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PubMed1997

Release of lipopolysaccharide from intracellular compartments containing Salmonella typhimurium to vesicles of the host epithelial cell.

The biological effects of bacterial lipopolysaccharide (LPS) on eucaryotic cells have traditionally been characterized following extracellular challenge of LPS on susceptible cells. In this study, we report the capacity of Salmonella typhimurium to release LPS once it is located in the intracellular environment of cultured epithelial cells. LPS is liberated from vacuolar compartments, where intracellular bacteria reside, to vesicles present in the host cell cytosol. The vesicle-associated LPS is detected in infected cells from the time when invading bacteria enter the host cell. Release of LPS is restricted to S. typhimurium-infected cells, with no LPS observed in neighboring uninfected cells, suggesting that dissemination of LPS occurs entirely within the intracellular environment of the infected cell. The amount of LPS present in host vesicles reaches a maximum when intracellular S. typhimurium cells start to proliferate, a time at which the entire host cell cytosol is filled with numerous vesicles containing LPS. All these data support the concept that intracellular bacterial pathogens might signal the host cell from intracellular locations by releasing bioactive bacterial components such as LPS.

Animals↗

PubMed2004

Vectorial proteomics reveal targeting, phosphorylation and specific fragmentation of polymerase I and transcript release factor (PTRF) at the surface of caveolae in human adipocytes.

Caveolae, the specialized invaginations of plasma membranes, formed sealed vesicles with outwards-orientated cytosolic surface after isolation from primary human adipocytes. This morphology allowed differential, vectorial identification of proteins at the opposite membrane surfaces by proteolysis and MS. Extracellular-exposed caveolae-specific proteins CD36 and copper-containing amine oxidase were concealed inside the vesicles and resisted trypsin treatment. The cytosol-orientated caveolins were efficiently digested by trypsin, producing peptides amenable to direct MS sequencing. Isolation of peripheral proteins associated with the cytosolic surface of caveolae revealed a set of proteins that contained nuclear localization signals, leucine-zipper domains and PEST (amino acid sequence enriched in proline, glutamic acid, serine and threonine) domains implicated in regulation by proteolysis. In particular, PTRF (polymerase I and transcript release factor) was found as a major caveolae-associated protein and its co-localization with caveolin was confirmed by immunofluorescence confocal microscopy. PTRF was present at the surface of caveolae in the intact form and in five different truncated forms. Peptides (44 and 45 amino acids long) comprising both the PEST domains were sequenced by nanospray-quadrupole-time-of-flight MS from the full-length PTRF, but were not found in the truncated forms of the protein. Two endogenous cleavage sites corresponding to calpain specificity were identified in PTRF; one of them was in a PEST domain. Both cleavage sites were flanked by mono- or diphosphorylated sequences. The phosphorylation sites were localized to Ser-36, Ser-40, Ser-365 and Ser-366 in PTRF. Caveolae of human adipocytes are proposed to function in targeting, relocation and proteolytic control of PTRF and other PEST-domain-containing signalling proteins.

Adipocytes↗

PubMed1982

The adverse effects of HEPES, TES, and BES zwitterion buffers on the ultrastructure of cultured chick embryo epiphyseal chondrocytes.

Chick embryo epiphyseal chondrocytes cultured in media containing HEPES, TES, and BES zwitterion buffers, used in combination or independently, consistently developed cytoplasmic vacuoles. This cytoplasmic vacuolation was resolved when the zwitterion buffered media was replaced by media containing bicarbonate:CO2 enriched air buffer. Vacuoles were infrequent or absent in cultures grown in bicarbonate:CO2 enriched air. Chondrocytes with an established extracellular matrix showed less vacuolation than fibroblastlike and polygonal shaped cells that lacked such a matrix. The granular endoplasmic reticulum and Golgi dictyosomes of zwitterion buffered chondrocytes were distended and contained a flocculent amorphous material. Cytoplasmic vacuoles (0.5 to 3.0 micron diam) formed by the fusion and intracellular accumulation of Golgi vesicles and vacuoles also contained a flocculent material enhanced by ruthenium red. Membrane bound extracellular vacuoles containing ruthenium red stained proteoglycan aggregates were common in the extracellular matrix of zwitterion buffered cultures but were generally absent from bicarbonate treated cultures. Electron dense calcium deposits seemed much larger and more numerous in the presence of zwitterion buffers. It is suggested that HEPES, TES, and BES buffers, used alone or in combination, may adversely affect cell membrane systems, and thus the transport or secretory mechanisms operative in cultured chondrocytes, or both, resulting in vacuole formation and the intracellular accumulation of synthesized export material. Although the mechanism by which HEPES, TES, and BES induce these changes remains unclear, the use of zwitterion buffers in biological preparations should be treated with caution.

Alkanesulfonates↗

PubMed1992

Immunohistochemical, autoradiographic and electron microscopic studies on the transformation of fibroblasts into chondrocytes in the mouse subfascia induced by bone morphogenetic protein.

Functional morphology on the transformation of fibroblasts into chondrocytes induced by bone morphogenetic protein (BMP) was studied by light and electron microscopy using 35S autoradiography and immunohistochemistry for S-100 protein and type-II collagen. A pellet containing BMP obtained from a murine osteosarcoma was transplanted into the mouse subfascia. By 3 days after implantation, many typical fibroblasts, which were free of the silver grains for 35S and devoid of both S-100 protein and type-II collagen, entered the pellet region. By 5 days, the fibroblasts in the pellet region became polygonal in shape, and cytoplasmic vesicles and vacuoles appeared, both containing a homogeneous substance of low electron density. At 5 days, autoradiography revealed many silver grains for 35S over the Golgi apparatus and vesicles and vacuoles of the cells in the pellet region as well as over the surrounding extracellular matrix. Moreover, the cells at 5 days displayed immunoreactivity to both proteins. The extracellular matrix around the cell began to show clear metachromasia and increased in amount with time. At 9 days all the cells in the pellet region were round or oval in shape and surrounded by an abundant cartilaginous matrix. The rough endoplasmic reticulum and Golgi apparatus were extremely well developed, and a large number of vacuoles and vesicles were seen in the cytoplasm. These cells showed intense immunoreactivity to both proteins, and strong accumulation of sulfur was visualized in the extracellular matrix by autoradiography. These results suggest that the fibroblasts in the pellet region change into chondroblasts by 5 days, and become typical chondrocytes by 9 days.

Animals↗

PubMed1980

Accumulation of CPC-precipitable material at apical cell surfaces during formation of the optic cup.

Accumulation of extracellular material at the apical surfaces of cells in the optic vesicle was studied by precipitation with cetylpyridinium chloride (CPC) and scanning electron microscopy. Treatment at low salt concentration to preserve all precipitable material indicated an initial appearance of surface material at the tme that the retinal primordium first formed. The amount of precipitate increased as the optic cup formed, particularly at the margins of the cup. Stability of the precipitate during subsequent washing at higher salt concentrations suggested that the apical cell surface material contained highly acidic glycosaminoglycans. The greatest resistance to extraction occurred during the period in which invagination was most pronounced.

Animals↗

PubMed1990

The mannose 6-phosphate receptor cytoplasmic domain is not sufficient to alter the cellular distribution of a chimeric EGF receptor.

Unlike most receptors, 300 kd mannose 6-phosphate receptors (MPRs) are localized primarily in the trans-Golgi network (TGN) and endosomes, and they cycle constitutively between these compartments. Yet, when present at the cell surface, MPRs are internalized together with other cell surface receptors in clathrin-coated vesicles. We constructed a chimeric receptor, comprised of human EGF receptor extracellular and transmembrane domains joined to the bovine MPR cytoplasmic domain, to test whether the MPR cytoplasmic domain contained sufficient information to direct a cell surface receptor into both of these transport pathways. The expressed protein was stable, bound EGF with high affinity, and was efficiently endocytosed and recycled back to the cell surface, in the presence or absence of EGF. If the cytoplasmic domain alone is responsible for sorting native MPRs, chimeric receptors might have been expected to be located primarily in the TGN and in endosomes at steady state. Surprisingly, under conditions in which essentially all endogenous MPRs were intracellular, greater than 85% of the chimeric receptors were located at the cell surface. These experiments demonstrate that the MPR cytoplasmic domain is not sufficient to alter the distribution of the EGF receptor, and suggest a role for extracellular and transmembrane domains in MPR routing.

Animals↗

PubMed1996

A central body region defined by a position-specific molecule in the planarian Dugesia (Girardia) tigrina: spatial and temporal variations during regeneration.

We have isolated a monoclonal antibody, TCEN49, that displays position-specific binding to the central body region of the planarian Dugesia (Girardia) tigrina. TCEN49 binds to all cell types of the central area with the exception of epithelial cells. Labeled cells are more related by position than by lineage. In the intact organism, boundaries between labeled and unlabeled regions are very sharp and in a dynamic state as they shift in relative position along the body axis during growth and degrowth. During regeneration, TCEN49 antigen is present in the area which will form the central region before it is morphologically apparent. Moreover, when animals are cut through the central region, TCEN49 label fades quickly from those regions determined to become head or tail well before morphogenesis takes place. Taken together, these data suggest that expression of TCEN49 antigen is very sensitive to positional information changes and that it could be involved in the expression of central-body positional identity. TCEN49 antigen is a glycoprotein with an apparent molecular weight of 4.5 kDa, as determined by gel immunoblotting. Immunogold light and electron microscopy demonstrate that the main location of TCEN49 antigen is intracellular (granular components of secretory cells and, to a lesser extent, clear cytoplasmic vesicles and heterochromatin blocks), though it is also detected in the extracellular matrix. TCEN49 antigen represents the first position-specific molecule reported in planarians to date.

Animals↗

PubMed1992

Immunolocalization of alkaline phosphatase in osteoblasts and matrix vesicles of human fetal bone.

A monoclonal antibody raised against alkaline phosphatase (ALP) of human osteosarcoma was used to localize this enzyme in human fetal bone tissue. For light microscopy, the presence of alkaline phosphatase in osteoblasts and osteocytes was demonstrated by use of an avidin-biotin immunoperoxidase procedure. Electron microscopic immunolocalization was accomplished with an indirect immunoperoxidase method which revealed a concentration of the enzyme on matrix vesicle and osteoblast plasma membranes. In addition, many vesicular protrusions arising from areas of plasma membrane on the lateral surfaces of adjacent osteoblasts were strongly immunolabeled. Immunostaining for ALP was absent in vesicles which contained fine crystallites. Alkaline-phosphatase-rich matrix vesicles may play a significant role in the mineralization of the extracellular matrix.

Alkaline Phosphatase↗

PubMed2003

Separation and quantification of chicken and bovine growth plate cartilage matrix vesicle lipids by high-performance liquid chromatography using evaporative light scattering detection.

Matrix vesicles (MV) are lipid bilayer-enclosed nanoscale structures that initiate extracellular mineral formation in most vertebrate species. Little attention has been given to differences between species in membrane lipid composition or to how new mineral is formed in MV. To explore more precisely the lipids of MV isolated from avian and bovine species, we developed a new high-performance liquid chromatography (HPLC) method used in combination with evaporative light scattering detection (ELSD) to quantify their lipid composition. HPLC analyses were performed on a Lichrosorb silica column using separate binary gradient elution systems for analyzing polar and nonpolar lipids. Standard mixtures of both phospholipids and nonpolar lipids were used to prepare calibration curves for each lipid, which were analyzed mathematically by polynomial regression for accurate quantitation. This new methodology provides high-resolution separations and quantitation of both the polar and the nonpolar lipids typically present in biological membranes, including lyso- (monoacyl-) phospholipids. We have applied this method to quantitate the phospholipid and nonpolar lipid composition of MV isolated from chicken and bovine growth plate cartilage. We also compared the ability of these MV to induce mineral formation. While the ability to induce mineralization and the lipid composition were generally similar, some significant differences between MV from these two disparate species were seen.

Animals↗

PubMed1984

Experimental brain tumors and edema in rats. III. Peritumorous edema.

In rats experimental brain tumors were produced by intracerebral implantation of a glioma cell clone. Peritumorous edema was investigated by light and electron microscopy, using exogenous tracers [human serum albumin (HSA), horseradish peroxidase] and immuno-histochemical methods for localization of endogenous serum proteins. An infiltration by serum proteins was observed in peritumoral grey and white matter. Peroxidase was found in pinocytotic vesicles of vascular endothelial cells, in pericytes and diffusely in the extracellular space. High concentrations of endogenous rat serum proteins and exogenous human serum albumin were also found in neurons of the grey matter and in oligodendrocytes of the white matter. A cellular uptake of extravasated proteins, therefore, may contribute to edema resolution.

Animals↗

PubMed1984

Experimental brain tumors and edema in rats. II. Tumor edema.

Experimentally induced brain tumors in rats with the morphological characteristics of low differentiated gliomas were investigated with special consideration of the distribution of extravasated serum proteins within the tumor itself. Protein extravasation across tumor vessels occurs by pinocytotic vesicles which enclose serum proteins at the luminal surface of the endothelial cells. These vesicles move to the abluminal cell surface where proteins are released after fusion between vesicles and the cell membrane. Serum proteins then spread through the extracellular space from where they are taken up in tumor cells by pinocytosis. They are then transmitted to and digested by lysosomes in which acid phosphatase activity could be demonstrated. Tracer studies (human serum albumin, horseradish peroxidase) revealed that proteins reach the lysosomes 15 min after systemic application. The cellular content of tracers is variable even when systemic circulation has lasted 120 min.

Animals↗

PubMed2000

Crossing the finish line of development: regulated secretion of Dictyostelium proteins.

The genesis of the spore coat of Dictyostelium represents an exquisite example of developmentally regulated protein secretion. The proteins that are destined to be assembled into the extracellular matrix of the spore coat are stored in unique prespore vesicles that are triggered to secrete their contents at terminal differentiation. The regulation of this process is being revealed by the identification of the individual proteins in these vesicles.

Animals↗

PubMed1991

A new role for the transferrin receptor in the release of iron from transferrin.

Iron removal by pyrophosphate from human serum diferric transferrin and the complex of transferrin with its receptor was studied in 0.05 M HEPES or MES buffers containing 0.1 M NaCl and 0.01 M CHAPS at 25 degrees C at pH 7.4, 6.4, and 5.6. At each pH, the concentration of pyrophosphate was adjusted to achieve rates of release amenable to study over a reasonable time course. Released iron was separated from protein-bound iron by poly(ethylene glycol) precipitation of aliquots drawn from the reaction mixture at various times during the course of a kinetic run. The amount of 59Fe label associated with the protein and pyrophosphate was determined from the radioactivity of precipitate and supernatant, respectively, in each aliquot. Iron removal of 0.05 M pyrophosphate at pH 7.4 from diferric transferrin bound to the receptor is considerably slower than that from free diferric transferrin, with observed pseudo-first-order rate constants of 0.020 and 0.191 min-1, respectively. For iron removal by 0.01 M pyrophosphate at pH 6.4, corresponding rate constants are 0.031 and 0.644 min-1. However, at pH 5.6, iron removal by 0.001 M pyrophosphate is faster from diferric transferrin bound to its receptor than from free transferrin (observed rate constants of 0.819 and 0.160 min-1, respectively). Thus, the transferrin receptor not only facilitates the removal of iron from diferric transferrin at the low pH that prevails in endocytic vesicles but may also reduce its accessibility to iron acceptors at extracellular pH, thereby minimizing the likelihood of nonspecific release of iron from transferrin at the cell surface.

Cell Compartmentation↗

PubMed2004

Tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone induces phosphorylation of mu- and m-calpain in association with increased secretion, cell migration, and invasion.

Mounting evidence indicates that cigarette smoking not only promotes tumorigenesis but also may increase the spread of cancer cells in the body. However, the intracellular mechanism(s) by which cigarette smoking promotes metastasis of human lung cancer remains enigmatic. Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is an important component in cigarette smoke and is formed by nitrosation of nicotine. mu- and m-calpain (calpain I and calpain II) are major members of the calpain family, which are ubiquitously expressed in both small cell lung cancer and non-small cell lung cancer cells. Our findings indicated that NNK potently induces phosphorylation of both mu- and m-calpain in association with their activation and increased migration as well as invasion of lung cancer cells. Treatment of cells with PD98059 blocked phosphorylation of m- and mu-calpain and resulted in suppression of NNK-induced cell migration and invasion. p44 MAPK/extracellular signal-regulated kinase 1 (ERK1) and p42 MAPK/ERK2 were activated by NNK, co-localized with mu- and m-calpain in cytoplasm, and directly phosphorylated mu- and m-calpain in vitro. These findings suggest a role for the ERK1/2 kinases as NNK-activated physiological calpain kinases. Specific knock-down of mu- and/or m-calpain expression by RNA interference blocked NNK-stimulated migration and invasion, suggesting that mu- and m-calpain may act as required targets in a NNK-induced metastatic signaling pathway. Furthermore, NNK promotes secretion of active mu- and m-calpain from lung cancer cells through vesicles, which may have the potential to cleave substrates in the extracellular matrix. Thus, NNK-induced cell migration and invasion may occur, at least in part, through a novel mechanism involving phosphorylation of calpains that leads to their activation and secretion, which may contribute to metastasis and/or progression of lung cancer.

Blotting, Western↗

PubMed2005

SLC18A2 promoter haplotypes and identification of a novel protective factor against alcoholism.

The vesicular monoamine transporter 2 (VMAT2, SLC18A2) takes up cytosolic monoamines into intracellular secretory vesicles, preventing their neurotoxicity in the cytosol and discharging them into extracellular space by exocytosis. It has been shown that one-copy deletion of the VMAT2 gene increases locomotion activity significantly in response to drug treatments and dopamine neuron death rate in response to neurotoxin treatments in knockout mice. Little is known about promoter polymorphisms and their influence on SLC18A2 promoter activity. We have re-sequenced a 17.4 kb DNA in the SLC18A2 promoter region for Caucasians and revealed 47 polymorphisms that confer 13 haplotypes. One of the haplotypes reaches a frequency as high as 65%, likely due to positive selection. In vitro analysis showed a 20% difference in promoter activity between two frequent haplotypes and identified some of the polymorphisms that influence promoter activity. Four haplotype-defining single nucleotide polymorphisms (hdSNPs) can define the frequent haplotypes and by genotyping these hdSNPs, we find that haplotypes with -14234G and -2504C of SLC18A2 promoter region represent a protective factor against alcoholism (P = 0.0038 by Fisher's exact tests). Therefore, SLC18A2 promoter haplotypes defined here create a foundation for transcriptional characterization of individuality and for association study on monoamine-related human diseases.

Alcoholism↗

PubMed2006

Membrane traffic in outer hair cells of the adult mammalian cochlea.

Outer hair cells (OHCs), the sensory-motor cells responsible for the extraordinary frequency selectivity and dynamic range of the cochlea, rapidly endocytose membrane and protein at their apical surface. Endocytosis and transcytosis in isolated OHCs from the mature guinea-pig cochlea were investigated using the amphipathic membrane probe FM1-43. We observed membrane transport from the apical surface to both the basolateral wall and the subnuclear pole. By double-labelling with DiOC6, a stain for endoplasmic reticulum, and aspiration of the plasma membrane, we showed that the basolateral target was the subsurface cisternae. The fluorescent signal was about three times weaker at the basal than at the apical pole. The speed of vesicle transport to the subnuclear pole was approximately 0.4 microm/s. Changing extracellular Ca2+ concentration from 25 microM to 2 mM accelerated rapid endocytosis. Extracellular application of BAPTA-AM (25 microM), an intracellular Ca2+ chelator, and TFP (20 microM), a specific inhibitor of calmodulin, reduced endocytic activity, as did depolarization of the whole cell. The presence of extracellular Cd2+ (200 microM), a Ca2+-channel blocker, had no effect on the voltage dependence of endocytosis at the apical pole, and inhibited the voltage dependence at the subnuclear pole. These results suggest that rapid endocytosis is a Ca2+/calmodulin-dependent process, with extracellular Ca2+ entering through voltage-gated Ca2+ channels at the basal pole. The two distinct destinations of endocytosed membrane are consistent with the functional polarization of the OHC, with the basolateral wall being dedicated to electromechanical transduction and the subnuclear pole being dedicated to electrochemical transduction processes.

Animals↗

PubMed2001

Egress of alphaherpesviruses: comparative ultrastructural study.

Egress of four important alphaherpesviruses, equine herpesvirus 1 (EHV-1), herpes simplex virus type 1 (HSV-1), infectious laryngotracheitis virus (ILTV), and pseudorabies virus (PrV), was investigated by electron microscopy of infected cell lines of different origins. In all virus-cell systems analyzed, similar observations were made concerning the different stages of virion morphogenesis. After intranuclear assembly, nucleocapsids bud at the inner leaflet of the nuclear membrane, resulting in enveloped particles in the perinuclear space that contain a sharply bordered rim of tegument and a smooth envelope surface. Egress from the perinuclear cisterna primarily occurs by fusion of the primary envelope with the outer leaflet of the nuclear membrane, which has been visualized for HSV-1 and EHV-1 for the first time. The resulting intracytoplasmic naked nucleocapsids are enveloped at membranes of the trans-Golgi network (TGN), as shown by immunogold labeling with a TGN-specific antiserum. Virions containing their final envelope differ in morphology from particles within the perinuclear cisterna by visible surface projections and a diffuse tegument. Particularly striking was the addition of a large amount of tegument material to ILTV capsids in the cytoplasm. Extracellular virions were morphologically identical to virions within Golgi-derived vesicles, but distinct from virions in the perinuclear space. Studies with gB- and gH-deleted PrV mutants indicated that these two glycoproteins, which are essential for virus entry and direct cell-to-cell spread, are dispensable for egress. Taken together, our studies indicate that the deenvelopment-reenvelopment process of herpesvirus maturation also occurs in EHV-1, HSV-1, and ILTV and that membrane fusion processes occurring during egress are substantially different from those during entry and direct viral cell-to-cell spread.

Alphaherpesvirinae↗

PubMed1991

Exocytosis in pheochromocytoma cells after exposure to a high concentration of potassium.

An electron-microscopic study revealed exocytosis of electron-dense granules in rat pheochromocytoma (PC12) cells after 1-min exposure to a high concentration of potassium medium. Numerous omega profiles indicative of the exocytosis were seen in the surface membrane of the PC12 cells. Most omega profiles showed clear or did not contain the granule contents. Sometimes, the granular, flocculent or dense materials were seen within the omega profiles that were exposed directly to the extracellular medium. Moreover, noncoated or coated omega profiles were observed. Coated vesicles were also visible under the surface membrane. The present study suggests that PC12 cells retain the exocytotic release mechanism for catecholamines.

Animals↗