Este trabajo fue apoyado por la Iniciativa de Investigación Universidad de Wisconsin-Milwaukee crecimiento, el Instituto de Investigación Biomédica de Wisconsin y Tecnologías de la Salud y la Fundación Bradley.

<ol>
	<li>Raicu, V., Diaspro, 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> Nanoscopy and Multidimensional Optical Fluorescence Microscopy. , (2010).</li><li>Lakowicz, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of Fluorescence Spectroscopy. , (2006).</li><li>Raicu, V., Popescu, 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> Integrated Molecular and Cellular Biophysics. , (2008).</li><li>Selvin, P. 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>

En esta presentación, se muestra cómo determinar el tamaño y la información estructural de un complejo de proteínas oligómero in vivo. Aunque los datos presentados se obtuvieron de un receptor específico de membrana (es decir, Ste2p) expresado en células de levadura, el método es que todo lo abarca, ya que se puede aplicar a cualquier tipo de proteína expresada en cualquier tipo de célula, la única condición es que las proteínas están etiquetados con los marcadores fluorescentes adecuados. Futura...

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
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<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

El estudio de las interacciones de proteínas en células vivas es un área importante de investigación debido a que la información acumulada tanto en las aplicaciones de los beneficios industriales, así como aumenta el conocimiento básico biológico fundamental. Förster (fluorescencia) la transferencia de energía por resonancia (FRET) entre una molécula de donantes en un estado electrónicamente excitado y una molécula de receptor de cerca ha sido frecuentemente utilizado para el estudio de las interacciones proteína-proteína en las células vivas. Las proteínas de interés se marcan con dos tipos diferentes de sondas fluorescentes y se expresa en las células biológicas. Las sondas fluorescentes son entonces emocionado, por lo general usando luz láser, y las propiedades espectrales de la emisión de fluorescencia que emanan de las sondas fluorescentes se recopila y analiza. La información sobre el grado de las interacciones proteína está incrustado en los datos de emisión espectral. Por lo general, la célula debe ser analizado un número de veces que el fin de acumular suficiente información espectral para cuantificar con exactitud el alcance de las interacciones de proteínas por cada región de interés dentro de la célula. Sin embargo, la composición molecular de estas regiones pueden cambiar durante el curso del proceso de adquisición, lo que limita la determinación espacial de los valores cuantitativos de la aparente eficiencia de FRET a un promedio de células enteras. Por medio de un espectro resuelto de dos fotones microscopio, podemos obtener un conjunto completo de imágenes espectral resuelto después de sólo un ciclo de excitación completa de la muestra de interés. De estos datos espectrales a nivel de píxeles, un mapa de FRET eficiencia a través de la celda se calcula. Mediante la aplicación de una teoría simple de FRET en los complejos oligoméricos a la distribución obtenidos experimentalmente la eficiencia de FRET en toda la célula, una sola exploración espectral resuelto revela información estequiométrica y estructural sobre el complejo oligómero en estudio. A continuación se describe el procedimiento de preparación de las células biológicas (la levadura Saccharomyces cerevisiae) que expresan receptores de membrana (estéril factor 2 α-receptores) marcado con dos tipos diferentes de sondas fluorescentes. Además, ilustran los factores críticos de la recogida de los datos de fluorescencia utilizando el espectro resuelto de dos fotones sistema de imágenes de microscopía. El uso de este protocolo podrá ser extendido a estudiar cualquier tipo de proteína que puede ser expresado en una célula viva con un marcador fluorescente que se le atribuye.

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

Mediante el empleo de un espectro resuelto de dos fotones sistema de imágenes de microscopía, a nivel de píxel de mapas de transferencia de energía por resonancia de Förster (FRET) se obtienen eficiencias de las células que expresan receptores de membrana, la hipótesis de que forma oligomérica homo-complejos. Mapas de la FRET eficiencia, son capaces de estimar la información sobre el complejo estequiométrico oligómero en estudio.

En la cuantificación in vivo de interacciones de proteínas G del receptor acoplado con espectralmente resuelve de dos fotones de Microscopía

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 vistas.  University of Wisconsin - Milwaukee. Mediante el empleo de un espectro resuelto de dos fotones sistema de imágenes de microscopía, a nivel de píxel de mapas de transferencia de energía por resonancia de Förster (FRET) se obtienen eficiencias de las células que expresan receptores de membrana, la hipótesis de que forma oligomérica homo-complejos. Mapas de la FRET eficiencia, son capaces de estimar la información sobre el complejo estequiométrico oligómero en estudio.

Video: 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.

Tinción de las proteínas en geles con Coomassie G-250, sin ácido disolvente orgánico y el acético

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

Investigación

Biología

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.

En vivo-La detección de interacciones proteína-proteína en la Micro-patrón superficies

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.

Imágenes de interacciones 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, ...

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.

La cuantificación de proteínas mediante el enriquecimiento de inmunoafinidad péptido acoplado a espectrometría de masas

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.

Receptor de imagen acoplados a proteína G (GPCR) mediada por eventos de señalización que la quimiotaxis de control de Dictyostelium discoideum</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.

La cuantificación de la actividad agonista en el G receptores acoplados a proteínas

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.

Codificados genéticamente-sondas moleculares para Estudiar G receptores acoplados a proteínas

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).

Caracterización de G receptores acoplados a proteína por un ensayo de movilización de calcio basado en fluorescencia

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.

Visualización de endocitosis mediada por clatrina de G receptores acoplados a proteínas en la Resolución Individual de eventos a través de TIRF de Microscopía

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.

Imaging G acoplados a proteínas mediada por el receptor de quimiotaxis y sus eventos de señalización en células HL60 neutrófilos como

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