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1.
Previous reports from our laboratories and others have hinted that the nucleus is a site for an autonomous signalling system acting through the activation of the inositol lipid cycle. Among phospholipases (PLC) it has been shown previously that PLCβ1 is specifically localised in the nucleus as well as at the plasma membrane. Using NIH 3T3 cells, it has been possible to obtain, with two purification strategies, in the presence or in the absence of Nonidet P-40, both intact nuclei still maintaining the outer membrane and nuclei completely stripped of their envelope. In these nuclei, we show that not only PLCβ1 is present, but also PLCβ2, PLCβ3 and PLCβ4. The more abounding isoform is PLCβ1 followed by PLCβ3, PLCβ2 and PLCβ4, respectively. All the isoforms are enriched in nuclear preparations free from nuclear envelope and cytoplasmatic debris, indicating that the actual localisation of the PLCβ isozymes is in the inner nuclear compartment.  相似文献   

2.
An extensive body of evidence links inositide-specific phospholipase C (PLC) to the nucleus and the main isoform located in the nucleus is PLCbeta(1). Constitutive overexpression of nuclear PLCbeta(1) has been previously shown to inhibit Friend erythroleukemia cells differentiation and to induce cell cycle progression targeting cyclin D3. The aim of this study was to identify new proteins regulated by PLCbeta(1) overexpression, given the role exerted by its signaling in the nucleus during cell growth and differentiation. To identify novel downstream effectors of nuclear PLCbeta(1)-dependent signaling in Friend erythroleukemia cells, we performed the high-resolution 2-DE-based proteomic analysis. Using a proteomic approach we found that SRp20, a member of the highly conserved SR family of splicing regulators, was down-regulated in cells overexpressing nuclear PLCbeta(1) as compared with wild-type cells. Reduction in SRp20 was confirmed by 2-D Western blotting. Moreover, we have shown that nuclear PLCbeta(1) is bound to the SRp20 splicing factor. Indeed, by immunoprecipitation and subcellular fractioning, we have demonstrated that endogenous PLCbeta(1) and SRp20 physically interact in the nucleus. Here we show the existence of a PLCbeta(1)-specific target, the splicing factor SRp20, whose expression is specifically down-regulated by the nuclear signaling evoked by PLCbeta(1).  相似文献   

3.
In the present work, we have analyzed the expression and subcellular localization of all the members of inositide-specific phospholipase C (PLCbeta) family in muscle differentiation, given that nuclear PLCbeta1 has been shown to be related to the differentiative process. Cell cultures of C2C12 myoblasts were induced to differentiate towards the phenotype of myotubes, which are also indicated as differentiated C2C12 cells. By means of immunochemical and immunocytochemical analysis, the expression and subcellular localization of PLCbeta1, beta2, beta3, beta4 have been assessed. As further characterization, we investigated the localization of PLCbeta isoenzymes in C2C12 cells by fusing their cDNA to enhanced green fluorescent protein (GFP). In myoblast culture, PLCbeta4 was the most expressed isoform in the cytoplasm, whereas PLCbeta1 and beta3 exhibited a lesser expression in this cell compartment. In nuclei of differentiated myotube culture, PLCbeta1 isoform was expressed at the highest extent. A marked decrease of PLCbeta4 expression in the cytoplasm of differentiated C2C12 cells was detected as compared to myoblasts. No relevant differences were evidenced as regards the expression of PLCbeta3 at both cytoplasmatic and nuclear level, whilst PLCbeta2 expression was almost undetectable. Therefore, we propose that the different subcellular expression of these PLC isoforms, namely the increase of nuclear PLCbeta1 and the decrease of cytoplasmatic PLCbeta4, during the establishment of myotube differentiation, is related to a spatial-temporal signaling event, involved in myogenic differentiation. Once again the subcellular localization appears to be a key step for the diverse signaling activity of PLCbetas.  相似文献   

4.
Although the outer nuclear membrane is continuous with the endoplasmic reticulum, it is possible to isolate nuclei both intact and free from endoplasmic reticulum contaminants. The outer and the inner nuclear membranes can be purified free from cross-contamination. Evidence in support of autonomous regulation of nuclear calcium signaling relies upon the investigations with isolated nuclei. Mechanisms for generating calcium signaling in the nucleus have been identified. Two calcium transporting systems, an ATP-dependant nuclear Ca(2+)-ATPase and an IP4-mediated inositol 1,3,4,5-tetrakisphosphate receptor, are located on the outer nuclear membrane. Thus, ATP and IP4, depending on external free calcium concentrations, are responsible for filling the nuclear envelope calcium pool. The inositol 1,4,5-trisphosphate receptor is located on the inner nuclear membrane with its ligand binding domain facing toward the nucleoplasm. Likewise, the ryanodine receptor is located on the inner nuclear membrane and its ligand cADP-ribose is generated within the nucleus. A 120 kDa protein fragment of nuclear PLC-gamma1 is stimulated in vivo by epidermal growth factor nuclear signaling coincident with the time course of nuclear membrane epidermal growth factor receptor activation. Stimulated 120 kDa protein fragment interacts with PIKE, a nuclear GTPase, and together they form a complex with PI[3]kinase serving as a module for nuclear PI[3]K stimulation. Thus, the nucleus has its own IP(3) generating system.  相似文献   

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When basidia of Cronartium asclepiadeum (Uredinales) develop basidiospores, nuclei migrate from the basidial cells into the basidiospores. A mitotic nuclear division yields two nuclei in the basidiospore. One of these nuclei degenerates if the basidiospore develops a secondary ballistospore. Several stages in the degeneration of the nuclei can be recognized: (1) Condensation of chromatin, (2) separation of the nucleus into a portion containing the chromatin and a portion containing the nucleoplasm by an invagination of the nuclear envelope, (3) reduction of nuclear volume, (4) enveloping and (5) twisting of nuclear membranes around the degenerating nucleus, (6) homogenization of the chromatin with reduction of the nuclear envelope, and reduction of the enclosing membrane complex from a multilamellar structure to a single membrane layer, and (7) invagination or splitting off of the spiralled membranes. The nuclear behavior during early developmental stages of secondary spore formation is similar to that of budding basidiomycetous yeasts. Basidiomycetes producing ballistospores may possibly have arisen from those whose reproducing phase is yeast-like.  相似文献   

7.
The cytoskeleton is connected to the nuclear interior by LINC (linker of nucleoskeleton and cytoskeleton) complexes located in the nuclear envelope. These complexes consist of SUN proteins and nesprins present in the inner and outer nuclear membrane respectively. Whereas SUN proteins can bind the nuclear lamina, members of the nesprin protein family connect the nucleus to different components of the cytoskeleton. Nesprin-1 and -2 can establish a direct link with actin filaments, whereas nesprin-4 associates indirectly with microtubules through its interaction with kinesin-1. Nesprin-3 is the only family member known that can link the nuclear envelope to intermediate filaments. This indirect interaction is mediated by the binding of nesprin-3 to the cytoskeletal linker protein plectin. Furthermore, nesprin-3 can connect the nucleus to microtubules by its interactions with BPAG1 (bullous pemphigoid antigen 1) and MACF (microtubule-actin cross-linking factor). In contrast with the active roles that nesprin-1, -2 and -4 have in actin- and microtubule-dependent nuclear positioning, the role of nesprin-3 is likely to be more passive. We suggest that it helps to stabilize the anchorage of the nucleus within the cytoplasm and maintain the structural integrity and shape of the nucleus.  相似文献   

8.
Protein prenylation is a posttranslational modification involving the covalent attachment of a prenyl lipid to a cysteine at or near the COOH terminus of a protein. It is required for membrane localization and efficient function of a number of cytoplasmic as well as nuclear proteins including the proto-oncogenic and activated forms of Ras. Farnesylation in conjunction with a nuclear localization signal has been shown to be necessary to target newly synthesized nuclear lamins to the inner nuclear envelope membrane. It is, however, not clear where in the cell isoprenylation of nuclear lamins takes place. In this study we describe in vivo and in vitro experiments on the isoprenylation of the Xenopus oocyte nuclear lamin B3. We show by kinetic analysis that newly synthesized lamins are isoprenylated in the cytosol of oocytes before uptake into the nucleus. From our data it can be concluded that isoprenylation of lamins in the nucleus, as it is observed under certain conditions of isoprene starvation, represents a default pathway rather than the physiological situation. We further analyzed the capacity of isolated nuclei to carry out isoprenylation of B3. Our results are in line with a dual localization of a protein farnesyltransferase in the cytosol and nuclei of amphibian oocytes. Implications for the possible functions of a nuclear protein farnesyltransferase as well as possible mechanisms of the selective inhibition of farnesylation of cytoplasmic proteins by peptidomimetics are discussed.  相似文献   

9.
The phospholipase C (PLC) isoform most important during agonist-activated IP(3) production in vascular smooth muscle is still unknown. When PLC activity in rat tail artery homogenate was determined, this activity was shown to be inhibited by an antibody directed against PLCbeta2. Antibodies directed against the gamma1, beta1, beta3 and delta1 isoforms of PLC failed to inhibit PLC activity in this tissue. Both PLCbeta2 and PLCgamma1 were isolated from rat tail artery by DEAE column chromatography and PLCbeta2 activity was shown to be 3-fold greater than PLCgamma1 activity. When rat tail artery was treated with norepinephrine (10 mM), PLCbeta2 was shown to translocate from cytosol to membranes. When subcellular fractions of rat tail artery were isolated by sucrose density gradient centrifugation, including nuclei, plasma membrane, and cytosol, PLCbeta2 was detected in the plasma membrane and the cytosol but not in the nuclei. PLCdelta1 and PLCgamma1 were found only in cytosol. This evidence is consistent with the model wherein an agonist such as norepinephrine can activate smooth muscle contraction via interaction with a plasma membrane receptor which can easily interact with a plasma membrane-associated isoform of PLC, such as PLCbeta2.  相似文献   

10.
The location of centromeric protein CENP-B and telomeric protein TRF2/MTBP in the mouse spermatogenic line has been studied using indirect immunofluorescent and immunoelectron microscopy. CENP-B localized to the heterochromatic parts of the nuclei at meiotic stages. A clearly distinct chromocenter forms in the nucleus at stages 3-4 of spermatid maturation; CENP-B localizes in it and in the area adjacent to the future acrosome. CENP-B localization in the subacrosomal area and in the chromocenters' periphery demonstrates that centromeres are organized in two groups in mouse spermatozoa, unlike human centromeres. TRF2/MTBP concentrates around the forming chromocenter at spermiogenesis early stages. The TRF2/MTBP main signal migrates into the area of acrosomal membrane at the course of spermatozoon maturation. TRF2/MTBP never localizes inside the synaptonemal complex but can be found in the areas where the synaptonemal complex attaches to the nuclear envelope. At the pachytene and diplotene stages when chromosomes separate from the nuclear envelope, some amount of the protein remains bound to the nuclear membrane while the other part reveals itself in chromosomes. TRF2/MTBP accumulates in the future acrosome from the very beginning of its formation. In the mature spermatozoon TRF2/MTBP decorates the acrosomal membrane as well as spreads in condensed chromatin.  相似文献   

11.
应用电镜观察了重组AcMNPV感染的Sf9细胞,结果表明该病毒的囊膜形成至少有两种形式:一是通过细胞质膜上出芽,二是在核内被膜结构包围而获得囊膜,此外通过核膜出芽也可能是病毒获得囊膜的一种方式。应用免疫荧光技术研究了该病毒在Sf9细胞内囊膜的形成及其与病毒囊膜蛋白gp64间的关系,结果表明gp64主要存在于细胞的质膜与核膜上。该存在方式使得通过出芽而获得囊膜的病毒粒子与核内包被产生的病毒粒子在囊膜成分上有很大差异。  相似文献   

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13.
双滴虫类是迄今所知的现存最原始的真核生物类群。以蓝氏贾第虫作为双滴虫类的代表,对其细胞核进行了电镜观察。除了未见有核仁外,还发现其核被膜的横切面上存在有缺口。在缺口的边缘处,核内膜与校外膜是相互连接着的,表明并非切片时所造成的假象。核被膜缺口处常有一核纤层样的薄层分隔核质与细胞质。用高锰酸钾固定细胞以求只保存膜结构时,核被膜缺口仍然可见,上述的薄层即未见到。核被膜缺口的发现证实了李靖炎(1979)的核被膜起源假说所作出的推断。  相似文献   

14.
The nucleus is a spherical dual‐membrane bound organelle that encapsulates genomic DNA. In eukaryotes, messenger RNAs (mRNA) are transcribed in the nucleus and transported through nuclear pores into the cytoplasm for translation into protein. In certain cell types and pathological conditions, nuclei harbor tubular invaginations of the nuclear envelope known as the “nucleoplasmic reticulum.” Nucleoplasmic reticulum expansion has recently been established as a mediator of neurodegeneration in tauopathies, including Alzheimer's disease. While the presence of pore‐lined, cytoplasm‐filled, nuclear envelope invaginations has been proposed to facilitate the rapid export of RNAs from the nucleus to the cytoplasm, the functional significance of nuclear envelope invaginations in regard to RNA export in any disorder is currently unknown . Here, we report that polyadenylated RNAs accumulate within and adjacent to tau‐induced nuclear envelope invaginations in a Drosophila model of tauopathy. Genetic or pharmacologic inhibition of RNA export machinery reduces accumulation of polyadenylated RNA within and adjacent to nuclear envelope invaginations and reduces tau‐induced neuronal death. These data are the first to point toward a possible role for RNA export through nuclear envelope invaginations in the pathogenesis of a neurodegenerative disorder and suggest that nucleocytoplasmic transport machinery may serve as a possible novel class of therapeutic targets for the treatment of tauopathies.  相似文献   

15.
These studies examined the importance of phospholipase Cbeta (PLCbeta) in the calcium responses of pituitary cells using PLCbeta3 knockout mice. Pituitary tissue from wild-type mice contained PLCbeta1 and PLCbeta3 but not PLCbeta2 or PLCbeta4. Both Galphaq/11 and Gbetagamma can activate PLCbeta3, whereas only Galphaq/11 activates PLCss1 effectively. In knockout mice, PLCbeta3 was absent, PLCbeta1 was not up-regulated, and PLCbeta2 and PLCbeta4 were not expressed. Since somatostatin inhibited influx of extracellular calcium in pituitary cells from wild-type and PLCbeta3 knockout mice, the somatostatin signal pathway was intact. However, somatostatin failed to increase intracellular calcium in pituitary cells from either wild-type or knockout mice under a variety of conditions, indicating that it did not stimulate PLCbeta3. In contrast, somatostatin increased intracellular calcium in aortic smooth muscle cells from wild-type mice, although it evoked no calcium response in cells from PLCbeta3 knockout animals These results show that somatostatin, like other Gi/Go-linked hormones, can stimulate a calcium transient by activating PLCbeta3 through Gbetagamma, but this response does not normally occur in pituitary cells. The densities of Gi and Go, as well as the relative concentrations of PLCbeta1 and PLCbeta3, were similar in cells that responded to somatostatin with an increase in calcium and pituitary cells. Calcium responses to 1 nM and 1 microM TRH and GnRH were identical in pituitary cells from wild-type and PLCbeta3 knockout mice, as were responses to other Gq-linked agonists. These results show that in pituitary cells, PLCbeta1 is sufficient to transmit signals from Gq-coupled hormones, whereas PLCbeta3 is required for the calcium-mobilizing actions of somatostatin observed in smooth muscle cells.  相似文献   

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18.
SYNOPSIS. The ultrastructure of interphase and mitotic nuclei of the epimastigote form of Trypanosoma cyclops Weinman is described. In the interphase nucleus the nucleolus is located centrally while at the periphery of the nucleus condensed chromatin is in contact with the nuclear envelope. The nucleolus fragments at the onset of mitosis, but granular material of presumptive nucleolar origin is often recognizable in the mitotic nucleus. Peripheral chromatin is in contact with the nuclear envelope throughout mitosis, and it seems reasonable to assume that the nuclear envelope is involved in its segregation to the daughter nuclei. Spindle microtubules extend between the poles of the dividing nucleus and terminate close to the nuclear envelope. The basal body and kinetoplast divide before the onset of mitosis and do not appear to have any morphologic involvement in that process. Spindle pole bodies, kinetochores, and chromosomal microtubules have not been observed.  相似文献   

19.
Abstract. To study whether an electrical potential difference exists across the nuclear envelope or inner nuclear membrane of plant cells, the authors have used an optical probe of membrane potential, the cationic fluorescent dye, DiOC6(3) (MW = 572.5). This dye was microinjected into the nucleoplasm of isolated Acetabularia nuclei (which are still surrounded by a thin layer of cytoplasm) and its subnuclear localization visualized by fluorescence microscopy. Striking differences, which seemed to be correlated with the developmental stage of the isolated nucleus, were observed. In nuclei isolated from cells at the stage of early cap stage formation, the dye was restricted to the nuclear envelope. In nuclei isolated from cells with intermediate or fully developed caps, there was increased nucleoplasmic staining, and the staining of the envelope was frequently diminished or abolished. In all nuclei, the dye remained within the nucleus after injection. Cytoplasmic staining was only observed when nuclei isolated from cells at the stage of early cap formation were incubated in a hyper- or hypo-tonic medium. Various ionophores, injected before the dye into the nucleoplasm, had no effect on the subsequent nuclear localization of DiOC6(3), although they did rapidly induce nucleolar condensation in nuclei isolated from cells at the stage of early cap formation. The results suggested that the electrical properties of Acetabularia nuclear envelopes or inner nuclear membranes change during cell maturation. Furthermore, the retention of the dye in the nucleoplasm under isotonic conditions indicated that the nuclear pores were not open channels for molecules of this size.  相似文献   

20.
Nuclear migration is a critical component of many cellular and developmental processes. The nuclear envelope forms a barrier between the cytoplasm, where mechanical forces are generated, and the nucleoskeleton. The LINC complex consists of KASH proteins in the outer nuclear membrane and SUN proteins in the inner nuclear membrane that bridge the nuclear envelope. How forces are transferred from the LINC complex to the nucleoskeleton is poorly understood. The Caenorhabditis elegans lamin, LMN-1, is required for nuclear migration and interacts with the nucleoplasmic domain of the SUN protein UNC-84. This interaction is weakened by the unc-84(P91S) missense mutation. These mutant nuclei have an intermediate nuclear migration defect—live imaging of nuclei or LMN-1::GFP shows that many nuclei migrate normally, others initiate migration before subsequently failing, and others fail to begin migration. At least one other component of the nucleoskeleton, the NET5/Samp1/Ima1 homologue SAMP-1, plays a role in nuclear migration. We propose a nut-and-bolt model to explain how forces are dissipated across the nuclear envelope during nuclear migration. In this model, SUN/KASH bridges serve as bolts through the nuclear envelope, and nucleoskeleton components LMN-1 and SAMP-1 act as both nuts and washers on the inside of the nucleus.  相似文献   

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