scholarly journals Cell Surface Assembly of HIV gp41 Six-Helix Bundles for Facile, Quantitative Measurements of Hetero-oligomeric Interactions

2012 ◽  
Vol 134 (36) ◽  
pp. 14642-14645 ◽  
Author(s):  
Xuebo Hu ◽  
Piyali Saha ◽  
Xiaoyue Chen ◽  
Dogeun Kim ◽  
Mahesh Devarasetty ◽  
...  
Keyword(s):  
Cell Surface ◽  
Quantitative Measurements ◽  
Surface Assembly ◽  
Hiv Gp41

scholarly journals Periplasmic depolymerase provides insight into ABC transporter-dependent secretion of bacterial capsular polysaccharides

2018 ◽  
Vol 115 (21) ◽  
pp. E4870-E4879 ◽  
Author(s):  
Sean D. Liston ◽  
Stephen A. McMahon ◽  
Audrey Le Bas ◽  
Michael D. L. Suits ◽  
James H. Naismith ◽  
...  
Keyword(s):  
Cell Surface ◽  
Abc Transporter ◽  
Pectate Lyase ◽  
Capsular Polysaccharides ◽  
Ph Optimum ◽  
Pectate Lyases ◽  
Surface Assembly ◽  
Binding Groove ◽  
Biochemical Analyses ◽  
Vi Antigen

Capsules are surface layers of hydrated capsular polysaccharides (CPSs) produced by many bacteria. The human pathogenSalmonella entericaserovar Typhi produces “Vi antigen” CPS, which contributes to virulence. In a conserved strategy used by bacteria with diverse CPS structures, translocation of Vi antigen to the cell surface is driven by an ATP-binding cassette (ABC) transporter. These transporters are engaged in heterooligomeric complexes proposed to form an enclosed translocation conduit to the cell surface, allowing the transporter to power the entire process. We identified Vi antigen biosynthesis genetic loci in genera of theBurkholderiales, which are paradoxically distinguished fromS.Typhi by encoding VexL, a predicted pectate lyase homolog. Biochemical analyses demonstrated that VexL is an unusual metal-independent endolyase with an acidic pH optimum that is specific for O-acetylated Vi antigen. A 1.22-Å crystal structure of the VexL-Vi antigen complex revealed features which distinguish common secreted catabolic pectate lyases from periplasmic VexL, which participates in cell-surface assembly. VexL possesses a right-handed parallel β-superhelix, of which one face forms an electropositive glycan-binding groove with an extensive hydrogen bonding network that includes Vi antigen acetyl groups and confers substrate specificity. VexL provided a probe to interrogate conserved features of the ABC transporter-dependent export model. When introduced intoS. Typhi, VexL localized to the periplasm and degraded Vi antigen. In contrast, a cytosolic derivative had no effect unless export was disrupted. These data provide evidence that CPS assembled in ABC transporter-dependent systems is actually exposed to the periplasm during envelope translocation.

A novel method for measuring protein expression at the cell surface

1993 ◽  
Vol 106 (4) ◽  
pp. 1201-1209
Author(s):  
W. Stoorvogel ◽  
V. Oorschot ◽  
B. Neve
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Protein Expression ◽  
Surface Expression ◽  
Surface Proteins ◽  
Proteinase K ◽  
Cell Surface Expression ◽  
Specific Cell ◽  
Quantitative Measurements ◽  
Dab Cytochemistry

All methods described in the literature that allow quantitative measurements of protein expression at the cell surface are applicable to subsets of surface-exposed proteins only. We developed a new method, involving 3,3′-diaminobenzidine (DAB) cytochemistry, which allowed determination of cell-surface expression of all plasma membrane proteins measured, in at least three different cell lines. Adherent cells were first brought into suspension by proteinase K and EDTA treatment at 0 degrees C removing many, but not all, surface-exposed proteins. Subsequently, horseradish peroxidase (HRP) was linked by means of its glycosyl residues to specific cell-surface-exposed sugar moieties using the multivalent lectin concanavalin A (ConA). The suspended cells were encapsulated by polymerized DAB, a process that was catalysed by plasma membrane-bound HRP. After cell lysis, and removal of nuclei and most of the DAB polymer by centrifugation, proteins were analysed by SDS-PAGE. Surface proteins encapsulated by non-pelleted DAB polymer were retained on top of the stacking gel. After 125I-labelling the cell surface, protease-resistant 125I-labelled proteins could be quantitatively coupled to DAB polymer. This process was completely dependent on the presence of ConA, HRP, DAB and H2O2. Surface 125I-labelled beta-Na+,K(+)-ATPase was resistant to proteinase K but could be completely removed using DAB cytochemistry. Intracellular ConA binding proteins were not affected. Other intracellular proteins, including endosomal asialoglycoprotein receptor and cation-independent mannose 6-phosphate/insulin-like growth factor II receptor were also not affected.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Identification of desmosomal surface components (desmocollins) and inhibition of desmosome formation by specific Fab'

1984 ◽  
Vol 70 (1) ◽  
pp. 41-60
Author(s):  
P. Cowin ◽  
D. Mattey ◽  
D. Garrod
Keyword(s):  
Cell Surface ◽  
Fluorescent Antibody ◽  
Protein A ◽  
Antibody Staining ◽  
Quantitative Measurements ◽  
Mdbk Cells ◽  
Cell Surface Staining ◽  
Lateral Constraint ◽  
Gold Labelling ◽  
Fluorescent Antibody Staining

Specific antibodies against the components of desmosomes, the adhesive junctions of epithelial cells, have been used to determine which components are located on the cell surface. Three criteria have been used: fluorescent antibody staining, immuno-gold labelling and electron microscopy, and quantitative measurements of antibody binding using [125I]protein A. When these techniques were applied to living Madin-Darby bovine kidney (MDBK) cells, antibodies against only two desmosomal components, glycoproteins of approximately 115 X 10(3) Mr and 100 X 10(3) Mr, bound to the cell surface. Antibodies against all other components, the 230 and 205 X 10(3) Mr proteins (desmoplakins), the 150 X 10(3) Mr glycoprotein and the 82 and 86 X 10(3) Mr proteins reacted in fluorescent antibody staining only after cells had been fixed and made permeable. MDBK cells were cultured in the presence of univalent fragments (Fab') of anti-desmosomal antibodies for periods from 24 h to 72 h. After these times cells were fixed, made permeable, and stained with anti-desmoplakin antibody to assay for desmosome formation. Fab' derived from anti-100 X 10(3) Mr protein specifically inhibited desmosome formation, whereas Fab's from anti-desmoplakin, anti-150 X 10(3) Mr and anti-82 and 86 X 10(3) Mr proteins were without effect. We conclude that the 100 X 10(3) Mr and the immunologically related 115 X 10(3) Mr components are located on the cell surface and are directly involved in cell-cell adhesion. We have named them desmocollins to denote that they are involved in the adhesive function of desmosomes. The modulation of desmocollin distribution during monolayer formation and establishment of epithelial polarity has also been studied. Fluorescent and immuno-gold labelling using Fab' or IgG at 4 degrees C revealed that desmocollins were initially evenly dispersed over the cell surface. Staining with IgG at 37 degrees C caused the desmocollins to “patch' but not to “cap'. With the establishment of confluency, desmocollins were gradually removed from the upper surfaces of the cells (or masked and rendered inaccessible to antibody) being confined to the lateral and probably basal regions of the cells. Treatment of confluent monolayers with 3 mM-EGTA rendered the desmocollins stainable, probably by causing their release from lateral constraint. Desmocollin staining at the cell surface was not appreciably reduced during 5 h of EGTA treatment, suggesting that desmocollins, unlike desmosomal plaques, may not be internalized after junction breakdown.

scholarly journals A role for phosphoinositide 3-kinase in the completion of macropinocytosis and phagocytosis by macrophages.

1996 ◽  
Vol 135 (5) ◽  
pp. 1249-1260 ◽  
Author(s):  
N Araki ◽  
M T Johnson ◽  
J A Swanson
Keyword(s):  
Cell Surface ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Fluid Phase ◽  
Video Data ◽  
Surface Receptors ◽  
Quantitative Measurements ◽  
Vesicular Membrane ◽  
Macrophage Colony ◽  
Phosphoinositide 3 Kinase

Phosphoinositide 3-kinase (PI 3-kinase) has been implicated in growth factor signal transduction and vesicular membrane traffic. It is thought to mediate the earliest steps leading from ligation of cell surface receptors to increased cell surface ruffling. We show here that inhibitors of PI 3-kinase inhibit endocytosis in macrophages, not by interfering with the initiation of the process but rather by preventing its completion. Consistent with earlier studies, the inhibitors wortmannin and LY294002 inhibited fluid-phase pinocytosis and Fc receptor-mediated phagocytosis, but they had little effect on the receptor-mediated endocytosis of diI-labeled, acetylated, low density lipoprotein. Large solute probes of endocytosis reported greater inhibition by wortmannin than smaller probes did, indicating that macropinocytosis was affected more than micropinocytosis. Since macropinocytosis and phagocytosis are actin-mediated processes, we expected that their inhibition by wortmannin resulted from deficient signaling from macrophage colony-stimulating factor (M-CSF) receptors or Fc receptors to the actin cytoskeleton. However, video microscopy showed cell surface ruffling in wortmannin-treated cells, and increased ruffling after addition of M-CSF or phorbol myristate acetate. Quantitative measurements of video data reported slightly diminished ruffling in wortmannin-treated cells. Remarkably, the ruffles that formed in wortmannin-treated macrophages all receded into the cytoplasm without closing into macropinosomes. Similarly, wortmannin and LY294002 did not inhibit the extension of actin-rich pseudopodia along IgG-opsonized sheep erythrocytes, but instead prevented them from closing into phagosomes. These findings indicate that PI 3-kinase is not necessary for receptor-mediated stimulation of pseudopod extension, but rather functions in the closure of macropinosomes and phagosomes into intracellular organelles.

Translocation of the Nuclear Auto Antigen La to cell Surface: Assembly and Disassembly with the Extracellular Matrix

Autoimmunity ◽  
1991 ◽  
Vol 9 (2) ◽  
pp. 99-107 ◽  
Author(s):  
M. Bachmann ◽  
S. Chang ◽  
A. Bernd ◽  
W. Mayet ◽  
K. H. Meyer Zum Buschenfelde ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Cell Surface ◽  
Surface Assembly

Influence of Calcium Ions on the Plant Cell Surface: Membrane Fusions and Conformational Changes

1974 ◽  
Vol 32 ◽  
pp. 154-155
Author(s):  
D. James Morré ◽  
Charles E. Bracker ◽  
William J. VanDerWoude
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Conformational Changes ◽  
Calcium Ions ◽  
Surface Membrane ◽  
Carboxyl Groups ◽  
Cross Linking ◽  
Ultrastructural Evidence ◽  
Glycine Max L ◽  
Wall Extensibility

Calcium ions in the concentration range 5-100 mM inhibit auxin-induced cell elongation and wall extensibility of plant stems. Inhibition of wall extensibility requires that the tissue be living; growth inhibition cannot be explained on the basis of cross-linking of carboxyl groups of cell wall uronides by calcium ions. In this study, ultrastructural evidence was sought for an interaction of calcium ions with some component other than the wall at the cell surface of soybean (Glycine max (L.) Merr.) hypocotyls.

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