Name: live cell imaging and 3d analysis of angiotensin receptor type 1a trafficking in transfected human embryonic kidney cells using confocal microscopy
Uploaded: 2017-03-27T13:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy at JoVE.co

The research work presented in this manuscript was supported by grant R01 HL121456-01A1 to Kathryn Sandberg. This work was supported in addition by the availability of a Leica SP8 AOBS in the Microscopy and Imaging Shared Resource (MISR) at Georgetown University Medical Center which is partially funded by P30-CA051008 from the National Institutes of Health of the United States of America. We gratefully acknowledge Leica SP8 imaging assistance by Peter Johnson, and the use of the Zeiss LSM880 in the laboratory of Thomas Coate, Department of Biology at Georgetown University with imaging assistance provided by Alma Arnold of Carl Zeiss Microscopy LLC. The content in this manuscript is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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The authors declare that they have no competing financial interests.

Preliminary experiments presented here illustrate that over a rather short time course of 24 min, no appreciable change in delivery of AT1aR-GFP to the lysosomes occurred. In general, delivery of internalized receptors to lysosomes could occur as early as 15-20 min. This experiment is consistent with the hypothesis that the bulk of the internalized AT1aR is targeted to large, perinuclear endosomes. Evidence that at least some AT1aR arrives in lysosomes was demonstrated by Li et al., who compared cells at 5 min and 30 min after Ang II treatment and found significant colocalization of LAMP1&#160;with AT1aR at 30 but not 5 min 6.
This paper provides a detailed description of cell preparation, imaging, and analysis steps and discusses the many inherent pitfalls in this advanced imaging technique. This method depends upon critical steps discussed below.
Cell Culture and Transfection
Since the good health of cells is critical for any live cell imaging experiment, it is important to avoid using cells beyond passage 11 because of changes in growth rates and transfection efficiency at higher passages. To maintain healthy cells throughout the experiment, the incubation period of transfection mix with cells should not exceed 5 h since liposome solution is toxic to cells evidenced by increased membrane blebbing. Temperature, humidity and CO2 control is significant before as well as during imaging. Drifts in temperature during imaging can cause relatively major z-section drifts and loss of focal plane stability. Hence extra caution is recommended during addition of Ang II to the cells during imaging to ensure that the focal plane is accurately maintained.
Imaging
An advantage of live-cell imaging over immunostaining using fixed cells is that the in vivo conditions can be closely controlled and artifacts associated with fixed cells can be avoided. However, live-cell imaging can be challenging to optimize for cellular processes that are extremely rapid, like receptor internalization. Instrument speed and the need to avoid photobleaching limit the choice of events that can be studied. Each imaging experiment might demand certain choices to be made in some parameters and compromises in others; examples are choices in pixel size, line accumulation, zoom factor, and scanning speed to get good quality data for analysis and avoid oversampling at the same time. Critical decisions in these factors depend upon balancing the need for obtaining adequate pixel size to distinguish individual vesicles and the need for obtaining sufficient temporal resolution of the events that occur. Thus some tradeoffs must be made in order to acquire information quickly enough as the cell begins to internalize GFP-labeled receptor. In our case, although modern confocal software can suggest the optimal pixel size, our experiments used a value less than the optimal pixel size according to the Nyquist equation 14. Optimal choices of settings for other experiments will also depend upon how bright the cells are since larger pixel size will enable faster accumulation of photons allowing a trade off with longer voxel dwell time to collect more photons.
For the analysis to be successful, the live-cell imaging also critically requires accurate focal stabilization during time-lapse imaging. Focal shifts as well as failure to image the entire cell can have profound impact upon quantification of receptor internalization as well as total cellular GFP intensity. The solution to this problem of focal shifts during earliest time points of imaging following the addition of Ang II has been provided by confocal manufacturers in the form of autofocus solutions whereby coverslip interferometry with an IR laser coupled with feedback to automated focus control is used to maintain relative distance from the objective to the coverslip surface identified by the IR reflection (e.g. Definite Focus or Adaptive Focus Control). The remaining problem of the microscopist is then to identify the correct z-range so that despite shape changes, the entire cell remains in the imaging volume. Temperature is another critical factor to maintain the focus/Z position during initial time points after addition of Ang II. The temperature of the objective as well as the live cell imaging chamber should be maintained at 37 &#176;C throughout the experiment.
Another critical component of imaging, is acquiring images quantitatively. If a HyD detector is used in the Photon Counting Mode, the imaging parameters must be set so that photon pileup does not occur. For PMTs and other detectors, the images must not contain saturated pixels (voxels). In both cases, a specialized look-up table (LUT) can be used to visualize the presence of saturated pixels and of course if they are present, the microscopist should reduce laser intensity and/or gain.
Analysis
Although the approach described here is applied to the AT1aR receptor and its colocalization with lysosomes, it is applicable to experiments in which the relative quantity of the receptor in subcellular compartments labeled with various fluorescent tags is at question. If the goal is to measure whether or not the receptor moves to the organelle, then using a Pearson&#39;s colocalization coefficient with a marker for the organelle is problematic. Every pixel where the organelle is identified will have a high amount of the marker so that one of the coefficients will always be near 100%. Thus, colocalization measurements could be misleading. On the other hand, measuring the percent of receptor on the organelle versus total amount of receptor is much more informative and clearer to interpret. A case in which protein colocalization might best be described using Pearson&#39;s correlation coefficient would be AT1aR receptor and LAMP1 whose interaction has been documented, albeit by a more sophisticated colocalization approach, namely FLIM/FRET 6.
The use of 3D imaging is more accurate than using representative slices of cells with analysis in 2D because of cellular movements that are inevitable during the time course (Live Imaging 2 movie). It is clear from the live-cell images shown in Figures 3, 4B, and 5A that the receptor is primarily localized at the plasma membrane, however, within a few minutes of exposure to Ang II it localizes to bright spots on the membrane and small vesicles adjacent to the membrane, probably coated pits and early endosomes (Figures 4B, 4C). With continued progression of time, the vesicles move through the cell accumulating in larger vesicles adjacent to the nucleus that have been described as multivesicular endosomes (MVEs, Figures 1, 5A) 3,15. A single slice through the cell over time would distort the internalization process and misrepresent the true events particularly if the slice did not include the cell surface at early times, and the MVEs at later times.
In this study, two approaches for image analysis can minimize effects of photobleaching of GFP as well as fluorescent dye on the quantitative outcome. As cells photobleach (albeit to a limited extent), the relative brightness of internalized vesicles relative to the entire cell intensity is maintained, assuming photobleaching occurs uniformly. Fluorescent dye-positive vesicles are also easily identified by selecting the standard deviation of the mean fluorescent intensity option for thresholding bright internalized vesicles. This distinguishes them from background even as the entire level of fluorescence declines. Thus, our measurements were not sensitive to the effects of modest photobleaching. However, strong photobleaching such as 50% loss of cell intensity over time might have a larger impact upon quantitative results. Alternatively, the effects of photobleaching could be measured independently in control experiments absent Ang II in the case of AT1aR-GFP or in adjacent untransfected cells in the case of fluorescent dyes. The effects of photobleaching could then be factored in during analysis.
Additional Concerns
Recently, cell biologists have conducted critical assessment and modification of fluorescent proteins for monitoring membrane trafficking in cells to avoid artifacts generated by the fluorescent protein itself, independent of the protein being tagged. Various fluorescent proteins are vulnerable to interactions with the local microenvironment leading to protein precipitation or crosslinking based on their pKa&#39;s and presence of the amino acid cysteine, respectively 16. Another outcome of these interactions may include loss of fluorescence 16. All of these artifacts affect quantitative measurements of trafficking. Constantini et al. have generated monomeric fluorescent proteins for imaging that lack cysteine to eliminate potential artifacts 16. Thus, depending upon the goal of the imaging experiments, switching from EGFP (non-monomeric) to a monomeric, cysteine-free form would be advantageous for studies of acidic and/or reducing vesicular microenvironments. Another example would be their improved utility for FRAP or FRET experiments where multimerization and crosslinking would impact protein diffusion and protein-protein interactions.
An additional caveat is that it is important to test whether or not EGFP labeling modifies the normal behavior of the receptor. One way to test this possibility is by determining if there are any differences in receptor affinity (Kd) and maximum receptor number (Bmax) by conducting radioligand binding assays on cells transfected with the wild type or the EGFP-tagged receptor.
Another potential artifact to consider when imaging lysosomes using LysoTracker Red together with GFP fusion proteins is that LysoTracker Red has been shown to be capable of photoconversion to green 17. The authors recommend that use of epifluorescence illumination be minimized, discuss occurrences of this artifact, and refer to successful instances of colocalization using this reagent. Additionally, other markers of the lysosomes can also be employed to verify colocalization results, for example, tagged LAMP1 6.
In future, the sensitivity and specificity of this assay can be examined for receptor targeting to lysosomes as well as other subcellular compartments by using inhibitors of vesicle transport and/or fusion, by inhibiting lysosome acidification using bafilomycin to block degradation but not accumulation 7,18, by using chloroquine to block fusion with lysosomes consequently blocking co-localization of receptor with lysosomes 2,5,6,7,18, by using positive controls such as the targeting of labelled Ang II to lysosomes 2, and by comparison to other well-described receptor and ligand systems such as transferrin or epidermal growth factor and their cognate receptors 3. An interesting positive control, albeit artefactual, in our experiments was a result observed in adjacent, untransfected, AT1aR-GFP negative cells. These cells apparently took up GFP released from transfected cells and targeted it to lysosomes where colocalization with fluorescent dye was quite prominent (not shown). This colocalization, however, was not sensitive to Ang II as expected.
In conclusion, the methods for transfection, imaging, and image analysis presented here provide a means by which receptor internalization to various subcellular compartments can be quantitatively traced within living cells over time. Relative comparisons between different receptors or selectively mutated receptors are possible with regard to kinetics and subcellular localization to specific compartments. Possible difficulties and artifacts associated with this technology are discussed. Nonetheless, fast live-imaging in 3D coupled with image analysis can be used to measure receptor uptake and rapid transitions through the cytoplasm to molecularly definable compartments. This sets the stage for measurement of differences resulting from mutation of receptor or application of agonists, antagonists, and inhibitors.

The overall goal is to obtain quantitative evidence in time and space of receptor colocalization with specific subcellular organelles after treatment with a receptor agonist. Thus, the immediate goal here is to capture time-lapse confocal images of lysosomes and a transmembrane spanning receptor in human embryonic kidney cells (HEK) following transfection and to subsequently assemble and analyze the imaging data in 3D to quantitatively measure the delivery of receptor to lysosomes. Investigating the simultaneous trafficking of a transmembrane receptor with lysosomes or other organelles during endocytosis when activated by receptor ligand can help determine how the transmembrane receptor is regulated under physiological and pathophysiological conditions.
AT1aR is presumed to be degraded in lysosomes following treatment with Ang II in model cells systems, primarily HEK293 1 although the majority of receptors are primarily localized in multivesicular recycling endosomes for later recycling to the plasma membrane while the bulk of the ligand, Ang II, is degraded in the lysosome 2,3,4,5. More recently, Li et al. demonstrated using F&#246;rster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) techniques that AT1aR colocalizes (on ~10 nm scale) with LAMP1, a lysosomal membrane protein suggesting that at least some of the receptor is targeted to and degraded by lysosomes 6. These authors noted that the lysosomal inhibitor chloroquine blocked AT1aR-LAMP1 association which is consistent with previous reports suggesting that an effect of chloroquine is to block fusion of late endosomes or autophagosomes with lysosomes. Other indirect approaches to determine AT1aR localization in lysosomes have utilized bafilomycin, a lysosomal inhibitor to infer AT1aR presence in lysosomes 3,4,5,6,7,8.
Live-cell imaging avoids potential effects of fixation on cell volume, and loss/dimming/redistribution of GFP-chimeric receptor. Previous studies have relied upon fixed cells to determine colocalization or co-occurrence of the receptor with lysosomes or using live cells labeled with receptor-binding surrogates such as &#946;-arrestin 1,2,3,4,5,6. Live-cell imaging of other GPCRs using spinning disk confocal or laser point scanning confocal microscopes equipped with GaASP or HyD detectors enabled investigators to observe the internalization and trafficking of receptors in individual cells imaged in z-stacks at 30 s intervals 9,10. However even when live cell z-stacks are rapidly collected, analysis may only occur after selection of a single plane within each stack, i.e. in 2D 10. While this might be sufficient for studying the internalized GPCR or tyrosine kinase receptor in question, the AT1aRs accumulates in vesicles that change size and position over time and thus are inherently more difficult to adequately sample using a representative single slice at each time point. To thoroughly determine the route of the labeled AT1aR through the cell and to detect each subpopulation of vesicles differing in size and position, we have devised a method for 3D imaging and 3D analysis to track these changes and to be used in conjunction with labeling of various intracellular compartments such as lysosomes.
Investigators can use this protocol for live-cell imaging to directly visualize the movement of AT1aR into the cell and its transfer to subcellular compartments following Ang II stimulation. Subcellular compartments are tagged with fluorescent protein chimeras or other fluorescent markers. This protocol can also be used as a first approach to localize receptors in subcellular organelles with a minimum resolution of 200 nm in order to compare mutated versus wild-type receptors or to detect changes following pharmacological treatments. The technique is accessible since it can be carried out on any confocal microscope equipped for live-cell imaging. The relative ease of this approach contrasts with increased expertise and equipment needed to carry out FRET/FLIM/BRET techniques which detect molecular interactions 6,11. These measurements define protein-protein interactions at high resolution (~10 nm) and localization within subcellular organelles is inferred. These more advanced techniques are used to follow up and further define molecular interactions of interest, rather than the passage of receptors through subcellular compartments, and they directly demonstrate protein-protein interactions at specific times and places within the cell 11. FLIM, a FRET technique independent of acceptor concentration, is most often performed on fixed samples since image acquisition is slower. In contrast, ratio FRET provides rapid imaging and high resolution colocalization of interacting proteins. The disadvantage of ratio FRET is that imaging utilizes widefield epi-fluorescence to gain imaging speed and to include the entire cell, resulting in reduced resolution and contrast of organelles 12. Bioluminescence resonance energy transfer (BRET) is another advanced technique that has been applied to GPCRs to measure the acquisition of molecular proximity over time 11. In this technique a protein fluorophore is molecularly divided and each half linked to one of two proteins in question. When the two proteins of interest bind each other, the parts of the chimeric tag re-assemble to acquire fluorescence and the increased fluorescence quantified over time.
Here, we present a straightforward technique for live-cell imaging coupled with image quantification to study trafficking of AT1aR in the cell upon Ang II stimulation and the potential change in delivery of internalized AT1aR to lysosomes following Ang II stimulation. The ultimate use for this technique is to characterize differences in membrane trafficking comparing wild type and mutated receptors. These differences will help to identify the mechanism(s) that impact physiological activities of AT1aR leading to regulation of blood pressure.

<table><tbody><tr><td>Angiotensin II (Ang II)</td><td>Sigma-Aldrich</td><td>A9525&amp;nbsp;</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle medium (DMEM 1x)</td><td>Gibco -ThermoFisher Scientific</td><td>11960-044</td><td>Warm in 37 &amp;deg;C water bath before use&amp;nbsp;</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Sigma-Aldrich</td><td>F2442</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Penicillin Streptomycin (Pen Strep)</td><td>Gibco -ThermoFisher Scientific</td><td>15140-122</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>L-Glutamine 200 mM (100x)</td><td>Gibco (life technologies)</td><td>25030-081</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Human Embryonic Kidney (HEK)-293 cells</td><td>American Type Culture Collection (ATCC)&amp;nbsp;</td><td>CRL-1573</td><td>Passage number 5 - 12</td></tr><tr><td>Nunc Lab-Tek II chambered 1.5 German Coverglass System</td><td>Sigma-Aldrich</td><td>155409</td><td></td></tr><tr><td>Lipofectamine 2000 Transfection Reagent</td><td>ThermoFisher Scientific</td><td>11668019</td><td>Store at 4 &amp;deg;C&amp;nbsp;</td></tr><tr><td>Opti-MEM, Reduced Serum Medium</td><td>Gibco- ThermoFisher Scientific</td><td>31985-070</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>PBS 10x&amp;nbsp;</td><td>Fisher BioReagents</td><td>BP3994</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Dulbecco&#039;s Modified Eagle medium (DMEM 1x)&amp;nbsp;</td><td>Gibco -ThermoFisher Scientific</td><td>31053-028</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>LysoTracker Red DND-99</td><td>molecular probes -ThermoFisher Scientific</td><td>L7528</td><td>Store aliquotes in dark at -20 &amp;deg;C .&amp;nbsp;</td></tr><tr><td>Volocity Ver. 6.3 Image Analysis software</td><td>PerkinElmer</td><td>Visualization and Quantitation Modules</td><td>For 3D image analysis of image stacks</td></tr><tr><td>Leica SP8 AOBS</td><td>Leica Microsystems</td><td>laser scanning confocal microscope</td><td>For image collection</td></tr><tr><td>Zeiss LSM 880</td><td>Carl Zeiss</td><td>laser scanning confocal microscope</td><td>For image collection</td></tr></tbody></table>

live cell imaging and 3d analysis of angiotensin receptor type 1a trafficking in transfected human embryonic kidney cells using confocal microscopy

Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in transfected human embryonic kidney-293 (HEK) cells following stimulation with angiotensin II (Ang II). HEK cells are transiently transfected with plasmid DNA containing AT1aR tagged with enhanced green fluorescent protein (EGFP). Lysosomes are identified with a red fluorescent dye. Live-cell images are captured on a laser scanning confocal microscope after Ang II stimulation and analyzed by software in three dimensions (3D, voxels) over time. Live-cell imaging enables investigations into receptor trafficking and avoids confounds associated with fixation, and in particular, the loss or artefactual displacement of EGFP-tagged membrane receptors. Thus, as individual cells are tracked through time, the subcellular localization of receptors can be imaged and measured. Images must be acquired sufficiently rapidly to capture rapid vesicle movement. Yet, at faster imaging speeds, the number of photons collected is reduced. Compromises must also be made in the selection of imaging parameters like voxel size in order to gain imaging speed. Significant applications of live-cell imaging are to study protein trafficking, migration, proliferation, cell cycle, apoptosis, autophagy and protein-protein interaction and dynamics, to name but a few.

In this representative set of experiments, HEK cells were transfected with angiotensin type 1a receptor (AT1aR-GFP) construct (E1,2,3-AT1aR) cloned into the pEGFP-N2 plasmid13. Time-lapse images of HEK were acquired and played as a movie (See Live Cell Imaging 1 and Live Cell Imaging 2 movies). The movies and stills show that after ligand stimulation, AT1aR-EGFPs are localized predominantly in the plasma membrane, but with the addition of Ang II these receptors become internalized within 2.5 min to form bright vesicles and at later times cluster in larger vesicles in a perinuclear zone (Figures 4B, 5, and 6A; Live Cell Imaging 2).
Quantification of total cell GFP intensity and total vesicle GFP intensity for the cell in Figure 5 provide an illustration of the complications inherent when Ang II induces ruffling and cell shape changes immediately following stimulation (Figure 5A, arrow at 0 min post-Ang II, upper row, orange indicates thresholded objects). At later times, the localization of GFP-containing vesicles in a perinuclear cluster also interferes (Figure 5A, red arrow at 29.5 min). Although all objects should be included in the total cell GFP intensity measurement, the ruffles should be excluded from vesicle measurements as they represent plasma membrane AT1aR-GFP. In contrast, the clustered perinuclear vesicles should not be excluded from the vesicle measurement (the latter are seen in an area of the cell enclosed in red circles in Figure 5A, arrow at 29.5 min post-Ang II, lower row). Attempts to accomplish this by size filtering (more than or less than 7 &#181;m3) was not useful as it could not discriminate ruffles and clustered vesicles (Figures 5A and 5B, compare Filter&#62;7, Filter&#60;7, and no filter). A possible alternative would be to draw a Regions of Interest (ROI) encompassing the cytoplasmic portion of the cell but excluding the ruffling edge and perimeter of the cell. Ultimately, this cell would not be included in quantitative results because there were saturated pixels of GFP present.
A second representative example is shown in Figure 6A illustrating a time course in which the autofocus was not used. The internalized GFP is shown as percent total cellular GFP (Figure 6B). The time points prior to the red arrow at 3 min are misleading as only part of the cell was in the imaging volume but 3 min after Ang II addition, the focal plane restabilized. The total GFP within the cell demonstrates that this cell did not photobleach appreciably over time (Figure 6C). Between 3 and approximately 12 min, percent GFP intensity in vesicles increases then levels off (Figure 6B). Next, lysosomes identified by fluorescent dye were used to identify ROIs within a transfected cell which were then quantified for the sum of GFP intensity (Figure 6D). After three minutes, the intensity of AT1aR-GFP in red-identified lysosomes does not vary over time (Figure 3D, n = 5, mean&#160;&#177; standard error of the mean). Thus there is no detectable increase in delivery of AT1aR-GFPs to the lysosomes for up to 24 minutes after Ang II administration.
<img alt="Figure 1" src="/files/ftp_upload/55177/55177fig1.jpg" /> 
Figure 1: Saturated Pixels Identified in a Lookup Table (LUT). The red arrow indicates saturated pixels shown in blue. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55177/55177fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55177/55177fig2.jpg" /> 
Figure 2: Overview of Image Quantification Software. Key items are indicated by numbers and discussed within the methods text. 1) Action item, 2) Selected image, 3) free-hand ROI tool, 4) Measurements tab, 5) Find Objects, 6) Population 1 Find Objects dialog box, 7) Arrow activating time lapse movie, 8) Measurements item, 9) A second Find Objects dialog box containing a Filter Population command. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55177/55177fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55177/55177fig3.jpg" /> 
Figure 3: Color Registration Correction. Images with uncorrected and corrected pixel registration are shown together with insets of several colocalizing vesicles containing AT1aR-GFP. The red pixels are shifted 1 pixel to the left in the XY plane of the uncorrected image. Insets show boxed areas at higher magnification. Scale bar = 3.3 &#181;m, voxel dimensions = 0.138 x 0.138 x 1 &#181;m3. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55177/55177fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55177/55177fig4.jpg" /> 
Figure 4: Illustration of Thresholding to Identify GFP- and Fluorescent Dye-containing Vesicles and GFP-whole Cells. (A) Identification of whole cells using AT1aR-GFP (GFP, top row) and thresholded GFP for the whole cell (ID Cell, bottom row) are shown at 0 and 2.5 min after Ang II addition. White boxes indicate inset areas shown in (B) and (C). (B) AT1aR-GFPs are compared at 0 and 2. 5 min after Ang II addition in single color images (top row). Vesicles identified by thresholding are shown in the bottom row (ID Ves, purple). (C) Two-color images (AT1aR-GFP /fluorescent dye) are shown at 0 and 2.5 min post Ang II addition. Lysosomes identified by thresholding are shown by red outlines. Scale bar = 7.0 &#181;m, voxel dimensions = 0.114 x 0.114 x 1.4 &#181;m3. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55177/55177fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/55177/55177fig5.jpg" /> 
Figure 5: Illustration of the Effects of Ruffles and Clustered Vesicles on Measurement of Internalized GFP Receptors. (A) AT1aR-GFP in Ang II treated cells at 0 (top row) and 2.5 min (bottom row) are shown in three filtering experiments where in the first column a filter was applied to select only objects greater than 7 &#956;m3, in the second column to select objects less 7 &#956;m3, and in the third column no filter was used. Thresholding level was constant in all three. Arrows indicate large thresholded objects, circles indicate an area of cytoplasm containing perinuclear vesicles at later time points. Thresholded areas are orange. (B) Quantification of GFP total intensity in vesicles is shown for thresholded areas with or without size filtering (by column in A) at two time points (by row in A). Voxel dimensions were 0.138 x 0.138 x 1 &#181;m3. Scale bar = 5.6 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55177/55177fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/55177/55177fig6.jpg" /> 
Figure 6: Tracking AT1aR- GFP Internalization and Potential Localization in Lysosomes. (A) Five selected time points of AT1aR-GFP with LysoTracker Red are shown from the accompanying movie (Live cell imaging 1 movie) at 0, 3, 6, 9, and 12 min post Ang II. The grid represents a unit scale of 6.56 &#956;m3, voxel dimensions were 0.297 x 0.297 x 1.2 &#181;m3, and the z-step = 1.2 &#181;m. (B) A plot of the percent total GFP in vesicles over time illustrates the rapid internalization of AT1aR-GFP. (C) A plot of the total GFP in the cell over time illustrates that relatively little photobleaching occurred during 24 min of imaging. (D) Lysosomes were identified by identifying LysoTracker Red-positive vesicles. The total GFP intensity within fluorescent dye-positive vesicle ROIs was measured at each time point (Normalized to the first time point with no compensation for photobleaching which was not independently measured). (C-D) Prior to the arrow, the measurements are not valid since the focus shifted. N = 5, Mean &#177; standard error of the mean. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55177/55177fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Movie 1" src="/files/ftp_upload/55177/55177mov1.jpg" /> 
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55177/Live_Cell_Movie_1.MOV">Movie 1: Live Cell Imaging 1: (Right click to download)</a> HEK cell transfected with AT1aR-GFP was imaged every 1 min for 24 min (25 frames) after the addition of 100 nM Ang II. Movie was acquired using a laser scanning confocal microscope with a Plan-Apochromat 63X/1.4 N.A oil objective, voxel dimensions of 0.279 x 0.279 x 1.2 &#181;m3; and 5 slices for a total of 4.794 &#181;m thickness imaged at 3.20 &#181;s/pixel. The movie rate is 2 frames/s. Unit = 6.56 &#181;m.
<img alt="Movie 2" src="/files/ftp_upload/55177/55177mov2.jpg" /> 
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55177/Live_Cell_Movie_2.mp4">Movie 2: Live Cell Imaging 2: (Right click to download)</a> HEK cell transfected with AT1aR-GFP was imaged every 30 s for 25 min (51 frames) after the addition of 100 nM Ang II. Movie was acquired using a laser scanning confocal microscope with a Plan-Apochromat 40X/1.4 N.A oil objective. Voxel dimensions were 0.11 x 0.11 x 1.4 &#181;m3. 7 slices for a total of 8.4 &#181;m thickness were imaged at 3.72 s/frame. The movie rate is 3 frames/s. Scale bar = 7 &#181;m.

Watch this Scientific Journal Video about Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy at JoVE.co

Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy

Here we present a protocol to image cells expressing green fluorescent protein-tagged angiotensin type 1a receptors during endocytosis initiated by angiotensin II treatment. This technique includes labeling lysosomes with a second fluorescent marker, and then utilizing software to analyze the co-localization of receptor and lysosome in three dimensions over time.

Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in ...

live-cell-imaging-3d-analysis-angiotensin-receptor-type-1a

Nanyang Technological University

Research

JoVE Journal

Biology

9.6K Views.  Georgetown University Medical Center.  Hier presenteren we een protocol om de afbeelding cellen die groen fluorescerend eiwit-tag soort angiotensine 1a receptoren tijdens endocytose geïnitieerd door angiotensine II behandeling. Deze techniek omvat labeling lysosomen met een tweede fluorescerende marker, en vervolgens met behulp van software om de co-lokalisatie van receptor en lysosomen te analyseren in drie dimensies in de tijd. 

Video: Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &beta;-arrestin

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to intracellular responses. GPCRs control diverse biological functions such as chemotaxis, intracellular calcium release, gene regulation in a ligand dependent manner via heterotrimeric G-proteins1-2. Ligand binding induces a series of conformational changes leading to activation of heterotrimeric G-proteins that modulate levels of second messengers such as cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3) and diacyl glycerol (DG). Concomitant with activation of the receptor ligand binding also initiates a series of events to attenuate the receptor signaling via desensitization, sequestration and/or internalization. The desensitization process of GPCRs occurs via receptor phosphorylation by G-protein receptor kinases (GRKs) and subsequent binding of &beta;-arrestins3. &beta;-arrestins are cytosolic proteins and translocate to membrane upon GPCR activation, binding to phosphorylated receptors (most cases) there by facilitating receptor internalization 4-6.
Leukotriene B4 (LTB4) is a pro-inflammatory lipid molecule derived from arachidonic acid pathway and mediates its actions via GPCRs, LTB4 receptor 1 (BLT1; a high affinity receptor) and LTB4 receptor 2 (BLT2; a low affinity receptor)7-9. The LTB4-BLT1 pathway has been shown to be critical in several inflammatory diseases including, asthma, arthritis and atherosclerosis10-17. The current paper describes the methodologies developed to monitor LTB4-induced leukocyte migration and the interactions of BLT1 with &beta;-arrestin and , receptor translocation in live cells using microscopy imaging techniques18-19.
Bone marrow derived dendritic cells from C57BL/6 mice were isolated and cultured as previously described 20-21. These cells were tested in live cell imaging methods to demonstrate LTB4 induced cell migration. The human BLT1 was tagged with red fluorescent protein (BLT1-RFP) at C-terminus and &beta;-arrestin1 tagged with green fluorescent protein (&beta;-arr-GFP) and transfected the both plasmids into Rat Basophilic Leukomia (RBL-2H3) cell lines18-19. The kinetics of interaction between these proteins and localization were monitored using live cell video microscopy. The methodologies in the current paper describe the use of microscopic techniques to investigate the functional responses of G-protein coupled receptors in live cells. The current paper also describes the use of Metamorph software to quantify the fluorescence intensities to determine the kinetics of receptor and cytosolic protein interactions.

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &#946;-arrestin

This paper describes the methodology to determine the chemotactic response of leukocytes to specific ligands and identify interactions between the cell surface receptors and cytosolic proteins using live cell imaging techniques.

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to ...

Immunology and Infection

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. 
Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.
By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

Bioengineering

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Developmental Biology

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and a working heart on the other. Thus, cardiomyocytes serve as good models for cardiac cellular physiology and pathophysiology, for pharmaceutical investigations as well as for the exploration of transgenic animal models. Here we describe a method of isolating the cells from the heart. Furthermore we show how a genetic manipulation on cardiac myocytes can be performed without breeding a transgenic animal: This is the combination of long term culture (1 week) and adenoviral gene transfer. The latter one is described from the construction of the virus to the transduction of the cells. It can be used for the expression of genetically encoded biosensors (GEBs), fluorescent fusion proteins, but also for protein over expression and down regulation, e.g. using RNAi. Here we provide an example for the expression of a fusion protein staining a subcellular structure (Golgi). Such protein expression can be visualized by confocal imaging of z-stacks for a 3D-reconstruction of subcellular structures. The protocol comprises state-of-the-art in cell culture, molecular biology and biophysics and thus provides an approach for exploring new horizons in cellular cardiology.

Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and ...

Receptor Autoradiography Protocol for the Localized Visualization of Angiotensin II Receptors

This protocol describes receptor binding patterns for Angiotensin II (Ang II) in the rat brain using a radioligand specific for Ang II receptors to perform receptor autoradiographic mapping.
Tissue specimens are harvested and stored at -80 &#176;C. A cryostat is used to coronally section the tissue (brain) and thaw-mount the sections onto charged slides. The slide-mounted tissue sections are incubated in 125I-SI-Ang II to radiolabel Ang II receptors. Adjacent slides are separated into two sets: &#39;non-specific binding&#39; (NSP) in the presence of a receptor saturating concentration of non-radiolabeled Ang II, or an AT1 Ang II receptor subtype (AT1R) selective Ang II receptor antagonist, and &#39;total binding&#39; with no AT1R antagonist. A saturating concentration of AT2 Ang II receptor subtype (AT2R) antagonist (PD123319, 10 &#181;M) is also present in the incubation buffer to limit 125I-SI-Ang II binding to the AT1R subtype. During a 30 min pre-incubation at ~22 &#176;C, NSP slides are exposed to 10 &#181;M PD123319 and losartan, while &#39;total binding&#39; slides are exposed to 10 &#181;M PD123319. Slides are then incubated with 125I-SI-Ang II in the presence of PD123319 for &#39;total binding&#39;, and PD123319 and losartan for NSP in assay buffer, followed by several &#39;washes&#39; in buffer, and water to remove salt and non-specifically bound radioligand. The slides are dried using blow-dryers, then exposed to autoradiography film using a specialized film and cassette. The film is developed and the images are scanned into a computer for visual and quantitative densitometry using a proprietary imaging system and a spreadsheet. An additional set of slides are thionin-stained for histological comparisons.
The advantage of using receptor autoradiography is the ability to visualize Ang II receptors in situ, within a section of a tissue specimen, and anatomically identify the region of the tissue by comparing it to an adjacent histological reference section.

Here we present a protocol to describe the localization of angiotensin II Type 1 receptors in the rat brain by quantitative, densitometric, in vitro receptor autoradiography using an iodine-125 labeled analog of angiotensin II.

This protocol describes receptor binding patterns for Angiotensin II (Ang II) in the rat brain using a radioligand specific for Ang II receptors to ...

Neuroscience

Visualizing Leukocyte Rolling and Adhesion in Angiotensin II-Infused Mice: Techniques and Pitfalls

Epifluorescence intravital video microscopy (IVM) of blood vessels is an established method to evaluate the activation of immune cells and their ability to role and adhere to the endothelial layer. Visualization of circulating cells by injection of fluorescent dyes or fluorophore-coupled antibodies is commonly used. Alternatively, fluorescent reporter mice can be used. Interactions of leukocytes, in particular lysozyme M+ (LysM+) monocytes, with the vessel wall play pivotal roles in promoting vascular dysfunction and arterial hypertension. We here present the technique to visualize and quantify leukocyte rolling and adhesion in carotid arteries in angiotensin II (AngII)-induced hypertension in mice by IVM.
The implantation of a catheter damages the vascular wall and leads to altered blood cell responses. We compared different injection techniques and administration routes to visualize leukocytes in a LysMCre+IRG+ mouse with widespread expression of red fluorescent protein and conditional expression of green fluorescent protein in LysM+ cells. To study LysM+ cell activation, we used AngII infused mice in which rolling and adhesion of leukocytes to the endothelium is increased. We either injected acridine orange using a jugular catheter or directly though the tail vein and compared the amount of rolling and adhering cells. We found that jugular catheter implantation per se increased the number of rolling and adhering LysM+ cells in sham-infused LysMCre+IRG+ mice compared to controls. This activation was augmented in AngII-infused mice. Interestingly, injecting acridine orange directly through the tail vein did not increase LysM+ cell adhesion or rolling in sham-infused mice. We thereby demonstrated the importance of transgenic reporter mice expressing fluorescent proteins to not interfere with in vivo processes during experimentation. Furthermore, tail vein injection of fluorescent tracers might be a possible alternative to jugular catheter injections.

This manuscript describes the use of transgenic reporter mice and different administration routes of fluorescent dyes in angiotensin II-induced hypertension using intravital video microscopy of blood vessels to evaluate the activation of immune cells and their ability to roll and adhere to the endothelium.

Epifluorescence intravital video microscopy (IVM) of blood vessels is an established method to evaluate the activation of immune cells and their ability ...

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology1 even in complex tissue sections2. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. 
Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells3, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgen&ouml;ssische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). 
We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single procedure. Due to advances in microscope technology it is relatively easy to capture live cell images for the purpose of investigating cellular events in real time with minimal concern regarding phototoxicity to the cells. This protocol describes how to take live cell timelapse images of primary rat neonatal cardiomyocytes using a confocal spinning disk microscope following lentiviral and adenoviral transduction to modulate properties of the cell. The application of two different types of viruses makes it easier to achieve an appropriate transduction rate and expression levels for two different genes. Well focused live cell images can be obtained using the microscope&#8217;s autofocus system, which maintains stable focus for long time periods. Applying this method, the functions of exogenously engineered proteins expressed in cultured primary cells can be analyzed. Additionally, this system can be used to examine the functions of genes through the use of siRNAs as well as of chemical modulators.

This protocol describes a method of live cell imaging using primary rat neonatal cardiomyocytes following lentiviral and adenoviral transduction using confocal spinning disk microscopy. This enables detailed observations of cellular processes in living cardiomyocytes.

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single ...

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 ...

Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy

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