The E-selectin-ligand ESL-1 is located in the Golgi as well as on microvilli on the cell surface

1997 ◽  
Vol 110 (6) ◽  
pp. 687-694 ◽  
Author(s):  
M. Steegmaier ◽  
E. Borges ◽  
J. Berger ◽  
H. Schwarz ◽  
D. Vestweber
Keyword(s):  
Flow Cytometry ◽  
Cell Surface ◽  
Surface Proteins ◽  
Intact Cells ◽  
Fgf Receptor ◽  
Cell Surface Biotinylation ◽  
Cell Surface Staining ◽  
Surface Staining ◽  
Selectin Ligand ◽  
Golgi Protein

Neutrophils and subsets of lymphocytes bind to E-selectin, a cytokine inducible adhesion molecule on endothelial cells. The E-selectin-ligand-1 (ESL-1) is a high affinity glycoprotein ligand which participates in the binding of mouse myeloid cells to E-selectin. The sequence of mouse ESL-1 is highly homologous to the cysteine rich FGF receptor (CFR) in chicken and the rat Golgi protein MG160. We have analysed the subcellular distribution of ESL-1 by indirect immunofluorescence, flow cytometry, various biochemical techniques and by immunogold scanning electron microscopy. We could localize ESL-1 in the Golgi as well as on the cell surface of 32Dc13 cells and neutrophils. Cell surface staining was confirmed by cell surface biotinylation and by cell surface immunoprecipitations in which antibodies only had access to surface proteins on intact cells. In addition, ESL-1(high) and ESL-1(low) expressing cells, sorted by flow cytometry, gave rise to high and low immunoprecipitation signals for ESL-1, respectively. Based on immunogold labeling of intact cells, we localized ESL-1 on microvilli of 32Dc13 cells and of the lymphoma cell line K46. Quantitative evaluation determined 80% of the total labeling for ESL-1 on microvilli of K46 cells while 69% of the labeling for the control antigen B220 was found on the planar cell surface. These data indicate that ESL-1 occurs at sites on the leukocyte cell surface which are destined for the initiation of cell contacts to the endothelium.

Intracellular cytokine detection by flow cytometry in pigs: Fixation, permeabilization and cell surface staining

2007 ◽  
Vol 327 (1-2) ◽  
pp. 18-29 ◽  
Author(s):  
Petra Zelnickova ◽  
Martin Faldyna ◽  
Hana Stepanova ◽  
Jaroslav Ondracek ◽  
Frantisek Kovaru
Keyword(s):  
Flow Cytometry ◽  
Cell Surface ◽  
Intracellular Cytokine ◽  
Cytokine Detection ◽  
Cell Surface Staining ◽  
Surface Staining

scholarly journals Flagellar adhesion in chlamydomonas induces synthesis of two high molecular weight cell surface proteins

1983 ◽  
Vol 96 (3) ◽  
pp. 589-597 ◽  
Author(s):  
WJ Snell ◽  
A Clausell ◽  
WS Moore
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Adhesion Molecules ◽  
Mating Type ◽  
Surface Proteins ◽  
Cell Wall Proteins ◽  
Intact Cells ◽  
Mating Reaction ◽  
Ionic Detergent ◽  
Flagellar Proteins

Because our previous studies (Snell, W.J., and W.S. Moore, 1980, J. Cell Biol. 84:203- 210) on the mating reaction of chlamydomonas reinhardtii showed that there was an adhesion-induced turnover of proteins whose synthesis is induced during aggregation. Analysis by SDS PAGE and autoradiography showed that proteins of 220,000 M(r) and 165, 000 M(r) (designated A(1) and A(2) respectively) consistently showed a high rate of synthesis only in flagella or flagellar membrane-enriched fractions prepared from aggregating gametes. Since the two proteins were soluble in the non-ionic detergent NP-40 and were removed from intact cells by a brief pronase treatment, it is likely that A(1) and A(2) are membrane proteins expose on the cell surface. A(1) and A(2) were each synthesized by gametes of both mating types (mt(-) and mt(+)) and synthesis of these two proteins could be detected in the normal mating reaction (wild type mt(-) and mt(+)), in mixtures of mt(-) and impotent mt(+) gametes (which could aggregate but not fuse), and in mixtures of gametes of a single mating type with isolated flagella of the opposite mating type. Cells aggregating in tunicamycin, an inhibitor of protein glycosylation, lost their adhesiveness during aggregation and did not synthesize the 220,000 M(r) protein but instead produced a protein (possibly an underglycosylated form of A(1)) of slightly lower mol wt. The 220,000 and 165,000 M(R) proteins appeared to be flagellar proteins and not cell wall proteins because A(1) and A(2) did not co-migrate with previously identified cell wall proteins, and synthesis of the two proteins could not be detected in flagella-less (bald-2) mutant cells. Analysis of the adhesive activity of sucrose gradient fraction of detergent (octyl glucoside)-solubilized flagellar membranes revealed that fractions containing A(1) and A(2) did not have detectable adhesive activity. The possibility remains that A(1) and A(2) are adhesion molecules whose activity could not be measured in the assay we used. Alternatively, the 220,000 and 165,000 M(r) proteins may be inactivated adhesion molecules or else they may be flagellar surface proteins involved only indirectly in the adhesion process.

scholarly journals Dynamic Regulation of a GPCR-Tetraspanin-G Protein Complex on Intact Cells: Central Role of CD81 in Facilitating GPR56-Gαq/11Association

2004 ◽  
Vol 15 (5) ◽  
pp. 2375-2387 ◽  
Author(s):  
Kevin D. Little ◽  
Martin E. Hemler ◽  
Christopher S. Stipp
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
Cell Activation ◽  
Surface Proteins ◽  
Membrane Microdomains ◽  
Cholesterol Depletion ◽  
Scaffolding Proteins ◽  
Intact Cells ◽  
Heterotrimeric G Protein ◽  
Vast Number

By means of a variety of intracellular scaffolding proteins, a vast number of heterotrimeric G protein–coupled receptors (GPCRs) may achieve specificity in signaling through a much smaller number of heterotrimeric G proteins. Members of the tetraspanin family organize extensive complexes of cell surface proteins and thus have the potential to act as GPCR scaffolds; however, tetraspanin-GPCR complexes had not previously been described. We now show that a GPCR, GPR56/TM7XN1, and heterotrimeric G protein subunits, Gαq, Gα11, and Gβ, associate specifically with tetraspanins and CD81, but not with other tetraspanins. CD9 Complexes of GPR56 with CD9 and CD81 remained intact when fully solubilized and were resistant to cholesterol depletion. Hence they do not depend on detergent-insoluble, raft-like membrane microdomains for stability. A central role for CD81 in promoting or stabilizing a GPR56-CD81-Gαq/11complex was revealed by CD81 immunodepletion and reexpression experiments. Finally, antibody engagement of cell surface CD81 or cell activation with phorbol ester revealed two distinct mechanisms by which GPR56-CD81-Gαq/11complexes can be dynamically regulated. These data reveal a potential role for tetraspanins CD9 and CD81 as GPCR scaffolding proteins.

scholarly journals Coupling of Cell Surface Biotinylation and SILAC-Based Quantitative Proteomics Identified Myoferlin as a Potential Therapeutic Target for Nasopharyngeal Carcinoma Metastasis

2021 ◽  
Vol 9 ◽  
Author(s):  
Maoyu Li ◽  
Fang Peng ◽  
Guoqiang Wang ◽  
Xujun Liang ◽  
Meiying Shao ◽  
...  
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Cell Surface ◽  
Therapeutic Target ◽  
Surface Proteins ◽  
Differentially Expressed ◽  
Attractive Potential ◽  
Potential Therapeutic Target ◽  
Cell Surface Proteins ◽  
Mesenchymal Transition ◽  
Cell Surface Biotinylation

Distant metastasis is a major cause of treatment failure in nasopharyngeal carcinoma (NPC) patients. Cell surface proteins represent attractive targets for cancer diagnosis or therapy. However, the cell surface proteins associated with NPC metastasis are poorly understood. To identify potential therapeutic targets for NPC metastasis, we isolated cell surface proteins from two isogenic NPC cell lines, 6-10B (low metastatic) and 5-8F (highly metastatic), through cell surface biotinylation. Stable isotope labeling by amino acids in cell culture (SILAC) based proteomics was applied to comprehensively characterize the cell surface proteins related with the metastatic phenotype. We identified 294 differentially expressed cell surface proteins, including the most upregulated protein myoferlin (MYOF), two receptor tyrosine kinases(RTKs) epidermal growth factor receptor (EGFR) and ephrin type-A receptor 2 (EPHA2) and several integrin family molecules. These differentially expressed proteins are enriched in multiple biological pathways such as the FAK-PI3K-mTOR pathway, focal adhesions, and integrin-mediated cell adhesion. The knockdown of MYOF effectively suppresses the proliferation, migration and invasion of NPC cells. Immunohistochemistry analysis also showed that MYOF is associated with NPC metastasis. We experimentally confirmed, for the first time, that MYOF can interact with EGFR and EPHA2. Moreover, MYOF knockdown could influence not only EGFR activity and its downstream epithelial–mesenchymal transition (EMT), but also EPHA2 ligand-independent activity. These findings suggest that MYOF might be an attractive potential therapeutic target that has double effects of simultaneously influencing EGFR and EPHA2 signaling pathway. In conclusion, this is the first study to profile the cell surface proteins associated with NPC metastasis and provide valuable resource for future researches.

scholarly journals Nuclear Translocation of fibroblast growth factor (FGF) receptors in response to FGF-2.

1996 ◽  
Vol 134 (2) ◽  
pp. 529-536 ◽  
Author(s):  
P A Maher
Keyword(s):  
Cell Surface ◽  
Nuclear Translocation ◽  
Cell Types ◽  
Surface Proteins ◽  
Nuclear Fraction ◽  
Fgf Receptor ◽  
Quiescent Cells ◽  
3T3 Fibroblasts ◽  
Different Cell Types ◽  
Dose Dependent Increase

Members of the FGF family of growth factors localize to the nuclei in a variety of different cell types. To determine whether FGF receptors are also present within nuclei and if this localization is regulated by FGFs, nuclei were prepared from quiescent and FGF-2-treated Swiss 3T3 fibroblasts and examined for the presence of FGF receptors by immunoblotting with an antibody produced against the extracellular domain of FGF receptor-1 (FGFR-1). Little or no FGFR-1 is detected in nuclei prepared from quiescent cells. When cells are treated with FGF-2, however, there is a time- and dose-dependent increase in the association of FGFR-1 immunoreactivity with the nucleus. In contrast, treatment with either EGF or 10% serum does not increase the association of FGFR-1 with the nucleus. When cell surface proteins are labeled with biotin, a biotinylated FGFR-1 is detected in the nuclear fraction prepared from FGF-2-treated, but not untreated, cells indicating that the nuclear-associated FGFR-1 immunoreactivity derives from the cell surface. The presence of FGFR-1 in the nuclei of FGF-2-treated cells was confirmed by immunostaining with a panel of different FGFR-1 antibodies, including one directed against the COOH-terminal domain of the protein. Fractionation of nuclei from FGF-2-treated cells indicates that nuclear FGFR-1 is localized to the nuclear matrix, suggesting that the receptor may play a role in regulating gene activity.

scholarly journals Comprehensive cell surface proteomics defines markers of classical, intermediate and non-classical monocytes

2020 ◽  
Author(s):  
Benjamin J. Ravenhill ◽  
Lior Soday ◽  
Jack Houghton ◽  
Robin Antrobus ◽  
Michael P. Weekes
Keyword(s):  
Cell Surface ◽  
Surface Proteins ◽  
Monocyte Subset ◽  
Protein Markers ◽  
Phenotypic Differences ◽  
Tandem Mass Tag ◽  
Cell Surface Biotinylation ◽  
Cd16 Expression ◽  
Surface Proteome ◽  
Classical Monocytes

AbstractMonocytes are a critical component of the cellular innate immune system, and can be subdivided into classical, intermediate and non-classical subsets on the basis of surface CD14 and CD16 expression. Classical monocytes play the canonical role of phagocytosis, and account for the majority of circulating cells. Intermediate and non-classical cells are known to exhibit varying levels of phagocytosis and cytokine secretion, and are differentially expanded in certain pathological states. Characterisation of cell surface proteins expressed by each subset is informative not only to improve understanding of phenotype, but may also provide biological insights into function. Here we use highly multiplexed Tandem-Mass-Tag (TMT)-based mass spectrometry with selective cell surface biotinylation to characterise the classical monocyte surface proteome, then interrogate the phenotypic differences between each monocyte subset to identify novel protein markers.

scholarly journals Cyclopropene derivatives of aminosugars for metabolic glycoengineering

2019 ◽  
Vol 15 ◽  
pp. 584-601 ◽  
Author(s):  
Jessica Hassenrück ◽  
Valentin Wittmann
Keyword(s):  
Cell Surface ◽  
Diels Alder ◽  
Inverse Electron Demand ◽  
Distinct Cell ◽  
Chemical Reporter ◽  
Cell Surface Staining ◽  
Surface Staining ◽  
Metabolic Glycoengineering ◽  
Derivatives Of ◽  
Electron Demand

Cyclopropenes have been proven valuable chemical reporter groups for metabolic glycoengineering (MGE). They readily react with tetrazines in an inverse electron-demand Diels–Alder (DAinv) reaction, a prime example of a bioorthogonal ligation reaction, allowing their visualization in biological systems. Here, we present a comparative study of six cyclopropene-modified hexosamine derivatives and their suitability for MGE. Three mannosamine derivatives in which the cyclopropene moiety is attached to the sugar by either an amide or a carbamate linkage and that differ by the presence or absence of a stabilizing methyl group at the double bond have been examined. We determined their DAinv reaction kinetics and their labeling intensities after metabolic incorporation. To determine the efficiencies by which the derivatives are metabolized to sialic acids, we synthesized and investigated the corresponding cyclopropane derivatives because cyclopropenes are not stable under the analysis conditions. From these experiments, it became obvious that N-(cycloprop-2-en-1-ylcarbonyl)-modified (Cp-modified) mannosamine has the highest metabolic acceptance. However, carbamate-linked N-(2-methylcycloprop-2-en-1-ylmethyloxycarbonyl)-modified (Cyoc-modified) mannosamine despite its lower metabolic acceptance results in the same cell-surface labeling intensity due to its superior reactivity in the DAinv reaction. Based on the high incorporation efficiency of the Cp derivative we synthesized and investigated two new Cp-modified glucosamine and galactosamine derivatives. Both compounds lead to comparable, distinct cell-surface staining after MGE. We further found that the amide-linked Cp-modified glucosamine derivative but not the Cyoc-modified glucosamine is metabolically converted to the corresponding sialic acid.

Unexpected interference in cell surface staining by monoclonal antibodies to unrelated antigens

2014 ◽  
pp. n/a-n/a ◽  
Author(s):  
Martina De Vita ◽  
Valentina Catzola ◽  
Alexia Buzzonetti ◽  
Marco Fossati ◽  
Alessandra Battaglia ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Surface ◽  
Cell Surface Staining ◽  
Surface Staining
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