この作品は、UW -ミルウォーキーの研究成長戦略大綱、生物医学と医療技術のためのウィスコンシン州研究所、そしてブラッドリー財団によってサポートされていました。

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+renaissance+of+fluorescence+resonance+energy+transfer.">The renaissance of fluorescence resonance energy transfer.</a> Nat Struct Biol. 7, 730-734 (2000).</li><li>Raicu, V., Jansma, D. B., Miller, R. J., Friesen, 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=Protein+interaction+quantified+in+vivo+by+spectrally+resolved+fluorescence+resonance+energy+transfer.">Protein interaction quantified in vivo by spectrally resolved fluorescence resonance energy transfer.</a> Biochem J. 385, 265-277 (2005).</li><li>Maurel, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-surface+protein-protein+interaction+analysis+with+time-resolved+FRET+and+snap-tag+technologies:+application+to+GPCR+oligomerization.">Cell-surface protein-protein interaction analysis with time-resolved FRET and snap-tag technologies: application to GPCR oligomerization.</a> Nat Methods. 5, 561-567 (2008).</li><li>Shyu, Y. J., Suarez, C. D., Hu, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+AP-1+NF-kappaB+ternary+complexes+in+living+cells+by+using+a+BiFC-based+FRET.">Visualization of AP-1 NF-kappaB ternary complexes in living cells by using a BiFC-based FRET.</a> Proc Natl Acad Sci USA. 105, 151-156 (2008).</li><li>Demarco, I. A., Periasamy, A., Booker, C. F., Day, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+dynamic+protein+interactions+with+photoquenching+FRET.">Monitoring dynamic protein interactions with photoquenching FRET.</a> Nat Methods. 3, 519-524 (2006).</li><li>Wallrabe, H., Periasamy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+protein+molecules+using+FRET+and+FLIM+microscopy.">Imaging protein molecules using FRET and FLIM microscopy.</a> Curr Opin Biotechnol. 16, 19-27 (2005).</li><li>Wlodarczyk, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+FRET+signals+in+the+presence+of+free+donors+and+acceptors.">Analysis of FRET signals in the presence of free donors and acceptors.</a> Biophys J. 94, 986-1000 (2008).</li><li>F&#246;rster, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimentelle+und+theoretische+Untersuchung+des+zwischenmolekularen+bergangs+von+Elektronenanregungsenergie.+Z.+Naturforsch.">Experimentelle und theoretische Untersuchung des zwischenmolekularen bergangs von Elektronenanregungsenergie. Z. Naturforsch.</a> A: Astrophys Phys Phys Chem. 4, 321-321 (1949).</li><li>Shaner, N. C., Steinbach, P. A., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+choosing+fluorescent+proteins.">A guide to choosing fluorescent proteins.</a> Nat Methods. 2, 905-909 (2005).</li><li>Giepmans, B. N., Adams, S. R., Ellisman, M. H., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fluorescent+toolbox+for+assessing+protein+location+and+function.">The fluorescent toolbox for assessing protein location and function.</a> Science. 312, 217-2124 (2006).</li><li>Zimmermann, T., Rietdorf, J., Girod, A., Georget, V., Pepperkok, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+imaging+and+linear+un-mixing+enables+improved+FRET+efficiency+with+a+novel+GFP2-YFP+FRET+pair.">Spectral imaging and linear un-mixing enables improved FRET efficiency with a novel GFP2-YFP FRET pair.</a> FEBS Lett. 531, 245-249 (2002).</li><li>Nagai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+variant+of+yellow+fluorescent+protein+with+fast+and+efficient+maturation+for+cell-biological+applications.">A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications.</a> Nat Biotechnol. 20, 87-90 (2002).</li><li>Rizzo, M. A., Springer, G. H., Granada, B., Piston, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+cyan+fluorescent+protein+variant+useful+for+FRET.">An improved cyan fluorescent protein variant useful for FRET.</a> Nat Biotechnol. 22, 445-449 (2004).</li><li>Raicu, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficiency+of+Resonance+Energy+Transfer+in+Homo-Oloigomeric+Complexes+of+Proteins.">Efficiency of Resonance Energy Transfer in Homo-Oloigomeric Complexes of Proteins.</a> J Biol Phys. 33, 109-127 (2007).</li><li>Raicu, V., Fung, R., Melnichuk, M., Chaturvedi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> , (2007).</li><li>Raicu, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+supramolecular+structure+and+spatial+distribution+of+protein+complexes+in+living+cells.">Determination of supramolecular structure and spatial distribution of protein complexes in living cells.</a> Nat Photonics. 3, 107-113 (2009).</li><li>Rath, S., Sullivan, A. P., Stoneman, M. R., Raicu, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microspectroscopic+method+for+determination+of+size+and+distribution+of+protein+complexes+in+vivo.">Microspectroscopic method for determination of size and distribution of protein complexes in vivo.</a> Proc of SPIE. 7378, 737829-737829 (2009).</li><li>Kurjan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pheromone+response+in+yeast.">Pheromone response in yeast.</a> Annu Rev Biochem. 61, 1097-1129 (1992).</li><li>Overton, M. C., Chinault, S. L., Blumer, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligomerization+of+G-protein-coupled+receptors:+lessons+from+the+yeast+Saccharomyces+cerevisiae.">Oligomerization of G-protein-coupled receptors: lessons from the yeast Saccharomyces cerevisiae.</a> Eukaryot Cell. 4, 1963-1970 (2005).</li></ol>

利害の衝突は宣言されません。

本発表では、我々はin vivoでのタンパク質のオリゴマー複合体についてのサイズと構造情報を決定する方法を示します。提示されたデータは、酵母細胞で発現し、特定の膜受容体（すなわちSte2p）から得ていたが、方法は、それがセルの任意のタイプで発現するタンパク質の任意の種類、その蛋白質であることのみを規定する適用可能なすべての点で網羅です適切な蛍光マーカーでタグ?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Peptone</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP1420</td>
<td>&nbsp;</td>
<tr>
<td>Yeast Extract</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP1422</td>
<td>&nbsp;</td>
<tr>
<td>Polyethlyene Glycol 4000</td>
<td>&nbsp;</td>
<td>Hampton Research</td>
<td>HR2-605</td>
<td>&nbsp;</td>
<tr>
<td>Yeast Nitrogen Base w/o (NH4)2SO4 and amino acids</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>DF0335-15-9</td>
<td>&nbsp;</td>
<tr>
<td>Yeast Synthetic Drop-out Medium Supplements</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>Y2001</td>
<td>&nbsp;</td>
<tr>
<td>D-Glucose</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>D16-1</td>
<td>&nbsp;</td>
<tr>
<td>Agar</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>S70210</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium Sulfate</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>A702-500</td>
<td>&nbsp;</td>
<tr>
<td>Potassium Chloride</td>
<td>&nbsp;</td>
<td>Acros Organics</td>
<td>424090010</td>
<td>&nbsp;</td>
<tr>
<td>Leucine</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP385</td>
<td>&nbsp;</td>
<tr>
<td>Histidine</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP382</td>
<td>&nbsp;</td>
<tr>
<td>Plan Achromat Infinity Corrected 100x Oil Immersion Objective NA=1.43</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Spectrally resolved two photon microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
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in vivo quantification of g protein coupled receptor interactions using spectrally resolved two-photon microscopy

情報が両方の利点を産業用途だけでなく、基本的な基本的な生物学的知識を高めるに蓄積されたので、生きた細胞内のタンパク質相互作用の研究は、研究の重要な領域です。電子励起状態におけるドナー分子と近隣のアクセプター分子との間のフェルスター（蛍光）共鳴エネルギー移動は、（FRET）頻繁に生細胞におけるタンパク質間相互作用の研究のために利用されている。興味のあるタンパク質は、蛍光プローブの2種類のタグ付けと生物細胞で発現されています。蛍光プローブは、一般的にレーザー光を使用して、励起され、蛍光プローブから発せられる蛍光発光のスペクトル特性を収集し、分析される。タンパク質相互作用の程度に関する情報は、分光放射データに埋め込まれています。一般的に、細胞を正確に細胞内で関心のある各地域のタンパク質相互作用の程度を定量化するために十分なスペクトル情報を蓄積するために、回数をスキャンする必要があります。しかし、これらの領域の分子組成は、細胞全体の平均には明らかに、FRET効率の定量的な値の空間的な決定を制限する、取得プロセスの過程で変更される可能性があります。スペクトル的に解決された二光子顕微鏡を用いて、我々は、関心のサンプルの唯一の完全な励起のスキャン後のスペクトル解決のイメージの完全なセットを取得することができます。このピクセルレベルのスペクトルデータから、細胞全体に効率のFRETのマップが計算されます。細胞全体に効率のFRETの実験で得られた分布するオリゴマー複合体におけるFRETの単純な理論を適用することにより、単一のスペクトル解決のスキャンは、研究の下でオリゴマー複合体に関する化学量論と構造情報を明らかにする。ここでは、蛍光プローブの2種類のタグが付けられた膜の受容体（滅菌2α-因子受容体）を発現する生物細胞（ 酵母 Saccharomyces cerevisiae）の準備の手順を説明します。さらに、我々はスペクトル解決二光子顕微鏡イメージングシステムを用いて蛍光データの収集に関与する重要な要素を示しています。このプロトコルの使用はそれに接続されている蛍光マーカーと生細胞内で発現可能なタンパク質の任意のタイプを研究するために拡張することができる。

Watch this Scientific Journal Video about In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy at JoVE.com

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

スペクトル的に解決された二光子顕微鏡イメージングシステムを採用することにより、フェルスター共鳴エネルギー移動（FRET）効率のピクセルレベルのマップは、ホモオリゴマー複合体を形成するために仮定膜受容体を発現する細胞に対して得られる。効率マップをFRETから、我々が検討されてオリゴマー複合体に関する化学量論的情報を推定することができます。

スペクトル的に解決された二光子顕微鏡を用いたGタンパク質共役受容体相互作用の in vivo定量で

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

in-vivo-quantification-g-protein-coupled-receptor-interactions-using

Research

JoVE Journal

Biology

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

13.2K ビュー.  University of Wisconsin - Milwaukee. スペクトル的に解決された二光子顕微鏡イメージングシステムを採用することにより、フェルスター共鳴エネルギー移動（FRET）効率のピクセルレベルのマップは、ホモオリゴマー複合体を形成するために仮定膜受容体を発現する細胞に対して得られる。効率マップをFRETから、我々が検討されてオリゴマー複合体に関する化学量論的情報を推定することが...

ビデオ: In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

クーマシー有機溶媒および酢酸のないG - 250でゲル中のタンパク質の染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

生物学

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of methodological options for addressing molecular interactions in cells have been developed, but most of these methods suffer from being rather indirect and therefore hardly quantitative. On the contrary, a few high-end quantitative approaches were introduced, which however are difficult to extend to high throughput. To combine high throughput capabilities with the possibility to extract quantitative information, we recently developed a new concept for identifying protein-protein interactions (Schwarzenbacher et al., 2008). Here, we describe a detailed protocol for the design and the construction of this system which allows for analyzing interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in-vivo. Cells are plated on micropatterned surfaces functionalized with antibodies against the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput capability, making it applicable as screening tool.

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

This video shows experiments with subsequent analysis of protein-protein interactions by the use of micro-patterned surfaces. The approach offers the possibility to detect protein interactions in living cells and combines high throughput capabilities with the possibility to extract quantitative information.

マイクロパターン化表面でのタンパク質間相互作用の in vivo検出

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of ...

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.

イメージングタンパク質間相互作用 in vivoで</em

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

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

質量分析法とあいまってペプチドイムノエンリッチメントを使用したタンパク質の定量化

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1. This guided cell migration is referred as chemotaxis, which is essential for various cells to carry out their functions such as trafficking of immune cells and patterning of neuronal cells 2, 3. A large family of G-protein coupled receptors (GPCRs) detects variable small peptides, known as chemokines, to direct cell migration in vivo 4. The final goal of chemotaxis research is to understand how a GPCR machinery senses chemokine gradients and controls signaling events leading to chemotaxis. To this end, we use imaging techniques to monitor, in real time, spatiotemporal concentrations of chemoattractants, cell movement in a gradient of chemoattractant, GPCR mediated activation of heterotrimeric G-protein, and intracellular signaling events involved in chemotaxis of eukaryotic cells 5-8. The simple eukaryotic organism, Dictyostelium discoideum, displays chemotaxic behaviors that are similar to those of leukocytes, and D. discoideum is a key model system for studying eukaryotic chemotaxis. As free-living amoebae, D. discoideum cells divide in rich medium. Upon starvation, cells enter a developmental program in which they aggregate through cAMP-mediated chemotaxis to form multicullular structures. Many components involved in chemotaxis to cAMP have been identified in D. discoideum. The binding of cAMP to a GPCR (cAR1) induces dissociation of heterotrimeric G-proteins into G&gamma; and G&beta;&gamma; subunits 7, 9, 10. G&beta;&gamma; subunits activate Ras, which in turn activates PI3K, converting PIP2 into PIP3 on the cell membrane 11-13. PIP3 serve as binding sites for proteins with pleckstrin Homology (PH) domains, thus recruiting these proteins to the membrane 14, 15. Activation of cAR1 receptors also controls the membrane associations of PTEN, which dephosphorylates PIP3 to PIP2 16, 17. The molecular mechanisms are evolutionarily conserved in chemokine GPCR-mediated chemotaxis of human cells such as neutrophils 18. We present following methods for studying chemotaxis of D. discoideum cells. 1. Preparation of chemotactic component cells. 2. Imaging chemotaxis of cells in a cAMP gradient. 3. Monitoring a GPCR induced activation of heterotrimeric G-protein in single live cells. 4. Imaging chemoattractant-triggered dynamic PIP3 responses in single live cells in real time. Our developed imaging methods can be applied to study chemotaxis of human leukocytes.

Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Here, we describe detailed live cell imaging methods for investigating chemotaxis. We present fluorescence microscopic methods to monitor spatiotemporal dynamics of signaling events in migrating cells. Measurement of signaling events permits us to further understand how a GPCR-signaling network achieves gradient sensing of chemoattractants and controls directional migration of eukaryotic cells.

イメージングG -タンパク質共役受容体（GPCR）を介するシグナル伝達事象その制御走キイロタマホコリカビ</em

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1.  This guided cell migration is referred as ...

Quantifying Agonist Activity at G Protein-coupled Receptors

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell or tissue. This process can be analyzed at the level of a single receptor, a population of receptors, or a downstream response. Here we describe how to analyze the downstream response to obtain an estimate of the agonist affinity constant for the active state of single receptors.
Receptors behave as quantal switches that alternate between active and inactive states (Figure 1). The active state interacts with specific G proteins or other signaling partners. In the absence of ligands, the inactive state predominates. The binding of agonist increases the probability that the receptor will switch into the active state because its affinity constant for the active state (Kb) is much greater than that for the inactive state (Ka). The summation of the random outputs of all of the receptors in the population yields a constant level of receptor activation in time. The reciprocal of the concentration of agonist eliciting half-maximal receptor activation is equivalent to the observed affinity constant (Kobs), and the fraction of agonist-receptor complexes in the active state is defined as efficacy (&#949;) (Figure 2).
Methods for analyzing the downstream responses of GPCRs have been developed that enable the estimation of the Kobs and relative efficacy of an agonist 1,2. In this report, we show how to modify this analysis to estimate the agonist Kb value relative to that of another agonist. For assays that exhibit constitutive activity, we show how to estimate Kb in absolute units of M-1.
Our method of analyzing agonist concentration-response curves 3,4 consists of global nonlinear regression using the operational model 5. We describe a procedure using the software application, Prism (GraphPad Software, Inc., San Diego, CA). The analysis yields an estimate of the product of Kobs and a parameter proportional to efficacy (&tau;). The estimate of &tau;Kobs of one agonist, divided by that of another, is a relative measure of Kb (RAi) 6. For any receptor exhibiting constitutive activity, it is possible to estimate a parameter proportional to the efficacy of the free receptor complex (&tau;sys). In this case, the Kb value of an agonist is equivalent to &tau;Kobs/&tau;sys 3.
Our method is useful for determining the selectivity of an agonist for receptor subtypes and for quantifying agonist-receptor signaling through different G proteins.

A method for estimating the affinity constant of an agonist for the active state (Kb) of a G protein-coupled receptor is described. The analysis provides absolute or relative measures of Kb depending on whether constitutive receptor activation is measurable. Our method applies to various responses downstream from receptor activation.

Gタンパク質共役受容体でアゴニスト活性を定量化

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell ...

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.

遺伝的にコードされた分子プローブは、Gタンパク質共役受容体を研究する

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

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous G&#945;16 construct is co-transfected. Following ligand binding, activation of the G&#945;16 subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous G&#945;16, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.

The here described fluorescence-based calcium mobilization assay is a medium-throughput reverse pharmacology screening system for the identification of functionally activating ligand(s) of orphan G protein-coupled receptors (GPCRs).

蛍光ベースのカルシウム動員アッセイによるGタンパク質共役受容体のキャラクタリゼーション

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors ...

Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological assays, measuring global changes in endocytosis, have identified over 30 known components participating in CME, and biochemical studies have generated an interaction map of many of these components. It is becoming increasingly clear, however, that CME is a highly dynamic process whose regulation is complex and delicate. In this manuscript, we describe the use of Total Internal Reflection Fluorescence (TIRF) microscopy to directly visualize the dynamics of components of the clathrin-mediated endocytic machinery, in real time in living cells, at the level of individual events that mediate this process. This approach is essential to elucidate the subtle changes that can alter endocytosis without globally blocking it, as is seen with physiological regulation. We will focus on using this technique to analyze an area of emerging interest, the role of cargo composition in modulating the dynamics of distinct clathrin-coated pits (CCPs). This protocol is compatible with a variety of widely available fluorescence probes, and may be applied to visualizing the dynamics of many cargo molecules that are internalized from the cell surface.

Clathrin-mediated endocytosis, a rapid and highly dynamic process internalizes many proteins, including signaling receptors. The protocol described here directly visualizes the kinetics of individual endocytic events. This is essential for understanding how core members of the endocytic machinery coordinate with each other, and how protein cargo influence this process.

TIRF顕微鏡を経由してシングルイベント解像度のGタンパク質共役型受容体のクラスリン依存性エンドサイトーシスの可視化

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological ...

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

イメージングGタンパク質共役型受容体媒介走化性及び好中球様HL60細胞におけるそのシグナル伝達のイベント

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many ...