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PubMed1998

Selenium deficiency reduces the abundance of mRNA for Se-dependent glutathione peroxidase 1 by a UGA-dependent mechanism likely to be nonsense codon-mediated decay of cytoplasmic mRNA.

The mammalian mRNA for selenium-dependent glutathione peroxidase 1 (Se-GPx1) contains a UGA codon that is recognized as a codon for the nonstandard amino acid selenocysteine (Sec). Inadequate concentrations of selenium (Se) result in a decrease in Se-GPx1 mRNA abundance by an uncharacterized mechanism that may be dependent on translation, independent of translation, or both. In this study, we have begun to elucidate this mechanism. We demonstrate using hepatocytes from rats fed either a Se-supplemented or Se-deficient diet for 9 to 13 weeks that Se deprivation results in an approximately 50-fold reduction in Se-GPx1 activity and an approximately 20-fold reduction in Se-GPx1 mRNA abundance. Reverse transcription-PCR analyses of nuclear and cytoplasmic fractions revealed that Se deprivation has no effect on the levels of either nuclear pre-mRNA or nuclear mRNA but reduces the level of cytoplasmic mRNA. The regulation of Se-GPx1 gene expression by Se was recapitulated in transient transfections of NIH 3T3 cells, and experiments were extended to examine the consequences of converting the Sec codon (TGA) to either a termination codon (TAA) or a cysteine codon (TGC). Regardless of the type of codon, an alteration in the Se concentration was of no consequence to the ratio of nuclear Se-GPx1 mRNA to nuclear Se-GPx1 pre-mRNA. The ratio of cytoplasmic Se-GPx1 mRNA to nuclear Se-GPx1 mRNA from the wild-type (TGA-containing) allele was reduced twofold when cells were deprived of Se for 48 h after transfection, which has been shown to be the extent of the reduction for the endogenous Se-GPx1 mRNA of cultured cells incubated as long as 20 days in Se-deficient medium. In contrast to the TGA allele, Se had no effect on expression of either the TAA allele or the TGC allele. Under Se-deficient conditions, the TAA and TGC alleles generated, respectively, 1.7-fold-less and 3-fold-more cytoplasmic Se-GPx1 mRNA relative to the amount of nuclear Se-GPx1 mRNA than the TGA allele. These results indicate that (i) under conditions of Se deprivation, the Sec codon reduces the abundance of cytoplasmic Se-GPx1 mRNA by a translation-dependent mechanism and (ii) there is no additional mechanism by which Se regulates Se-GPx1 mRNA production. These data suggest that the inefficient incorporation of Sec at the UGA codon during mRNA translation augments the nonsense-codon-mediated decay of cytoplasmic Se-GPx1 mRNA.

3T3 Cells↗
PubMed1998

Cytoplasmic tail regulates the intercellular adhesion function of the epithelial cell adhesion molecule.

Ep-CAM, an epithelium-specific cell-cell adhesion molecule (CAM) not structurally related to the major families of CAMs, contains a cytoplasmic domain of 26 amino acids. The chemical disruption of the actin microfilaments, but not of the microtubuli or intermediate filaments, affected the localization of Ep-CAM at the cell-cell boundaries, suggesting that the molecule interacts with the actin-based cytoskeleton. Mutated forms of Ep-CAM were generated with the cytoplasmic domain truncated at various lengths. All of the mutants were transported to the cell surface in the transfectants; however, the mutant lacking the complete cytoplasmic domain was not able to localize to the cell-cell boundaries, in contrast to mutants with partial deletions. Both the disruption of the actin microfilaments and a complete truncation of the cytoplasmic tail strongly affected the ability of Ep-CAM to mediate aggregation of L cells. The capability of direct aggregation was reduced for the partially truncated mutants but remained cytochalasin D sensitive. The tail truncation did not affect the ability of the transfectants to adhere to solid-phase-adsorbed Ep-CAM, suggesting that the ability to form stable adhesions and not the ligand specificity of the molecule was affected by the truncation. The formation of intercellular adhesions mediated by Ep-CAM induced a redistribution to the cell-cell boundaries of alpha-actinin, but not of vinculin, talin, filamin, spectrin, or catenins. Coprecipitation demonstrated direct association of Ep-CAM with alpha-actinin. Binding of alpha-actinin to purified mutated and wild-type Ep-CAMs and to peptides representing different domains of the cytoplasmic tail of Ep-CAM demonstrates two binding sites for alpha-actinin at positions 289 to 296 and 304 to 314 of the amino acid sequence. The results demonstrate that the cytoplasmic domain of Ep-CAM regulates the adhesion function of the molecule through interaction with the actin cytoskeleton via alpha-actinin.

Actinin↗
PubMed2002

Oxygen-dependent ubiquitination and degradation of hypoxia-inducible factor requires nuclear-cytoplasmic trafficking of the von Hippel-Lindau tumor suppressor protein.

It is becoming increasingly evident that the degradation of nuclear proteins requires nuclear-cytoplasmic trafficking of both the substrate proteins, as well as the E3 ubiquitin-ligases. Here, we show that nuclear-cytoplasmic trafficking of the von Hippel-Lindau tumor suppressor protein (VHL) is required for oxygen-dependent ubiquitination and degradation of the alpha subunits of hypoxia-inducible factor (HIF-alpha). VHL engages in a constitutive transcription-sensitive nuclear-cytoplasmic shuttle unaffected by oxygen tension or levels of nuclear substrate HIF-alpha. Ubiquitinated forms of HIF-alpha, as well as VHL/ubiquitinated HIF-alpha complexes, are found solely in the nuclear compartment of normoxic or reoxygenated VHL-competent cells. HIF-alpha localizes exclusively in the nucleus of hypoxic cells but is exported to the cytoplasm upon reoxygenation. Oxygen-dependent nuclear ubiquitination and nuclear export of HIF-alpha can be prevented by treatment with an HIF-specific prolyl hydroxylase inhibitor. Treatment with inhibitors of RNA polymerase II activity, which interfere with the ability of VHL to engage in nuclear export, also prevents cytoplasmic accumulation of HIF-alpha in reoxygenated cells. This caused a marked increase in the HIF-alpha half-life without affecting its nuclear ubiquitination. We present a model by which VHL-mediated ubiquitination of HIF-alpha and its subsequent degradation are dependent upon dynamic nuclear-cytoplasmic trafficking of both the E3 ubiquitin-ligase and the nuclear substrate protein.

Active Transport, Cell Nucleus↗
PubMed2004

Brap2 functions as a cytoplasmic retention protein for p21 during monocyte differentiation.

The cell cycle inhibitor p21 plays an important role in monocytic cell differentiation, during which it translocates from the nucleus to cytoplasm. This process involves the negative regulation of the p21 nuclear localization signal (NLS). Here, we sought to determine the relationship between the cytoplasmic translocation of p21 and another molecule, Brap2, a cytoplasmic protein which binds the NLS of BRCA1 and was recently reported to inactivate KSR in the Ras-activating signal pathway under the name of IMP. We report that p21 and Brap2 directly interact, both in vitro and in vivo, in a manner requiring the NLS of p21 and the C-terminal portion of Brap2. When it is cotransfected with Brap2, p21 is expressed in the cytoplasm. Monocytic differentiation of the promyelomonocytic cell lines U937 and HL60 is associated with the upregulation of Brap2 expression concomitantly with the upregulation and cytoplasmic relocalization of p21. Our results underscore the role played by Brap2 in the process of cytoplasmic translocation of p21 during monocyte differentiation.

Apoptosis↗
PubMed1987

Effect of secretagogues on cytoplasmic free calcium in alveolar type II epithelial cells.

Pulmonary surfactant is synthesized and secreted by alveolar type II epithelial cells. Although intracellular calcium and other second messengers have been implicated in secretion by type II cells, this is the first report on measurement of cytoplasmic free calcium concentration ([Ca2+]i). Known secretagogues, 12-O-tetradecanoylphorbol-13-acetate (TPA) and terbutaline, were tested to see if they caused rapid increases in cytoplasmic calcium. Ionomycin, a calcium ionophore, was used to increase cytoplasmic free calcium concentration, to determine if a rapid increase in cytoplasmic free calcium would stimulate secretion, and to measure interactions with other secretagogues. Ionomycin increased both [Ca2+]i and pulmonary surfactant secretion from alveolar type II cells. A low concentration of ionomycin (100 nM) greatly enhanced secretion stimulated by terbutaline or by 8-bromo-cAMP but only had an additive effect on secretion stimulated by TPA. Terbutaline transiently increased [Ca2+]i by 24% over control basal condition, and the increase in [Ca2+]i produced by terbutaline occurred in the absence of extracellular calcium. TPA itself did not change [Ca2+]i. However, TPA completely inhibited the terbutaline-induced increase of [Ca2+]i but not the increase due to ionomycin. When alveolar type II cells were loaded with 2-(2-bis-[carboxymethyl]-amino-5-methyl-phenoxy)-methyl-6-methoxy-8-bis carboxymethylaminoquinoline (quin2) in calcium-free buffer, [Ca2+]i decreased from 143 +/- 10 to 31 +/- 8 nM. Lowering [Ca2+]i inhibited TPA- or terbutaline-induced secretion by 22 and 40%, respectively. Although the precise role of cytoplasmic free calcium on surfactant secretion cannot be established on the basis of current data, our results indicate that an increase in cytoplasmic free calcium produced by ionomycin stimulates secretion and that an increase in [Ca2+]i affects cAMP-induced secretion more than protein kinase C-mediated secretion in alveolar type II cells.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1997

Cytoplasmic codiffusion of fatty acids is not specific for fatty acid binding protein.

The intracellular movement of fatty acids is thought to be facilitated through codiffusion with fatty acid binding protein (FABP). Previous work suggested that FABP decreases fatty acid binding to immobile membranes, causing faster cytoplasmic diffusion. However, the specificity for binding to FABP has not been addressed. The aim of the current study was to determine whether specific FABP binding is required or whether binding to other proteins will produce the same effect. A model cytoplasm consisted of a fatty acid, proteins, and liposomes to simulate intracellular membranes. Laser photobleaching (fluorescence recovery after photobleaching) was used to measure the movement of the fluorescent fatty acid 12-N-methyl-7-nitrobenzo-2-oxa-1,3-diazoaminostearate (NBD-stearate) in model cytoplasm, in normal and permeabilized Hep G2 cells, and after incubation of permeabilized cells with bovine serum albumin (BSA) or FABP. Increasing protein in the model cytoplasm increased the diffusion rate in proportion to the extent of protein binding. Cell permeabilization reduced diffusion of NBD-stearate to < 5% of controls. Incubation of permeabilized cells with FABP or BSA resulted in a concentration-dependent increase in the NBD-stearate diffusion rate. BSA was more effective than FABP in binding NBD-stearate and increasing its diffusion rate after permeabilization. Proteins like FABP promote the diffusion of fatty acids. Removal of these proteins drastically reduces cytoplasmic diffusion. Substitution with BSA reestablishes the diffusive flux, suggesting that specific binding to FABP is not required. These data support a role for intracellular binding proteins in facilitating the cytoplasmic movement of fatty acids.

4-Chloro-7-nitrobenzofurazan↗
PubMed2000

Agonist-induced cytoplasmic volume changes in cultured rabbit parietal cells.

Concomitant Na(+)/H(+) and Cl(-)/HCO(3)(-) exchange activation occurs during stimulation of acid secretion in cultured rabbit parietal cells, possibly related to a necessity for volume regulation during the secretory process. We investigated whether cytoplasmic volume changes occur during secretagogue stimulation of cultured rabbit parietal cells. Cells were loaded with the fluorescent dye calcein, and the calcein concentration within a defined cytoplasmic volume was recorded by confocal microscopy. Forskolin at 10(-5) M, carbachol at 10(-4) M, and hyperosmolarity (400 mosmol) resulted in a rapid increase in the cytoplasmic dye concentration by 21 +/- 6, 9 +/- 4, and 23 +/- 5%, respectively, indicative of cell shrinkage, followed by recovery to baseline within several minutes, indicative of regulatory volume increase (RVI). Depolarization by 5 mM barium resulted in a decrease of the cytoplasmic dye concentration by 10 +/- 2%, indicative of cell swelling, with recovery within 15 min, and completely prevented forskolin- or carbachol-induced cytoplasmic shrinkage. Na(+)/H(+) exchange inhibitors slightly reduced the initial cell shrinkage and significantly slowed the RVI, whereas 100 microM bumetanide had no significant effect on either parameter. We conclude that acid secretagoguges induce a rapid loss of parietal cell cytoplasmic volume, followed by RVI, which is predominantly mediated by Na(+)/H(+) and Cl(-)/HCO(3)(-) exchange.

Amiloride↗
PubMed1986

Cytoplasmic calcium in individual proximal tubular cells in culture.

The development of high quantal yield Ca2+-sensitive fluorescent indicators has made microspectrofluorometric monitoring of cytoplasmic calcium feasible. The detailed technique to monitor cytoplasmic calcium concentration in individual proximal tubular cells grown on glass cover slips is described. Manipulations of cytoplasmic calcium concentration by means of a Ca2+ ionophore or Ca2+-free medium resulted in corresponding changes of fura-2 fluorescence. Parathyroid hormone elicited a fivefold increase in cytoplasmic calcium concentration, with the subsequent complete recovery of this parameter in 3 min. This effect of parathyroid hormone was abolished by perfusion of the cells with Ca2+-free medium. Repeated pulses of parathyroid hormone spaced at an interval of 20 min, caused refractoriness of adenosine 3',5'-cyclic monophosphate response, whereas cytoplasmic calcium transients remained unaltered. When the frequency of the sequential pulses with parathyroid hormone was increased (5 min intervals), the amplitude of calcium transients was diminished, and the recovery of basal level of cytoplasmic calcium was incomplete and protracted. These observations may have application to disordered renal cell calcium metabolism in hyperparathyroid states.

Animals↗
PubMed2004

Cellular dynamics visualized in live cells in vitro and in vivo by differential dual-color nuclear-cytoplasmic fluorescent-protein expression.

We report here the genetic engineering of dual-color fluorescent cells with one color in the nucleus and the other in the cytoplasm that enables real-time nuclear-cytoplasmic dynamics to be visualized in living cells in vivo as well as in vitro. To obtain the dual-color cells, red fluorescent protein (RFP) was expressed in the cytoplasm of HT-1080 human fibrosarcoma cells, and green fluorescent protein (GFP) linked to histone H2B was expressed in the nucleus. Nuclear GFP expression enabled visualization of nuclear dynamics, whereas simultaneous cytoplasmic RFP expression enabled visualization of nuclear cytoplasmic ratios as well as simultaneous cell and nuclear shape changes. Thus, total cellular dynamics can be visualized in the living dual-color cells in real time. The parental HT-1080 and the derived dual-color clones had similar cell proliferation rates, suggesting that expression of GFP and/or RFP does not affect cell cycle progression. The cell cycle position of individual living cells was readily visualized by the nuclear-cytoplasmic ratio and nuclear morphology. Real-time induction of apoptosis was observed by nuclear size changes and progressive nuclear fragmentation. Mitotic cells were visualized by whole-body imaging after injection in the mouse ear. Common carotid artery injection of dual-color cells and a reversible skin flap enabled the external visualization of the dual-color cells in microvessels in the mouse brain where extreme elongation of the cell body as well as the nucleus occurred. Dual-color cells in various positions of the cell cycle were visualized in excised mouse lungs after tail-vein injection of the dual-color cells. In the lung, the dual-color cells were observed frequently juxtaposing their nuclei, suggesting a potential novel form of cell-cell communication. The dual-color cells thus are a useful tool for visualizing living-cell dynamics in vivo as well as in vitro. Drugs that could specifically perturb these processes can now be readily screened in real time in vivo.

Animals↗
PubMed1997

Cytoplasmic localization of endothelial constitutive nitric oxide synthase in endometrial carcinomas.

BACKGROUND: Production of nitric oxide by nitric oxide synthase (NOS) has been implicated in numerous physiologic and pathophysiologic processes including mutagenesis. This study was designed to examine the expression of the endothelial constitutive isoform of NOS (ecNOS) in endometrial carcinomas. METHODS: Fifty endometrial carcinomas (42 endometrioid, 4 serous papillary, 2 clear cell, and 2 adenosquamous carcinomas) and 21 normal endometrial gland tissue specimens (5 cases of proliferative, 5 early secretory, 5 mid-secretory, and 5 late secretory and 1 menstrual phase endometrium), previously formalin fixed and paraffin embedded, were immunostained using a commercially available anti-ecNOS monoclonal antibody. Localization of ecNOS staining to the plasma membrane, cytoplasm and nuclei was graded with respect to overall staining intensity (0-3+ scale) and frequency (percentage of immunoreactive cells). RESULTS: Relatively little staining for ecNOS was localized to the plasma membrane in either normal or neoplastic tissues. Normal and hyperplastic endometrial glands demonstrated moderate cytoplasmic and weak nuclear staining in a small percentage of cells. While ecNOS expression was most prominent in epithelial cells, weak expression was also rarely noted in endometrial stroma, blood vessel walls, and endothelium. We found a broad range of ecNOS expression in endometrial carcinomas, predominantly localized to the cytoplasm and nuclei. No statistically significant difference in ecNOS staining frequency or intensity was found between different histologic subtypes of endometrial carcinomas. No apparent correlation was found between ecNOS expression and tumor stage, grade, extension to the lower uterine segment or cervix, nodal or distant metastases, recurrence, or final patient status among patients with endometrioid adenocarcinomas. Endometrioid tumors invading more than 1/2 of myometrial thickness (n = 18) had significantly higher cytoplasmic staining intensity than those tumors limited to the inner 1/2 of myometrium (n = 27; 2.0 vs. 1.3, p < 0.04). Furthermore, a trend toward shorter disease-free survival was noted with increased staining intensity and decreased staining frequency. CONCLUSIONS: Cytoplasmic and nuclear expression of ecNOS, which is primarily limited to the glandular elements of normal endometrium, is also found to be expressed in endometrial carcinoma. Increased ecNOS staining intensity and decreased frequency tends to correlate with decreased disease-free survival. Lastly, increased cytoplasmic ecNOS staining intensity correlates with increased myometrial invasion.

Adult↗
PubMed1992

Influence of exogenous factors and antibiotics on the cytoplasmic membrane proteins of Staphylococcus aureus.

The influence of various nutrient media, growth temperature, phase of growth and subinhibitory concentrations of antibiotics from various groups on the cytoplasmic membrane proteins was studied in 6 clinical S. aureus isolates; further study included the comparison of the cytoplasmic membrane proteins in antibiotic-sensitive wild-type (parent) strains with their resistant counterparts. Resistant clones could be selected with a frequency ranging from 10(-7) to 10(-8) from the parent strain at twice the MIC. The following was observed from the study of six clinical isolates: (a) there was no uniform pattern of cytoplasmic membrane proteins among the isolates; (b) during the phase of growth, the number of high-molecular-weight proteins showed a decrease according to the duration of growth; (c) the addition of 1% w/v glucose to the growth medium resulted in the loss of an approximately 75 kD protein in five of the six strains; (d) subinhibitory concentrations of various antibiotics (1/4 of the MIC) resulted in major changes of cytoplasmic membrane proteins, when the strains were exposed to oxacillin, whereas this was not the case for erythromycin, rifampicin, gentamicin, trimethoprim, fleroxacin and vancomycin; (e) the resistant clones differed from their sensitive parent strains in their cytoplasmic membrane protein patterns. The alterations were not characteristic either for the strain or for the antibiotic considered, thus suggesting an 'unspecific' defense mechanism characterized by individual changes of the cytoplasmic membrane proteins.

Anti-Bacterial Agents↗
PubMed1985

Discordance between surface and cytoplasmic expression of the Leu-4 (T3) antigen in thymocytes and in blast cells from childhood T lymphoblastic malignancies.

We have examined the expression of the Leu-4 (T3) antigen on the cell surface and in the cytoplasm of blast cells from 23 patients with T cell acute lymphoblastic leukemia and T cell lymphoblastic lymphoma. In the majority of cases (17), the Leu-4 antigen was absent from the cell surface; however, in 16 of these 17 cases, blast cells demonstrated cytoplasmic expression of Leu-4. This discordance between surface and cytoplasmic expression of Leu-4 was also found in thymocytes and appeared to be restricted to Leu-4, in that tests of other T cell antigens rarely revealed discordance between surface and cytoplasmic expression. To study further the cytoplasmic determinant identified by anti-Leu-4 in malignant T lymphoblasts, immunoprecipitation studies were performed that utilized biosynthetic labeling of established T cell lines derived from T lymphoblastic malignancies. By one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, identical Leu-4 polypeptide families were immunoprecipitated from surface Leu-4+ and surface Leu-4-/cytoplasmic Leu-4+ cell lines. Because T lymphoblastic malignancies represent proliferations of immature T cells, and because the cases studied demonstrated surface phenotypes corresponding to all of the proposed stages of T cell ontogeny, it appears that cytoplasmic expression of Leu-4 occurs early in T cell development. The reason for the failure of these immature T cells to transport the Leu-4 molecule to their surface remains to be elucidated.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1986

Relationship of superoxide production to cytoplasmic free calcium in human monocytes.

Calcium has been proposed as an intracellular second messenger for activation of secretion, phagocytosis, and the oxidative burst of neutrophils. We have examined the role of calcium in human monocyte activation. Concanavalin A (Con A)-stimulated monocytes displayed an increment in cytoplasmic ionized calcium at 31 +/- 6 s and the onset of superoxide production at 61 +/- 9 s. The increase in cytoplasmic calcium invariably preceded the onset of superoxide production. If the external calcium concentration was reduced to less than 28 nM by the addition of 10 mM EGTA, superoxide production was not diminished at 5 min; however, superoxide production decreased thereafter. The Con A-evoked increment in cytoplasmic ionized calcium was blunted upon the addition of EGTA and decreased further with time. Both the production of superoxide and the Con A-evoked increment in cytoplasmic ionized calcium displayed a 50% inhibition after 15 min of calcium depletion and were completely inhibited after 60 min. Total cell calcium fell from 0.7 to 0.5 fmol/cell, and the basal level of ionized calcium fell from 83 to 30 nM after 60 min. Histidine, a strong chelator of divalent cations other than calcium and magnesium, had no effect on monocyte superoxide production or on ionized calcium concentrations, indicating that EGTA inhibition was due to cell calcium depletion. In calcium-depleted cells, Con A did not evoke superoxide production until calcium was restored to the incubation medium. The restoration of calcium to Con A-treated, calcium-depleted monocytes permitted a rapid rise in the cytoplasmic ionized calcium, and the production of superoxide within 9 s. These data suggest that an increase in ionized cytoplasmic calcium is necessary for the activation of monocyte superoxide production by Con A. The rise in ionized calcium in response to Con A results, in part, from an internal redistribution of calcium, which is sufficient to permit superoxide generation.

Aminoquinolines↗
PubMed1989

Regulation of the cytoplasmic accumulation of 5-methyltetrahydrofolate in MA104 cells is independent of folate receptor regulation.

To better understand how the folate receptor (also known as the membrane folate binder) is able to deliver 5-methyltetrahydrofolic acid to the cytoplasm of folate-depleted MA104 cells, we have examined the kinetics of movement from the cell surface into the cytoplasm. Bound 5-methyltetrahydrofolic acid was transferred into an acid-resistant membrane compartment at the rate of 0.9-1.0 pmol/10(6) cells per h. This folate appeared in the cytoplasm at the same rate. Furthermore, cytoplasmic 5-methyltetrahydrofolic acid became polyglutamated at the rate of 0.6-0.7 pmol/10(6) cells per h. As soon as intracellular 5-methyltetrahydrofolate reached 5-7 pmol/10(6) cells, however, cytoplasmic accumulation was markedly inhibited even though the folate receptor remained functional. Therefore, the acute regulation of 5-methyltetrahydrofolic acid accumulation appears to be achieved by controlling the movement of the vitamin from the receptor into the cytoplasm of the cell.

Animals↗
PubMed1983

Immunological characterization of an anti-actin antibody specific for cytoplasmic actins and its use for the immunocytological localization of actin in Aplysia nervous tissue.

Previous immunochemical and immunocytochemical studies have shown that an antibody to actin prepared from body wall muscle of the marine mollusc Aplysia californica is specific for vertebrate cytoplasmic actins. The ability of this anti-actin to distinguish between different forms of actin most likely reflects the recognition of amino acid sequences unique to cytoplasmic actins. We have confirmed the specificity of this antibody for cytoplasmic actins using nervous tissue as a source of cytoplasmic actin in further immunochemical studies. In addition to binding cytoplasmic actin in purified preparations, the antibody removed actin selectively from crude extracts of nervous tissue of some but not all of the species tested. Our results also suggest that tissue-specific differences in the distribution of cytoplasmic actins may exist. Immunofluorescence studies of Aplysia nervous tissue stained with anti-actin revealed that actin is present in the cell body and axonal processes of Aplysia neurons. Although the function of actin in nerve cells is not understood, the observed pattern of immunofluorescence staining is consistent with the idea that actin may be involved in movement within the axoplasm.

Actins↗
PubMed2003

Filamin A binding to the cytoplasmic tail of glycoprotein Ibalpha regulates von Willebrand factor-induced platelet activation.

We examined the hypothesis that filamin A binding to the cytoplasmic tail of platelet glycoprotein Ibalpha (GpIbalpha) is regulated by pathologic shear stress and modulates von Willebrand factor (VWF)-induced platelet activation. To begin, we examined filamin binding to GpIbalpha in Chinese hamster ovary cells coexpressing mutant human GpIb-IX and wild-type human filamin A. We observed that many different deletions and truncations N-terminal to GpIbalpha's cytoplasmic domain residue 594 disrupted filamin A binding, but that binding was unaffected by 14 different point mutations in hydrophilic residues between amino acids 557 and 593. To try to narrow GpIbalpha's filamin A-binding domain, we next measured the effect of several cytoplasmic domain peptides on human filamin A binding to a GST-GpIbalpha cytoplasmic domain fusion protein. One peptide (residues 557-575; designated "A4 peptide") inhibited filamin A binding to the GST-GpIbalpha cytoplasmic domain fusion protein and competed with GpIbalpha for binding to filamin A. When the A4 peptide was delivered to intact human platelets using a carrier peptide, we observed the dose-dependent inhibition of VWF-induced platelet aggregation in response to both ristocetin and shear stress. The effect of the A4 peptide on shear-induced platelet aggregation was accompanied by the attenuation of shear-induced filamin A binding to GpIbalpha and diminished shear-dependent protein tyrosine phosphorylation. These results suggest that shear-dependent VWF-induced platelet activation affects filamin A binding to GpIb-IX-V, and that filamin A binding to the cytoplasmic tail of GpIbalpha regulates proaggregatory tyrosine kinase signaling.

Amino Acid Sequence↗
PubMed2004

Transforming growth factor-beta induces Cdk2 relocalization to the cytoplasm coincident with dephosphorylation of retinoblastoma tumor suppressor protein.

BACKGROUND: The transforming growth factor-beta (TGF-beta) signaling pathway functions to prevent tumorigenesis, and loss of sensitivity to TGF-beta-mediated cell cycle arrest is nearly ubiquitous among human cancers. Our previous studies demonstrated that rapamycin potentiates TGF-beta-induced cell cycle arrest in nontransformed epithelial cells and partially restores TGF-beta-induced growth arrest of some human cancer cell lines. Growth arrest correlated with increased binding of p21 and p27 to cyclin-dependent kinase-2 (Cdk2), and inhibition of Cdk2 kinase activity. However, it was unclear how TGF-beta caused increased binding of p21 and p27 to Cdk2. METHODS: Cell fractionation and immunofluorescence microscopy experiments were performed to examine the effect of TGF-beta on the intracellular localization of Cdk2, p21, and p27. Kinase assays were performed on cytoplasmic and nuclear extracts to determine how TGF-beta altered Cdk2 activity in both subcellular compartments. RESULTS: In breast epithelial cells treatment with TGF-beta induced a decrease in nuclear Cdk2 concentrations and relocalization of Cdk2 to the cytoplasm. Cdk2 relocalization to the cytoplasm correlated with dephosphorylation of nuclear retinoblastoma tumor suppressor protein and decreased nuclear Cdk2 activity. In these epithelial cell lines, p21 and p27 were localized primarily in the cytoplasm. Decreases in nuclear Cdk2 concentrations correlated with increased binding of Cdk2 to cytoplasmic p21 and p27. CONCLUSION: Cooperative growth arrest induced by treatment with TGF-beta + rapamycin causes inhibition of nuclear Cdk2 activity through multiple mechanisms, including Cdk2 relocalization to the cytoplasm, increased p27 and p21 binding to Cdk2, and increased phosphorylation of nuclear Cdk2 on its inhibitory site, Tyr15.

Active Transport, Cell Nucleus↗
PubMed1979

Properties of nuclear and cytoplasmic estrogen receptor in the rabbit corpus luteum: evidence for translocation.

Nuclear and cytoplasmic estrogen receptors have been identified and characterized in the rabbit corpus luteum, and validated methods are described for the measurement of both unoccupied and total estrogen receptor. Binding was specific for the biologically active estrogens. Equilibrium binding analysis of cytoplasmic estrogen receptor yielded saturable, high affinity binding sites with a Kd of 5.4 x 10(-11) M. Two types of binding sites were found in the nuclear fraction: one with high affinity (Kd = 8.9 x 10(-11) M) and low capacity, the other with low affinity (Kd = 2.7 x 10(-8) M) and high capacity. In the presence of 0.4 M KCl, the sedimentation coefficients of nuclear and cytoplasmic estrogen receptors are 3.4S, while the value is 6.8S for cytoplasmic receptor in buffer without KCl. Twenty minutes after an iv injection of 2 micrograms 17 beta-estradiol, the available and total cytoplasmic estrogen receptors were depleted. This depletion was accompanied by concomitant and stoichiometric accumulation of receptor in the nucleus, indicating an apparent translocation of the receptor. In corpora lutea of normal rabbits, approximately 80% of the total nuclear receptor is unoccupied. Evidence is presented to suggest that nuclear receptor sites might be ordinarily occupied with estradiol, but during isolation of the nuclear fraction these sites become available or unoccupied. The identification of nuclear estrogen receptor and the phenomenon of translocation of cytoplasmic receptor to the nucleus suggest a similarity of estrogen action in the rabbit corpus luteum and other estrogen target tissues.

Animals↗