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PubMed2003

The M-PMV cytoplasmic targeting-retention signal directs nascent Gag polypeptides to a pericentriolar region of the cell.

Intracytoplasmic protein targeting in mammalian cells is critical for organelle function as well as virus assembly, but the signals that mediate it are poorly defined. We show here that Mason-Pfizer monkey virus specifically targets Gag precursor proteins to the pericentriolar region of the cytoplasm in a microtubule dependent process through interactions between a short peptide signal, known as the cytoplasmic targeting-retention signal, and the dynein/dynactin motor complex. The Gag molecules are concentrated in pericentriolar microdomains, where they assemble to form immature capsids. Depletion of Gag from this region by cycloheximide treatment, coupled with the presence of ribosomal clusters that are in close vicinity to the assembling capsids, suggests that the dominant N-terminal cytoplasmic targeting-retention signal functions in a cotranslational manner. Transport of the capsids out of the pericentriolar assembly site requires the env-gene product, and a functional vesicular transport system. A single point mutation that renders the cytoplasmic targeting-retention signal defective abrogates pericentriolar targeting of Gag molecules. Thus the previously defined cytoplasmic targeting-retention signal appears to act as a cotranslational intracellular targeting signal that concentrates Gag proteins at the centriole for assembly of capsids.

Animals↗
PubMed2004

Nuclear-cytoplasmic male-sterility in diploid dandelions.

Male-sterility was found in diploid dandelions from two widely separated populations from France, and its inheritance was analysed by crossing a diploid male-sterile dandelion to diploid sexuals and triploid apomicts. Nuclear genetic variation, found in full-sib families, segregated for male-fertility, partial male-sterility, and full male-sterility, and also segregated for small-sized versus normally sized pollen. The crossing results are best explained by a cytoplasmic male-sterility factor in combination with two dominant restorer genes. Involvement of the cytoplasmic male-sterility factor was further investigated by chloroplast haplotyping. Male-sterility was exclusively associated with a rare chloroplast haplotype (designated 16b). This haplotype was found in seven male-sterile plants and one (apparently restored) male-fertile individual but does not occur in 110 co-existing male-fertile plants and not in several hundreds of individuals previously haplotyped. Apomicts with cytoplasmic male sterility were generated in some test crosses. This raises the question as to whether the male sterility found in natural dandelion apomicts, is of cytoplasmic or of nuclear genetic nature. As many breeding systems in Taraxacum are involved in shaping population structure, it will be difficult to predict the evolutionary consequences of nuclear-cytoplasmic male-sterility for this species complex.

Cell Nucleus↗
PubMed2000

Cytoplasmic and nuclear localization of the 130 and 160 kDa Bcr proteins.

Formation of the Bcr-Abl chimeric protein is the molecular hallmark of Philadelphia-positive leukemia. Normal Bcr is a complex protein which has been found in the cytoplasm, has serine kinase activity, and has been implicated in cellular signal transduction. However, we have recently demonstrated that Bcr can also associate with condensed chromatin. Since two major Bcr proteins have been characterized (p160Bcr and p130Bcr), we sought to determine if different forms of Bcr localized to the nucleus vs the cytoplasm. Metabolic labeling and Western blotting experiments were performed using nuclear and cytoplasmic extracts of three human Philadelphia-negative leukemia/lymphoma cell lines (KG-1, HL-60, and Jurkat). Both methodologies showed that p160Bcr and p130Bcr localized to the cytoplasm, but the p130 form predominated in the nucleus. These results suggest that Bcr serves both nuclear and cytoplasmic functions, and that different forms of Bcr may be preferentially involved in these distinct activities.

Acute Disease↗
PubMed1997

Cytoplasmic retention of the p53 tumor suppressor gene product is observed in the hepatitis B virus X gene-transfected cells.

It has been suggested that hepatitis B virus (HBV) X gene activates X gene expression by disrupting the function of p53 tumor suppressor gene (Takada et al., 1996). To find out their connection, effect of X protein expression on the nuclear localization of p53 protein in human hepatoma cells was examined by the immunofluorescent double-staining technique. The location of transiently-expressed p53 protein was examined in X gene-transfected cells, where X protein was detected in the cytoplasm. The nuclear location of transiently-expressed p53 protein was changed to the cytoplasm by X protein co-expression. Endogenous p53 protein was also observed in the cytoplasm by X protein expression. The transcriptional activation domain of X protein and the carboxy-terminal region of p53 protein were found mutually responsible for the cytoplasmic retention of p53 protein in X gene-transfected cells. Therefore, the cytoplasmic retention of p53 protein may be closely correlated to the function of X protein expressed in transfected cells.

Biological Transport↗
PubMed1998

Cytoplasmic retention of mutant tsp53 is dependent on an intermediate filament protein (vimentin) scaffold.

The temperature-sensitive mutant tsp53val135 accumulates in the cytoplasm of cells kept at the non-permissive temperature (39 degrees C), but is rapidly transported into the cell nucleus at the permissive temperature (30 degrees C). tsp53 thus may serve as a model for analysing cellular parameters influencing the subcellular location of p53. Here we provide evidence that retention of tsp53 in the cytoplasm at the non-permissive temperature is due to cytoskeletal anchorage of the p53 protein. Two sublines of C6 rat glioma cells differing in their expression of the intermediate filament protein vimentin (vimentin expressing or vimentin negative cells) were stably transfected with a vector encoding tsp53. Whereas cells of vimentin expressing C6 subclones retained tsp53 in the cytoplasm at the non-permissive temperature, cells of vimentin negative subclones exclusively harbored the tsp53 within their nuclei. Intermediate filament deficient cells that had been reconstituted with a full length vimentin protein again showed a cytoplasmic localization of tsp53, whereas in cells expressing a C-terminally truncated (tail-less) vimentin tsp53 localized to the nucleus. We conclude that cytoplasmic sequestration of tsp53 requires an intact intermediate filament system.

Actins↗
PubMed2000

MDM2-dependent ubiquitination of nuclear and cytoplasmic P53.

Wild-type p53 is stabilized and accumulates in the nucleus of DNA damaged cells. The effect of stabilizing p53 is to inhibit cell growth, either through a G1 cell cycle arrest or apoptotic cell death. MDM2 can inhibit p53 activity, in part, by promoting its rapid degradation through the ubiquitin proteolysis pathway. In the current study, MDM2-mediated degradation of p53 was partially inhibited in cells treated with leptomycin B (LMB), a specific inhibitor of nuclear export. In contrast, levels of ubiquitinated p53 increased in LMB-treated cells, indicating that nuclear export is not required for p53 ubiquitination. To investigate this further, p53 mutants were generated which localize to either the nucleus or cytoplasm, and their susceptibility to MDM2-mediated ubiquitination was assessed. p53 mutants that localized to either the nucleus or the cytoplasm were efficiently ubiquitinated, and their steady-state levels decreased, when coexpressed with MDM2. In addition, an MDM2-mutant that localized to the cytoplasm was able to ubiquitinate and degrade a p53 mutant which was similarly localized in the cytoplasm. Our results indicate that nuclear export is not required for p53 ubiquitination, and that p53 proteins that localize to either the nucleus or cytoplasm can be ubiquitinated and degraded by MDM2.

Animals↗
PubMed1970

Cerebral-cortex hexokinase. Comparison of properties of solubilized mitochondrial and cytoplasmic activities.

1. Cerebral-cortex mitochondria, after purification by using high-density sucrose solutions, were extracted with Triton X-100. The total hexokinase activity of the intact mitochondria was increased by 50-80% in the Triton extracts. 2. Triton X-100 was removed from mitochondrial extracts by a combination of ammonium sulphate fractionation and DEAE-cellulose chromatography. Mitochondrial hexokinase remained soluble after removal of extractant. 3. The behaviour of solubilized mitochondrial hexokinase was compared with soluble cytoplasmic hexokinase from the same samples of cerebral cortex on identical columns of DEAE-cellulose. Two peaks were eluted from each source of hexokinase. The distribution between hexokinase peaks was similar for the two sources. Peak I (approx. 80% of the total hexokinase) from each was eluted at identical concentrations of potassium chloride and slight differences were observed in the elution profiles for peak II. 4. The purified mitochondrial hexokinase showed the following kinetic properties: peak I, K(m)(ATP) 0.60mm, K(m)(glucose) 0.042mm; peak II, K(m)(ATP) 0.66mm, K(m)(glucose) 0.043mm. The purified cytoplasmic hexokinase Michaelis constants were: peak I, K(m)(ATP) 0.56mm, K(m)(glucose) 0.048mm; peak II, K(m)(ATP) 0.68mm, K(m)(glucose) 0.062mm. 5. Although no significant differences between mitochondrial and cytoplasmic hexokinases were noted in chromatographic behaviour or in the kinetic properties studied, the purified mitochondrial enzyme was activated slightly (approx. 20%) by Triton X-100, in contrast with the cytoplasmic enzyme, which was not affected. 6. The results, taken to indicate basic similarity between mitochondrial and cytoplasmic hexokinases, are discussed in relation to the role of the two sources of enzyme in the metabolism of the tissue.

Animals↗
PubMed1973

Steroid-binding properties and stabilization of cytoplasmic glucocorticoid receptors from rat thymus cells.

1. A competitive binding assay was adapted for determination of the specific binding of glucocorticoids to cytoplasmic receptors from rat thymus cells. The steroid-receptor complexes prepared by incubation of a cytoplasmic fraction from rat thymus cells with [1,2-(3)H(2)]cortisol or with [1,2,4-(3)H(3)]triamcinolone acetonide had rates of dissociation at 37 degrees C similar to those from intact cells. 2. The cytoplasmic receptor was unstable at 3 degrees C, but the rate of inactivation was decreased in the presence of 2.5mm-EDTA. The steroid-receptor complex was stable. 3. Rate constants for association and for dissociation, and association constants, were determined for the interactions of cortisol, cortexolone, dexamethasone and triamcinolone acetonide with the cytoplasmic receptor at 3 degrees C. Differences in the association constants for different steroids could largely be accounted for by the differences in the rate constants for dissociation, but the rate constants for association did not vary greatly; the implications of these findings for the nature of the steroid-binding site are discussed. 4. A cytoplasmic fraction prepared from cells which had been incubated at 37 degrees C under anaerobic conditions bound much less [1,2-(3)H(2)]cortisol than did a fraction from aerobic cells, but the binding capacity was restored after exposure of the anaerobic cells to O(2). 5. The specific binding of [1,2-(3)H(2)]-cortisol to intact thymus cells incubated aerobically was not affected by the presence of 0.1mm-cycloheximide, nor did this concentration of cycloheximide inhibit the recovery of specific binding observed when anaerobic cells were transferred to an aerobic atmosphere. 6. The energy dependence of specific binding of cortisol to the receptor is discussed with reference to possible mechanisms.

17-Hydroxycorticosteroids↗
PubMed2005

Involvement of a cytoplasmic-tail serine cluster in urotensin II receptor internalization.

Most G-protein-coupled receptors that undergo agonist-dependent internalization require the presence of specific cytoplasmic-tail residues to initiate interactions with proteins of the endocytic machinery. Here we show that the UT receptor (urotensin II receptor) undergoes internalization, and that specific serine residues of the receptor's cytoplasmic tail participate in this process. We first observed a time-dependent increase in internalization of the UT receptor expressed in COS-7 cells following binding of the agonist urotensin II. This sequestration was significantly reduced in the presence of sucrose, demonstrating that the agonist-activated UT receptor is internalized in part by clathrin-coated pits. Moreover, the sequestered receptor was co-localized in endocytic vesicles with beta-arrestin1 and beta-arrestin2. To assess whether specific regions of the receptor's cytoplasmic tail were involved in internalization, five UT receptor mutants were constructed. In four constructs the receptor's cytoplasmic tail was truncated at various positions (UTDelta367, UTDelta363, UTDelta350 and UTDelta336), and in the other four adjacent serine residues at positions 364-367 were replaced by Ala (Mut4S). Each mutant, except UTDelta367, demonstrated a significantly reduced internalization rate, thereby revealing the importance of specific serine residues within the cytoplasmic tail of the UT receptor for its ability to be internalized efficiently.

Amino Acid Sequence↗
PubMed2001

The C-terminal half of Hsp90 is responsible for its cytoplasmic localization.

With some exceptions, research so far has shown heat shock protein (Hsp) 90 to be a cytoplasmic protein. Here, we studied the sequence determinants which dictate the subcellular localization of Hsp90. By constructing hybrid molecules between a nuclear protein, progesterone receptor (PR), and parts of Hsp90, we demonstrated that the C-terminal but not the N-terminal half of Hsp90 can prevent nuclear translocation of the PR. Studies with an antibody raised against a region which contains the major nuclear localization signal (NLS) of the PR suggest that the inhibition of nuclear localization is not due to steric hindrance of the NLS of the PR by Hsp90 sequences in hybrid molecules. In order to characterize further the cytoplasmic anchoring of Hsp90 we constructed four chimeric molecules between the C-terminal half of Hsp90 and estrogen receptor (ER) with different numbers of nuclear localization protosignals (proto-NLS). When the C-terminal half of Hsp90 was fused with ER containing no or one proto-NLS, the hybrid molecule was located exclusively in the cytoplasm. When the nuclear translocation signal was strengthened by adding two or three protosignals, the hybrid molecule was exclusively nuclear. These results suggest that the C-terminal half of Hsp90 contains a sequence which is responsible for the cytoplasmic localization of the protein. Further deletions of the molecule suggested that the cytoplasmic anchoring signal is located between amino acids 333 and 664.

Amino Acid Sequence↗
PubMed2002

Pistillody, homeotic transformation of stamens into pistil-like structures, caused by nuclear-cytoplasm interaction in wheat.

Homeotic transformation of stamens into pistil-like structures (pistillody) has been observed in a cytoplasmic substitution (alloplasmic) line of wheat (Triticum aestivum L.) cv. Norin 26, which has the cytoplasm of a wild relative species, Aegilops crassa L. On the other hand, an alloplasmic line of wheat cv. Chinese Spring (CS) with Ae. crassa cytoplasm has normal flowers. This is due to the presence in the CS nucleus of a fertility-restoring gene, Rfd1. Deletion mapping analysis revealed that Rfd1 is located on the middle part of the long arm of chromosome 7B. To investigate the function of the Rfd1 gene by a loss-of-function strategy, we produced alloplasmic lines of CS ditelosomic 7BS [(cr)-CSdt7BS] and CS monotelodisomic 7BS [(cr)-CSmd7BS] with the Ae. crassa cytoplasm, and characterized their phenotypes. The line (cr)-CSdt7BS without Rfd1 exhibited pistillody in all florets, and also female sterility. Scanning electron microscopy of the young spikes revealed that the pistillody was induced at an early stage of stamen development. The pistillate stamens often developed incomplete ovule-like structures with integuments instead of tapetum and pollen grains. It is possible that MADS box genes are associated with the induction of pistillody, because the expression of wheat APETALA3 homologue (WAP3) was reduced in the young spikes of (cr)-CSdt7BS. In addition, a histological study indicated that the female sterility in (cr)-CSdt7BS is due to the abnormality of the ovule, which fails to form an inner epidermis and integuments in the chalaza region. The line (cr)-CSmd7BS, hemizygous for Rfd1, showed partial pistillody (51%) and restored female fertility up to 72%. These results suggest that the induction of both pistillody and ovule deficiency caused by the Ae. crassa cytoplasm is inhibited by the Rfd1 gene in a dose-dependent manner.

Cell Differentiation↗
PubMed2000

Cell density-dependent VP-16 sensitivity of leukaemic cells is accompanied by the translocation of topoisomerase IIalpha from the nucleus to the cytoplasm.

The resistance of several leukaemic and myeloma cell lines (CCRF, L1210, HL-60, KG-1a and RPMI 8226) to VP-16 was found to increase with cell density and to be maximal (3.5- to 39-fold) in plateau phase cell cultures, as measured by clonogenic and MTT assays. Non-transformed confluent Flow 2000 human fibroblasts and Chinese hamster ovary (CHO) cells were also five- and 15-fold resistant to VP-16 respectively. The transition from log to plateau phase was accompanied by a drastic decrease in topoisomerase (topo) IIalpha content in CHO cells and human fibroblasts, while the leukaemic cells maintained constant cellular levels of topo IIalpha and topo IIbeta. However, the nuclear topo IIalpha content was found to decrease as a result of translocation of the enzyme to the cytoplasmic compartment in the leukaemic cells. This was confirmed by subcellular fractionation experiments, Western blotting analyses and immunocytochemistry studies. The quantity of topo IIalpha in plateau phase cytoplasmic fractions ranged from 18% in L1210 cells to 50% in HL-60 and 8226 cells, as measured by both immunoblotting and quantification of the label in immunofluorescent images. The cytoplasmic fraction from plateau phase cells retained topo II catalytic activity, as measured by the decatenation of kinetoplast DNA. The nuclear-cytoplasmic ratio of topo IIalpha may be critical in determining the sensitivity of leukaemic cells to topo II inhibitors. Cytoplasmic trafficking of topo IIalpha was observed in plasma cells obtained from patients with multiple myeloma, and perhaps contributes to drug resistance in this disease.

Antineoplastic Agents, Phytogenic↗
PubMed1997

Cytoplasmic detection of a novel protein containing a nuclear localization sequence by human autoantibodies.

A great diversity of antibodies directed to cell proteins has been described in sera of patients with autoimmune diseases. Most of these sera recognize nuclear components, but some others are directed against cytoplasmic autoantigens. Some of the antibodies directed to cytoplasmic autoantigens are well characterized, such as anti-mitochrondial, anti-ribosomal, anti-microsomal and anti-Golgi complex autoantibodies, but the target of many others remains unknown. In the last 5 years we have selected 32 sera with a characteristic speckled cytoplasmic pattern in indirect immunofluorescence (IIF) assay among a total of more than 31,000 sera from patients with any kind of autoimmune manifestation who attend our Connective Tissue Disease Clinic. Using a human cDNA expression library, we have identified a new autoantibody specificity named RCD-8 in five of these sera, directed to one cytoplasmic autoantigen. Affinity-purified antibodies eluted from a positive clone reproduced the same IIF cytoplasmic staining pattern as native serum and reacted with one single band of 160 kD on an immunoblot of HeLa cell extract. The sequence was found homologous to an autoantigen recently reported named Ge-1, and contains a nuclear localization sequence (NLS), an active protein domain made by a contiguous stretch of amino acids which allows the selective entry of the protein into the nucleus. The five patients whose sera exhibited this new autoantibody specificity displayed different autoimmune pathological profiles.

Amino Acid Sequence↗
PubMed2001

Differentiation of antineutrophil nuclear antibodies in inflammatory bowel and autoimmune liver diseases from antineutrophil cytoplasmic antibodies (p-ANCA) using immunofluorescence microscopy.

Perinuclear antineutrophil cytoplasmic antibodies (p-ANCA) directed against cytoplasmic proteins of neutrophils have been studied extensively in patients with systemic vasculitides. Recent data indicate that antineutrophil antibodies in sera from patients with chronic inflammatory bowel diseases (IBD) or autoimmune liver disorders, currently called 'atypical p-ANCA', recognize a nuclear target antigen, rendering the term 'ANCA' inaccurate. Specific microscopic criteria to distinguish atypical p-ANCA from p-ANCA are lacking. We used planar and confocal laser scanning indirect immunofluorescence microscopy to examine the labelling characteristics of ethanol-, methanol- and formaldehyde-fixed neutrophils by antineutrophil antibodies in 153 serum samples from patients with IBD, autoimmune liver disorders, systemic vasculitides or healthy blood donors. On ethanol- or methanol-fixed neutrophils, multiple intranuclear fluorescent foci together with either a rim-like peripheral nuclear staining ('type A') or a combined cytoplasmic and peripheral nuclear staining ('type B') was noted exclusively with atypical p-ANCA in sera from patients with IBD or autoimmune liver disorders. Intranuclear foci, which probably corresponded to invaginations of the nuclear envelope, were not labelled by p-ANCA from patients with microscopic polyangiitis or cytoplasmic ANCA (c-ANCA) from patients with Wegener's granulomatosis. On formaldehyde-fixed neutrophils, atypical p-ANCA gave a fine rim-like staining of the nuclear periphery, whereas ANCA diffusely labelled the cytoplasm. To distinguish reliably between the patterns produced by atypical p-ANCA or p-ANCA, particularly p-ANCA, careful indirect immunofluorescence microscopy on ethanol- as well as on formaldehyde-fixed neutrophils is necessary, with particular emphasis on the presence of multiple intranuclear fluorescent foci.

Adolescent↗
PubMed2003

Detection of cytoplasmic CD antigens within normal human peripheral blood leucocytes.

Polymorphonuclear neutrophils (PMNs) are capable of synthesizing various pro-inflammatory cytokines which may indirectly influence specific immune responses. PMNs may also have the capacity to present foreign peptides to helper T cells (Th cells). In support of this hypothesis, recent studies have shown that neutrophils, when activated by the correct combination of cytokines, can be induced to express cell surface major histocompatibility complex (MHC) Class II (DR) antigen, CD80 (B7.1) and CD86 (B7.2): molecules required for antigen presentation and subsequent T-cell activation. In this study we have used normal "resting" human peripheral blood neutrophils and demonstrated, using a mild fixation and permeabilization protocol, significant cytoplasmic "stores" of these molecules known to be important in antigen presentation. Cytoplasmic MHC Class II antigen was found with two out of 20 normal donors tested whereas cytoplasmic CD80 and CD86 were found to a variable extent within all normal donors. Surprisingly, we also found several other neutrophil cytoplasmic CD antigens more commonly associated with B cells, i.e. CD20, CD21 (CR2/EBV-R) and CD22 (BL-CAM). All of these antigens were confined to the "resting" cell cytoplasm and were never found to be expressed on the cell surface. To exclude the possibility that these antigens were absorbed from plasma and to provide evidence for active synthesis, we used a novel whole blood in situ hybridization flow cytometry assay method to detect mRNA specific for these antigens within normal PMNs. We also conducted real-time polymerase chain reactions to confirm these findings using CD22 as a good example of an "inappropriately expressed" CD antigen. These observations therefore provide support for the hypothesis that human PMNs have the potential to express molecules required for antigen presentation and cell signalling.

Antigen Presentation↗
PubMed1997

Identification of nitric oxide-sensitive and -insensitive forms of cytoplasmic guanylate cyclase.

Cytoplasmic, nitric oxide-activated guanylate cyclases are expressed in many regions of the mammalian brain and are thought to participate in functions as diverse as synaptogenesis and long-term potentiation. In this study, we have characterized cytoplasmic guanylate cyclases in the nervous system of an invertebrate, the American lobster. Cytoplasmic cyclase specific activity is higher in lobster nerve cord than in any other lobster tissue tested, and considerably higher than in typical rat tissues (cerebellum, lung, and liver). However, nitric oxide donors have minimal effects on lobster nerve cord cyclic GMP production, when applied either to intact tissue or to cytoplasmic extracts. Parallel immunocytochemical studies, using an anti-cyclic GMP antibody, reveal that only a small subset of lobster neurons responds to nitric oxide with a significant elevation of cyclic GMP levels. HPLC analysis of nerve cord cytoplasm reveals two chromatographically separable cyclases, a minor nitric oxide-sensitive form whose retention time is identical to that of the conventional mammalian enzyme and a more abundant nitric oxide-insensitive form that appears to be novel. The physiological function and phylogenetic distribution of this nitric oxide-insensitive enzyme, and the signaling mechanisms that regulate its activity, are not known.

Animals↗
PubMed2000

C-Terminus of desmoyokin/AHNAK protein is responsible for its translocation between the nucleus and cytoplasm.

We previously demonstrated that desmoyokin gene is identical to AHNAK gene, which is downregulated in neuroblastomas. Whereas desmoyokin/AHNAK protein is distributed in the nucleus and cytoplasm in nonepithelial tissues, it is distributed in the cell membrane in epithelial tissues. It is present diffusely in the cytoplasm and nucleus of epithelial cell lines cultured in low calcium condition. Low to normal calcium shift translocates it to the cell boundary. In this study, we investigated which domain(s) of desmoyokin/AHNAK protein are responsible for its different distribution. We constructed three different eukaryotic expression plasmids, pN-DY, pM-DY, and pC-DY, which expressed N-terminus, central domain, and C-terminus of this molecule, respectively, when transfected into COS-7 cells, normal human keratinocytes, and HeLa cells. In normal calcium medium, whereas N-terminus and central domain of desmoyokin/AHNAK protein were present mainly in the cytoplasm, C-terminus was present in the nucleus, cytoplasm, and weakly in the cell membrane. In low calcium medium, C-terminus was present exclusively in the nucleus, and a part of the molecules translocated from the nucleus to the cytoplasm, 3 h after the shift to normal calcium medium or 3 h after addition of protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate in low calcium medium. Calcium shift showed no effects on the distribution of N-terminus and central domain. These results suggested that C-terminus, but neither N-terminus nor central domain, is responsible for the translocation of this protein into the nucleus. This study may also suggest that C-terminus play a role in the translocation to the cell membrane, although further evidence is necessary.

Animals↗
PubMed2000

Calcium signalling in sarcoplasmic reticulum, cytoplasm and mitochondria during activation of rabbit aorta myocytes.

This study investigated the relationship between cytoplasmic, mitochondrial, and sarcoplasmic reticulum (SR) [Ca(2+)] in rabbit aorta smooth muscle cells, following cell activation. Smooth muscle cells were loaded with the Ca(2+)-sensitive fluorescent indicator Mag-Fura-2-AM, and then either permeabilized by exposure to saponin, or dialyzed with a patch pipette in the whole-cell configuration to remove cytoplasmic indicator. When the intracellular solution contained millimolar EGTA or BAPTA, activation of SR Ca(2+)release through IP(3)or ryanodine receptors induced a decrease in the [Ca(2+)] reported by Mag-Fura-2. However, when EGTA was present at < or =100 microM, the same stimuli caused an increase in the [Ca(2+)] reported by Mag-Fura-2. The increase in [Ca(2+)] caused by phenylephrine or caffeine was delayed, and prolonged, with respect to the cytoplasmic Ca(2+)transient. Evidence is presented that this Mag-Fura-2 signal reflected a rise in mitochondrial [Ca(2+)]. Agents that inhibit mitochondrial function, such as FCCP or cyanide in combination with oligomycin B, converted the increase in organelle Mag-Fura-2 fluorescence to a decrease, while also prolonging the cytoplasmic Ca(2+)transient. There was considerable similarity between the localization of Mag-Fura-2 fluorescence and the mitochondria-selective indicator tetramethylrhodamine ethyl ester. Thus, we propose that there is close functional integration between the SR and mitochondria in aorta smooth muscle cells, with mitochondria taking up Ca(2+)from the cytoplasm following cell activation.

Animals↗