This work was supported by a grant from CIHR (MOP394808) and a Canada Research Chair to C.M.C. E.W.O. was the recipient of a Post-Graduate Scholarship from NSERC.

Note: This protocol is broadly compatible with various monolayer-plated cell/tissue culture models, drug treatment regimens, and labelling targets. Thus in actual use, many specific parameters will vary based on experimental design. Here, references to these user-defined parameters are generic. Example conditions, as used to obtain the representative results, are included in italics.
1. Solutions
<ol>
	<li>Prepare washing buffer by mixing 0.1M Tris-Buffered Hypertonic (300 mM) Saline and 0.0...

<ol>
	<li>Drake, M. T., Shenoy, S. K., 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=Trafficking+of+G+protein-coupled+receptors.">Trafficking of G protein-coupled receptors.</a> Circ. Res. 99 (6), 570-582 (2006).</li><li>Hanyaloglu, A. C., von Zastrow, M. <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+GPCRs+by+endocytic+membrane+trafficking+and+its+potential+implications.">Regulation of GPCRs by endocytic membrane trafficking and its potential implications.</a> Annu. Rev. Pharmacol. Toxicol. 48, 537-568 (2008).</li><li>Hislop, J. N., von Zastrow, M. <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+ubiquitination+in+endocytic+trafficking+of+G-protein-coupled+receptors.">Role of ubiquitination in endocytic trafficking of G-protein-coupled receptors.</a> Traffic. 12 (2), 137-148 (2011).</li><li>Nagi, K., Pi&#241;eyro, G. <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+opioid+receptor+signalling:+implications+for+the+development+of+analgesic+tolerance.">Regulation of opioid receptor signalling: implications for the development of analgesic tolerance.</a> Mol. Brain. 4 (1), 25 (2011).</li><li>Whistler, J. L., Enquist, 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=Modulation+of+postendocytic+sorting+of+G+protein-coupled+receptors.">Modulation of postendocytic sorting of G protein-coupled receptors.</a> Science. 297 (5581), 615-620 (2002).</li><li>Von Zastrow, M. <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+opioid+receptors+by+endocytic+membrane+traffic:+mechanisms+and+translational+implications.">Regulation of opioid receptors by endocytic membrane traffic: mechanisms and translational implications.</a> Drug Alcohol Depend. 108 (3), 166-171 (2010).</li><li>Milan-Lobo, L., Whistler, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heteromerization+of+the+&#181;-+and+&#948;-opioid+receptors+produces+ligand-biased+antagonism+and+alters+&#181;-receptor+trafficking.">Heteromerization of the &#181;- and &#948;-opioid receptors produces ligand-biased antagonism and alters &#181;-receptor trafficking.</a> J. Pharmacol. Exp. Ther. 337 (3), 868-875 (2011).</li><li>Wang, H. -. B., Guan, J. -. S., Bao, L., Zhang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+subcellular+distribution+of+delta-opioid+receptor+fused+with+various+tags+in+PC12+cells.">Distinct subcellular distribution of delta-opioid receptor fused with various tags in PC12 cells.</a> Neurochem. Res. 33 (10), 2028-2034 (2008).</li><li>Scherrer, G., Tryoen-T&#243;th, P., 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=Knockin+mice+expressing+fluorescent+delta-opioid+receptors+uncover+G+protein-coupled+receptor+dynamics+in+vivo.">Knockin mice expressing fluorescent delta-opioid receptors uncover G protein-coupled receptor dynamics in vivo.</a> Proc. Natl. Acad. Sci. 103 (25), 9691-9696 (2006).</li><li>He, S. -. Q., Zhang, Z. -. N., 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=Facilitation+of+&#181;-opioid+receptor+activity+by+preventing+&#948;-opioid+receptor-mediated+codegradation.">Facilitation of &#181;-opioid receptor activity by preventing &#948;-opioid receptor-mediated codegradation.</a> Neuron. 69 (1), 120-131 (2011).</li><li>French, A. P., Mills, S., Swarup, R., Bennett, M. J., Pridmore, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colocalization+of+fluorescent+markers+in+confocal+microscope+images+of+plant+cells.">Colocalization of fluorescent markers in confocal microscope images of plant cells.</a> Nat. Protoc. 3 (4), 619-628 (2008).</li><li>Ong, E. W., Xue, L., Olmstead, M. C., Cahill, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+morphine+treatment+alters+&#948;+opioid+receptor+post-internalization+trafficking.">Prolonged morphine treatment alters &#948; opioid receptor post-internalization trafficking.</a> Br. J. Pharmacol. 172 (2), 615-629 (2014).</li><li>Schnell, U., Dijk, F., Sjollema, K. a. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunolabeling+artifacts+and+the+need+for+live-cell+imaging.">Immunolabeling artifacts and the need for live-cell imaging.</a> Nature Methods. 9 (2), 152-158 (2012).</li><li>Rozenfeld, R., Gupta, A., Devi, L. A. <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+targeting+of+mu-delta+opioid+receptor+heterodimers+by+RTP4.">Cell surface targeting of mu-delta opioid receptor heterodimers by RTP4.</a> Proc. Natl. Acad. Sci. 105 (41), 16045-16050 (2008).</li><li>Gupta, A., Mulder, 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=Increased+abundance+of+opioid+receptor+heteromers+after+chronic+morphine+administration.">Increased abundance of opioid receptor heteromers after chronic morphine administration.</a> Sci. Signal. 3 (131), ra54 (2010).</li><li>Seibenhener, M. L., Wooten, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+hippocampal+neurons+from+prenatal+mice.">Isolation and culture of hippocampal neurons from prenatal mice.</a> J. Vis. Exp. (65), 1-7 (2012).</li><li>Bolte, S., Cordeli&#232;res, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guided+tour+into+subcellular+colocalization+analysis+in+light+microscopy.">A guided tour into subcellular colocalization analysis in light microscopy.</a> J. Microsc. 224 (Pt 3), 213-232 (2006).</li><li>Field, A., Miles, J., Field, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Discovering Statistics Using R. , (2012).</li><li>Mattioli, T. -. A. M., Milne, B., Cahill, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-low+dose+naltrexone+attenuates+chronic+morphine-induced+gliosis+in+rats.">Ultra-low dose naltrexone attenuates chronic morphine-induced gliosis in rats.</a> Mol. Pain. 6, 22 (2010).</li><li>Vogt, R. F., Phillips, D. L., Henderson, L. O., Whitfield, W., Spierto, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+differences+among+various+proteins+as+blocking+agents+for+ELISA+microtiter+plates.">Quantitative differences among various proteins as blocking agents for ELISA microtiter plates.</a> J. Immunol. Methods. 101 (1), 43-50 (1987).</li><li>Johnson, I., Spence, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Molecular Probes Handbook. , (2010).</li><li>Morris, T. J., Stanley, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+immunocytochemical+staining+with+multiple+rabbit+polyclonal+antibodies.">A simple method for immunocytochemical staining with multiple rabbit polyclonal antibodies.</a> J. Neurosci. Methods. 127 (2), 149-155 (2003).</li><li>Pawley, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Biological Confocal Microscopy. , (2006).</li><li>North, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeing+is+believing?+A+beginners&#8217;+guide+to+practical+pitfalls+in+image+acquisition.">Seeing is believing? A beginners&#8217; guide to practical pitfalls in image acquisition.</a> J. Cell Biol. 172 (1), 9-18 (2006).</li><li>Zinchuk, V., Zinchuk, O., Okada, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+colocalization+analysis+of+multicolor+confocal+immunofluorescence+microscopy+images:+pushing+pixels+to+explore+biological+phenomena.">Quantitative colocalization analysis of multicolor confocal immunofluorescence microscopy images: pushing pixels to explore biological phenomena.</a> Acta Histochem. Cytochem. 40 (4), 101-111 (2007).</li></ol>

The authors have nothing to disclose.

We have optimized this protocol for the analysis of primary cultures of adult dorsal root ganglion neurons (primary sensory neurons). It can also be used, with little or no modification, for monolayer-plated cultured cells broadly. The colocalizational analysis is also possible in tissue slices and other such preparations11, however the drug treatment and tissue fixation/preparation components would not be appropriate.
Of interest, the ICC methods presented here can also be used, wi...

Receptors, especially G protein coupled receptors (GPCRs), are routinely trafficked intracellularly, to and from the cell surface1. These complexly orchestrated and tightly controlled processes dictate cells&#8217; available receptor complements and regulate receptor temporal activity, desensitization, and resensitization2&#8211;4. Importantly, these processes are responsive to cellular environments including drug-induced receptor activity or inactivity. That is, the actions of ligands at receptors can alter intracellular trafficking of those receptors, thereby altering cell responsiveness. In this manner, external ligands exert yet more effects upon cell function, even beyond classical messenger-to-effector cascades5,6.
Examining such changes in induced receptor trafficking is difficult. All available techniques involve limitations. Biotin protection assays have been used to monitor surface receptors populations. These receptors are biotinylated and a timecourse of immunoprecipitations is performed to quantify the reduction in biotinylated receptors over time. This technique essentially monitors the gradual degradation of an initial, labeled, population of receptors7, and is very useful in constructing time courses of this process. Unfortunately, this assay is unable to monitor any process other than degradation of the original pool of receptors, such as internalization, recycling, or new receptors. Also, the addition of an antibody in the 150kDa range to a receptor in the 50kDa range can alter the receptor&#8217;s trafficking8,9, and this technique may be difficult to use with low expression-level receptors.
Other procedures use various methods to identify intracellular trafficking compartments (e.g., endosomes, etc.) and assess their colocalization with the receptors of interest. This includes the use of heterologous systems expressing fluorescent-protein-tagged chimeric constructs of the receptors and compartment markers (e.g., Rab-family GTPases). This potentially enables the use of live-cell imaging, removing issues related to fixation and permeabilization. While powerful, such a strategy suffers from the same limitations of heterologous systems in general: tag and expression level effects on trafficking behavior and incompatibility with more physiologically representative cell types. More popularly, dyes are used to easily label intracellular compartments (e.g., lysosomes, ostensibly)10. Dyes, however, can lack specificity (all acidic organelles in the case of dyes for lysosomes) and do not assess trafficking through other compartments. Still, these techniques allow considerable control over the system and experimental conditions and may benefit from the colocalization analysis methods presented here, below.
The method we present here refines the tracking of receptor trafficking by colocalization. Using immunocytochemistry (ICC) to label appropriate markers, it is possible to identify multiple distinct intracellular compartments. This also allows the use of physiologically-relevant primary cell cultures in place of heterologous systems. This ICC protocol involves fixing the cells of interest prior to labeling; this permits labeling at a specific timepoint following drug treatment(s). This produces a &#8216;snapshot&#8217; of global receptor-compartment associations at that timepoint. With multiple timepoints, a timecourse of trafficking changes can also be constructed.
Briefly, cells are drug-treated, labeled for the receptor and intracellular compartment of interest, confocally imaged, and the photomicrographs are analyzed to mathematically quantify colocalization of the receptor and compartment11. In our use, we examined the colocalization of a receptor with Rab5, Rab11, and Lysosomal-associated membrane protein 1 (LAMP1). These markers identify early endosomes, recycling endosomes, and lysosomes, respectively. These colocalization measures act as proxies for the overarching processes of internalization, recycling, and degradation12.
As with all techniques, some limitations should be considered. Due to the need to image every individual neuron analyzed, this technique can become quite labor-intensive depending on the number of conditions and timepoints involved. All immunolabeling must also contend with the effects on cellular ultrastructure, protein localization, and epitope accessibility caused by fixation and permeabilization13.
Though originally optimized for use with primary cultures of primary sensory neurons, this method is broadly compatible with other monolayer-plated culture models.
The use of a mathematically quantified measure of colocalization is, notably, far more methodologically rigorous than previous techniques used to assess receptor trafficking changes, which have often relied on vague, subjective measures such as visually-inspected multi-channel overlays14.
This technique is particularly useful for its broad compatibility with in vivo interventions (prior to primary culture generation), in vitro interventions (during culture growth), and various labeling targets15. As such, it may be adapted to many different research questions.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Trizma Base</td>
			<td>Sigma Aldrich</td>
			<td>T1503-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>S9888-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Fisher Scientific</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Sigma Aldrich</td>
			<td align="right">258148</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin from Bovine Serum</td>
			<td>Sigma Aldrich</td>
			<td>A7906-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin from cold water fish skin</td>
			<td>Sigma Aldrich</td>
			<td>G7041-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Costar Cell Culture Plates: 24-well</td>
			<td>Fisher Scientific</td>
			<td align="right">720084</td>
			<td></td>
		</tr>
		<tr>
			<td>12 circle Microscope Cover Glass</td>
			<td>Fisher Scientific</td>
			<td align="right">1254580</td>
			<td></td>
		</tr>
		<tr>
			<td>Aqua/Poly-Mount</td>
			<td>Polysciences</td>
			<td>18606-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate Monobasic</td>
			<td>Sigma Aldrich</td>
			<td>S9638-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate Dibasic</td>
			<td>Sigma Aldrich</td>
			<td>S9763-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Polysciences</td>
			<td>00380-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps - Standard/Dumoxel</td>
			<td>Fine Science Tools</td>
			<td>11251-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-DOR antibody</td>
			<td>MyBioSource</td>
			<td>MBS316175</td>
			<td>Used at 1:1500</td>
		</tr>
		<tr>
			<td>Mouse anti-Rab5 antibody</td>
			<td>Sigma Aldrich</td>
			<td>R7904</td>
			<td>Used at 1:750</td>
		</tr>
		<tr>
			<td>Mouse anti-Rab11 antibody</td>
			<td>Millipore</td>
			<td>05-853</td>
			<td>Used at 1:500</td>
		</tr>
		<tr>
			<td>Goat anti-LAMP1 antibody</td>
			<td>Santa Cruz</td>
			<td>SC8098</td>
			<td>Used at 1:750</td>
		</tr>
		<tr>
			<td>Donkey anti-rabbit Alexa 488 conjugated antibody</td>
			<td>Life Technologies</td>
			<td>A-21206</td>
			<td>Used at 1:200 to 1:2000</td>
		</tr>
		<tr>
			<td>Goat anti-mouse Alexa 594 conjugated antibody</td>
			<td>Life Technologies</td>
			<td>A-11005</td>
			<td>Used at 1:200 to 1:2000</td>
		</tr>
		<tr>
			<td>Donkey anti-goat Alexa 594 conjugated antibody</td>
			<td>Life Technologies</td>
			<td>A-11058</td>
			<td>Used at 1:200 to 1:2000</td>
		</tr>
	</tbody>
</table>

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.

Using this technique, it is possible to quantify changes in receptor post-internalization trafficking following both chronic/prolonged and acute drug treatments. After drug treatments, fixation, and labeling, high-resolution two-channel photomicrographs are captured of each cell of interest. Representative images may be combined with false-color colocalization to generate illustrative figures (Figure 1). Subsequent colocalizational analysis, as described, yield quantitative scores of target-target coloca...

Watch this Scientific Journal Video about Tracking Drug-induced Changes in Receptor Post-internalization Trafficking by Colocalizational Analysis at JoVE.com

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

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 ...

tracking-drug-induced-changes-receptor-post-internalization

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Biology

8.7K 조회수.  University of California Irvine.  수용체는 리간드 매매 시그널링 및 세포 반응성을 조절하고, 그 자체의 리간드 - 유도 된 세포 신호 전달을 포함한 조건에 응답한다. 여기, 우리는 양적 immunolabeling 및 colocalizational 분석을 사용하여 약물 유발 수용체 인신 매매를 평가하기위한 강력하고 유연한 기술을 설명합니다. 