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.

Day One
1. Cell Seeding
<ol>
	<li>Prepare complete Dulbecco&#39;s modified Eagle Medium (DMEM). Add 50 mL of fetal bovine serum, 5 mL of penicillin/streptomycin, 5 mL of L-glutamine to 450 mL of DMEM to make 500 mL of complete DMEM supplemented with fetal bovine serum (10%), penicillin/streptomycin (1%) and L-glutamine (1%).</li>
	<li>Seed human embryonic kidney (HEK) cells, passage number 6-11 at 50,000/well in an 8 well chambered coverglass system in complete DMEM.</li>
	<li>Maintain the cham...

<ol>
	<li>Dale, L. B., Seachrist, J. L., Babwah, A. V., Ferguson, S. S. <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+angiotensin+II+type+1A+receptor+intracellular+retention,+degradation,+and+recycling+by+Rab5,+Rab7,+and+Rab11+GTPases.">Regulation of angiotensin II type 1A receptor intracellular retention, degradation, and recycling by Rab5, Rab7, and Rab11 GTPases.</a> J Biol Chem. 279 (13), 13110-13118 (2004).</li><li>Hein, L., Meinel, L., Pratt, R. E., Dzau, V. J., Kobilka, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+trafficking+of+angiotensin+II+and+its+AT1+and+AT2+receptors:+evidence+for+selective+sorting+of+receptor+and+ligand.">Intracellular trafficking of angiotensin II and its AT1 and AT2 receptors: evidence for selective sorting of receptor and ligand.</a> Mol Endocrinol. 11 (9), 1266-1277 (1997).</li><li>Hunyady, L., 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=Differential+PI+3-kinase+dependence+of+early+and+late+phases+of+recycling+of+the+internalized+AT1+angiotensin+receptor.">Differential PI 3-kinase dependence of early and late phases of recycling of the internalized AT1 angiotensin receptor.</a> J Cell Biol. 157 (7), 1211-1222 (2002).</li><li>Lazari, M. F., Porto, C. S., Freymuller, E., Abreu, L. C., Picarelli, Z. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor-mediated+endocytosis+of+angiotensin+II+in+rat+myometrial+cells.">Receptor-mediated endocytosis of angiotensin II in rat myometrial cells.</a> Biochem Pharmacol. 54 (3), 399-408 (1997).</li><li>Li, H., Li, H. F., Felder, R. A., Periasamy, A., Jose, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rab4+and+Rab11+coordinately+regulate+the+recycling+of+angiotensin+II+type+I+receptor+as+demonstrated+by+fluorescence+resonance+energy+transfer+microscopy.">Rab4 and Rab11 coordinately regulate the recycling of angiotensin II type I receptor as demonstrated by fluorescence resonance energy transfer microscopy.</a> J Biomed Opt. 13 (3), 031206 (2008).</li><li>Li, H., 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=Actin+cytoskeleton-dependent+Rab+GTPase-regulated+angiotensin+type+I+receptor+lysosomal+degradation+studied+by+fluorescence+lifetime+imaging+microscopy.">Actin cytoskeleton-dependent Rab GTPase-regulated angiotensin type I receptor lysosomal degradation studied by fluorescence lifetime imaging microscopy.</a> J Biomed Opt. 15 (5), 056003 (2010).</li><li>Yamamoto, A., 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=Bafilomycin+A1+prevents+maturation+of+autophagic+vacuoles+by+inhibiting+fusion+between+autophagosomes+and+lysosomes+in+rat+hepatoma+cell+line,+H-4-II-E+cells.">Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H-4-II-E cells.</a> Cell Struct Funct. 23 (1), 33-42 (1998).</li><li>Deliu, E., Tica, A. A., Motoc, D., Brailoiu, G. C., Brailoiu, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+angiotensin+II+activates+rat+myometrium.">Intracellular angiotensin II activates rat myometrium.</a> Am J Physiol Cell Physiol. 301 (3), C559-C565 (2011).</li><li>Fortian, A., Sorkin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+fluorescence+imaging+reveals+high+stoichiometry+of+Grb2+binding+to+the+EGF+receptor+sustained+during+endocytosis.">Live-cell fluorescence imaging reveals high stoichiometry of Grb2 binding to the EGF receptor sustained during endocytosis.</a> J Cell Sci. 127 (Pt 2), 432-444 (2014).</li><li>Aymerich, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+G-protein-coupled+receptor+imaging+to+understand+and+quantify+receptor+dynamics.">Real-time G-protein-coupled receptor imaging to understand and quantify receptor dynamics.</a> ScientificWorldJournal. 11, 1995-2010 (2011).</li><li>Jaeger, W. C., Armstrong, S. P., Hill, S. J., Pfleger, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysical+Detection+of+Diversity+and+Bias+in+GPCR+Function.">Biophysical Detection of Diversity and Bias in GPCR Function.</a> Front Endocrinol (Lausanne). 5, 26 (2014).</li><li>Inuzuka, T., 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=Attenuation+of+ligand-induced+activation+of+angiotensin+II+type+1+receptor+signaling+by+the+type+2+receptor+via+protein+kinase.">Attenuation of ligand-induced activation of angiotensin II type 1 receptor signaling by the type 2 receptor via protein kinase.</a> C. Sci Rep. 6, 21613 (2016).</li><li>Liu, 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=Selective+inhibition+of+angiotensin+receptor+signaling+through+Erk1/2+pathway+by+a+novel+peptide.">Selective inhibition of angiotensin receptor signaling through Erk1/2 pathway by a novel peptide.</a> Am J Physiol Regul Integr Comp Physiol. 306 (8), R619-R626 (2014).</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'+guide+to+practical+pitfalls+in+image+acquisition.">Seeing is believing? A beginners' guide to practical pitfalls in image acquisition.</a> J Cell Biol. 172 (1), 9-18 (2006).</li><li>Mueller, S. C., Hubbard, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor-mediated+endocytosis+of+asialoglycoproteins+by+rat+hepatocytes:+receptor-positive+and+receptor-negative+endosomes.">Receptor-mediated endocytosis of asialoglycoproteins by rat hepatocytes: receptor-positive and receptor-negative endosomes.</a> J Cell Biol. 102 (3), 932-942 (1986).</li><li>Costantini, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+palette+of+fluorescent+proteins+optimized+for+diverse+cellular+environments.">A palette of fluorescent proteins optimized for diverse cellular environments.</a> Nat Commun. 6, 7670 (2015).</li><li>Freundt, E. C., Czapiga, M., Lenardo, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoconversion+of+Lysotracker+Red+to+a+green+fluorescent+molecule.">Photoconversion of Lysotracker Red to a green fluorescent molecule.</a> Cell Res. 17 (11), 956-958 (2007).</li><li>Yoshimori, T., Yamamoto, A., Moriyama, Y., Futai, M., Tashiro, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bafilomycin+A1,+a+specific+inhibitor+of+vacuolar-type+H(+)-ATPase,+inhibits+acidification+and+protein+degradation+in+lysosomes+of+cultured+cells.">Bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells.</a> J Biol Chem. 266 (26), 17707-17712 (1991).</li></ol>

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.</e...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Angiotensin II (Ang II)</td>
			<td>Sigma-Aldrich</td>
			<td>A9525&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle medium (DMEM 1X)</td>
			<td>Gibco -ThermoFisher Scientific</td>
			<td>11960-044</td>
			<td>Warm in 37 &#176;C water bath before use&#160;</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Sigma-Aldrich</td>
			<td>F2442</td>
			<td>Warm in 37 &#176;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 &#176;C water bath before use</td>
		</tr>
		<tr>
			<td>L-Glutamine 200mM (100X)</td>
			<td>Gibco (life technologies)</td>
			<td>25030-081</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr>
			<td>Human Embryonic Kidney (HEK)-293 cells</td>
			<td>American Type Culture Collection (ATCC)&#160;</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 &#176;C&#160;</td>
		</tr>
		<tr>
			<td>Opti-MEM, Reduced Serum Medium</td>
			<td>Gibco- ThermoFisher Scientific</td>
			<td>31985-070</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr>
			<td>PBS 10X&#160;</td>
			<td>Fisher BioReagents</td>
			<td>BP3994</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle medium (DMEM 1X)&#160;</td>
			<td>Gibco -ThermoFisher Scientific</td>
			<td>31053-028</td>
			<td>Warm in 37 &#176;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 &#176;C .&#160;</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 p...

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 ...

The overall goal of this experiment is to obtain quantitative spatial and temporal evidence of Angiotensin type one receptor lysosome colocalization after Angiotensin two treatment. This method was devised to detect differences in the subcellular processing of Y type versus mutant receptors, As during the internalization process, that utters in response to ligand simulation. To advantages of this technique are that life cell imaging, eliminates artifacts from fixed and detergent permeabilized cells and that dynamic changes in receptor localization can be assessed in real time.Begin by selecting an appropriate 1.4 numerical aperture objective and centering the objective over the microscope stage. Add a drop of hight viscosity, low auto florescence immersion oil onto the objective and transfer the chamber with the tranfected cells to the microscope stage. Gently raise the objective until the oil just touches the bottom of the first slide and locate the dim cells.With a healthy cell of interest, in the center of the field of view, switch to confocal mode and confirm that the cell is well spread, with its ventral side juxtaposed closely with the cover slip. Open the Z Stack dialog box, click Live, focus on the top of the cell, and then click Begin. Then focus to the bottom of the cell, enter Z step size 1.00 micrometer, and then click End.Then select XYZT Mode under Acquisition settings at the top. Lastly, set the time interval for the scan and the length of time for scanning to observe the targeting of GFP tagged Angiotensin Type 1A Receptor to the lysosomes. Then to begin the ligand stimulation, add three microliters of a 100 X stock solution of ligand to the well that is being imaged and click Start to image the response in 3D for the desired period and frequency.To analyze the live cell images, open the appropriate 3D image quantification software. In this protocol, Velocity. Right click within the image field and select Properties.Enter the appropriate XY and Z image pixel properties for creating a measurement sequence to analyze the GFP tagged receptor vesicle, and hole cell intensities in response to ligand treatment. Under the 2D menu, select the extended focus mode to observe the cell. Next, use the freestyle region tool, to draw a region of interest around the cell, and right click the image to select Crop to Selection.A new image item will be generated. Click on the image name to select the cropped cell image and open the measurement tab. To measure the GFP expression of the internalized vesicles, drag Find Objects from Finding into the sequence box above.Set the find objects channel to GFP and open the settings icon to set the threshold using to standard deviation and the lower limit to six, to select objects with the brightest GFP intensities. Set the minimum object size to zero micrometers cubed. Then, click measure, and select the channels and the types of measurements.To define the thresholding and filters for identifying the whole cell and to measure the total GFP intensity, drag a second Find Objects box. Next, set the Find Objects channel to GFP and set the standard deviation to zero, to select objects with any GFP intensity and minimum object size to two micrometers cubed. Drag Filter Population to the Population two Find Objects box and select a volume of micrometers cubed, greater than 100 to select the whole cell and eliminate all smaller objects.Then, select measurements and Make Measurement item, to make measurement at all time points. To analyze the measured GFP expression in vesicles, and the whole cell over time, click on the measurement item file name below the image name. To extract the data for GFP and vesicles from the total data, open the raw tab and select population one.Then, open the analyzed tab and right clicking on the data, select analysis, followed by restricting analysis to select population one. To measure the amount of GFP in the whole cell, change both to population two. Under Analyze These Data and Summarized By, select sum GFP color channel, and sum.Then, under Organize the data by, and row, select time point. To extract the data for the amount of GFP in lysosomes, in the Find Objects for population one, set the find objects channel to the red channel. In the wheel icon within the dialog box, set the threshold using to standard deviation, the lower limit to six, to select red objects with the brightest intensities, and the minimum object size to 0.017 micrometers cubed.Select measurements, and make make measurement item, to make measurement at all time points. To analyze the measured GFP expression in lysosomes, click on the measurement item file name below the image name. Select population one under the raw tab, and then select population one and sum GFP color channel and sum under the analyze tab, right clicking as before to measure the amount of GFP in the red vesicles.Before ligand stimulation, GFP tagged Angiotensin Type 1A Receptors are localized predominantly in the plasma membrane. But within 2.5 minutes of the addition of Angiotensin two, these receptors become internalized, forming bright vesicles and later clustering and large paranuclearzome vesicles. Angiotensin two induced ruffling complicates the quantification of the total vesicle GFP intensity as observed for these representative GFP expression measurement data.Size filtering does not discriminate between the larger ruffles and clustered vesicles, but does reveal that GFP in smaller objects increases sooner than GFP in larger objects. In this representative time course of GFP expression without autofocus, the internalized GFP was measured as the percent total cellular GFP. Analysis of the total GFP within the cell revealed that this cell did not photo bleach appreciably over time.Quantification of the GFP in the lysosomes of transfected cells also indicates that the intensity of the Angiotensin Type 1A Receptor present within the lysosomes, does not vary for up to 24 minutes after Angiotensin two administration. During this procedure, it's important to remember to monitor the health of the cells prior to and during the imaging, since the procedure is time consuming, the experiment should be aborted if the cell cultures are abnormal in any way. Following this procedure, immunostaining and FRET or FLIM can be used to detect and confirm the subcellular localization of the receptors.Western blots can be performed to address the identity and changes in signaling molecules involved during the receptor trafficking. After watching this video, you should have a good understanding of how to perform live cell imaging and analysis to measure the ligand induced transmembrane receptor targeting to lysosomes as well as to other subcellular compartments.

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

Research

JoVE Journal

Biology

9.6K visualizações.  Georgetown University Medical Center. The overall goal of this experiment is to obtain quantitative spatial and temporal evidence of Angiotensin type one receptor lysosome colocalization after Angiotensin two treatment. This method was devised to detect differences in the subcellular processing of Y type versus mutant receptors, As during the internalization process, that utters in response to ligand simulation. To advantages of this technique are that life cell imaging, eliminates artifacts from fixed and detergent permeabilized...

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