Research in the Lowe-Krentz lab is supported by research grant HL54269 from the National Institutes of Health to LLK.

<ol>
	<li>Pugh, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmembrane+Protein+184A+Is+a+Receptor+Required+for+Vascular+Smooth+Muscle+Cell+Responses+to+Heparin.">Transmembrane Protein 184A Is a Receptor Required for Vascular Smooth Muscle Cell Responses to Heparin.</a> J Biol Chem. 291, 5326-5341 (2016).</li><li>Daher, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Toxoplasma+TgPH1,+a+pleckstrin+homology+domain-containing+protein+that+binds+to+the+phosphoinositide+PI(3,5)P.">Identification of Toxoplasma TgPH1, a pleckstrin homology domain-containing protein that binds to the phosphoinositide PI(3,5)P.</a> Mol Biochem Parasitol. , (2016).</li><li>Vit, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+identification+of+membrane+proteins+based+on+analysis+of+trypsin-protected+transmembrane+segments.">Large-scale identification of membrane proteins based on analysis of trypsin-protected transmembrane segments.</a> J Proteomics. , (2016).</li><li>Attwood, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topology+based+identification+and+comprehensive+classification+of+four-transmembrane+helix+containing+proteins+(4TMs)+in+the+human+genome.">Topology based identification and comprehensive classification of four-transmembrane helix containing proteins (4TMs) in the human genome.</a> BMC genomics. 17, 268 (2016).</li><li>Zou, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-Wide+Identification+of+Jatropha+curcas+Aquaporin+Genes+and+the+Comparative+Analysis+Provides+Insights+into+the+Gene+Family+Expansion+and+Evolution+in+Hevea+brasiliensis.">Genome-Wide Identification of Jatropha curcas Aquaporin Genes and the Comparative Analysis Provides Insights into the Gene Family Expansion and Evolution in Hevea brasiliensis.</a> Front Plant Sci. 7, 395 (2016).</li><li>Gilotti, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparin+responses+in+vascular+smooth+muscle+cells+involve+cGMP-dependent+protein+kinase+(PKG).">Heparin responses in vascular smooth muscle cells involve cGMP-dependent protein kinase (PKG).</a> J Cell Physiol. 229, 2142-2152 (2014).</li><li>Farwell, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparin+Decreases+in+Tumor+Necrosis+Factor+alpha+(TNFalpha)-induced+Endothelial+Stress+Responses+Require+Transmembrane+Protein+184A+and+Induction+of+Dual+Specificity+Phosphatase+1.">Heparin Decreases in Tumor Necrosis Factor alpha (TNFalpha)-induced Endothelial Stress Responses Require Transmembrane Protein 184A and Induction of Dual Specificity Phosphatase 1.</a> J Biol Chem. 291, 5342-5354 (2016).</li><li>Xu, D., Esko, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demystifying+heparan+sulfate-protein+interactions.">Demystifying heparan sulfate-protein interactions.</a> Annu Rev Biochem. 83, 129-157 (2014).</li><li>Chiodelli, P., Bugatti, A., Urbinati, C., Rusnati, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparin/Heparan+sulfate+proteoglycans+glycomic+interactome+in+angiogenesis:+biological+implications+and+therapeutical+use.">Heparin/Heparan sulfate proteoglycans glycomic interactome in angiogenesis: biological implications and therapeutical use.</a> Molecules. 20, 6342-6388 (2015).</li><li>Slee, J. B., Lowe-Krentz, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+realignment+and+cofilin+regulation+are+essential+for+barrier+integrity+during+shear+stress.">Actin realignment and cofilin regulation are essential for barrier integrity during shear stress.</a> J Cell Biochem. 114, 782-795 (2013).</li><li>Patton, W. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+heparin-binding+protein+using+monoclonal+antibodies+that+block+heparin+binding+to+porcine+aortic+endothelial+cells.">Identification of a heparin-binding protein using monoclonal antibodies that block heparin binding to porcine aortic endothelial cells.</a> The Biochemical journal. 311, 461-469 (1995).</li><li>Doggett, T. M., Breslin, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+the+actin+cytoskeleton+in+live+endothelial+cells+expressing+GFP-actin.">Study of the actin cytoskeleton in live endothelial cells expressing GFP-actin.</a> J Vis Exp. , (2011).</li><li>Skalamera, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+genome+scale+lentiviral+vector+library+for+EF1alpha+promoter-driven+expression+of+human+ORFs+and+identification+of+human+genes+affecting+viral+titer.">Generation of a genome scale lentiviral vector library for EF1alpha promoter-driven expression of human ORFs and identification of human genes affecting viral titer.</a> PloS one. 7, 51733 (2012).</li><li>Castro, M., Nikolaev, V. O., Palm, D., Lohse, M. J., Vilardaga, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turn-on+switch+in+parathyroid+hormone+receptor+by+a+two-step+parathyroid+hormone+binding+mechanism.">Turn-on switch in parathyroid hormone receptor by a two-step parathyroid hormone binding mechanism.</a> Proc Natl Acad Sci U S A. 102, 16084-16089 (2005).</li><li>Albertazzi, L., Arosio, D., Marchetti, L., Ricci, F., Beltram, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+FRET+analysis+with+the+EGFP-mCherry+fluorescent+protein+pair.">Quantitative FRET analysis with the EGFP-mCherry fluorescent protein pair.</a> Photochem Photobiol. 85, 287-297 (2009).</li><li>Wang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Domain+organization+of+the+ATP-sensitive+potassium+channel+complex+examined+by+fluorescence+resonance+energy+transfer.">Domain organization of the ATP-sensitive potassium channel complex examined by fluorescence resonance energy transfer.</a> J Biol Chem. 288, 4378-4388 (2013).</li><li>Christiansen, E., Hudson, B. D., Hansen, A. H., Milligan, G., Ulven, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+Characterization+of+a+Potent+Free+Fatty+Acid+Receptor+1+(FFA1)+Fluorescent+Tracer.">Development and Characterization of a Potent Free Fatty Acid Receptor 1 (FFA1) Fluorescent Tracer.</a> J Med Chem. 59, 4849-4858 (2016).</li><li>Chiang, C. F., Okou, D. T., Griffin, T. B., Verret, C. R., Green Williams, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=fluorescent+protein+rendered+susceptible+to+proteolysis:+positions+for+protease-sensitive+insertions.">fluorescent protein rendered susceptible to proteolysis: positions for protease-sensitive insertions.</a> Arch Biochem Biophys. 394, 229-235 (2001).</li></ol>

Os autores não têm nada a revelar.

Os protocolos aqui relatados foram concebidos para proporcionar provas de confirmação para a identificação de um receptor TMEM184A como heparina em células vasculares 1. knockdown técnicas são rotineiramente utilizado como um mecanismo para confirmar a identificação de novas proteínas. No entanto, alguma perda funcional após knockdown não é geralmente suficiente a prova de que uma proteína candidata é realmente o receptor correcto (ou outra proteína funcional). É também importan...

Identificação de proteínas candidatas para novos funções muitas vezes depende protocolos de isolamento à base de afinidade, seguido por determinação da sequência parcial. Exemplos recentes de proteínas recentemente identificados incluem proteína transmembranar 184A (TMEM184A), um receptor de heparina identificado após interacções de afinidade de heparina 1, e TgPH1, um proteína do domínio de homologia que se liga plecstrina PI fosfoinositida (3,5) P 2 2. Outro novo identificação de proteínas envolve a análise de sequência directa de péptidos, tais como que por Vit, et ai. que usou peptídeos transmembrana para identificar produtos de proteínas de genes previamente descaracterizados 3. Do mesmo modo, a identificação de sequências de proteína nova pode ser realizada utilizando bioinformática procura de famílias de proteínas previamente caracterizadas como a identificação de novas proteínas 4TM 4. Exame de sequências de genes da família aquaporin tem alpor isso permitiu a identificação de novos membros com novas funções 5. Após a identificação, análise da função da proteína é tipicamente um passo seguinte, que pode, por vezes, ser examinada utilizando um ensaio específico da função da proteína, tais como no caso aquaporina. Quando possível, a função de uma proteína recentemente identificada pode ser examinada com enzimática específica ou ensaios in vitro de função semelhante. Porque muitas funções de novas proteínas dependem de interacções complexas que ocorrem apenas em células intactas ou organismos, em ensaios in vitro não são sempre eficazes. No entanto, os ensaios in vivo devem ser concebidos de tal maneira que eles dependem da sequência do gene. Em cultura de células, e / ou organismos modelo simples, knockdown pode fornecer elementos de prova para a identificação / função da proteína 6. Com novas proteínas identificadas como notado acima, é muitas vezes insuficiente para derrubar simplesmente uma proteína para confirmar a função, umad o desenho de ensaios funcionais in vivo que dependem da sequência do gene torna-se importante para a caracterização de novas proteínas. A recente identificação de TMEM184A como um receptor de heparina (que modula a proliferação no músculo liso vascular e respostas inflamatórias em células endoteliais), utilizando cromatografia de afinidade e de MALDI MS 1, 7 fornecido uma oportunidade para desenvolver um conjunto de ensaios após knockdown produziu resultados consistentes com a identificação . Uma revisão recente confirmou que a heparina interage especificamente com muitos factores de crescimento, os seus receptores, os componentes da matriz extracelular, adesão celular, receptores e outras proteínas 8. No sistema vascular, a heparina e os proteoglicanos de sulfato de heparano (contendo cadeias de sulfato de heparano de uma estrutura semelhante à heparina) interagem com várias centenas de proteínas 9. Para confirmar funcionalmente that TMEM184A estava envolvido com a captação de heparina e de ligação, as técnicas que empregaram a construção de gene para TMEM184A foram desenvolvidos. O presente relatório inclui um conjunto de ensaios com base em um GFP-TMEM184A construção para utilização em confirmando a identidade do TMEM184A como um receptor de heparina.

<table><tbody><tr><td>GFP-TMEM184A construct</td><td>OriGene</td><td>RG213192</td><td></td></tr><tr><td>Rhodamine-Heparin</td><td>Creative PEGWorks</td><td>HP-204</td><td>Light Sensitive</td></tr><tr><td>Fluorescein-Heparin</td><td>Creative PEGWorks</td><td>HP-201</td><td>Light Sensitive</td></tr><tr><td>Mowiol</td><td>EMD Millipore</td><td>475904-100GM</td><td></td></tr><tr><td>Paraformaldehyde (PFA, methanol free)</td><td>Thermo Sci Pierce Biotech, available through Fisher Scientific</td><td>PI28908 at Fisher</td><td>Use in Fume Hood</td></tr><tr><td>Reacti-bind neutravidin plates (Avidin coated black 96-well dishes)</td><td>Thermo Sci Pierce Biotech, through Fisher Scientific</td><td>PI15510 at Fisher</td><td>Pay attention to shelf-life</td></tr><tr><td>Black 96-well plates</td><td>Corning Life Sciences Plastic, purchased through Fisher Scientific</td><td>064432 at Fisher</td><td></td></tr><tr><td>A7r5 vascular smooth muscle cell line</td><td>ATCC</td><td>CRL 1444</td><td>Can be exchanged into MEM medium&lt;sup&gt;1&lt;/sup&gt;</td></tr><tr><td>BAOEC bovine aortic endothelial cells</td><td>Cell Applications, Inc.</td><td>B304-05</td><td>Culture as recommended initially, can be exchanged into MEM medium for continuing culture&lt;sup&gt;1,7&lt;/sup&gt;</td></tr><tr><td>BAOSMC bovine aortic smooth muscle cells</td><td>Cell Applications, Inc.</td><td>B354-05</td><td>Culture as recommended initially, can be exchanged into MEM medium for continuing culture&lt;sup&gt;1&lt;/sup&gt;</td></tr><tr><td>RAOEC rat aortic endothelial cells</td><td>Cell Applications, Inc.</td><td>R304-05a</td><td>Culture as recommended initially, can be exchanged into MEM medium for continuing culture&lt;sup&gt;7&lt;/sup&gt;</td></tr><tr><td>Biotinylated anti-GFP</td><td>Thermo Sci Pierce Biotech, through Fisher Scientific</td><td>MA5-15256-BTIN</td><td></td></tr><tr><td>Streptavidin-coated beads</td><td>Sigma</td><td>S1638</td><td></td></tr><tr><td>HeBS</td><td>Available from Bio-Rad</td><td></td><td>Can be prepared in the lab. The pH is 6.8</td></tr><tr><td>TMEM184A antibody to the N-terminus</td><td>Santa Cruz Biotechnology</td><td>sc292006</td><td>Only known TMEM184A antibody to N-terminal region.</td></tr><tr><td>TMEM184A antibody to the C-terminus</td><td>Obtained from ProSci Inc, Poway, CA&amp;nbsp;</td><td>Pro Sci 5681</td><td>ProSci used in &lt;strong&gt;Figure 1&lt;/strong&gt;</td></tr><tr><td>GFP antibodies</td><td>Santa Cruz Biotechnology</td><td>sc9996</td><td>Used in &lt;strong&gt;Figure 5&lt;/strong&gt;</td></tr><tr><td>Secondary antibodies, labeled with TRITC or Cy3</td><td>Jackson ImmunoResearch Laboratories, Inc, West Grove, PA</td><td>711 025 152 (donkey anti-rabbit, TRITC)&lt;br/&gt; 715 165 150 (donkey anti-mouse, Cy3)</td><td>Minimal cross-reactivity to minimize any nonspecific staining.</td></tr><tr><td>CHAPS</td><td>Purchased from Sigma</td><td>C5849</td><td>Note that this specific catalog number has been discontinued.&amp;nbsp; Supplier will provide information regarding replacement.</td></tr><tr><td>Live imaging 35-mm dishes</td><td>MatTek (Ashland MA)</td><td>P35G-1.0 &amp;ndash; 20 mm - C</td><td></td></tr><tr><td>Confocal Microscope</td><td>Zeiss</td><td>LSM 510 Meta with a 63X oil-immersion lens</td><td>Used for images and live-imaging in &lt;strong&gt;Figures 1, 2 and 3&lt;/strong&gt;</td></tr><tr><td>Confocal Microscope</td><td>Nikon</td><td>C2+ confocal with a 60X oil-immersion lens</td><td>Used for images in &lt;strong&gt;Figure 5&lt;/strong&gt;</td></tr><tr><td>Confocal Microscope</td><td>Zeiss</td><td>Zeiss LSM 880 with a 63X oil-immersion lens</td><td>Used for images in &lt;strong&gt;Figure 2C&lt;/strong&gt;</td></tr><tr><td>Electroporation equipment</td><td>Bio-Rad</td><td>Gene Pulser X-Cell System</td><td></td></tr><tr><td>Electroporation cuvettes</td><td>Available from MidSci</td><td>EC2L</td><td>Can also be obtained from equipment supplier</td></tr><tr><td>Plate reader</td><td>TECAN</td><td>TECAN Infinite&amp;reg; m200 Pro plate reader</td><td>Readings in the middle of the wells rather than at the surface.</td></tr><tr><td>Computer program for measuring staining intensity</td><td>Image J</td><td>https://imagej.nih.gov/ij/&lt;br/&gt; Program and information available on-line</td><td>Any appropriate program can be used. See https://theolb.readthedocs.io/en/latest/imaging/measuring-cell-fluorescence-using-imagej.html for additional detail&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Cell Culture trypsin solution</td><td>Sigma</td><td>T4174</td><td>purchased as a 10x solution</td></tr></tbody></table>

using a gfp-tagged tmem184a construct for confirmation of heparin receptor identity

Quando novas proteínas são identificados através de análise de isolamento e bioinformática à base de afinidade, eles são muitas vezes não caracterizados em grande parte. Os anticorpos contra os péptidos específicos dentro da sequência prevista permitir que algumas experiências de localização. No entanto, outros possíveis interacções com os anticorpos muitas vezes não podem ser excluídos. Esta situação constituiu uma oportunidade para desenvolver um conjunto de ensaios dependentes da sequência da proteína. Especificamente, foi obtida uma construção contendo a sequência de gene acoplado à sequência codificante de GFP na extremidade C-terminal da proteína e utilizados para estes fins. Experimentos para caracterizar localização, afinidade do ligando, e ganho de função foram originalmente concebidos e realizados para confirmar a identificação de TMEM184A como um receptor heparina 1. Além disso, a construção pode ser utilizada para estudos abordando questões de topologia de membrana e as interacções proteína-ligando detalhados. O presente relatório apresenta arange de protocolos experimentais com base na GFP-TMEM184A expresso em células vasculares que poderia ser facilmente adaptada para outras novas proteínas.

Embora, em teoria, a transfecção de qualquer construção de ADN para as células pode ser realizada com os reagentes de transfecção lipofílicos, relatórios anteriores indicam transfecção mais eficaz de GFP construções em células endoteliais utilizando electroporação 12. O protocolo aqui fornecida tipicamente conseguida GFP-construção de expressão em mais de 80% das células endoteliais derivados de primários e células de músculo liso utilizados...

Watch this Scientific Journal Video about Using a GFP-tagged TMEM184A Construct for Confirmation of Heparin Receptor Identity at JoVE.com

Using a GFP-tagged TMEM184A Construct for Confirmation of Heparin Receptor Identity

Uma construção que codifica TMEM184A com uma marcação GFP no carboxi-terminal concebido para expressão eucariótica, foi empregue em ensaios desenhados para confirmar a identificação de um receptor TMEM184A como heparina em células vasculares.

Usando um TMEM184A GFP Construir para a confirmação da Heparina Receptor Identity

When novel proteins are identified through affinity-based isolation and bioinformatics analysis, they are often largely uncharacterized. Antibodies ...

using-gfp-tagged-tmem184a-construct-for-confirmation-heparin-receptor

Research

JoVE Journal

Genetics

7.9K visualizações.  Lehigh University.  Uma construção que codifica TMEM184A com uma marcação GFP no carboxi-terminal concebido para expressão eucariótica, foi empregue em ensaios desenhados para confirmar a identificação de um receptor TMEM184A como heparina em células vasculares. 

Vídeo: Using a GFP-tagged TMEM184A Construct for Confirmation of Heparin Receptor Identity

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium homeostasis, particularly store-operated calcium entry, which is critical to maintaining proper function of synaptic vesicle release and intracellular signaling pathways. Accordingly, TRPC channels have been implicated in a variety of human diseases including cardiovascular disorders such as cardiac hypertrophy, neurodegenerative disorders such as Parkinson&#39;s disease, and neurologic disorders such as spinocerebellar ataxia. Therefore, TRPC channels represent a potential pharmacologic target in human diseases. However, the molecular mechanisms of gating in these channels are still unclear. The difficulty in obtaining large quantities of stable, homogeneous, and purified protein has been a limiting factor in structure determination studies, particularly for mammalian membrane proteins such as the TRPC ion channels. Here, we present a protocol for the large-scale expression of mammalian ion channel membrane proteins using a modified baculovirus gene transfer system and the purification of these proteins by affinity and size-exclusion chromatography. We further present a protocol to collect single-particle cryo-electron microscopy images from purified protein and to use these images to determine the protein structure. Structure determination is a powerful method for understanding the mechanisms of gating and function in ion channels.

This protocol describes techniques used to determine ion channel structures by cryo-electron microscopy, including a baculovirus system used to efficiently express genes in mammalian cells with minimum effort and toxicity, protein extraction, purification, and quality checking, sample grid preparation and screening, as well as data collection and processing.

Expressão e purificação de TRPC3 os humanos sensíveis lipídico cação canal para determinação estrutural por microscopia de Single-particle Cryo-elétron

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium ...

Bioquímica

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and reactive moieties onto proteins directly in the live cell. Each ncAA is incorporated by a dedicated orthogonal suppressor-tRNA/amino-acyl-tRNA-synthetase (AARS) pair that is imported into the host organism. The incorporation efficiency of different ncAAs can greatly differ, and be unsatisfactory in some cases. Orthogonal pairs can be improved by manipulating either the AARS or the tRNA. However, directed evolution of tRNA or AARS using large libraries and dead/alive selection methods are not feasible in mammalian cells. Here, a facile and robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs in mammalian cells is presented. The assay allows screening tens to hundreds of AARS/tRNA variants with a moderate effort and within a reasonable time. Use of this assay to generate new tRNAs that significantly improve the efficiency of the pyrrolysine orthogonal system is described, along with the application of ncAAs to the study of G-protein coupled receptors (GPCRs), which are challenging objects for ncAA mutagenesis. First, by systematically incorporating a photo-crosslinking ncAA throughout the extracellular surface of a receptor, binding sites of different ligands on the intact receptor are mapped directly in the live cell. Second, by incorporating last-generation ncAAs into a GPCR, ultrafast catalyst-free receptor labeling with a fluorescent dye is demonstrated, which exploits bioorthogonal strain-promoted inverse Diels Alder cycloaddition (SPIEDAC) on the live cell. As ncAAs can be generally applied to any protein independently on its size, the method is of general interest for a number of applications. In addition, ncAA incorporation does not require any special equipment and is easily performed in standard biochemistry labs.

A facile fluorescence assay is presented to evaluate the efficiency of amino-acyl-tRNA-synthetase/tRNA pairs incorporating non-canonical amino-acids (ncAAs) into proteins expressed in mammalian cells. The application of ncAAs to study G-protein coupled receptors (GPCRs) is described, including photo-crosslinking mapping of binding sites and bioorthogonal GPCR labeling on live cells.

Otimizando a incorporação genética do produto químico sondas em GPCRs para mapeamento de foto-reticulação e opaco química em células de mamíferos vivas

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and ...

Química

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen Presenting Cells (APCs). This is despite T-cell antigen receptors (TCRs) exerting usually a moderate affinity (&#181;M range) to antigen when binding is measured in vitro1. In view of the molecular and cellular parameters contributing to T-cell antigen sensitivity, a microscopy-based methodology has been developed as a means to monitor TCR-pMHC binding in situ, as it occurs within the synapse of a live T-cell and an artificial and functionalized glass-supported planar lipid bilayer (SLB), which mimics the cell membrane of an Antigen presenting Cell (APC) 2. Measurements are based on F&#246;rster Resonance Energy Transfer (FRET) between a blue- and red-shifted fluorescent dye attached to the TCR and the pMHC. Because the efficiency of FRET is inversely proportional to the sixth power of the inter-dye distance, one can employ FRET signals to visualize synaptic TCR-pMHC binding. The sensitive of the microscopy approach supports detection of single molecule FRET events. This allows to determine the affinity and off-rate of synaptic TCR-pMHC interactions and in turn to interpolate the on-rate of binding. Analogous assays could be applied to measure other receptor-ligand interactions in their native environment.

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

This manuscript describes how to conduct (single molecule) F&#246;rster Resonance Energy Transfer (FRET)- based assays to measure the binding dynamics between T-cell antigen receptor (TCR) and antigenic peptide-loaded MHC molecules as they occur within the immunological synapse of a T-cell in contact with a functionalized planar supported lipid bilayer.

Medição de TCR-pMHC Binding<em&gt; In Situ</em&gt; Usando a microscopia Ensaio de FRET

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen ...

Bioengenharia

<meta charset="utf8"></head><body>Marcação de proteína fluorescente em larvas de peixe-zebra usando CRISPR/Cas9 Knock-In

Fluorescence Labeling to Visualize Low-Expressed Proteins in Zebrafish

CRISPR/Cas9-mediated knock-in (KI) technology allows for easier fluorescent-protein tagging in zebrafish (Danio rerio), a preferred model organism for in vivo imaging due to its transparency during the early developmental stage. Here, we provide a detailed protocol for performing high-efficiency fluorescence gene KI, rapid screening for KI founders, and low-abundance protein tracing in zebrafish larvae, which will lay a critical foundation for subsequent physio-pathological studies in zebrafish. The current protocol includes complete steps for the sgRNA design for the gene of interest, sgRNA in vitro transcription, Cas9 mRNA in vitro transcription, in vivo sgRNA screen for the one with the highest efficiency, donor plasmid design and construction, microinjection in zebrafish larvae, KI founder screen and zebrafish live imaging. Critical steps, troubleshooting tips, quality control methods, and advantages and applications of this protocol are included and discussed. This protocol assures quick and accurate results at a low cost and has been validated by multiple trials.

<meta charset="utf8"></head><body>Marcação de fluorescência para visualizar proteínas de baixa expressão em peixe-zebra

Here, we present a protocol to label proteins with a fluorescence protein tag in zebrafish larvae, a newly developed and modified in vivo system especially useful for visualizing low-abundance proteins in zebrafish.

Marcação de fluorescência para visualizar proteínas de baixa expressão em peixe-zebra

CRISPR/Cas9-mediated knock-in (KI) technology allows for easier fluorescent-protein tagging in zebrafish (Danio rerio), a preferred model organism for in ...

Genética

Using an &#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell surface mobility1-7. To study receptor cell surface localization and endocytosis, the technique described here combines the use of fluorescent &#945;-bungarotoxin with cells expressing constructs containing an &#945;-bungarotoxin (Bgt) binding site (BBS). The BBS (WRYYESSLEPYPD) is based on the &#945; subunit of the muscle nicotinic acetylcholine receptor, which binds Bgt with high affinity8,9. Incorporation of the BBS site allows surface localization and measurements of receptor insertion or removal with application of exogenous fluorescent Bgt, as previously described in the tracking of GABAA and metabotropic GABAB receptors2,10. In addition to the BBS site, we inserted a pH-sensitive GFP (pHGFP11) between amino acids 4 and 5 of the mature GABAAR subunit by standard molecular biology and PCR cloning strategies (see Figure 1)12. The BBS is 3&#39; of the pH-sensitive GFP reporter, separated by a 13-amino acid alanine/proline linker. For trafficking studies described in this publication that are based on fixed samples, the pHGFP serves as a reporter of total tagged GABAAR subunit protein levels, allowing normalization of the Bgt labeled receptor population to total receptor population. This minimizes cell to cell Bgt staining signal variability resulting from higher or lower baseline expression of the tagged GABAAR subunits. Furthermore the pHGFP tag enables easy identification of construct expressing cells for live or fixed imaging experiments.

Here we demonstrate the use of fluorescent Alexa dye coupled to &#945;-bungarotoxin to measure GABA A receptor surface localization and endocytosis in hippocampal neurons. Through the use of constructs bearing a short extracellular tag that binds &#945;-bungarotoxin, analysis of plasma membrane protein endocytic trafficking can be achieved.

Usando um α-Bungarotoxina Binding Tag Site para Estudar GABA um receptor de membrana Localização e Tráfico

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell ...

Neurociência

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

Imagem interações proteína-proteína In vivo</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

Biologia

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes.

We genetically-encode the unnatural amino acid, p-azido-L-phenylalanine at various targeted positions in GPCRs and show the versatility of the azido group in different applications. These include a targeted photocrosslinking technology to identify residues in the ligand-binding pocket of a GPCR, and site-specific bioorthogonal modification of GPCRs with a peptide-epitope tag or fluorescent probe.

Geneticamente codificados Molecular Probes para estudar G receptores acoplados à proteína

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these multi-spanning transmembrane proteins, with or without change in channel gating, is often postulated to contribute significantly in this process. It has thus become critical to develop a method to quantify the change of the relative cell surface expression of cardiac ion channels on a large scale. Herein, a detailed protocol is provided to determine the relative total and cell surface expression of cardiac L-type calcium channels CaV1.2 and membrane-associated subunits in tsA-201 cells using two-color fluorescent cytometry assays. Compared with other microscopy-based or immunoblotting-based qualitative methods, flow cytometry experiments are fast, reproducible, and large-volume assays that deliver quantifiable end-points on large samples of live cells (ranging from 104 to 106 cells) with similar cellular characteristics in a single flow. Constructs were designed to constitutively express mCherry at the intracellular C-terminus (thus allowing a rapid assessment of the total protein expression) and express an extracellular-facing hemagglutinin (HA) epitope to estimate the cell surface expression of membrane proteins using an anti-HA fluorescence conjugated antibody. To avoid false negative, experiments were also conducted in permeabilized cells to confirm the accessibility and proper expression of the HA epitope. The detailed procedure provides: (1) design of tagged DNA (deoxyribonucleic acid) constructs, (2) lipid-mediated transfection of constructs in tsA-201 cells, (3) culture, harvest, and staining of non-permeabilized and permeabilized cells, and (4) acquisition and analysis of fluorescent signals. Additionally, the basic principles of flow cytometry are explained and the experimental design, including the choice of fluorophores, titration of the HA antibody and control experiments, is thoroughly discussed. This specific approach offers objective relative quantification of the total and cell surface expression of ion channels that can be extended to study ion pumps and plasma membrane transporters.

Inherited cardiac arrhythmias are often caused by mutations that alter the surface delivery of one or more ion channels. Here, we adapt flow cytometry assays to provide a quantification of the relative total and cell surface protein expression of recombinant ion channels expressed in tsA-201 cells.

Determinação da superfície celular Relativa e Expressão total de canais recombinante de iões utilizando Citometria de Fluxo

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these ...

Usando um TMEM184A GFP Construir para a confirmação da Heparina Receptor Identity

Resumo

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Usando um α-Bungarotoxina Binding Tag Site para Estudar GABA um receptor de membrana Localização e Tráfico

Imagem interações proteína-proteína In vivo

Geneticamente codificados Molecular Probes para estudar G receptores acoplados à proteína

Determinação da superfície celular Relativa e Expressão total de canais recombinante de iões utilizando Citometria de Fluxo