Investigation of Neurotransmitter Receptors in Brain Slices Using Cell Surface Biotinylation

2016 ◽  
pp. 39-48 ◽  
Author(s):  
Elek Molnár
Keyword(s):  
Cell Surface ◽  
Brain Slices ◽  
Neurotransmitter Receptors ◽  
Cell Surface Biotinylation

scholarly journals A Cell Surface Biotinylation Assay to Reveal Membrane-associated Neuronal Cues: Negr1 Regulates Dendritic Arborization

2013 ◽  
Vol 13 (3) ◽  
pp. 733-748 ◽  
Author(s):  
Francesca Pischedda ◽  
Joanna Szczurkowska ◽  
Maria Daniela Cirnaru ◽  
Florian Giesert ◽  
Elena Vezzoli ◽  
...  
Keyword(s):  
Cell Surface ◽  
Dendritic Arborization ◽  
Cell Surface Biotinylation ◽  
A Cell

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.

scholarly journals Targeting neurotransmitter receptors with nanoparticles in vivo allows single-molecule tracking in acute brain slices

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Juan A. Varela ◽  
Julien P. Dupuis ◽  
Laetitia Etchepare ◽  
Agnès Espana ◽  
Laurent Cognet ◽  
...  
Keyword(s):  
Single Molecule ◽  
Brain Slices ◽  
Neurotransmitter Receptors ◽  
Single Molecule Tracking

scholarly journals MAGI-1 interacts with Slo1 channel proteins and suppresses Slo1 expression on the cell surface

2009 ◽  
Vol 297 (1) ◽  
pp. C55-C65 ◽  
Author(s):  
Lon D. Ridgway ◽  
Eun Young Kim ◽  
Stuart E. Dryer
Keyword(s):  
Cell Surface ◽  
Outward Current ◽  
Cell Types ◽  
Surface Expression ◽  
Scaffolding Protein ◽  
Actin Binding ◽  
Differentiated Cells ◽  
Cell Surface Biotinylation ◽  
Bait Construct ◽  
Wide Range

Large conductance Ca2+-activated K+ (BKCa) channels encoded by the Slo1 gene (also known as KCNMA1) are physiologically important in a wide range of cell types and form complexes with a number of other proteins that affect their function. We performed a yeast two-hybrid screen to identify proteins that interact with BKCa channels using a bait construct derived from domains in the extreme COOH-terminus of Slo1. A protein known as membrane-associated guanylate kinase with inverted orientation protein-1 (MAGI-1) was identified in this screen. MAGI-1 is a scaffolding protein that allows formation of complexes between certain transmembrane proteins, actin-binding proteins, and other regulatory proteins. MAGI-1 is expressed in a number of tissues, including podocytes and the brain. The interaction between MAGI-1 and BKCa channels was confirmed by coimmunoprecipitation and glutathione S-transferase pull-down assays in differentiated cells of a podocyte cell line and in human embryonic kidneys (HEK)293T cells transiently coexpressing MAGI-1a and three different COOH-terminal Slo1 variants. Coexpression of MAGI-1 with Slo1 channels in HEK-293T cells results in a significant reduction in the surface expression of Slo1, as assessed by cell-surface biotinylation assays, confocal microscopy, and whole cell recordings. Partial knockdown of endogenous MAGI-1 expression by small interfering RNA (siRNA) in differentiated podocytes increased the surface expression of endogenous Slo1 as assessed by electrophysiology and cell-surface biotinylation assays, whereas overexpression of MAGI-1a reduced steady-state voltage-evoked outward current through podocyte BKCa channels. These data suggest that MAGI-1 plays a role in regulation of surface expression of BKCa channels in the kidney and possibly in other tissues.

Differential surface localization and temperature-dependent expression of the Candida albicans CSH1 protein

Microbiology ◽  
2004 ◽  
Vol 150 (2) ◽  
pp. 285-292 ◽  
Author(s):  
David R. Singleton ◽  
Kevin C. Hazen
Keyword(s):  
Candida Albicans ◽  
Cell Surface ◽  
Cell Surface Hydrophobicity ◽  
Surface Hydrophobicity ◽  
Growth Conditions ◽  
Northern Blotting ◽  
Temperature Dependent ◽  
Cell Surface Biotinylation ◽  
Extracellular Compartment ◽  
Primary Amino

Cell-surface hydrophobicity (CSH) in Candida albicans contributes to virulence and can be conveniently regulated in planktonic cultures by altering growth temperature. The CSH1 gene is the first candidate gene that has been demonstrated to play a role in affecting the CSH phenotype. However, the primary amino acid sequence of the CSH1 gene product suggests that the protein should be restricted to the cytoplasm. A majority of the protein appears to demonstrate that localization. Cell-surface biotinylation and limited glucanase digestion were used to determine and estimate the relative amount of Csh1p in the extracellular compartment in comparison to the cytoplasmic pool. Additionally, Western and Northern blotting were used to assess expression of the CSH1 gene under different growth conditions. Compared with cells grown at 23 °C, the total cellular levels of Csh1p are significantly greater at elevated growth temperatures. Detection of Csh1p on the cell surface correlates with the level of overall protein expression. The temperature-dependent regulation and surface presentation of Csh1p suggests a mechanism for regulating the CSH phenotype.

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 TRAIL Induces Nuclear Translocation and Chromatin Localization of TRAIL Death Receptors

Cancers ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 1167 ◽  
Author(s):  
Ufuk Mert ◽  
Alshaimaa Adawy ◽  
Elisabeth Scharff ◽  
Pierre Teichmann ◽  
Anna Willms ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Nuclear Export ◽  
Nuclear Translocation ◽  
Nuclear Accumulation ◽  
Nuclear Trafficking ◽  
Surface Receptors ◽  
Tnf Superfamily ◽  
Cell Surface Biotinylation ◽  
Intracellular Compartments

Binding of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to the plasma membrane TRAIL-R1/-R2 selectively kills tumor cells. This discovery led to evaluation of TRAIL-R1/-R2 as targets for anti-cancer therapy, yet the corresponding clinical trials were disappointing. Meanwhile, it emerged that many cancer cells are TRAIL-resistant and that TRAIL-R1/-R2-triggering may lead to tumor-promoting effects. Intriguingly, recent studies uncovered specific functions of long ignored intracellular TRAIL-R1/-R2, with tumor-promoting functions of nuclear (n)TRAIL-R2 as the regulator of let-7-maturation. As nuclear trafficking of TRAIL-Rs is not well understood, we addressed this issue in our present study. Cell surface biotinylation and tracking of biotinylated proteins in intracellular compartments revealed that nTRAIL-Rs originate from the plasma membrane. Nuclear TRAIL-Rs-trafficking is a fast process, requiring clathrin-dependent endocytosis and it is TRAIL-dependent. Immunoprecipitation and immunofluorescence approaches revealed an interaction of nTRAIL-R2 with the nucleo-cytoplasmic shuttle protein Exportin-1/CRM-1. Mutation of a putative nuclear export sequence (NES) in TRAIL-R2 or the inhibition of CRM-1 by Leptomycin-B resulted in the nuclear accumulation of TRAIL-R2. In addition, TRAIL-R1 and TRAIL-R2 constitutively localize to chromatin, which is strongly enhanced by TRAIL-treatment. Our data highlight the novel role for surface-activated TRAIL-Rs by direct trafficking and signaling into the nucleus, a previously unknown signaling principle for cell surface receptors that belong to the TNF-superfamily.

scholarly journals Mutant voltage-gated Na+ channels can exert a dominant negative effect through coupled gating

2018 ◽  
Vol 315 (5) ◽  
pp. H1250-H1257 ◽  
Author(s):  
Jérôme Clatot ◽  
Yang Zheng ◽  
Aurore Girardeau ◽  
Haiyan Liu ◽  
Kenneth R. Laurita ◽  
...  
Keyword(s):  
Cell Surface ◽  
Single Channel ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Wild Type ◽  
Voltage Gated ◽  
Cell Surface Biotinylation ◽  
Negative Effect ◽  
Coupled Gating ◽  
Α Subunits

Mutations in voltage-gated Na+ channels have been linked to several channelopathies leading to a wide variety of diseases including cardiac arrhythmias, epilepsy, and myotonia. We have previously demonstrated that voltage-gated Na+ channel (Nav)1.5 trafficking-deficient mutant channels could lead to a dominant negative effect by impairing trafficking of the wild-type (WT) channel. We also reported that voltage-gated Na+ channels associate as dimers with coupled gating properties. Here, we hypothesized that the dominant negative effect of mutant Na+ channels could also occur through coupled gating. This was tested using cell surface biotinylation and single channel recordings to measure the gating probability and coupled gating of the dimers. As previously reported, coexpression of Nav1.5-L325R with WT channels led to a dominant negative effect, as reflected by a 75% reduction in current density. Surprisingly, cell surface biotinylation showed that Nav1.5-L325R mutant is capable of trafficking, with 40% of Nav1.5-L325R reaching the cell surface when expressed alone. Importantly, even though a dominant negative effect on the Na+ current is observed when WT and Nav1.5-L325R are expressed together, the total Nav channel cell surface expression was not significantly altered compared with WT channels alone. Thus, the trafficking deficiency could not explain the 75% decrease in inward Na+ current. Interestingly, single channel recordings showed that Nav1.5-L325R exerted a dominant negative effect on the WT channel at the gating level. Both coupled gating and gating probability of WT:L325R dimers were drastically impaired. We conclude that dominant negative suppression exerted by Nav1.5 mutants can also be caused by impairing the WT gating probability, a mechanism resulting from the dimerization and coupled gating of voltage-gated Na+ channel α-subunits. NEW & NOTEWORTHY The presence of dominant negative mutations in the Na+ channel gene leading to Brugada syndrome was supported by our recent findings that Na+ channel α-subunits form dimers. Up until now, the dominant negative effect was thought to be caused by the interaction of the wild-type Na+ channel with trafficking-deficient mutant channels. However, the present study demonstrates that coupled gating of voltage-gated Na+ channels can also be responsible for the dominant negative effect leading to arrhythmias.

Cell surface biotinylation of GLUT4

1997 ◽  
Vol 25 (3) ◽  
pp. 470S-470S ◽  
Author(s):  
FRANCOISE KOUMANOV ◽  
JING YANG ◽  
ALISON JONES ◽  
YASUMARU HATANAKA ◽  
GEOFFREY D. HOLMAN
Keyword(s):  
Cell Surface ◽  
Cell Surface Biotinylation

Vasopressin-inducible ubiquitin-specific protease 10 increases ENaC cell surface expression by deubiquitylating and stabilizing sorting nexin 3

2008 ◽  
Vol 295 (4) ◽  
pp. F889-F900 ◽  
Author(s):  
Sheerazed Boulkroun ◽  
Dorothée Ruffieux-Daidié ◽  
Jean-Jacques Vitagliano ◽  
Olivier Poirot ◽  
Roch-Philippe Charles ◽  
...  
Keyword(s):  
Cell Surface ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Long Term Effects ◽  
Hek 293 ◽  
Fourfold Increase ◽  
Sorting Nexin ◽  
Cell Surface Biotinylation ◽  
Specific Protease ◽  
Ubiquitin Specific Protease

Adjustment of Na+ balance in extracellular fluids is achieved by regulated Na+ transport involving the amiloride-sensitive epithelial Na+ channel (ENaC) in the distal nephron. In this context, ENaC is controlled by a number of hormones, including vasopressin, which promotes rapid translocation of water and Na+ channels to the plasma membrane and long-term effects on transcription of vasopressin-induced and -reduced transcripts. We have identified a mRNA encoding the deubiquitylating enzyme ubiquitin-specific protease 10 (Usp10), whose expression is increased by vasopressin at both the mRNA and the protein level. Coexpression of Usp10 in ENaC-transfected HEK-293 cells causes a more than fivefold increase in amiloride-sensitive Na+ currents, as measured by whole cell patch clamping. This is accompanied by a three- to fourfold increase in surface expression of α- and γ-ENaC, as shown by cell surface biotinylation experiments. Although ENaC is well known to be regulated by its direct ubiquitylation, Usp10 does not affect the ubiquitylation level of ENaC, suggesting an indirect effect. A two-hybrid screen identified sorting nexin 3 (SNX3) as a novel substrate of Usp10. We show that it is a ubiquitylated protein that is degraded by the proteasome; interaction with Usp10 leads to its deubiquitylation and stabilization. When coexpressed with ENaC, SNX3 increases the channel's cell surface expression, similarly to Usp10. In mCCDcl1 cells, vasopressin increases SNX3 protein but not mRNA, supporting the idea that the vasopressin-induced Usp10 deubiquitylates and stabilizes endogenous SNX3 and consequently promotes cell surface expression of ENaC.

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