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1.
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.  相似文献   

2.
In the interphase nucleus, the chromatin associated with the nuclear envelope is represented by a layer of anchorosomes, granules with a diameter of 20–25 nm. Biochemically, the fraction of chromatin directly associated with the nuclear envelope is characterized by resistance against decondensing influences, a low level of DNA methylation, and presence of specific acid-soluble proteins. However, the mechanisms lying at the base of chromatin-nuclear envelope interaction have been insufficiently studied. Specifically, it is unknown whether anchorosomes are constant structures or subject to reversible disassembly, when the contacts between chromatin and 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.  相似文献   

3.
The granular particles of chromatin peripheral layer, were isolated together, with the nuclear envelope by treatment of nuclei with nuclease. These particles differ from total chromatin by a decreased content of histone H1, a specific set of minor acid-soluble proteins and a low DNA methylation level. Taking account of the fact that these particles facilitate chromatin interaction with the nuclear envelope, the latter were termed as "anchorosomes". Using UV-induced cross-linking of DNA to the proteins, it was found that all anchorosome-specific acid-soluble proteins can directly interact with anchorosomal DNA. Treatment of anchorosomes with staphylococcal nuclease and electron microscopic data showed that anchorosomes have a nucleosomal organization. Five to ten per cent of anchorosomal DNA appear to be firmly bound to nuclear lamina. This DNA cannot be separated from the lamina by treatment with 2 M NaCl, 1% SDS or heparin (1 mg/ml). The bulk of DNA in the laminal fraction after treatment with the above reagents is protected from hydrolysis with DNAase I by anchorosomal proteins and thus has a high molecular weight (10,000-30,000 base pairs). After treatment of anchorosomes with 0.6 M or 2 M NaCl, DNAase I splits this DNA, predominantly to minor fragments.  相似文献   

4.
Specific interactions of chromatin with the nuclear envelope (NE) in early embryos of Drosophila melanogaster have been mapped and analyzed. Using fluorescence in situ hybridization, the three-dimensional positions of 42 DNA probes, primarily to chromosome 2L, have been mapped in nuclei of intact Drosophila embryos, revealing five euchromatic and two heterochromatic regions associated with the NE. These results predict that there are approximately 15 NE contacts per chromosome arm, which delimit large chromatin loops of approximately 1-2 Mb. These NE association sites do not strictly correlate with scaffold-attachment regions, heterochromatin, or binding sites of known chromatin proteins. Pairs of neighboring probes surrounding one NE association site were used to delimit the NE association site more precisely, suggesting that peripheral localization of a large stretch of chromatin is likely to result from NE association at a single discrete site. These NE interactions are not established until after telophase, by which time the nuclear envelope has reassembled around the chromosomes, and they are thus unlikely to be involved in binding of NE vesicles to chromosomes following mitosis. Analysis of positions of these probes also reveals that the interphase nucleus is strongly polarized in a Rabl configuration which, together with specific targeting to the NE or to the nuclear interior, results in each locus occupying a highly determined position within the nucleus.  相似文献   

5.
In interphase, chromosomes occupy defined nuclear volumes known as chromosome territories. To probe the biological consequences of the described nonrandom spatial positioning of chromosome territories in human lymphocytes, we performed an extensive FISH-based analysis of ionizing radiation-induced interchanges involving chromosomes 1, 4, 18 and 19. Since the probability of exchange formation depends strongly on the spatial distance between the damage sites in the genome, a preferential formation of exchanges between proximally positioned chromosomes is expected. Here we show that the spectrum of interchanges deviates significantly from one expected based on random chromosome positioning. Moreover, the observed exchange interactions between specific chromosome pairs as well as the interactions between homologous chromosomes are consistent with the proposed gene density-related radial distribution of chromosome territories. The differences between expected and observed exchange frequencies are more pronounced after exposure to densely ionizing neutrons than after exposure to sparsely ionizing X rays. These experiments demonstrate that the spatial positioning of interphase chromosomes affects the spectrum of chromosome rearrangements.  相似文献   

6.
Summary In 1885 Carl Rabl published his theory on the internal structure of the interphase nucleus. We have tested two predictions of this theory in fibroblasts grown in vitro from a female Chinese hamster, namely (1) the Rabl-orientation of interphase chromosomes and (2) the stability of the chromosome arrangement established in telophase throughout the subsequent interphase. Tests were carried out by premature chromosome condensation (PCC) and laser-UV-microirradiation of the interphase nucleus. Rabl-orientation of chromosomes was observed in G1 PCCs and G2 PCCs. The cell nucleus was microirradiated in G1 at one or two sites and pulse-labelled with 3H-thymidine for 2h. Cells were processed for autoradiography either immediately thereafter or after an additional growth period of 10 to 60h. Autoradiographs show unscheduled DNA synthesis (UDS) in the microirradiated nuclear part(s). The distribution of labelled chromatin was evaluated in autoradiographs from 1035 cells after microirradiation of a single nuclear site and from 253 cells after microirradiation of two sites. After 30 to 60h postincubation the labelled regions still appeared coherent although the average size of the labelled nuclear area fr increased from 14.2% (0h) to 26.5% (60h). The relative distance dr, i.e. the distance between two microirradiated sites divided by the diameter of the whole nucleus, showed a slight decrease with increasing incubation time. Nine metaphase figures were evaluated for UDS-label after microirradiation of the nuclear edge in G1. An average of 4.3 chromosomes per cell were labelled. Several chromosomes showed joint labelling of both distal chromosome arms including the telomeres, while the centromeric region was free from label. This label pattern is interpreted as the result of a V-shaped orientation of these particular chromosomes in the interphase nucleus with their telomeric regions close to each other at the nuclear edge. Our data support the tested predictions of the Rabl-model. Small time-dependent changes of the nuclear space occupied by single chromosomes and of their relative positions in the interphase nucleus seem possible, while the territorial organization of interphase chromosomes and their arrangement in general is maintained during interphase. The present limitations of the methods used for this study are discussed.Part of this work is included in the doctoral thesis of H. Baumann to be submitted to the Faculty of Biology of the University of HeidelbergPart of this work is included in the doctoral thesis of V. Teuber to be submitted to the Faculty of Medicine of the University of Freiburg i. Br.  相似文献   

7.
Multinucleate (MN) cells were induced in PtK1 cells by colcemid treatment. A large percentage of cells developed nuclear asynchrony both in relation to DNA synthesis and mitosis within one cell cycle. Asynchrony could be traced even in metaphase and anaphase cells in which interphase nuclei, PCC of S-phase nuclei and less condensed prophase-like chromosomes could be observed along with normally condensed chromosomes. The occurrence of such abnormalities in these large MN cells may be explained on the basis of an uneven distribution of inducer molecules of DNA synthesis and mitosis due to cytoplasmic compartmentation. The less condensed form of all the chromosomes except chromosome 4 could be traced in asynchronous metaphase. The failure of the less condensed chromosomes to undergo complete condensation does not always appear to result from late entry of nuclei containing these chromosomes into G2 phase. It is likely that chromosome 4 carries gene(s) for chromosome condensation, as this chromosome itself never appears in a less condensed form. The inducers for chromosome condensation may not always be available at equal concentrations to all chromosomes located in separate nuclei, thus they may sometimes fail to undergo complete condensation before other nuclei reach the end of prophase, when the nuclear envelopes of all nuclei present in the cell break down simultaneously.  相似文献   

8.
Ki-67 protein has been widely used as a proliferation marker for human tumor cells for decades. In recent studies, multiple molecular functions of this large protein have become better understood. Ki-67 has roles in both interphase and mitotic cells, and its cellular distribution dramatically changes during cell cycle progression. These localizations correlate with distinct functions. For example, during interphase, Ki-67 is required for normal cellular distribution of heterochromatin antigens and for the nucleolar association of heterochromatin. During mitosis, Ki-67 is essential for formation of the perichromosomal layer (PCL), a ribonucleoprotein sheath coating the condensed chromosomes. In this structure, Ki-67 acts to prevent aggregation of mitotic chromosomes. Here, we present an overview of functional roles of Ki-67 across the cell cycle and also describe recent experiments that clarify its role in regulating cell cycle progression in human cells.  相似文献   

9.
10.
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.  相似文献   

11.
In Chinese hamster Don cells, fusion of an interphase cell with a metaphase cell resulted either in prophasing of the interphase nucleus, including loss of the nuclear envelope (NE), or in the formation of a double membrane around the metaphase chromosomes. Only one of these phenomena occurred in a given interphase-metaphase (I–M) binucleate cell. At pH 7.4, there was about an equal probability that either event could occur amongst the population of I–M cells. The effect of pH changes in the medium containing the fused cells was examined. At pH 6.6, prophasing was the predominant event; at pH 8.0, membrane formation predominated. It was found that the rate of progression of a mononucleate cell from G2 to metaphase was appreciably faster at pH 6.6 than at pH 8.0. Conversely, the progression from metaphase to G1 was faster at pH 8.0 than at pH 6.6. These results with the mononucleate cells strengthen the hypothesis that structural changes in I–M cells are reflections of normal mitotic phenomena. Additional evidence for this hypothesis was produced by electron microscope examination after direct fixation in chrom-osmium. The double membrane around the chromosomes of the I–M cell was indistinguishable from the normal NE. The results obtained by varying the pH of the medium containing the fused cells provide an indication that disruption or formation of the NE of Don cells depends on the balance reached between disruptive and formative processes.  相似文献   

12.
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.  相似文献   

13.
14.
During mitosis in metazoans, segregated chromosomes become enclosed by the nuclear envelope (NE), a double membrane that is continuous with the endoplasmic reticulum (ER). Recent in vitro data suggest that NE formation occurs by chromatin-mediated reorganization of the tubular ER; however, the basic principles of such a membrane-reshaping process remain uncharacterized. Here, we present a quantitative analysis of nuclear membrane assembly in mammalian cells using time-lapse microscopy. From the initial recruitment of ER tubules to chromatin, the formation of a membrane-enclosed, transport-competent nucleus occurs within ~12 min. Overexpression of the ER tubule-forming proteins reticulon 3, reticulon 4, and DP1 inhibits NE formation and nuclear expansion, whereas their knockdown accelerates nuclear assembly. This suggests that the transition from membrane tubules to sheets is rate-limiting for nuclear assembly. Our results provide evidence that ER-shaping proteins are directly involved in the reconstruction of the nuclear compartment and that morphological restructuring of the ER is the principal mechanism of NE formation in vivo.  相似文献   

15.
In interphase cells of the SPEV culture treated with Triton X-100, 2 M NaCl, and DNAse, in the presence of 2 mM CuCl2, we clearly revealed a stabilized nuclear protein material (NPM) composed of a peripheral lamina, residual nucleolus, and internal fibrillar network. This network is formed by thin fibrils 10–20 nm in diameter, which are also revealed in the nonhistone matrix of mitotic chromosomes at all stages of mitosis. In mitotic chromosomes, NPM is represented as a network of the 10–20-nm-thick fibrils without any features of the central-axial structures. Beginning from the middle prophase, it is possible to see approached sister chromatids in contact with each other in certain sites, similar to centromeres. At these sites, the thickness of fibrils increases up to 40–50 nm, whereas the fibrils themselves are disposed more tightly; this structure can be seen in the chromosome until telophase. At the end of telophase, the decondensation of chromosomes and formation of two new nuclei whose NPM is analogous to NPM of usual interphase nucleus are observed. Thus, the NPM elements can perform the role of a skeleton in both the interphase nucleus and mitotic chromosomes.  相似文献   

16.
Plant-specific mitotic targeting of RanGAP requires a functional WPP domain   总被引:6,自引:0,他引:6  
The small GTPase Ran is involved in nucleocytoplasmic transport, spindle formation, nuclear envelope (NE) formation, and cell-cycle control. In vertebrates, these functions are controlled by a three-dimensional gradient of Ran-GTP to Ran-GDP, established by the spatial separation of Ran GTPase-activating protein (RanGAP) and the Ran guanine nucleotide exchange factor RCC1. While this spatial separation is established by the NE during interphase, it is orchestrated during mitosis by association of RCC1 with the chromosomes and RanGAP with the spindle and kinetochores. SUMOylation of vertebrate RanGAP1 is required for NE, spindle, and centromere association. Arabidopsis RanGAP1 (AtRanGAP1) lacks the SUMOylated C-terminal domain of vertebrate RanGAP, but contains a plant-specific N-terminal domain (WPP domain), which is necessary and sufficient for its targeting to the NE in interphase. Here we show that the human and plant RanGAP-targeting domains are kingdom specific. AtRanGAP1 has a mitotic trafficking pattern uniquely different from that of vertebrate RanGAP, which includes targeting to the outward-growing rim of the cell plate. The WPP domain is necessary and sufficient for this targeting. Point mutations in conserved residues of the WPP domain also abolish targeting to the nuclear rim and the cell plate, suggesting that the same mechanism is involved in both targeting events. These results indicate that plant and animal RanGAPs undergo different migration patterns during cell division, which require their kingdom-specific targeting domains.  相似文献   

17.
The nuclear lamins are directed from the cytoplasm to chromosomes as part of the maturation pathway of the interphase nucleoskeleton. In mitosis, the three polypeptides lamin A, B and C were found in the cytoplasm from prophase until anaphase and shifted to chromosomal surfaces at telophase (Ely, D'Arcy and Jost, 1978; Gerace, Blum and Blobel, 1978). We show here that early events in nucleoskeleton formation could be regulated by extracellular pH. When exponentially growing tissue culture cells and cells arrested in mitosis were exposed to different extracellular pH values, three patterns of distribution of lamins were observed in mitotic cells: exclusively cytoplasmic distribution of mitotic lamins at low pH (6.8 to 7.3); a premature association of a lamin subfraction with metaphase chromosomes at intermediate pH 7.5; a more prominent relocation of lamins onto chromosomes in metaphase and in disorganized metaphase at pH 8.0. Reassembly of lamins occurred at telomeric ends of mitotic chromosomes followed by a lateral fusion to form a nuclear cage. Using immunogold localization, we show that pH-induced, premature, partial deposition of lamins onto condensed chromosomes may occur prior to the formation of the bilamellar nuclear envelope. These results suggest that the pH-induced redistribution of lamins acts to trigger early events of mitosis to interphase transition.  相似文献   

18.
Barrier-to-autointegration factor (BAF) is a conserved 10 kDa DNA-binding protein. BAF interacts with LEM-domain proteins including emerin, LAP2 beta, and MAN1 in the inner nuclear membrane. Using fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP), we compared the mobility of BAF to its partners emerin, LAP2 beta, and MAN1 in living HeLa cells. Like endogenous BAF, GFP-BAF was enriched at the nuclear envelope, and found inside the nucleus and in the cytoplasm during interphase. At every location, FRAP and FLIP analysis showed that GFP-BAF diffused rapidly; the halftimes for recovery in a 0.8 microm square area were 260 ms at the nuclear envelope, and even faster inside the nucleus and in the cytoplasm. GFP-fused emerin, LAP2 beta, and MAN1 were all relatively immobile, with recovery halftimes of about 1 min, for a 2 microm square area. Thus, BAF is dynamic and mobile during interphase, in stark contrast to its nuclear envelope partners. FLIP results further showed that rapidly diffusing cytoplasmic and nuclear pools of GFP-BAF were distinctly regulated, with nuclear GFP-BAF unable to replenish cytoplasmic BAF. Fluorescence resonance energy transfer (FRET) results showed that CFP-BAF binds directly to YFP-emerin at the inner nuclear membrane of living cells. We propose a "touch-and-go" model in which BAF binds emerin frequently but transiently during interphase. These findings contrast with the slow mobility of both GFP-BAF and GFP-emerin during telophase, when they colocalized at the 'core' region of telophase chromosomes at early stages of nuclear assembly.  相似文献   

19.
The nucleus of spermatocytes provides during the first meiotic prophase an interesting model for investigating relationships of the nuclear envelope (NE) with components of the nuclear interior. During the pachytene stage, meiotic chromosomes are synapsed via synaptonemal complexes (SCs) and attached through both ends to the nuclear periphery. This association is dynamic because chromosomes move during the process of synapsis and desynapsis that takes place during meiotic prophase. The NE of spermatocytes possesses some peculiarities (e.g., lower stability than in somatic cells, expression of short meiosis-specific lamin isoforms called C2 and B3) that could be critically involved in this process. For better understanding of the association of chromosomes with the nuclear periphery, in the present study we have investigated the distribution of NE proteins in relation to SC attachment sites. A major outcome was the finding that lamin C2 is distributed in the form of discontinuous domains at the NE of spermatocytes and that SC attachment sites are embedded in these domains. Lamin C2 appears to form part of larger structures as suggested by cell fractionation experiments. According to these results, we propose that the C2-containing domains represent local reinforcements of the NE that are involved in the proper attachment of SCs.  相似文献   

20.
FORMATION AND DISTRIBUTION OF NUCLEAR PORE COMPLEXES IN INTERPHASE   总被引:4,自引:0,他引:4       下载免费PDF全文
The possibility of nuclear pore formation in the interphase nucleus was investigated in control and phytohemagglutinin (PHA) stimulated lymphocytes by the freeze-etching technique. 48 hr after the addition of PHA, the newly formed blasts which had not as yet divided had at least twice the number of pores per nucleus as controls. This clearly demonstrates that in lymphocytes nuclear pore formation can take place during interphase. It has generally been assumed that the distribution of nuclear pore complexes in somatic animal cells is random. However, we have utilized freeze etched rat kidney cells and a computer program to evaluate pore distribution. We find a minimum pore center-to-center spacing of approximately 1300 A and multiples thereof with high frequency. This is strong evidence for a nonrandom distribution of nuclear pores. The nonrandomness may be related to an underlying chromosomal organization in interphase. Using three criteria for identifying prospective pore sites (membrane specialization, nonrandomness, and alteration of heterochromatin distribution), we have found forming pores in sectioned material from cultured human melanoma cells. While nuclear pore formation may take place in conjunction with reformation of the nuclear membrane, a mechanism also exists for their formation during interphase.  相似文献   

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