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 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.
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 resensitization2–4. 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’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 ‘snapshot’ 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.
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
2. Cell Culture
Note: Appropriate cell culture protocols will vary based on cell type(s) used. These procedures must be separately optimized. Detailed cell culture methodologies are readily available, including16. A similar procedure was used to obtain the representative results, with the notable differences:
3. Drug Treatment of Cultured Cells
Note: Multiple sequential, or overlapping, drug treatments are possible. The drugs, doses/concentrations, and durations of exposure used will depend on the specific experiment.
4. Fixation and Immunocytochemistry
Note:The specific labeling targets will vary by experiment. In our use, we labeled delta opioid receptors (DOR) and Rab5, Rab 11, and LAMP1, as discussed. The specific antibodies used will depend on the specific experiment. Optimum labeling conditions are dependent on the specific antibody(ies) used. A much fuller discussion of this topic is below.
5. Microscope Settings
6. Imaging
7. Colocalization Analysis
8. Data Normalization
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 colocalization (e.g., receptor-compartment marker). These data may then be used to compare changes in, in this case, receptor trafficking induced by the drug treatment(s) (Figure 2).
Figure 1. Representative photomicrographs of receptor and compartment-marker labelling with false-color colocalization maps. Primary cultures of dorsal root ganglia sensory neurons were exposed to prolonged (48 hr) and acute (1 hr) drug treatments (left axis labels). Only one prolonged condition is shown as representative results. The cells were then immunolabeled for delta opioid receptors (DOR) and a marker of recycling endosomes (Rab 11) with distinct primary-secondary (fluorophore conjugated) antibody pairs (top axis labels). The cells were imaged by two-channel sequential confocal microscopy (left column, center column). Representative images show the two labelled targets in two neurons. One neuron was treated with prolonged morphine followed by acute vehicle (top). The other neuron was treated with prolonged morphine followed by acute deltorphin II (DELT, a DOR agonist; bottom). In addition to subsequent quantitative colocalizational analysis, these representative images were processed to generate false-color colocalization maps (right column). Scale bars show 10 µm. Adapted from 12 under the provisions of CC BY-NC 3.0. Please click here to view a larger version of this figure.
Figure 2. Quantitative colocalization scores can be used to compare changes in receptor post-internalization trafficking. Primary cultures of dorsal root ganglia sensory neurons were exposed to prolonged (48 hr) and acute (1 hr) drug treatments (x-axis labels). Only one prolonged condition is shown as representative results. The cells were then immunolabelled for DOR and markers of early endosomes, recycling endosomes, and lysosomes. After imaging colocalization scores were determined, normalized, and pooled across 3 – 4 independent replicates. Data were then analyzed by two-way analysis of variance (ANOVA) with Tukey’s HSD post-hoc. Data are presented as mean +/- 95% confidence interval. * denotes p < 0.05. As is evident, this technique allowed the identification of changes in DOR post-internalization trafficking induced by acute treatment with deltorphin II, but not SNC80 (two different DOR agonists). Adapted from 12 under the provisions of CC BY-NC 3.0. Please click here to view a larger version of this figure.
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, with appropriate fixation and tissue preparation, to perform double-labeled immunohistochemistry in tissue slices, irrespective of subsequent analysis (e.g., 19).
This protocol is broadly compatible with different labeling targets. Each coverslip of plated cells will be double-labeled. Typically, this will be for the receptor of interest and a marker of one of the compartments of interest. There will typically be multiple labeling conditions in order to examine receptor colocalization with multiple different compartments.
Antibodies are inherently variable. Appropriate labeling protocols will vary between different antibodies. This includes appropriate antibody concentrations, buffer recipes, and timepoints. It should also be noted that different lots of the same antibody are best considered to be different antibodies. As such, labeling methods and antibody specificity should be validated, and if necessary optimized, for each combination of antibody and target tissue. The methods used here are a useful starting point. When validating labeling, it is important to perform appropriate controls: this includes samples processed without the addition of secondary antibodies (to control for autofluorescence) and without primary antibodies (to control for non-specific secondary labeling).
There are many factors which influence the design of labeling methods. Some considerations of note affecting the methods presented here include the use of Tris buffers, hypertonic saline, Polysorbate, BSA, and cold fish skin gelatin. Phosphate buffers can cause higher non-specific labeling and may interact with some lesser-used antibody conjugates. However, Tris buffers are, as noted, temperature sensitive. Hypertonic salt concentrations reduce non-specific labelling by disrupting ionic interactions. It is possible to further increase salt concentrations beyond what is specified in this protocol, if desired. Polysorbate 20 is used as a relatively gentle surfactant/detergent. This is preferred over Triton X-100, which has been reported to significantly disrupt, or even dissolve, membranes and thereby distort subcellular structure. The inclusion of low-concentration Polysorbate 20 in all buffers is useful in reducing non-specific labelling, as it improves the thoroughness of washes. BSA is a commonly used blocking agent intended to occupy non-specific protein binding sites in the target tissue. Some have reported that BSA may aggregate and lead to non-specific punctate labelling. If this issue arises, it is possible to replace BSA with non-fat dry milk or additional cold fish skin gelatin. Cold fish skin gelatin is a non-mammalian protein source also intended to occupy non-specific protein binding sites while presenting low reactivity to antibodies directed against mammalian proteins20.
Though not specified in this protocol, it is typical to add, to the blocking buffer, normal serum from the species in which the secondary antibody was raised. Typical concentrations are 1 – 3%. As discussed below, care in species selection must be exercised to avoid cross-reactivities.
When detecting two or more fluorescently-labeled targets, the choice of appropriate fluorophore combinations is particularly important. It is essential to have good spectral separation in order to avoid crosstalk. Further, the choice of fluorophores will depend on the configuration of the microscope to be used (available filters, laser lines, etc.). Fluorophore-conjugated antibody suppliers will offer advice on the best combinations of their products (e.g., 21). We find 488 nm- and 594 nm-excited fluorophores to be best pair for double-labeling and colocalizational analysis.
Secondary antibodies are typically species-reactive. That is, they will label any antibodies produced by the target species. Double-labeling ICC therefore requires that care be taken in choosing the host species of the primary and secondary antibodies in order to avoid cross-reactivity. For example, if the primaries are raised in rabbit and goat, it would not be appropriate to use a goat-raised secondary. If antibody availability does not allow for such species separation, there are protocols available to accomplish labelling with multiple same-host primary antibodies (e.g., 22), though substantially more optimization and validation should be expected.
As this method quantifies colocalization in photomicrographs, it is fundamentally necessary that all microscopy be consistent, of high quality, and accurately representative of the samples imaged. This includes both the hardware used (optics quality, etc.) and the particular procedures and parameters chosen. There are excellent resources available for guidance on appropriate microscopy23,24, including microscopy specifically for colocalizational analysis11,17,25.
Though of considerable methodological superiority to visual overlay methods, quantitative measures of colocalization do not as nicely lend themselves to the generation of illustrative figures for publication. Such illustrative figures are often helpful for readers and their absence may be criticized by reviewers. We have found that the inclusion of false-color ‘heatmaps’ visualizing colocalization to be helpful in constructing figures. The ImageJ plugin “Colocalization Colormap” (available from https://sites.google.com/site/colocalizationcolormap/) is useful in generating these images. It is important to note, however, that these images would be strictly illustrative in the context of the technique described here.
The authors have nothing to disclose.
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.
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 1:1500 |
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 1:2000 |
Goat anti-mouse Alexa 594 conjugated antibody | Life Technologies | A-11005 | Used at 1:200 to 1:2000 |
Donkey anti-goat Alexa 594 conjugated antibody | Life Technologies | A-11058 | Used at 1:200 to 1:2000 |