metabolism , Heterochromatin/metabolism , Nuclear Proteins/chemistry , Phosphoproteins/chemistry , Cell Cycle/genetics , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Chondroitin Sulfate Proteoglycans/genetics , Chondroitin Sulfate Proteoglycans/metabolism , Chromatids/ultrastructure , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , DNA-Binding Proteins , Euchromatin/ultrastructure , Gene Expression , HeLa Cells , Heterochromatin/ultrastructure , Humans , Microscopy, Immunoelectron , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Phosphoproteins/genetics , Phosphoproteins/metabolism , Protein Binding , Protein MultimerizationABSTRACT
Tight association of peripheral chromatin with nuclear lamina unavoidably creates topological constraints during replication. Additional complications are associated with high stability of lamina meshwork, which may hinder an access of replication factors to the sites of DNA synthesis in highly condensed template with limited mobility. In the current work we studied structural organization and dynamics of lamina as a function of replicative status of associated peripheral heterochromatin. The studies of molecular mobility of laminas at various stages of S-phase in vivo and using super-resolution microscopy showed no correlation between lamina dynamics and replicative status of attached heterochromatin. These data support the hypothesis that lamina-chromatin interactions during S-phase are regulated at the level of adapter proteins. Ultrastructural studies have demonstrated that temporal break of lamina-chromatin connections during replication does not cause noticeable spatial separation of replicating domains from nuclear periphery.
Subject(s)
DNA Replication , DNA/metabolism , Fibroblasts/metabolism , Heterochromatin/metabolism , Nuclear Lamina/metabolism , Animals , CHO Cells , Cell Line, Tumor , Cricetulus , Fibroblasts/cytology , Gene Expression , Heterochromatin/ultrastructure , Humans , Lamin Type A/genetics , Lamin Type A/metabolism , Lamin Type B/genetics , Lamin Type B/metabolism , Nuclear Lamina/ultrastructureABSTRACT
In vitro radioligand assay revealed interaction of afobazole with sigma(1)-receptors (Ki=5.9x10(-6) M). Translocation of sigma(1)-receptors from the endoplasmic reticulum to the outer membrane was demonstrated by confocal microscopy. Experiments were performed on the model of HT-22 immortalized hippocampal cells after incubation with afobazole in a concentration of 10(-8) M.
Subject(s)
Anti-Anxiety Agents/pharmacology , Benzimidazoles/pharmacology , Morpholines/pharmacology , Neuroprotective Agents/pharmacology , Receptors, sigma/drug effects , Animals , Cell Line, Transformed , Fluorescent Antibody Technique , Hippocampus/cytology , Hippocampus/drug effects , Humans , Jurkat Cells , Mice , Radioligand Assay , Sigma-1 ReceptorABSTRACT
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.
Subject(s)
Chromosomes/metabolism , Nuclear Matrix/metabolism , Tissue Fixation/methods , Animals , Antigens, Neoplasm/isolation & purification , Antigens, Neoplasm/metabolism , Carrier Proteins/isolation & purification , Carrier Proteins/metabolism , Cell Cycle Proteins , Chromosomes/ultrastructure , DNA Topoisomerases, Type II/isolation & purification , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/isolation & purification , DNA-Binding Proteins/metabolism , Humans , Matrix Attachment Regions , Mice , Microscopy, Electron , Nuclear Matrix/ultrastructure , Nuclear Proteins/isolation & purification , Nuclear Proteins/metabolism , Sodium Chloride/chemistry , Swine , Xenopus Proteins/isolation & purification , Xenopus Proteins/metabolism , Xenopus laevisABSTRACT
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.
Subject(s)
Cell Nucleus/metabolism , Chromosomes/ultrastructure , Nuclear Envelope/metabolism , Nuclear Proteins/metabolism , Cell Line , Cell Nucleus/ultrastructure , DNA-Binding Proteins/metabolism , Fluorescent Antibody Technique , HeLa Cells , Humans , Hypotonic Solutions , Interphase , Lamin Type A/metabolism , Lamin Type B/metabolism , Membrane Proteins/metabolism , Microscopy, Electron , Mitosis , Nuclear Envelope/ultrastructure , Osmolar ConcentrationABSTRACT
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.
