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
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.

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. 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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>

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

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Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles1,2. Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture3-6. Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue3,6. High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture6. By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6&#160;cultures in parallel.

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

We describe the dissection and ex vivo culture of mouse embryonic skin. The culture system maintains an air-liquid interface across the tissue surface and allows imaging on an inverted microscope. Melanoblasts, a component of the developing skin, are fluorescently labeled allowing their behavior to be observed using confocal microscopy.

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

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 ...

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of membrane-bound transcription factors controlling the expression of genes important for the synthesis of cholesterol, fatty acids and phospholipids, are frequently upregulated in these diseases. In the process of SREBP nuclear translocation, SREBP-cleavage activating protein (SCAP) plays a central role in the trafficking of SREBP from the endoplasmic reticulum (ER) to the Golgi and in subsequent proteolysis activation. Recently, we uncovered that glucose-mediated N-glycosylation of SCAP is a prerequisite condition for the exit of SCAP/SREBP from the ER and movement to the Golgi. N-glycosylation stabilizes SCAP and directs SCAP/SREBP trafficking. Here, we describe a protocol for the isolation of membrane fractions in human cells and for the preparation of the samples for the detection of SCAP N-glycosylation and total protein by using western blot. We further provide a method to monitor SCAP trafficking by using confocal microscopy. This protocol is appropriate for the investigation of SCAP N-glycosylation and trafficking in mammalian cells.

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

We describe a modified method for membrane fraction isolation from human cells and sample preparation for the detection of SCAP N-glycosylation and total protein by using western blot. We further introduce a GFP-labeling method to monitor SCAP trafficking using confocal microscopy. This protocol can be used in regular biology laboratories.