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At least 883 records · Page 49Linked to original sources
PubMed1997

Immunolocalization of cytoplasmic and myelin mcalpain in transfected Schwann cells: II. Effect of withdrawal of growth factors.

We have examined the reversal of the regulatory effect of growth factors on calpain/calpastatin activity in transfected Schwann cells (tSc) after their subsequent withdrawal. Removal of nerve growth factor (NGF) or cyclic adenosine monophosphate (cAMP) from tSc resulted in a smaller loss of mu calpain (37%) and mcalpain (36.5 %) activity compared to treated cells from which the growth factors were not withdrawn. The mu calpain activity increased approximately 12% following withdrawal of acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) at 24 hr, while the increased mcalpain activity was more than 30-40% compared with that of cells that were continuously treated. The activity of both isoforms returned to their normal levels (untreated) at 48-72 hr following withdrawal of various growth factors, including NGF, cAMP, aFGF, bFGF, platelet-derived growth factor aa (PDGFaa), and PDGFbb. The inhibitory activity of calpastatin was greater than control following withdrawal of NGF, cAMP, PDGFaa, or PDGFbb at 24 hr and this inhibitory activity was less with treatment by aFGF and bFGF. The control activity was restored at 48 hr following withdrawal of these factors. The intensity of the cytoplasmic calpain immunoreactivity was significantly decreased in the nuclear and non-nuclear regions of the cytoplasm, respectively, following withdrawal of cAMP at 144 hr. Removal of bFGF from the medium resulted in an increase of cytoplasmic calpain immunoreactivity in the nuclear regions and cytoplasm, while there was dramatic loss of myelin calpain immunoreactivity from both the nuclear region and cytoplasm. The changes in calpain activity and immunoreactivity in tSc following withdrawal of growth factors suggest that release of calpain from membrane to cytosol may be regulated by these factors.

Animals↗
PubMed2000

Frequent nuclear localization of ICAD and cytoplasmic co-expression of caspase-8 and caspase-3 in human lymphomas.

Lymphoma cells often display in vitro resistance to FAS-induced apoptosis, in which caspases act as crucial cell death effectors. Following FAS stimulation, caspase-8 activates caspase-3, which in turn activates the caspase-activated DNAse (CAD) by proteolysis of its inhibitor (ICAD). To investigate the mechanism of FAS resistance, the expression of caspase-8 was analysed by immunohistochemistry, together with that of the substrates caspase-3 and ICAD, in 52 representative samples from non Hodgkin's lymphoma (NHL), 12 from Hodgkin's disease (HD), and eight benign lymphoid tissues. In benign tissues, caspase-8 was co-expressed with caspase-3 in the cytoplasm in germinal centre (GC) cells and was co-expressed with ICAD in the nuclei of the mantle and marginal zone cells. ICAD expression was weak or absent in GC cells. Cytoplasmic staining for both caspase-8 and caspase-3 was present in 11/12 cases of diffuse large cell B-NHL. Caspase-8 positivity was nuclear and cytoplasmic in 9/9 follicular NHLs, in 5/5 mantle cell NHLs and in 6/6 marginal zone NHLs. Five out of six peripheral T-cell NHLs expressed cytoplasmic caspase-8. Ten out of the 12 HD cases lacked significant cytoplasmic staining for caspase-3 and caspase-8 in the majority of Reed-Sternberg cells. All lymphoma cases exhibited predominant nuclear ICAD positivity. Subcellular fractionation analysis of three lymphoma samples and normal mantle zone cells confirmed that ICAD and caspase-8 were at least partly localized in the nucleus. These results show that the profile of caspase-8 expression is correlated with histological lymphoma subtypes; that caspase-8 is co-expressed with caspase-3 in GC cells and their neoplastic counterparts; that ICAD has an immunohistochemical nuclear localization in vivo; and that caspase-8 and ICAD can be co-expressed in the nuclei of mantle zone and marginal zone cells; their unexpected nuclear localization allows a reappraisal of the biochemical cascade of caspase activation.

Apoptosis Regulatory Proteins↗
PubMed1975

Cortical cytoplasm and configurational changes of RGC-6 cells exposed to cytochalasin B.

RGC-6 cells, grown to confluency in monolayer cultures, are typically fusiform. In sections of these cells, fixed in situ and examined in the electron microscope, a discontinuous zone of cortical cytoplasm (5-4 nm wide), was found subjacent to the plasma membrane of the free surface. It was composed largely of 5-7 nm microfilaments distributed in an apparently irregular network and appeared to comprise a discrete zone separating the plasma membrane from the underlying endoplasm. Following incubation with cytodhalasin B (5 mug/ml), the configuration of the cells changed markedly. The perikaryal cytoplasm appeared to swell, while the cellular processes became attenuated and often terminated in flower-like tips. Although more discontinuous and somewhat less dense, the fine structure of the cortical cytoplasm appeared little affected by incubation with cytochalasin B for one hour. By 48 hours no zone of cortical cytoplasm was found. Subplasmalemmal microfilaments were no longer identifiable and endoplasmic organelles were found in direct contact with the plasma membrane of the free surface, which appeared to have collapsed onto the underlying endoplasm. The change in cell configuration resulting from exposure to cytochalasin B occurred at 37 degrees C but not at 4 degrees C, suggesting a dependency on metabolic activity. It did not, however, appeared related directly to protein synthesis. Whereas incorporation of 3H-leucine was decreased to 46.9% and 62.9% of control values after one hour or two hours exposure to cytochalasin B, respectively, no change was found after 24 or 48 hours. It is suggested that the effect of cytochalasin B on cell form, which appeared not to be a direct effect on either protein synthesis or subplasmalemmal microfilaments, may represent a primary response of the cortical cytoplasmic matrix.

Animals↗
PubMed2002

Accumulation of 14-3-3 proteins in glial cytoplasmic inclusions in multiple system atrophy.

Glial cytoplasmic inclusions are the pathological hallmark of multiple system atrophy. However, the molecular mechanisms underlying the formation of glial cytoplasmic inclusions remain unclear. Alpha-synuclein, a major component of glial cytoplasmic inclusions, has the ability to interact with 14-3-3 proteins, which mediate several types of signal transduction pathways. To elucidate the role of these 14-3-3 proteins in patients with multiple system atrophy, we performed immunohistochemical studies on 14-3-3 in brain tissue specimens from 7 control subjects and from 15 patients with multiple system atrophy. In both control and multiple system atrophy cases, 14-3-3 immunoreactivity was observed mainly in the neuronal somata and proximal processes, as well as the nerve fibers. Even in the severely affected regions of patients with multiple system atrophy, 14-3-3 immunoreactivity generally was spared in the surviving neurons, some of which were strongly immunolabeled. In addition, numerous glial cytoplasmic inclusions were intensely immunostained, and neuronal cytoplasmic inclusions and dystrophic neurites were also immunoreactive for 14-3-3. Our results suggest that an aberrant accumulation of 14-3-3 proteins may occur in brains affected by multiple system atrophy, and that 14-3-3 proteins may be associated with the pathogenesis of multiple system atrophy.

14-3-3 Proteins↗
PubMed1982

Evolution of Sertoli cell processes invading the cytoplasm of rat spermatids.

Sertoli cell processes invaginating the cytoplasm of rat spermatids were investigated in thin or thick sections stained respectively with ferrocyanide-reduced osmium (Karnovsky, 1971) and the Ur-Pb-Cu metallic impregnation method (Thiéry and Rambourg, 1976). Two types of Sertoli cell processes were observed: Some, seen in steps 11-15 of spermiogenesis, were associated with a tight network of ER cisternae located within the spermatid cytoplasm subjacent to the plasma membrane facing the Seertoli cell process; others, seen from step 11 until the end of spermiogenesis, were not associated with such a spermatid subsurface ER network. Following their formation, the ER-associated Sertoli cell processes were cleaved into double-walled vesicles. Concomitantly, the enclosed Sertoli cell cytoplasm underwent autolysis followed by the dissolution of the Sertoli cell plasma membrane leaving within the spermatid cytoplasm many empty-looking vacuoles delimited only by the plasma membrane of the spermatid. Thereafter, i.e., in steps 15 and 16 of spermiogenesis, these vacuoles clustered, and with the collapse of the vacuoles they gave rise to a single compact mass of interlaced ER cisternae and small vacuoles. With the dissolution of ER cisternae, in step 18, this mass was finally dispersed. These observations suggested that the ER-associated Sertoli cell processes may constitute a pathway for the delivery of material from the Sertoli cells into spermatids. In contrast, the Sertoli cell processes not associated with a network of ER cisternae did not show cleavage or autolysis and appear to be involved mainly in holding the cytoplasm of the spermatid in place.

Acrosome↗
PubMedDate not supplied

Cytoplasmic factors that affect the intensity and stability of bioluminescence from firefly luciferase in living mammalian cells.

In order to improve calibration of firefly luciferase signals obtained by injecting the enzyme into single, isolated heart and liver cells we have investigated why the luminescence from cells is greatly depressed compared with in vitro (in mammalian ionic milieu) and why the decay of the intracellular signal is remarkably slow. We have shown that inorganic pyrophosphatase greatly depresses the signal in vitro and that micromolar concentrations of inorganic pyrophosphate, comparable with that in cytoplasm, reverse this inhibition and stabilize the signal, eliminating its decay. Higher concentrations of pyrophosphate depress the signal by inhibiting ATP-binding to luciferase. Luciferase-injected cells exposed to extracellular luciferin concentrations above about 100 mumol/l (corresponding to a cytoplasmic level of c. 5-10 mumol/l because of a transplasmalemmal gradient) show a gradual, irreversible loss of signal. We attribute this phenomenon (which is not seen in vitro) to the gradual accumulation of a luminescently inactive, irreversible, luciferase-oxyluciferin complex. At low luciferin levels this complex is prevented from forming by cytoplasmic pyrophosphate. Above c. 100 mumol/l extracellular luciferin, the pyrophosphate level in the cytoplasm fails to fully prevent the complex forming. In vitro this phenomenon does not occur because the luciferase concentrations and hence oxyluciferin levels are orders of magnitude lower than in cells injected with concentrated luciferase solutions, which have a cytoplasmic luciferase concentration of approximately 2-4 mumol/l.

Animals↗
PubMed1988

Subcellular compartmentalization by local differentiation of cytoplasmic structure.

The compartmentalization of eukaryotic cells by internal membranes and the subcellular localization of endogenous macromolecules by specific binding mechanisms are familiar concepts. In this report we present evidence that the cytoplasmic ground substance, which surrounds and contains the membrane-bound compartments, may also be compartmentalized by local differentiations of its submicroscopic structure that sort subcellular particles on the basis of size. The subcellular distribution of size-fractionated, fluorescent tracer particles was studied in living cells by ratio imaging and fluorescence recovery after photobleaching (FRAP). Large and small particles showed different distributions within the cytoplasmic volume, suggesting that the large particles were relatively excluded from some domains. While the structural basis for this phenomenon is not yet understood in detail, ratio imaging of large and small particles can be used as an empirical tool to identify cytoplasmic compartments for further study. The cytoplasmic diffusion coefficient (Dcyto) and % mobile fraction of the large particles showed considerable spatial variation over the projected area of the cell, while Dcyto and % mobile fraction of the small particles did not. A model is presented to account for this difference. Based on this model, a method is proposed by which FRAP can be used to detect sol-gel transitions in the cytoplasmic ground substance of living cells.

Animals↗
PubMed1979

Membrane specializations and cytoplasmic channels of Schwann cells in mammalian peripheral nerve as seen in freeze-fracture replicas.

Mammalian Schwann cells in rat, rabbit and human fetal nerves were studied using several cryoprotective agents for electron microscopic study of freeze-fracture replicas. The findings in fixed and unfixed tissue reveal surface plasmalemma caveolar specializations and the outer layer membrane junctional complexes found in non-mammalian species. The plasmalemma also reveals a complex arrangement of contours outlining cytoplasmic channel networks distinct from the long-recognized Schmidt-Lanterman incisures and paranodal cytoplasmic loops. A specialized interconnected channel system in the outer "loose" myelin layer displays relatively uniform dimensions comparable in diameter to nodal microvilli, paranodal loops and some incisures. An adaxonal tubular channel system constituting the "axon-Schwann network" is found in the internodal region in addition to other variants of the adaxonal Schwann plasmalemma. The several forms of sequestration of Schwann cell cytoplasm presumably underlie the specialized needs of cytoplasmic continuity in a dynamic functional entity in which large domains of cytoplasm have been displaced by the formation of compact myelin.

Animals↗
PubMed1993

The lack of CD8 alpha cytoplasmic domain resulted in a dramatic decrease in efficiency in thymic maturation but only a moderate reduction in cytotoxic function of CD8+ T lymphocytes.

The glycoprotein CD8 is believed to play an important role in the maturation and function of MHC class I-restricted T lymphocytes. CD8 has been proposed to function as a co-receptor of the TcR to participate in signal transduction, possibly through its cytoplasmic domain that binds to protein tyrosine kinase p56lck. A T cell-specific transgene encoding CD8 alpha truncated at the cytoplasmic domain ("tailless CD8 alpha"), was introduced into CD8 alpha-deficient mice. This animal model was used to study the role of the CD8 cytoplasmic domain in T cell ontogeny and function. "Tailless CD8 alpha" was expressed on the cell surface of thymocytes and peripheral T cells. A small population of peripheral CD4- T cells (6% of T lymphocytes) was found to have cell surface expression of "tailless CD8 alpha" and endogenous CD8 beta, indicating that these cells may belong to the CD8+ T cell lineage. A consistent result was obtained from CD8 alpha-deficient mice bearing the "tailless CD8 alpha" and the MHC class I-restricted 2C TcR transgenes. A small population of CD4- T cells expressing CD8 beta, the "tailless CD8 alpha" and the 2C TcR transgenes was present in the periphery of these mice in a selecting background, but was absent in a deleting background. When "tailless CD8 alpha" mice were infected with lymphocytic choriomeningitis virus (LCMV), the peripheral CD8+ CD4- T cell subset expanded dramatically and a significant LCMV-specific cytolytic activity was detected. The results suggest that the cytoplasmic portion of CD8 alpha is not absolutely required but dramatically enhances the efficiency of thymic maturation of CD8+ T cells. The lack of CD8 alpha cytoplasmic domain in peripheral CD8+ T cells does not abolish the generation of cytotoxicity in response to an in vivo LCMV infection, although the cytolytic activity is slightly reduced compared to that in control mice.

Animals↗
PubMed1989

The cytoplasmic domain of the cell adhesion molecule uvomorulin associates with three independent proteins structurally related in different species.

Uvomorulin belongs to the group of Ca2+-dependent cell adhesion molecules, which are integral membrane proteins with several structural features in common. In particular, the cytoplasmic part of these proteins is highly conserved in different species, suggesting a common biological function. To test this assumption we transfected a uvomorulin full-length cDNA into uvomorulin-negative mouse NIH 3T3 and L cells. Immunoprecipitations with anti-uvomorulin antibodies detected, in addition to uvomorulin, three independent proteins of 102, 88 and 80 kd which are of host origin and which form complexes with uvomorulin. Using cDNA constructs coding for uvomorulin with cytoplasmic or extracellular deletions it is shown that the 102, 88 and 80 kd proteins complex with the cytoplasmic domain of uvomorulin. Peptide pattern analysis revealed that these three proteins are identical in different mouse cells. When uvomorulin cDNA was introduced into cell lines from other species, such as human HeLa and avian fibroblasts, the expressed uvomorulin was also associated with endogenous 102, 88 and 80 kd proteins and, moreover, each of these proteins showed structural similarities to the respective mouse molecule. A panel of antibodies specific for known cytoplasmic proteins of mol. wts similar to those of the three proteins did not react with any of the described components. This suggests that the 102, 88 and 80 kd proteins constitute a new group of proteins for which we propose the nomenclature of catenin alpha, beta and gamma respectively. The characterization of these proteins provides a first molecular basis for a possible cytoplasmic anchorage of uvomorulin to the cytoskeleton.

Animals↗
PubMed1990

Cloning and sequence analysis of beta-4 cDNA: an integrin subunit that contains a unique 118 kd cytoplasmic domain.

The alpha 6 beta 4 complex is a member of the integrin superfamily of adhesion receptors. A human keratinocyte lambda gt11 cDNA library was screened using a monoclonal antibody directed against the beta 4 subunit. Two cDNAs were selected and subsequently used to isolate a complete set of overlapping cDNA clones. The beta 4 subunit consists of 1778 amino acids with a 683 amino acid extracellular domain, a 23 amino acid transmembrane domain and an exceptionally long cytoplasmic domain of 1072 residues. The deduced amino-terminal sequence is in good agreement with the published amino-terminal sequence of purified beta 4. The extracellular domain contains five potential N-linked glycosylation sites and four cysteine-rich homologous repeat sequences. The extracellular part of the beta 4 subunit sequence shows 35% identify with other integrin beta subunits, but is the most different among this class of molecules. The transmembrane region is poorly conserved, whereas the cytoplasmic domain shows no substantial identity in any region to the cytoplasmic tails of the known beta sequences or to other protein sequences. The exceptionally long cytoplasmic domain suggests distinct interactions of the beta 4 subunit with cytoplasmic proteins.

Amino Acid Sequence↗
PubMed1991

Autonomous roles for the cytoplasmic domains of the CD2 and CD4 T cell surface antigens.

CD2 and CD4 are single chain transmembrane T cell surface molecules that are involved in signal transduction. Chimaeric constructs from rat CD2 and CD4 antigens were expressed in the Jurkat human T cell line to examine the role of extracellular, transmembrane and cytoplasmic domains in mediating functions controlled by CD2 and CD4. The results show that the large rise in concentration of cytoplasmic free Ca2+ mediated via CD2 crosslinking is controlled by the cytoplasmic domain and does not require the CD2 transmembrane and extracellular domains. Similarly the CD4 cytoplasmic domain alone was shown to encode the specificity for binding to the p56lck tyrosine kinase and to control down-modulation of CD4 after treatment with phorbol ester. Evidence was obtained that down-modulation of CD4 occurs when p56lck dissociates from the cytoplasmic domain due to phosphorylation of Ser 405.

Amino Acid Sequence↗
PubMed1993

The (YXXL/I)2 signalling motif found in the cytoplasmic segments of the bovine leukaemia virus envelope protein and Epstein-Barr virus latent membrane protein 2A can elicit early and late lymphocyte activation events.

The cytoplasmic domains of the transducing subunits associated with B and T cell antigen receptors contain a common amino acid motif consisting of two precisely spaced Tyr-X-X-Leu/Ile sequences (where X corresponds to a variable residue). Expression of a single copy of this motif suffices to initiate B or T cell activation. The bovine leukaemia virus (BLV) is a B cell lymphotropic retrovirus which causes a non-neoplasic proliferation of B cells. The cytoplasmic domain of the BLV transmembrane envelope glycoprotein, gp30, possesses two overlapping copies of the Tyr-X-X-Leu/Ile-containing motif which could participate in the induction of B cell activation. Similarly, the N-terminal cytoplasmic domain of the latent membrane protein 2A (LMP2A) of the Epstein-Barr virus (EBV) contains a single copy of the Tyr-X-X-Leu/Ile-containing motif which could play a critical role in B cell transformation. To determine whether these two virus-encoded cytoplasmic domains are endowed with signalling functions, we constructed chimeric proteins by replacing the cytoplasmic tail of CD8-alpha with that of either BLV gp30 or EBV LMP2A. We show here that, once separately expressed in B or T cell lines, these chimeras are capable of triggering both calcium responses and cytokine production when cross-linked with an antibody to CD8-alpha. Furthermore, using site-directed mutagenesis, we demonstrated unequivocally that this signalling function may be accounted for by the Tyr-X-X-Leu/Ile motifs they contain.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Two independent targeting signals in the cytoplasmic domain determine trans-Golgi network localization and endosomal trafficking of the proprotein convertase furin.

Furin, a subtilisin-like eukaryotic endoprotease, is responsible for proteolytic cleavage of cellular and viral proteins transported via the constitutive secretory pathway. Cleavage occurs at the C-terminus of basic amino acid sequences, such as R-X-K/R-R and R-X-X-R. Furin was found predominantly in the trans-Golgi network (TGN), but also in clathrin-coated vesicles dispatched from the TGN, on the plasma membrane as an integral membrane protein and in the medium as an anchorless enzyme. When furin was vectorially expressed in normal rat kidney (NRK) cells it accumulated in the TGN similarly to the endogenous glycoprotein TGN38, often used as a TGN marker protein. The signals determining TGN targeting of furin were investigated by mutational analysis of the cytoplasmic tail of furin and by using the hemagglutinin (HA) of fowl plague virus, a protein with cell surface destination, as a reporter molecule, in which membrane anchor and cytoplasmic tail were replaced by the respective domains of furin. The membrane-spanning domain of furin grafted to HA does not localize the chimeric molecule to the TGN, whereas the cytoplasmic domain does. Results obtained on furin mutants with substitutions and deletions of amino acids in the cytoplasmic tail indicate that wild-type furin is concentrated in the TGN by a mechanism involving two independent targeting signals, which consist of the acidic peptide CPSDSEEDEG783 and the tetrapeptide YKGL765. The acidic signal in the cytoplasmic domain of a HA-furin chimera is necessary and sufficient to localize the reporter molecule to the TGN, whereas YKGL is a determinant for targeting to the endosomes. The data support the concept that the acidic signal, which is the dominant one, retains furin in the TGN, whereas the YKGL motif acts as a retrieval signal for furin that has escaped to the cell surface.

Amino Acid Sequence↗
PubMed1984

An improved purification method for cytoplasmic dynein.

An improved method has been devised for the purification of cytoplasmic dynein from sea urchin eggs (Strongylocentrotus droebachiensis and S purpuratus). This protocol introduces three changes over a previously published procedure (Hisanaga and Sakai: J Biochem 93:87, 1983)--the substitution of diethylaminoethyl (DEAE)-cellulose for hydroxylapatite chromatography, the elimination of sucrose density gradient centrifugation, and the use of phosphocellulose chromatography. These changes reduce the time and increase the efficiency of the purification procedure. The purified egg cytoplasmic dynein has enzymatic properties in common with axonemal dynein, including ionic specificity (Ca++ATPase/Mg++ ATPase = 0.8) and inhibition by sodium vanadate and erythro-9-2,3-hydroxynonyl adenine (EHNA). As assayed by silver staining of polyacrylamide gels, the cytoplasmic dynein is composed of two high molecular weight polypeptides (greater than 300 kilodaltons) that comigrate with flagellar dynein heavy chains, and lesser amounts of three lower molecular weight bands. None of these polypeptides appears to contain bound carbohydrate. The purification procedure can be modified slightly to allow the preparation of cytoplasmic dynein in only 2 days from as little as 3-5 ml of packed eggs, a 20-fold reduction over the previous method. This more rapid and efficient method will facilitate the investigation of cytoplasmic dynein in other systems where starting material is limited, including tissue culture cells and nerve axoplasm.

Adenosine Triphosphatases↗
PubMed1989

Comparative study of nuclear and cytoplasmic glycogen isolated from mutant HD33 ascites cells.

Mutant cells of the HD33 subline of the Ehrlich-Lettré ascites tumor synthesize and store glycogen mainly intranuclearly, when growing in vivo, and exclusively in the cytoplasm, when permanently cultivated as a suspension cell strain. To investigate whether there exist differences between glycogen of nuclear and cytoplasmic origin, the ultrastructure and the biophysical and biochemical properties of glycogen from in vivo and in vitro grown HD33 ascites cells were compared. Pronounced heterogeneity and differences in glycogen particle ultrastructure were evident in situ and after isolation of the native, high-molecular polysaccharide. Nuclear glycogen contains a fraction of heavier molecules (up to 2 X 10(9)) and larger particles (up to 340 nm) which could not be found in the cytoplasmic preparations, which contained only particles smaller than 140 nm. The subparticles of beta-type are similar in both nuclear and cytoplasmic glycogen. The absorption spectra and glucose analysis after degradation with phosphorylase and debranching enzyme indicate that nuclear glycogen has a higher degree of branching, associated with a decrease in the average chain length between the branching points, and shorter external polyglucosidic chains than cytoplasmic glycogen. This is the first report about the analysis and properties of isolated nuclear glycogen.

Animals↗
PubMed1987

Cytoplasmic maturation of the snRNAs.

The snRNAs are abundant and stable components of the interphase nucleus. Aqueous and non-aqueous cell fractionation demonstrate that the snRNAs appear transiently in the cytoplasm shortly after transcription, before returning permanently to the interphase nucleus. In pulse label and chase experiments, the newly synthesized snRNA species appear in the cytoplasm after 1 min of labeling and then return to the interphase nucleus after approximately 15 min in the cytoplasm. In order to study the maturation and intracellular transport of these particles, a battery of metabolic inhibitors and alterations in cell culture conditions were investigated for their ability to interfere with the return of the newly synthesized snRNAs to the nucleus. A wide range of inhibitors of the cytoskeleton did not interfere with this process. Only the inhibition of protein synthesis and exposure of cells to medium of at least twice the normal tonicity block the return of the snRNAs to the nucleus. Immunofluorescent staining of cells exposed to hypertonic medium identifies discrete foci in the cytoplasm that stain with the Sm antiserum, directed against proteins associated with the snRNAs. Using a detergent extraction procedure that preserves the cytoskeleton, the newly synthesized snRNAs in the cytoplasm fractionate as soluble complexes. These data are consistent with the hypothesis that the snRNAs partition into the interphase nucleus because of a preferential solubility and the existence of specific binding sites.

Autoantigens↗
PubMed1989

Heterogeneity of the changes in cytoplasmic pH upon serum stimulation of quiescent fibroblasts.

Addition of mitogens to quiescent cells results in rapid ionic changes in the cytoplasm, including pH. We studied the changes in cytoplasmic pH in single Swiss 3T3 cells upon serum stimulation using fluorescence ratio imaging microscopy. Quiescence was attained using two approaches, serum deprivation of subconfluent cells and confluence. All measurements were made in the presence of bicarbonate and the absence of other organic buffers. We also used BCECF coupled to dextran to avoid several artifacts associated with using BCECF-AM, including leakage and phototoxicity. Analysis of the changes in cytoplasmic pH demonstrated a dramatic heterogeneity in the responses of single cells. There were six basic classes of responses, 1) a fast alkalinization, reaching a maximum pH in approximately 2-5 min; 2) a slow alkalinization, reaching a maximum pH in 10-20 min; 3) a very slow alkalinization, not reaching a plateau pH within the measurement time; 4) no apparent change in pH during the measurement time; 5) an early transient acidification, followed by either a fast or slow alkalinization; and 6) an acidification, followed by alkalinization and then by a decrease to some intermediate pH. Subconfluent cells exhibited greater heterogeneity in response than confluent cells, with no single dominant class of response. The dominant (55%) response for confluent cells was a gradual alkalinization of approximately 0.01 pH units/min. A larger proportion (52%) of subconfluent cells exhibited an early transient acidification compared to confluent cells (7%). A significant proportion of both types of cells (23% subconfluent, 36% confluent) exhibited no change in cytoplasmic pH upon stimulation. In general, the kinetics of changes in cytoplasmic pH were significantly different from the published results with population averaging methods.

Animals↗