scholarly journals Salmonella typhimurium induces selective aggregation and internalization of host cell surface proteins during invasion of epithelial cells

1994 ◽  
Vol 107 (7) ◽  
pp. 2005-2020 ◽  
Author(s):  
F. Garcia-del Portillo ◽  
M.G. Pucciarelli ◽  
W.A. Jefferies ◽  
B.B. Finlay
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Epithelial Cells ◽  
Cell Surface ◽  
Heavy Chain ◽  
Surface Proteins ◽  
Class I ◽  
Plasma Membrane Proteins ◽  
Class I Mhc ◽  
Host Plasma Membrane

Salmonella interact with eucaryotic membranes to trigger internalization into non-phagocytic cells. In this study we examined the distribution of host plasma membrane proteins during S. typhimurium invasion of epithelial cells. Entry of S. typhimurium into HeLa epithelial cells produced extensive aggregation of cell surface class I MHC heavy chain, beta 2-microglobulin, fibronectin-receptor (alpha 5 beta 1 integrin), and hyaluronate receptor (CD-44). Other cell surface proteins such as transferrin-receptor or Thy-1 were aggregated by S. typhimurium to a much lesser extent. Capping of these plasma membrane proteins was observed in membrane ruffles localized to invading S. typhimurium and in the area surrounding these structures. In contrast, membrane ruffling induced by epidermal growth factor only produced minor aggregations of surface proteins, localized exclusively in the membrane ruffle. This result suggests that extensive redistribution of these proteins requires a signal related to bacterial invasion. This bacteria-induced process was associated with rearrangement of polymerized actin but not microtubules, since preincubation of epithelial cells with cytochalasin D blocked aggregation of these proteins while nocodazole treatment did not. Of the host surface proteins aggregated by S. typhimurium, only class I MHC heavy chain was predominantly present in the bacteria-containing vacuoles. No extensive aggregation of host plasma membrane proteins was detected when HeLa epithelial cells were infected with invasive bacteria that do not induce membrane ruffling, including Yersinia enterocolitica, a bacterium that triggers internalization via binding to beta 1 integrin, and a S. typhimurium invasion mutant that utilizes the Yersinia-internalization route. In contrast to the situation with S. typhimurium, class I MHC heavy chain was not selectively internalized into vacuoles containing these other bacteria. Extensive aggregation of host plasma membrane proteins was also not observed when other S. typhimurium mutants that are defective for invasion were used. The amount of internalized host plasma membrane proteins in the bacteria-containing vacuoles decreased over time with all invasive bacteria examined, indicating that modification of the composition of these vacuoles occurs. Therefore, our data show that S. typhimurium induces selective aggregation and internalization of host plasma membrane proteins, processes associated with the specific invasion strategy used by this bacterium to enter into epithelial cells.

Apical plasma membrane proteins are not obligatorily stored in secretory granules in exocrine cells

1994 ◽  
Vol 107 (8) ◽  
pp. 2271-2277 ◽  
Author(s):  
V. Colomer ◽  
M.J. Rindler ◽  
A.W. Lowe
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Epithelial Cells ◽  
Cell Surface ◽  
Secretory Granules ◽  
Apical Plasma Membrane ◽  
Cell Fractionation ◽  
Plasma Membrane Proteins ◽  
Exocrine Cells ◽  
Ar42j Cells

Exocrine cells are epithelial cells in which secretory granules undergo fusion with the apical plasma membrane upon secretagogue stimulation. Several apical plasma membrane proteins have been found in secretory granules in cells from pancreas and salivary glands raising the possibility that incorporation into secretory granules followed by exocytosis of the granules accounts for their insertion into the apical plasma membrane. To test this hypothesis, we have expressed the influenza hemagglutinin (HA) in pancreatic AR42J cells, which make zymogen-like granules upon incubation with dexamethasone. The influenza virus HA is known to be specifically targeted to the apical plasma membrane of epithelial cells that lack a regulated pathway and is also known to be excluded from secretory granules in virally-infected pituitary AtT20 cells. Localization of the protein by immunofluorescence microscopy revealed that it accumulated at the plasma membrane of the transfected AR42J cells. HA was not observed in the amylase-rich secretory granules. By immunolabeling of ultrathin cryosections of the transfected cells, HA was also found exclusively on the cell surface, with label over secretory granules not exceeding that seen in control, untransfected cells. In addition, in cell fractionation experiments performed on radiolabeled AR42J cell transformants, HA was not detectable in the secretory granule fractions. These results indicate that HA is not efficiently stored in mature secretory granules and is likely to reach the cell surface via constitutive transport pathways.

Differential partitioning of plasma membrane proteins into the triton X-100-insoluble cytoskeleton fraction during concanavalin A-induced receptor redistribution

1989 ◽  
Vol 92 (1) ◽  
pp. 85-91
Author(s):  
W.F. Patton ◽  
M.R. Dhanak ◽  
B.S. Jacobson
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Cell Surface ◽  
Concanavalin A ◽  
Temporal Order ◽  
Surface Glycoprotein ◽  
Plasma Membrane Proteins ◽  
Cell Surface Glycoprotein ◽  
Extensive Cell ◽  
Triton X

The plasma membrane proteins of Dictyostelium discoideum were characterized with respect to their partitioning into the Triton-insoluble cytoskeleton fraction of the cell during concanavalin A-induced capping. Two fractions of plasma membrane-associated concanavalin A were identified; one that immediately associated with the cytoskeleton fraction via cell surface glycoproteins, and one that partitioned with the cytoskeleton only after extensive cell surface glycoprotein cross-linking. Three major classes of polypeptides were found in the plasma membrane that differed with respect to their partitioning properties into the cytoskeleton fraction. The temporal order of association of the polypeptides with the cytoskeleton during concanavalin A-induced capping corresponded to the strength of their association with the cytoskeleton fraction as determined by pH and ionic strength elution from unligated cytoskeletons.

scholarly journals Azaspiracid-1 Inhibits Endocytosis of Plasma Membrane Proteins in Epithelial Cells

2010 ◽  
Vol 117 (1) ◽  
pp. 109-121 ◽  
Author(s):  
Mirella Bellocci ◽  
Gian Luca Sala ◽  
Federica Callegari ◽  
Gian Paolo Rossini
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Epithelial Cells ◽  
Plasma Membrane Proteins

scholarly journals Intermediates in the Assembly and Degradation of Class I Major Histocompatibility Complex (MHC) Molecules Probed with Free Heavy Chain–specific Monoclonal Antibodies

1996 ◽  
Vol 184 (6) ◽  
pp. 2251-2260 ◽  
Author(s):  
Robert P. Machold ◽  
Hidde L. Ploegh
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Surface ◽  
Heavy Chain ◽  
Class I ◽  
Class I Mhc ◽  
Intact Cells ◽  
Β2 Microglobulin ◽  
Heavy Chains ◽  
Mhc Molecules ◽  
Cooperative Process

Unassembled (free) heavy chains appear during two stages of the class I MHC molecule's existence: immediately after translation but before assembly with peptide and β2-microglobulin, and later, upon disintegration of the heterotrimeric complex. To characterize the structures of folding and degradation intermediates of the class I heavy chain, three monoclonal antibodies have been produced that recognize epitopes along the H-2Kb heavy chain which are obscured upon proper folding and subsequent assembly with β2-microglobulin (KU1: residues 49-54; KU2: residues 23-30; KU4: residues 193-198). The Kb heavy chain is inserted into the lumen of the endoplasmic reticulum in an unfolded state reactive with KU1, KU2, and KU4. Shortly after completion of the polypeptide chain, reactivity with KU1, KU2 and KU4 is lost synchronously, suggesting that folding of the class I heavy chain is a rapid, cooperative process. Perturbation of the folding environment in intact cells with the reducing agent dithiothreitol or the trimming glucosidase inhibitor N-7-oxadecyl-deoxynojirimycin prolongs the presence of mAb-reactive Kb heavy chains. At the cell surface, a pool of free Kb heavy chains appears after 60–120 min of chase, whose subsequent degradation, but not their initial appearance, is impaired in the presence of concanamycin B, an inhibitor of vacuolar acidification. Thus, free heavy chains that arise at the cell surface are destroyed after internalization.

scholarly journals Kinetics of intracellular transport and sorting of lysosomal membrane and plasma membrane proteins.

1987 ◽  
Vol 105 (3) ◽  
pp. 1227-1240 ◽  
Author(s):  
S A Green ◽  
K P Zimmer ◽  
G Griffiths ◽  
I Mellman
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
Intracellular Transport ◽  
Rat Kidney ◽  
Fc Receptor ◽  
Lysosomal Membrane ◽  
Plasma Membrane Proteins ◽  
Kinetics Of

We have used monospecific antisera to two lysosomal membrane glycoproteins, lgp120 and a similar protein, lgp110, to compare the biosynthesis and intracellular transport of lysosomal membrane components, plasma membrane proteins, and lysosomal enzymes. In J774 cells and NRK cells, newly synthesized lysosomal membrane and plasma membrane proteins (the IgG1/IgG2b Fc receptor or influenza virus hemagglutinin) were transported through the Golgi apparatus (defined by acquisition of resistance to endo-beta-N-acetylglucosaminidase H) with the same kinetics (t1/2 = 11-14 min). In addition, immunoelectron microscopy of normal rat kidney cells showed that lgp120 and vesicular stomatitis virus G-protein were present in the same Golgi cisternae demonstrating that lysosomal and plasma membrane proteins were not sorted either before or during transport through the Golgi apparatus. To define the site at which sorting occurred, we compared the kinetics of transport of lysosomal and plasma membrane proteins and a lysosomal enzyme to their respective destinations. Newly synthesized proteins were detected in dense lysosomes (lgp's and beta-glucuronidase) or on the cell surface (Fc receptor or hemagglutinin) after the same lag period (20-25 min), and accumulated at their final destinations with similar kinetics (t1/2 = 30-45 min), suggesting that these two lgp's are not transported to the plasma membrane before reaching lysosomes. This was further supported by measurements of the transport of membrane-bound endocytic markers from the cell surface to lysosomes, which exhibited additional lag periods of 5-15 min and half-times of 1.5-2 h. The time required for transport of newly synthesized plasma membrane proteins to the cell surface, and for the transport of plasma membrane markers from the cell surface to lysosomes would appear too long to account for the rapid transport of lgp's from the Golgi apparatus to lysosomes. Thus, the observed kinetics suggest that lysosomal membrane proteins are sorted from plasma membrane proteins at a post-Golgi intracellular site, possibly the trans Golgi network, before their delivery to lysosomes.

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