Showing papers on "Cellular compartment published in 1992"

Characterization of the Saccharomyces Golgi complex through the cell cycle by immunoelectron microscopy.

Abstract: The membrane compartments responsible for Golgi functions in wild-type Saccharomyces cerevisiae were identified and characterized by immunoelectron microscopy. Using improved fixation methods, Golgi compartments were identified by labeling with antibodies specific for alpha 1-6 mannose linkages, the Sec7 protein, or the Ypt1 protein. The compartments labeled by each of these antibodies appear as disk-like structures that are apparently surrounded by small vesicles. Yeast Golgi typically are seen as single, isolated cisternae, generally not arranged into parallel stacks. The location of the Golgi structures was monitored by immunoelectron microscopy through the yeast cell cycle. Several Golgi compartments, apparently randomly distributed, were always observed in mother cells. During the initiation of new daughter cells, additional Golgi structures cluster just below the site of bud emergence. These Golgi enter daughter cells at an early stage, raising the possibility that much of the bud's growth might be due to secretory vesicles formed as well as consumed entirely within the daughter. During cytokinesis, the Golgi compartments are concentrated near the site of cell wall synthesis. Clustering of Golgi both at the site of bud formation and at the cell septum suggests that these organelles might be directed toward sites of rapid cell surface growth.

Isoform-specific subcellular targeting of glucose transporters in mouse fibroblasts.

Abstract: GLUT1, the erythrocyte glucose transporter, and GLUT4, the adipose/muscle transporter, were each expressed in NIH-3T3 cells by retrovirus-mediated gene transfer. In fibroblasts overexpressing GLUT1, basal as well as insulin-stimulated deoxyglucose uptake was increased. Expression of GLUT4 was without affect on either basal or hormone stimulated hexose uptake. Localization of each of the transporters by indirect immunofluorescence revealed that, whereas GLUT1 was found primarily on the cell surface, GLUT4 was directed to vesicles in a perinuclear distribution and throughout the cytoplasm. The GLUT4-containing compartment represented neither Golgi complex nor lysosomes, as evidenced by the failure of lgp110 or Golgi mannosidase to co-localize. However, there was substantial overlap between the distribution of GLUT4 and the transferrin receptor, and some colocalization of the transporter isoform with the manose-6-phosphate receptor. In addition, when FITC-wheat germ agglutinin bound to the cell surface was allowed to internalize at 37 degrees C, it concentrated in vesicular structures coincident with GLUT4 immunoreactivity. These data establish that GLUT1 and GLUT4 contain within their amino acid sequences information which dictates targeting to distinct cellular compartments. Moreover, GLUT4 can be recognized by those cellular factors which direct membrane proteins to the endosomal pathway.

Effects of cinnamic acid derivatives on in vitro growth of Plasmodium falciparum and on the permeability of the membrane of malaria-infected erythrocytes.

Abstract: Cinnamic acid derivatives (CADs) are known inhibitors of monocarboxylate transport across plasma and mitochondrial membranes. All derivatives were found to inhibit the growth of intraerythrocytic Plasmodium falciparum in culture, which is in correlation with their hydrophobic character. Parasites at the ring and trophozoite stages were equally susceptible to the different derivatives. This result could be attributed to their inhibition of the transport of lactate, the major product of parasite energy metabolism. However, unexpectedly, it was found that all derivatives also inhibit the translocation of carbohydrates and amino acids across the new permeability pathways induced in the host cell membrane by the parasite. This impediment correlated strictly with CADs' effect on parasite growth. Parasites residing in cells permeabilized by means of Sendai virus were less susceptible to the different drugs, a result which implies that in addition to the direct effect on parasite viability, the drugs may have inhibited some process in the host cell whose function may be vital for parasite growth. The effect of CADs on the ATP levels in infected cells, in virus-treated cells, and in the two cellular compartments of the infected cell revealed that the drugs caused a significant decline in ATP level in the parasite compartment, while they provoked only a small effect on ATP level in the intact cells and the host cell compartment. These observations suggest that CADs inhibit ATP production in the parasite and its utilization by the host cell.

The effect of cyclosporine administration on the cellular distribution and content of cyclophilin.

Abstract: Cyclophilin (CYP), an intracellular protein sharing amino acid sequence identity with the enzyme peptidyl-prolyl cis-trans isomerase has become the leading candidate for the receptor responsible for cyclosporine biological effects. Avid binding of CYP to cyclosporine and immunosuppressive cyclosporine metabolites has been demonstrated, while nonimmunosuppressive cyclosporine metabolites have tended not to bind to cyclophilin. A previous immunohistochemical analysis documented that CYP localized principally to the cytoplasmic cellular compartment, but nuclear staining was observed among some cells. This study was undertaken to more precisely define the ultrastructural distribution of CYP, and to determine whether CYP cellular content was affected by CsA therapy. Untreated Wistar rats or those receiving 7 days of CsA (15 mg/kg/day, i.p.) were anesthetized, perfusion-fixed in situ, and sacrificed. Analyses of lymph node, spleen, thymus, kidney, liver, heart, brain, and ileum used an affinity purified, rabbit anticyclophilin IgG. Transmission electron microscopy was performed after staining with anti-CYP using a horseradish peroxidase/biotin/avidin technique. Quantitative immunofluorescence was measured by confocal microscopy using anti-CYP, with a biotin/avidin/phycoerythrin technique. Cyclophilin localized to the cytoplasmic compartment--however, association with mitochondria endoplasmic reticulum, Golgi, and with the nuclear membrane among lymphocytes, as well as cells from kidney, liver and ileum--was documented. Cyclophilin was not identified within the nucleus proper. Tissues obtained from animals receiving CsA exhibited a generalized increase in CYP content compared with tissues from untreated controls, suggesting the possibility that CsA may exert a regulatory influence upon CYP gene activation. Collectively, the data were consistent with the hypothesis that CYP exerts a central role in cellular metabolism, and that CsA-mediated biologic effects result from the CsA/CYP interaction.

Ca2+-induced fusion of phospholipid-vesicles containing free fatty-acids - modulation by transmembrane ph gradients

Abstract: The influence of a transmembrane pH gradient on the Ca2+-induced fusion of phospholipid vesicles, containing free fatty acids, has been investigated. Large unilamellar vesicles composed of an equimolar mixture of cardiolipin, dioleoylphosphatidylcholine, and cholesterol, containing 20 mol 7% oleic acid, were employed. Fusion was measured using a kinetic assay for lipid mixing, based on fluorescence resonance energy transfer. At pH 7.5, but not at pH 6.0, in the absence of a pH gradient, oleic acid stimulates the fusion of the vesicles by shifting the Ca2+ threshold concentration required for aggregation and fusion of the vesicles from about 13 mM to 10 mM. In the presence of a pH gradient (at an external pH of 7.5 and a vesicle interior pH of 10.5), the vesicles exhibit fusion characteristics similar to vesicles that do not contain oleic acid at all, consistent with an effective sequestration of the fatty acid to the inner monolayer of the vesicle bilayer induced by the imposed pH gradient. The kinetics of the fusion process upon simultaneous generation of the pH gradient across the vesicle bilayer and initiation of the fusion reaction show that the inward movement of oleic acid in response to the pH gradient is extremely fast, occurring well within 1 s. Conversely, dissipation of an imposed pH gradient, by addition of a proton ionophore during the course of the fusion process, results in a rapid enhancement of the rate of fusion due to reequilibration of the oleic acid between the two bilayers leaflets. Membrane fusion is a fundamental process in cell biology. It plays a key role in cell-cell fusion phenomena such as fertilization and myogenesis. It is also the basis of intracellular trafficking and sorting processes, involving fusion of shuttle vesicles derived from one cellular compartment with the lim- iting membrane of another compartment or, as in the process of exocytosis, with the plasma membrane of the cell. Obvi- ously, these membrane fusion processes must be highly specific and strictly controlled, at the level of the initial recognition and attachment of the interacting membranes as well as that of the actual fusion reaction. However, very little is known about the molecular mechanisms involved. Much of our current knowledge of the molecular mecha- nisms of membrane fusion has been derived from investigation of fusion in lipid vesicle (liposome) systems (for reviews, see

Effect of L-Carnitine on the Cellular Distribution of Carnitine and Its Acyl Derivatives in the Ischemic Heart.

Abstract: SUMMARY The purpose of this study was to investigate the cellular distribution of carnitine and its acyl derivatives in the normal and ischemic myocardium, and the effects of exogenous l-carnitine on this distribution and mitochondrial function in the ischemic dog heart. Under nonischemic conditions, about 93% of the total cellular carnitine was located in the cytosolic compartment and 6.5% in the mitochondrial compartment. Sixty minutes of ischemia induced a decrease in the cytosolic free carnitine content, but caused the accumulation of long-chain acylcarnitine in the cytosolic and mitochondrial compartments. Treatment with l-carnitine (30 or 100mg/kg, i.v.) inhibited the mitochondrial accumulation of long-chain acylcarnitine. Free fatty acid (FFA) metabolism in the mitochondria differs from that in the cytosol. So, it is necessary to investigate the changes in FFA metabolism in both of these cellular compartments. Our results suggest that l-carnitine has a protective effect on the ischemic heart by selectively reducing mitochondrial accumulation of long-chain acylcarnitine.

Introduction of Large Molecules into Viable Fibroblasts by Electroporation: Optimization of Loading and Identification of Labeled Cellular Compartments

Abstract: Access to the cell cytoplasm in viable cells may permit direct labeling or manipulation of intracellular molecules and metabolic processes. One method to gain access to the cell cytoplasm is by electroporation, a technique that transiently creates pores in cell membranes by means of applied electrical fields. We used electroporation to introduce large-molecular-mass dextrans and proteins as probes of the cytoplasmic compartment in human gingival fibroblasts. Electrical field strength and pulse decay time were optimized to obtain cellular viability >60%. Analysis by confocal microscopy and by fluorescence spectrophotometry demonstrated that a large proportion of high-molecular-mass probe was membrane-bound after electroporation. Trypsinization did not affect membrane-bound FITC-destran but eliminated protein probe incorporated into the membrane, thereby permitting measurement of only intracellular, cytoplasmic label. Proteins of up to 66 hDa were incorporated at intracellular concentrations of 10-l’ M. After electroporation under optimal conditions, incorporated anti-vimentin antibodies were capable of binding to vimentin. Cells electroporated in the presence of RNase A exhibited significant reductions of cellular RNA. Electroporation appears to be a useful approach to probe or perturb specific cellular processes by introduction of functional molecular species into the cytoplasm of viable cells. Q

Translocation of proteins across and integration of membrane proteins into the rough endoplasmic reticulum

Abstract: One of the major topics addressed in the meeting “Proteases and Protease Inhibitors: Emerging Roles in the Pathogenesis of Alzheimer’s Disease” is the proteolytic processing reactions that lead to the production of the p protein from the amyloid precursor protein (APP). Eukaryotic cells produce numerous proteolytic enzymes that could be potentially responsible for the proteolytic cleavages that liberate the /3 protein from APP. Both the amyloid precursor protein and the proteolytic enzymes are restricted to specific cellular compartments; hence a consideration of the membrane topology and intracellular location of APP may allow investigators to identify proteolytic enzymes that are accessible to the regions of APP that are cleaved during /3 protein formation. The membrane orientation of integral proteins like APP is established during synthesis when the protein is integrated into the rough endoplasmic reticulum (RER). The focus of this paper will be the mechanisms responsible for the asymmetric integration of membrane proteins into the RER. Cotranslational protein modification reactions that occur during glycoprotein biosynthesis will also be described. Newly synthesized polypeptides that are mishandled by the translocation apparatus are probably degraded by proteases that reside within the cytoplasm and the endoplasmic reticulum. Proteins located within the lumen of the endoplasmic reticulum, Golgi apparatus, trans-Golgi network, endosome and lysosome and virtually all secreted proteins are cotranslationally translocated across the rough endoplasmic reticulum (RER). The majority of the integral membrane proteins of the rough and smooth endoplasmic reticulum, Golgi apparatus, endosome, lysosome and plasma membrane are synthesized by the membrane-bound ribosomes of the RER.1,2 Integral membrane proteins can be separated into several classes based upon the orientation and number of membrane spanning segment^.^.^ Type I and type I1 membrane proteins both contain a single membrane-spanning segment (FIG. 1). When initially integrated into the RER, the amino terminus of type I membrane proteins is located within the membrane lumen, while type I1 integral membrane proteins are integrated in the opposite orientation. Type I membrane proteins can have the bulk of the protein oriented towards either face of the membrane, unlike type I1 proteins, which typically have small Nterminal cytoplasmic domains. Based upon the protein sequence derived from sequencing a cDNA clone, the APP protein appears to be a typical type I membrane protein. Proteins with multiple membrane-spanning segments (type 111) can be oriented with the N-terminus facing either the cytoplasm or the lumen. Proteins anchored to the