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

Summary

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.

Abstract

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.

Introduction

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’ available receptor complements and regulate receptor temporal activity, desensitization, and resensitization^2–4. 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 e....

Protocol

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

1. Prepare washing buffer by mixing 0.1M Tris-Buffered Hypertonic (300 mM) Saline and 0.05% Polysorbate 20; pH 7.4 at RT.
2. Prepare blocking buffer. To 0.05 M Tris-Buffered Hypertonic (300 mM) Saline add 0.05% Polysorbate 20, 3% Bo....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Trizma Base	Sigma Aldrich	T1503-500G
Sodium Chloride	Sigma Aldrich	S9888-500G
Tween 20	Fisher Scientific	BP337-500
Hydrochloric Acid	Sigma Aldrich	258148
Albumin from Bovine Serum	Sigma Aldrich	A7906-100G
Gelatin from cold water fish skin	Sigma Aldrich	G7041-100G
Corning Costar Cell Culture Plates: 24-well	Fisher Scientific	720084
12 circle Microscope Cover Glass	Fisher Scientific	1254580
Aqua/Poly-Mount	Polysciences	18606-20
Sodium Phosphate Monobasic	Sigma Aldrich	S9638-500G
Sodium Phosphate Dibasic	Sigma Aldrich	S9763-500G
Paraformaldehyde	Polysciences	00380-1
Dumont #5 Forceps - Standard/Dumoxel	Fine Science Tools	11251-30
Rabbit anti-DOR antibody	MyBioSource	MBS316175	Used at 1:1,500
Mouse anti-Rab5 antibody	Sigma Aldrich	R7904	Used at 1:750
Mouse anti-Rab11 antibody	Millipore	05-853	Used at 1:500
Goat anti-LAMP1 antibody	Santa Cruz	SC8098	Used at 1:750
Donkey anti-rabbit Alexa 488 conjugated antibody	Life Technologies	A-21206	Used at 1:200 to 1:2,000
Goat anti-mouse Alexa 594 conjugated antibody	Life Technologies	A-11005	Used at 1:200 to 1:2,000
Donkey anti-goat Alexa 594 conjugated antibody	Life Technologies	A-11058	Used at 1:200 to 1:2,000