Name: detection of ligand-activated g protein-coupled receptor internalization by confocal microscopy
Uploaded: 2017-04-09T14:00:00
Description: Watch this Scientific Journal Video about Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy at JoVE.com

The authors of this paper would like to thank Prof. Jiayan Xie for technical assistance and equipment usage. This work was financially supported by the National Natural Science Foundation of China (41406137).

1. Cell Preparation
<ol>
	<li>Cell recovery
	<ol>
		<li>Transfer the cryopreserved human embryonic kidney 293 (HEK293) cells from a liquid nitrogen tank to a 37 &#176;C water bath and shake continuously for 1 - 2 min.</li>
		<li>Add 10 mL of equilibrated growth medium (Dulbecco&#39;s modified Eagle&#39;s medium (DMEM), 10% fetal bovine serum (FBS), and 1% penicillin-streptomycin solution) to a 10-cm cell culture dish. Gently add the thawed cells to the equilibrated growth medium.</li>
		<li>Incubate th...

<ol>
	<li>Mellman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocytosis+and+molecular+sorting.">Endocytosis and molecular sorting.</a> Annu. Rev. Cell Dev. Biol. 12, 575-625 (1996).</li><li>Hausott, B., Klimaschewski, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+turnover+and+receptor+trafficking+in+regenerating+axons.">Membrane turnover and receptor trafficking in regenerating axons.</a> Eur J Neuro. 43, 309-317 (2016).</li><li>Kallal, L., Benovic, J. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+vectors+for+the+expression+of+green+fluorescent+protein+fusion+proteins+in+transgenic+plants.">Cloning vectors for the expression of green fluorescent protein fusion proteins in transgenic plants.</a> Gene. 221, 35-43 (1998).</li><li>Webb, C. D., Decatur, A., Teleman, A., Losick, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+green+fluorescent+protein+for+visualization+of+cell-specific+gene+expression+and+subcellular+protein+localization+during+sporulation+in+Bacillus+subtilis.">Use of green fluorescent protein for visualization of cell-specific gene expression and subcellular protein localization during sporulation in Bacillus subtilis.</a> J Bacteriol. 177, 5906-5911 (1995).</li><li>Kain, S. R., 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=Green+fluorescent+protein+as+a+reporter+of+gene+expression+and+protein+localization.">Green fluorescent protein as a reporter of gene expression and protein localization.</a> BioTechniques. 19, 650-655 (1995).</li><li>Fukunaga, S., Setoguchi, S., Hirasawa, A., Tsujimoto, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+ligand-mediated+internalization+of+G+protein-coupled+receptor+as+a+novel+pharmacological+approach.">Monitoring ligand-mediated internalization of G protein-coupled receptor as a novel pharmacological approach.</a> Life Sci. 80, 17-23 (2006).</li><li>Yang, 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=Agonist-Activated+Bombyx+Corazonin+Receptor+Is+Internalized+via+an+Arrestin-Dependent+and+Clathrin-Independent+Pathway.">Agonist-Activated Bombyx Corazonin Receptor Is Internalized via an Arrestin-Dependent and Clathrin-Independent Pathway.</a> Biochemistry. 55, 3874-3887 (2016).</li><li>Kohout, T. A., Lefkowitz, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+G+protein-coupled+receptor+kinases+and+arrestins+during+receptor+desensitization.">Regulation of G protein-coupled receptor kinases and arrestins during receptor desensitization.</a> Mol Pharmacol. 63, 9-18 (2003).</li><li>Ferguson, S. 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=Role+of+beta-arrestin+in+mediating+agonist-promoted+G+protein-coupled+receptor+internalization.">Role of beta-arrestin in mediating agonist-promoted G protein-coupled receptor internalization.</a> Science. 271, 363-366 (1996).</li><li>Stojilkovic, S. S., Reinhart, J., Catt, K. 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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=Identification+and+Expression+Profile+of+the+Gonadotropin-Releasing+Hormone+Receptor+in+Common+Chinese+Cuttlefish,+Sepiella+japonica.">Identification and Expression Profile of the Gonadotropin-Releasing Hormone Receptor in Common Chinese Cuttlefish, Sepiella japonica.</a> J Exp Zool A Ecol Genet Physiol. , (2016).</li><li>Yang, 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=Specific+activation+of+the+G+protein-coupled+receptor+BNGR-A21+by+the+neuropeptide+corazonin+from+the+silkworm,+Bombyx+mori,+dually+couples+to+the+G(q)+and+G(s)+signaling+cascades.">Specific activation of the G protein-coupled receptor BNGR-A21 by the neuropeptide corazonin from the silkworm, Bombyx mori, dually couples to the G(q) and G(s) signaling cascades.</a> J Biol Chem. 288, 11662-11675 (2013).</li><li>Lu, Z. 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=Cloning,+Characterization,+and+Expression+Profile+of+Estrogen+Receptor+in+Common+Chinese+Cuttlefish,+Sepiella+japonica.">Cloning, Characterization, and Expression Profile of Estrogen Receptor in Common Chinese Cuttlefish, Sepiella japonica.</a> J Exp Zool A Ecol Genet Physiol. 325, 181-193 (2016).</li></ol>

The protocol presented here provides an efficient and easily interpretable method to detect the subcellular localization and internalization of expressed GPCR fusion protein in HEK293 cells. The technique can be easily adapted for many different genes and cell types, such as for the cellular localization and internalization of the corazonin receptor from Bombyx mori in HEK293 and BmN cells16.
The successful application of this powerful assay relies on a few key...

Cells possess cargo trafficking machinery to transport extracellular materials-such as ligands, microorganisms, nutrients, and transmembrane proteins-into the cell for information, energy, and other purposes1,2. It is critical for cellular homeostasis, tissue function, and overall cell survival. The cell-based expression of fluorescent fusion proteins is a powerful way to study the localization and internalization of transmembrane receptors, such as G protein-coupled receptors (GPCR), in signaling pathways3.
The expression of fusion proteins tagged with green fluorescent protein (GFP) from jellyfish Aequorea victoria, combined with direct fluorescence detection techniques, has gained wide acceptance in protein-targeting research4,5,6. GFP-based detection has the advantage of allowing the real-time imaging of living cells while avoiding fixation artifacts7. A series of versatile cloning vectors has been constructed to facilitate the expression of protein fusions to GFP in various cells8,9. The pEGFP-N1 vector is a widely used and commercialized plasmid vector encoding an enhanced green fluorescent protein (EGFP), which has been optimized from wild-type GFP for brighter fluorescence and higher expression in mammalian cells. To observe the fluorescent signal of EGFP expressed in mammalian cells, the fluorescence microscopy should be conducted with the excitation at 488 nm and the emission at 507 nm, preferably with confocal microscopy.
Monitoring the subcellular localization and ligand-mediated internalization of receptors is a common technique to investigate the signal transduction and function of GPCRs10,11. In most cases, internalization leads to desensitization of the GPCRs (the attenuation of the stimulus response in spite of the presence of agonists)12,13. The internalized receptors can be incorporated into the lysosome and degraded, or they can be recycled back to the cell surface, depending on the nature of the receptors and the cell lines used in the experiments.
The gonadotropin-releasing hormone receptors (GnRHRs) belong to the GPCR family and have a typical GPCR protein structure, with an extracellular amino terminal, an intracellular -COOH terminal, and seven transmembrane (TM) domains14. The transfection of recombinant plasmid SjGnRHR-EGFP (with the GnRHR gene from Sepiella japonica) and the confocal microscopy detection of EGFP-tagged SjGnRHR (expressed in HEK293 cells) was reported previously, and GFP fluorescence was established as a reporter for transmembrane protein localization assays15. Now, the internalization of SjGnRHR, activated by S. japonica gonadotropin-releasing hormone (SjGnRH), was demonstrated in time- and dose-dependent manners by confocal microscopy.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">HEK293 cell line&#160;</td>
			<td style="width:236px;"></td>
			<td style="width:119px;"></td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">DMEM/High glucose</td>
			<td style="width:236px;">Hyclone</td>
			<td style="width:119px;">SH30022.01</td>
			<td>High glucose 4.0 mM L-glutamine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Foetal Bovine Serum (FBS)</td>
			<td style="width:236px;">PuFei</td>
			<td style="width:119px;">1101-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Phosphate Buffered Saline (PBS)</td>
			<td style="width:236px;">Genom</td>
			<td style="width:119px;">GNM10010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Penicillin-Streptomycin</td>
			<td style="width:236px;">Genom</td>
			<td style="width:119px;">GNM15140</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">0.25% Trypsin &#38; 0.02% EDTA</td>
			<td style="width:236px;">Genom</td>
			<td style="width:119px;">GNM25200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Opti-MEM (1X)</td>
			<td style="width:236px;">GIBCO, by Life Technologies</td>
			<td style="width:119px;">31985-062</td>
			<td>reduced serum media</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;width:300px;">Lipofectamine 2000 Transfection Reagent</td>
			<td style="width:236px;">Invitrogen</td>
			<td style="width:119px;">11668027</td>
			<td>Reagent 1</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;width:300px;">X-tremeGENE HP DNA Transfection Reagent</td>
			<td style="width:236px;">Roche</td>
			<td style="width:119px;">6366236001</td>
			<td>Reagent 2</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:300px;">DAPI Staining Solution</td>
			<td style="width:236px;">Beyotime</td>
			<td style="width:119px;">C1006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">DiI</td>
			<td style="width:236px;">Beyotime</td>
			<td style="width:119px;">C1036</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Antifade Mounting Medium</td>
			<td style="width:236px;">Beyotime</td>
			<td style="width:119px;">P0126</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:300px;">Paraformaldehyde</td>
			<td style="width:236px;">Sinopharm Chemical Reagent Co.(SCRC)</td>
			<td style="width:119px;">80096618</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">100 mm cell culture</td>
			<td style="width:236px;">CORNING</td>
			<td style="width:119px;">430167</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">6-well plate&#160;</td>
			<td style="width:236px;">ExCell Bio</td>
			<td style="width:119px;">CS016-0092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">12-well plate&#160;</td>
			<td style="width:236px;">ExCell Bio</td>
			<td style="width:119px;">CS016-0093</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Cover Glass</td>
			<td style="width:236px;">NEST</td>
			<td style="width:119px;">801007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Microscope Slides</td>
			<td style="width:236px;">Citoglas</td>
			<td style="width:119px;">10127105P-G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Thermo Scientific Forma CO2 Incubator</td>
			<td style="width:236px;">Thermo</td>
			<td style="width:119px;">3111</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">TCS SP5II laser scanning confocal microscope</td>
			<td style="width:236px;">Leica&#160;</td>
			<td style="width:119px;">5100001311</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of ligand-activated g protein-coupled receptor internalization by confocal microscopy

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent fusion proteins, such as green fluorescent protein (GFP), to determine their expression, localization, and function. The subcellular localization of target proteins is important for identification, characterization, and functional analyses. Internalization is one of the predominant mechanisms controlling G protein-coupled receptor (GPCR) signaling to ensure the appropriate cellular responses to stimuli. Here, we describe an experimental method to detect the subcellular localization and internalization of GPCR in HEK293 cells with confocal microscopy. In addition, this experiment provides some details about cell culture and transfection. This protocol is compatible with a variety of widely available fluorescent markers and is applicable to the visualization of the subcellular localization of a majority of proteins, as well as of the internalization of GPCR. This technique should enable researchers to efficiently manipulate GPCR gene expression in mammalian cell lines and should facilitate studies on GPCR subcellular localization and internalization.

Figure 1 shows an example of a confocal microscope system. Figure 2 presents the expression of pEGFP-N1 in HEK293 cells. The GFP signal was detected in the cytoplasm and nucleus. Figure 3 shows the subcellular localization of GnRHR from S. japonica in HEK293 cells, consistent with our previously published results15. The fusion protein with the EGFP tag at the C-terminus of SjGnRHR (SjGnRH...

Watch this Scientific Journal Video about Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy at JoVE.com

Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy

This protocol describes confocal microscopy detection of G protein-coupled receptor (GPCR) internalization in mammalian cells. It includes the basic cell culture, transfection, and confocal microscopy procedure and provides an efficient and easily interpretable method to detect the subcellular localization and internalization of fusion-expressed GPCR.

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent ...

The overall goal of this experiment is to provide an efficient and easily-interpretable method to detect the subcellular localization and internalization of expressed GPCR fusion proteins in mammalian cells. This method can help answer the key questions in the subcellular protein localization field, such as receptor internalization and recycling. The main advantage of this technique is that it can be used easily and efficiently.So this method can provide insight in monitoring and locating GPCRs. And it can also apply to other systems, such as cytoplasmic and nuclear protein. After recovering and passaging HEK293 cells according to the text protocol, when the cells have grown to near confluence, discard the equilibrated growth medium, add two milliliters of PBS, and swirl the dish to wash the cells.Discard the PBS, and add one milliliter of trypsin EDTA. To completely digest the cells, carefully swirl the dish and aspirate off the solution. Then, put the dish in this 37 degrees Celsius water-jacketed C02 incubator for three minutes.While the cells are incubating, prepare a six well plate by adding two milliliters of equilibrated growth medium to each well. Then, when the cells have lifted from the bottom of the dish, add one milliliter of equilibrated growth medium to the dish. Mix by pipetting and immediately transfer 0.1 milliliters of cell suspension into each well of the prepared six well plate.Gently pipette the suspension in the wells, and incubate the cells at 37 degrees Celsius and 5%CO2. The following day, ensure that the cells are 85-95%confluent. Then, to two sterile microcentrifuge tubes, add three micrograms of plasmid DNA and 100 microliters of reduced serum medium to one tube.Meanwhile, add nine microliters of reagent one and 100 microliters of reduced serum medium to another tube. Incubate the tubes at room temperature for five minutes. Combine the diluted plasmid mixture with diluted reagent one.Then, gently mix the solution, and incubate it at room temperature for 20 minutes. Aspirate the medium of the cells and add 1.5 milliliters of fresh DMEM without FBS. Then, add dropwise 215 microliters of DNA transfection reagent mixture to each well, and gently mix by swirling the plate.Incubate the plate at 37 degrees Celsius and 5%CO2 for four to eight hours. Then, replace the medium with fresh DMEM with 10%FBS. Incubate the cells for another 24 to 48 hours before running any experiments.Transfection efficiency may vary greatly in different cell lines. Therefore, the optimization of the sit and stab in deposition may be necessary depending on the particular cell type used. Remove the growth medium from the six well culture plate.Then, use one milliliter of PBS to wash the cell monolayer. Add 0.5 milliliters of trypsin EDTA, swirl the dish, and aspirate off the solution. Then, incubate the culture plate at 37 degrees Celsius for three minutes.During the incubation, insert 15 millimeter diameter sterile coverslips into each well of a 12 well plate. Resuspend the cells in a small volume of fresh complete growth medium, and transfer the required number of cells to the 12 well plate with coverslips. Then, incubate the cells for 16 to 24 hours.After 16 to 24 hours of incubation, when the cells have grown to near confluence, add five micromolar of the membrane probe dill to each well, and place the cells back in the incubator for 30 minutes. Remove the equilibrated growth medium and use cold PBS to wash the cells twice. Then, add one milliliter of 4%PFA to each well, and agitate the cells for 15 minutes to fix the cells.With PBS, wash the fixed cells twice, then add 200 to 300 microliters of DAPI to each well, and incubate the plate for 10 minutes at room temperature. After washing the cells with PBS twice, carefully remove the coverslips from the plate, and wick off the excess buffer. Invert the coverslips onto microscope slides loaded with one drop of mounting medium.Then, remove the excess mounting medium from the slides. Using cells that have grown to near confluence, remove the equilibrated growth medium and add fresh warmed DMEM. Incubate the cells for one hour.Treat the cells with different concentrations of ligand for 30 minutes. And treat the cells with 10 to the negative six molar ligand for the different times shown here. Use cold PBS to wash the cells twice, then add one milliliter of 4%PFA in PBS to each well, before gently agitating the cells for 15 minutes.After washing the cells twice in PBS, carefully remove the coverslips from the wells of the plate, and mount them as just demonstrated earlier in this video. To carry out confocal microscopy, turn on the hardware of the confocal microscope, including the mercury lamp power, PC microscope, scanner, and laser. Launch the software, then check the configuration and select the laser.Turn on the different units of confocal microscopy system according to the instructions. Place a prepared slide on the microscope stage, and use the stage controller to position the sample over the objective lens. For an oil immersion lens, add one drop of cedar oil on to the coverslip, and orient the slide with a coverslip facing the lens.Under fluorescence, find the cells of interest, then switch to scan mode, and choose the laser power and spectral range of the emission fluorophore. Capture high-quality images by tuning the laser intensity and other parameters. This figure illustrates the expression of pEGFP-N1 in HEK293 cells.The GFP signal was detected in the cytoplasm and the nucleus. The subcellular localization in HEK293 cells of gonadotropin-releasing hormone receptors from S-Japonica, or SjGnRHR, is shown here. EGFP receptors are in green, and Dil staining, which highlights the cell membrane, is in red.The receptors are seen localizing to the plasma membrane. To visualize the internalization of SjGnRHRs, cells were treated with gonadotropin-releasing hormone at different concentrations. As seen here, the internalization of the receptors proceeded in a dose-dependent manner, and the receptor was completely internalized from the cell's surface with the 10 to the minus six molar ligand.When treated with 10 to the minus six molar ligand for different time periods, internalized SjGnRHRs were detectable 10 minutes after agonist simulation, and extreme internalization occurred by 60 minutes. As demonstrated in HEK293 cells, in both experiments, the fluorescent SjGnRHR EGFP fusion protein primarily localized to the plasma membrane, and was dramatically and rapidly internalized into the cells. While attempting this procedure, it's important to remember to keep the cells under the right conditions without bacteria, fungi, and mycoplasma contamination.Following this procedure, and the methods like cell checking and immobilization can be performed in order to answer additional questions like the second in pass V of the protein receptors. After each development, the technology paved the way for researchers in the field of the cell biology and biochemistry to explore the localization and the internalization of transmembrane receptors in various cell lines.

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Biology

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

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

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

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

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

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

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.

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.

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.

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).

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

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

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.

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

Tracking Drug-induced Changes in Receptor Post-internalization Trafficking by Colocalizational Analysis

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and overall cell responsiveness to ligands and is, itself, influenced by intra- and extracellular conditions, including ligand-induced signaling. Optimized for use with monolayer-plated cultured cells, but extendable to free-floating tissue slices, this protocol uses immunolabelling and colocalizational analysis to track changes in intracellular receptor trafficking following both chronic/prolonged and acute interventions, including exogenous drug treatment. After drug treatment, cells are double-immunolabelled for the receptor and for markers for the intracellular compartments of interest. Sequential confocal microscopy is then used to capture two-channel photomicrographs of individual cells, which are subjected to computerized colocalizational analysis to yield quantitative colocalization scores. These scores are normalized to permit pooling of independent replicates prior to statistical analysis. Representative photomicrographs may also be processed to generate illustrative figures. Here, we describe a powerful and flexible technique for quantitatively assessing induced receptor trafficking.

Receptor trafficking modulates signaling and cell responsiveness to ligands and is, itself, responsive to cell conditions, including ligand-induced signaling. Here, we describe a powerful and flexible technique for quantitatively assessing drug-induced receptor trafficking using immunolabeling and colocalizational analysis.

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and ...