Metabolic Biotinylation of Secreted and Cell Surface Proteins from Mammalian Cells

2001 ◽  
Vol 281 (4) ◽  
pp. 993-1000 ◽  
Author(s):  
M.Brandon Parrott ◽  
Michael A. Barry
Keyword(s):  
Cell Surface ◽  
Mammalian Cells ◽  
Surface Proteins ◽  
Cell Surface Proteins

Detection and Quantification of β2AR Internalization in Living Cells Using FAP-Based Biosensor Technology

2010 ◽  
Vol 15 (6) ◽  
pp. 703-709 ◽  
Author(s):  
Gregory W. Fisher ◽  
Sally A. Adler ◽  
Margaret H. Fuhrman ◽  
Alan S. Waggoner ◽  
Marcel P. Bruchez ◽  
...  
Keyword(s):  
Cell Surface ◽  
Adrenergic Receptor ◽  
High Throughput Screening ◽  
Mammalian Cells ◽  
Living Cells ◽  
Surface Proteins ◽  
Receptor Internalization ◽  
Cell Interior ◽  
Cell Surface Proteins ◽  
Signaling Systems

Ligand-dependent receptor internalization is a feature of numerous signaling systems. In this article, the authors describe a new kind of live-cell biosensor of receptor internalization that takes advantage of fluorogen-activating protein (FAP) technology. Recombinant genes that express the human beta2 adrenergic receptor (β2AR) with FAP domains at their extracellular N-termini were transduced into mammalian cells. Exposure of the cells to membrane-impermeant fluorogens led to a strong fluorescent signal from the cell surface. Agonist-dependent translocation of the receptor from the surface to the cell interior was readily observed and quantified by fluorescence microscopy or flow cytometry in a homogeneous format without wash or separation steps. The approach described here is generalizable to other receptors and cell surface proteins and is adaptable to a variety of fluorescence-based high-throughput screening platforms.

scholarly journals CELL SURFACE PROTEINS OF CRYPTOCOCCUS NEOFORMANS MEDIATE ADHERENCE AND INTERNALISATION INTO MAMMALIAN CELLS

2019 ◽  
Vol 22 (2) ◽  
pp. 24-30
Author(s):  
Khi Khi Choo ◽  
Voon Kin Chin ◽  
Pei Pei Chong ◽  
Siong Hock Ho ◽  
Voon Chen Yong
Keyword(s):  
Cell Surface ◽  
Cryptococcus Neoformans ◽  
Mammalian Cells ◽  
Surface Proteins ◽  
Cell Surface Proteins

What do nanometer molecular trajectories tell us about heterogeneous cell surface domaines?

1995 ◽  
Vol 53 ◽  
pp. 786-787
Author(s):  
Watt W. Webb
Keyword(s):  
Cell Surface ◽  
Lipid Membranes ◽  
Surface Proteins ◽  
Protein Diffusion ◽  
Coated Pits ◽  
Cell Surface Proteins ◽  
Membrane Heterogeneity ◽  
Fluorescence Photobleaching Recovery ◽  
Photobleaching Recovery ◽  
Plasma Membrane Heterogeneity

Plasma membrane heterogeneity is implicit in the existence of specialized cell surface organelles which are necessary for cellular function; coated pits, post and pre-synaptic terminals, microvillae, caveolae, tight junctions, focal contacts and endothelial polarization are examples. The persistence of these discrete molecular aggregates depends on localized restraint of the constituent molecules within specific domaines in the cell surface by strong intermolecular bonds and/or anchorage to extended cytoskeleton. The observed plasticity of many of organelles and the dynamical modulation of domaines induced by cellular signaling evidence evanescent intermolecular interactions even in conspicuous aggregates. There is also strong evidence that universal restraints on the mobility of cell surface proteins persist virtually everywhere in cell surfaces, not only in the discrete organelles. Diffusion of cell surface proteins is slowed by several orders of magnitude relative to corresponding protein diffusion coefficients in isolated lipid membranes as has been determined by various ensemble average methods of measurement such as fluorescence photobleaching recovery(FPR).

scholarly journals Cell Surface Expression of Nrg1 Protein in Candida auris

2021 ◽  
Vol 7 (4) ◽  
pp. 262
Author(s):  
Anuja Paudyal ◽  
Govindsamy Vediyappan
Keyword(s):  
Cell Surface ◽  
Growth Conditions ◽  
Zinc Ion ◽  
Surface Proteins ◽  
Cell Surface Expression ◽  
Unexpected Finding ◽  
Candida Auris ◽  
Cell Surface Proteins ◽  
Biofilm Growth

Candida auris is an emerging antifungal resistant human fungal pathogen increasingly reported in healthcare facilities. It persists in hospital environments, and on skin surfaces, and can form biofilms readily. Here, we investigated the cell surface proteins from C. auris biofilms grown in a synthetic sweat medium mimicking human skin conditions. Cell surface proteins from both biofilm and planktonic control cells were extracted with a buffer containing β-mercaptoethanol and resolved by 2-D gel electrophoresis. Some of the differentially expressed proteins were excised and identified by mass spectrometry. C. albicans orthologs Spe3p, Tdh3p, Sod2p, Ywp1p, and Mdh1p were overexpressed in biofilm cells when compared to the planktonic cells of C. auris. Interestingly, several proteins with zinc ion binding activity were detected. Nrg1p is a zinc-binding transcription factor that negatively regulates hyphal growth in C. albicans. C. auris does not produce true hypha under standard in vitro growth conditions, and the role of Nrg1p in C. auris is currently unknown. Western blot analyses of cell surface and cytosolic proteins of C. auris against anti-CalNrg1 antibody revealed the Nrg1p in both locations. Cell surface localization of Nrg1p in C. auris, an unexpected finding, was further confirmed by immunofluorescence microscopy. Nrg1p expression is uniform across all four clades of C. auris and is dependent on growth conditions. Taken together, the data indicate that C. auris produces several unique proteins during its biofilm growth, which may assist in the skin-colonizing lifestyle of the fungus during its pathogenesis.

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