The authors greatly acknowledge Pierre Bourdoncle, Thomas Guilbert and Louise Rimbault of the Cochin Imaging Facility. This work was supported by Inserm, CNRS and Ligue Nationale contre le Cancer (Equipe labellis&#233;e).

1. Preparation of T Lymphocytes
<ol>
	<li>Prepare primary mouse or human T cells as described10,11.</li>
	<li>Cultivate human T cells in complete RPMI medium with 10% human serum AB at a density of 2 - 3 x 106 cells/ml. 
	NOTE: The use of fetal calf serum (FCS) instead of human serum can elicit spontaneous polarization of a large fraction of human T cells in culture. Keep these cells in culture for 4 - 5 days and further process them at any time during this period.</li>
	<li>Maintain mouse T cells in complete RPMI medium supplemented with 10% FCS at a similar cell density. Use right away or the next day, after an O/N culture at 37 &#176;C in the presence of 10 ng/ml IL7.</li>
	<li>Cultivate the CEM human T cell line in complete RPMI supplemented with 10% FCS.</li>
</ol>
2. Chemokine Stimulation
<ol>
	<li>Wash 5 x 105 cells per experimental condition in warm HBSS medium supplemented with 10 mM Hepes. For some experiments, co-transfect primary human T cells the day before with 2 &#181;g pmaxGFP and 8 &#181;g pcDNA3.1+ (empty vector, control), PKC&#950; kinase dead, or N-terminal Par6 plasmids.</li>
	<li>Resuspend the cell pellet in warm HBSS-Hepes medium (use 0.3 ml x number of experimental conditions).</li>
	<li>Distribute the cells in 1.5 ml microcentrifuge tubes. 
	NOTE: Therefore, one tube corresponding to a single experimental condition will contain 5 x 105 cells in 0.3 ml HBSS-Hepes medium. For some experiments, add 500 nM Latrunculin A or vehicle (DMSO) and incubate the cells for 30 min before chemokine stimulation.</li>
	<li>Add 10 to 500 ng/ml CCL19 or CXCL12 chemokine in each tube. 
	NOTE: We usually use 10 - 200 ng/ml chemokine for human T cells. CEM cells do not express the CCR7 receptor that binds CCL19. Therefore, CEM cells will only be able to respond to a CXCL12 stimulation. Moreover, use higher chemokine concentrations (300 - 500 ng/ml) to polarize mouse T cells.</li>
	<li>Flip the tubes several times and incubate them in a 37 &#176;C water bath for 8 or 10 min for primary T cells or CEM, respectively.</li>
	<li>Stop the polarization process by adding to each tube 0.3 ml of a warm solution containing 2% paraformaldehyde (PFA) and 10 mM Hepes in PBS. Flip the tubes and incubate them at 37 &#176;C for 5 min.</li>
</ol>
3. Stainings
<ol>
	<li>Transfer the cells from the microcentrifuge tubes to flow cytometry tubes.</li>
	<li>Fill the tubes completely with a RT solution containing 1% bovine serum albumin (BSA), 0.5 mM EDTA and 10 mM Hepes in PBS.</li>
	<li>Centrifuge the tubes at 460 x g for 4 min.</li>
	<li>Discard the supernatant and resuspend the cells in 100 &#181;l of a RT solution containing 5% FCS in PBS.</li>
	<li>Add 5 &#181;l of FITC-anti-HLA-ABC in each tube, vortex them, and incubate them for 30 min at RT, away from light.</li>
	<li>Wash cells once with PBS containing 5% FCS, and once with PBS only.</li>
	<li>At RT: add 500 &#181;l 1% PFA, incubate for 10 min, then wash cells with a solution containing 1% BSA, 0.5 mM EDTA and 10 mM HEPES in PBS.</li>
	<li>Discard the supernatant, fill the tubes completely with a solution of PBS containing 0.2% BSA, 0.1% saponin, 0.5 mM EDTA and 10 mM Hepes (permeabilization buffer).</li>
	<li>Wash cells twice in this medium.</li>
	<li>Flip the tubes to remove the medium and vortex them to dissociate the cell pellet.</li>
	<li>Add 0.25 &#181;g/ml TRITC-Phalloidin and/or a 1:100 dilution of anti-P-ERM antibody to each tube, vortex them and incubate for 30 min at RT.</li>
	<li>Wash cells with the permeabilization buffer. Incubate with a fluorescent secondary antibody if necessary.</li>
	<li>Resuspend the cells in 200 &#181;l PBS and transfer them to microcentrifuge tubes. 
	NOTE: Cells are ready for acquisition. Alternatively, while keeping a maximum total volume of 200 &#181;l per tube, add concentrated PFA to a final 1% concentration in order to preserve the stainings if the cells are not to be used the same day for further processing.</li>
</ol>
4. Images Acquisition and Analysis
<ol>
	<li>Switch on the apparatus (see Table of Specific Reagents/Equipment) and let it calibrate itself according to the manufacturer&#8217;s instructions. 
	NOTE: The INSPIRE software was used with a 40X objective (0.75 NA). The IDEAS 3.0 software was used for all the reported quantifications.</li>
	<li>Prepare a series of acquisition windows which can be saved in a template for future experiments. Draw histograms that quantify the RMS gradient values. From the &#34;In Focus&#34; population, selected on the RMS gradient histogram, delimitate the &#34;Single cell&#34; population on a histogram of the Area. Once the microcentrifuge tube has been inserted in the machine, adjust the laser power, gate on individual cells and acquire 5,000 T lymphocytes.</li>
	<li>In the Ideas software, open the acquisition file and create a histogram that will show the value of the gradient RMS value for bright field images (Channel 1) obtained for each individual cell. Draw a gate on the RMS gradient histogram that considers cells exhibiting RMS gradient value above 57. 
	NOTE: The RMS gradient allows taking into account in the analysis only the T lymphocytes that are in the focal plane. We have determined empirically that individual cells exhibiting a RMS gradient value superior to 57 are in the focal plane, and can be accurately considered.</li>
	<li>In the Analysis/Masks menu, create a new mask, called Erode(M01,2) from the morphology mask on the brightfield images M01, eroded of 2 pixels. Then, in the Analysis/Features menu, create a new feature of the Area, calculated on the Erode(M01,2) mask. From the cells selected in the previous gate, open a histogram showing the Area of cells using this newly created mask. 
	NOTE: The morphology masks that are available by default are much bigger than the actual size of the cell. Thus, it is more precise to erode it up to 2 pixels.</li>
	<li>Draw a gate on the individual T cells, excluding the calibration beads and debris (small Area) and the doublets (large Area). Adjust this gate at each acquisition. 
	NOTE: Variations in cell size will affect the position of the gate. As mouse T cells are smaller than human T cells which are themselves smaller than CEM cells, the area of each cell type will be proportional to its size.</li>
	<li>Considering the single, in-focus cells that were gated at the previous steps, open a dot plot consisting of the Raw Max Pixel on the HLA-ABC staining (Channel 2, x axis) as a function of the Raw Max Pixel on the phalloidin staining (Channel 4, y axis). Create a gate to exclude the unstained or saturated fluorescent events. Open two histograms showing the Raw Max Pixel for HLA-ABC (Channel 2) and phalloidin (Channel 4) stainings.</li>
	<li>In the Analysis/Masks menu, create a new mask, called Erode(M02,2) from the morphology mask of the HLA-ABC stained cells (Channel 2), eroded of 2 pixels. Then, in the Analysis/Features menu, create a new feature consisting of the Circularity parameter, calculated on the Erode(M02,2). 
	NOTE: This parameter measures the deviation of the cell shape from a circle. Therefore, a perfectly round T cell will have a high circularity value whereas elongated polarized cells will exhibit a low circularity index.</li>
	<li>Subsequently, create another histogram that will report the values of the Circularity feature from the previously gated cells. Use this histogram to directly plot the distribution of an individual, in-focus cell population according to the value of the Circularity for each individual cell.</li>
	<li>Looking at the shape of the histogram for non-stimulated cells, draw a gate for polarized cells, starting at the lowest circularity value up to the circularity limit value where most of the non-stimulated cells are plotted. Look at the statistics display for the percentage of polarized cells among the gated population (%Gated). 
	NOTE: We have set this gate for Circularity index values below 13.</li>
	<li>In order to look at the polarization of the actin staining in the cells, plot a histogram for the Bright Detail Similarity R3 value, comparing the HLA-ABC staining (Channel 2) and the phalloidin staining (Channel 4). 
	NOTE: The bigger the value of this feature, the more superimposed the two stainings are.</li>
	<li>Using the same gating strategy as before, plot a gate on the non-stimulated cells for the segregated staining that shows the percentage of cells with polarized actin staining.</li>
	<li>When completed, the percentage of cells exhibiting a segregation in the distribution of the MHC Class I and phalloidin stainings, the percentage of polarized cells together with the mean values of the Circularity and Similarity indexes of all the cells &#177; SD are shown in the corresponding statistics panels.</li>
</ol>

<ol><li>Rossi, D., Zlotnik, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+biology+of+chemokines+and+their+receptors%5BTitle%5D)+AND+%22Annu+Rev+Immunol%22%5BJournal%5D)">The biology of chemokines and their receptors.</a> Annu Rev Immunol. 18, 217-242 (2000).</li>
<li>Thelen, M., Stein, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((How+chemokines+invite+leukocytes+to+dance%5BTitle%5D)+AND+%22Nat+Immunol%22%5BJournal%5D)">How chemokines invite leukocytes to dance.</a> Nat Immunol. 9 (9), 953-959 (2008).</li>
<li>Rougerie, P., Rho Delon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((GTPases%3A+masters+of+T+lymphocyte+migration+and+activation%5BTitle%5D)+AND+%22Immunol+Lett%22%5BJournal%5D)">GTPases: masters of T lymphocyte migration and activation.</a> Immunol Lett. 142 (1-2), 1-13 (2012).</li>
<li>Sanchez-Madrid, F., del Pozo, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Leukocyte+polarization+in+cell+migration+and+immune+interactions%5BTitle%5D)+AND+%22Embo+J%22%5BJournal%5D)">Leukocyte polarization in cell migration and immune interactions.</a> Embo J. 18 (3), 501-511 (1999).</li>
<li>Adams, D. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hepatocyte+growth+factor+and+macrophage+inflammatory+protein+1+beta%3A+structurally+distinct+cytokines+that+induce+rapid+cytoskeletal+changes+and+subset-preferential+migration+in+T+cells%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Hepatocyte growth factor and macrophage inflammatory protein 1 beta: structurally distinct cytokines that induce rapid cytoskeletal changes and subset-preferential migration in T cells.</a> Proc Natl Acad Sci U S A. 91 (15), 7144-7148 (1994).</li>
<li>Rougerie, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fam65b+is+a+new+transcriptional+target+of+FOXO1+that+regulates+RhoA+signaling+for+T+lymphocyte+migration%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">Fam65b is a new transcriptional target of FOXO1 that regulates RhoA signaling for T lymphocyte migration.</a> J Immunol. 190 (2), 748-7455 (2013).</li>
<li>Real, E., Faure, S., Donnadieu, E., Delon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cutting+edge%3A+Atypical+PKCs+regulate+T+lymphocyte+polarity+and+scanning+behavior%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">Cutting edge: Atypical PKCs regulate T lymphocyte polarity and scanning behavior.</a> J Immunol. 179 (9), 5649-5652 (2007).</li>
<li>Negulescu, P. A., Krasieva, T. B., Khan, A., Kerschbaum, H. H., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Polarity+of+T+cell+shape%2C+motility%2C+and+sensitivity+to+antigen%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">Polarity of T cell shape, motility, and sensitivity to antigen.</a> Immunity. 4 (5), 421-430 (1996).</li>
<li>Zuba-Surma, E. K., Kucia, M., Abdel-Latif, A., Lillard, J. W., Ratajczak, M. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+ImageStream+System%3A+a+key+step+to+a+new+era+in+imaging%5BTitle%5D)+AND+%22Folia+Histochem+Cytobiol%22%5BJournal%5D)">The ImageStream System: a key step to a new era in imaging.</a> Folia Histochem Cytobiol. 45 (4), 279-290 (2007).</li>
<li>Matheu, M. P., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+CD4%2B+T+cells+from+mouse+lymph+nodes+using+Miltenyi+MACS+purification%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Isolation of CD4+ T cells from mouse lymph nodes using Miltenyi MACS purification.</a> J Vis Exp. (9), 409(2007).</li>
<li>James, E. A., LaFond, R., Durinovic-Bello, I., Kwok, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualizing+antigen+specific+CD4%2B+T+cells+using+MHC+class+II+tetramers%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Visualizing antigen specific CD4+ T cells using MHC class II tetramers.</a> J Vis Exp. (25), (2009).</li>
<li>Wabnitz, G. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((L-plastin+phosphorylation%3A+a+novel+target+for+the+immunosuppressive+drug+dexamethasone+in+primary+human+T+cells%5BTitle%5D)+AND+%22Eur+J+Immunol%22%5BJournal%5D)">L-plastin phosphorylation: a novel target for the immunosuppressive drug dexamethasone in primary human T cells.</a> Eur J Immunol. 41 (11), 3157-3169 (2011).</li>
<li>Freeley, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((L-plastin+regulates+polarization+and+migration+in+chemokine-stimulated+human+T+lymphocytes%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">L-plastin regulates polarization and migration in chemokine-stimulated human T lymphocytes.</a> J Immunol. 188 (12), 6357-6370 (2012).</li>
</ol>

The authors declare that they have no competing financial interests.

Using a recent technology of imaging flow cytometry, a rapid and informative gating strategy to analyze cellular and molecular events induced by chemokine stimulation is presented. From a single experiment, one can obtain two main types of information: the changes in cell morphology induced by chemokine stimulation and the subcellular distribution of different proteins during the polarization process. Interestingly, evidence is also provided for the possibility to analyze a large number of cells, which allows statistical robustness and relevant analysis of a small fraction of the whole cell population. As reported, this technique can also be used for analyzing molecular redistribution events in the context of the immunological synapse12. In addition, a similar set up has already been used for SDF1-stimulated T cells but no analysis of the degree of co-localization between two markers has been provided13.
Although this technique can thus be suitable for many cell types, adherent cells cannot be directly analyzed and cells in suspension need to be fixed prior the acquisition. In addition to these intrinsic limitations of the machine, it is critical to keep the cells at 37 &#176;C when stimulating them. Temperature is indeed key to achieve proper T cell polarization since cytoskeletal changes, such as actin polymerization that drives shape changes, are very dependent on the temperature. Our experiments with latrunculin A treated cells confirm that proper actin remodeling induced by chemokine stimulation is indeed crucial for a cell to achieve its fully polarized state. In addition to the data provided here with this drug, we also tested the imaging flow cytometry technology with human primary T lymphocytes transfected with mutant proteins that inhibit the Par6/PKC&#950; signaling pathway. Consistently with our previously published results7, it is shown here that this polarity complex favors T cell polarization.
Upon chemokine stimulation, T cells lose their round morphology to adopt a polarized shape. We have found the Circularity parameter to be the most accurate to quantify those morphological alterations. However, other parameters describing shape changes are proposed by the analysis software, such as the Aspect Ratio or the Elongatedness. Therefore, one can easily test the contribution of a particular signaling pathway or a specific protein in this process.
In this study, we have always observed that the percentage of cells exhibiting a morphological polarization is higher than the one for molecular polarization. This indicates that a fraction of cells that adopt a change in morphology do not present large molecular redistributions of the markers studied. This could arise from the fact that some cells lose their round shape so they exhibit a lower Circularity index although they do not clearly show a leading edge and a uropod. These partially polarized T cells could still fall in the gate of morphologically polarized T cells without presenting obvious molecular redistributions of typical markers. Moreover, differences in the chemokine signaling threshold that could be higher for markers redistribution than for shape alterations could also explain these differences.
It is also shown here that the HLA-ABC marker is not relocalized in polarized T cells and there is no change in the intensity of this staining upon CCL19 stimulation. Conversely, a confirmation is provided here that F-actin staining increases upon CCL19 treatment (Figures 1 and 2) as already known, since chemokine stimulation activates actin polymerization5. Moreover, it is shown that actin filaments get relocalized at the front of polarized cells. Consequently, the Bright Detail Similarity index that measures the extent of co-localization between both markers tends to decrease upon CCL19 stimulation. As a comparison, such an analysis is provided here for the degree of F-actin co-localization with phospho-ERM proteins that get excluded from the cell front. The percentage of chemokine-stimulated T cells exhibiting F-actin &#8211; MHC Class I segregation is much lower than the one for F-actin &#8211; phospho-ERM (14% vs. 48%, respectively) since F-actin relocalizes at the front and phospho-ERM proteins at the rear of polarized T cells, whereas the repartition of MHC Class I molecules is not affected by chemokine stimulation. This Bright Detail Similarity index can thus be systematically used to compare the distribution of two markers in polarized T cells. Therefore, this technique is very appropriate to quantify the degree of co-localization or co-exclusion of two different markers on a large number of cells or, potentially, on rare events present in a broad cell population.

Chemokines are small soluble proteins that attract cells to specialized locations1. Therefore, they participate in the correct positioning of cells in tissues, a crucial function in development and physiology. The immune system is no exception to this rule as it relies on the action of many different cell types which act in concert to mount an effective immune response. By controlling the specific location of one immune cell type in a given state, chemokines are pre-required before foreign antigens can be detected and neutralized.
In T lymphocytes in particular, chemokines bind to specific surface receptors which, upon engagement, elicit many intracellular signals (calcium rise, ERK phosphorylation, Rho GTPases activation, increase in integrins affinity and cytoskeletal alterations) that favor T cell motility2,3. At the cellular level, one can observe morphological alterations elicited upon chemokine stimulation. These changes in cell shapes are especially dramatic in T cells: resting T cells have a bead-like round morphology when travelling in the blood stream. However, the sensing of the presence of chemokines in inflammatory sites or at proximity of lymphoid organs is going to change the shape of T cells which now adopt a typical &#8220;hand-mirror&#8221; morphology consisting of a bipolar form: a leading edge at the front and a trailing edge, or uropod, at the back4. In addition, intracellular components can segregate into these two opposite regions of a polarized T cell to sustain migration. For example, actin filaments polymerization increases upon chemokine stimulation5 and polymerized actin accumulates at the front of a polarized T cell2. On the other hand, several proteins, such as phosphorylated proteins of the Ezrin-Radixin-Moesin (ERM) family which link the plasma membrane to the cortical F-actin cytoskeleton, re-localize at the uropod of polarized T cells6. Interestingly, we and others have shown that this polarization process is required for T cell migration. Indeed, any treatment that interferes with polarization will inhibit cellular motility. For instance, inhibition of the activity of the members of the atypical protein kinases C (PKC) family, PKC&#950; and PKC&#953; blocks T cell polarization and their migratory scanning process of dendritic cells7. T cell polarization is also regulated by Rho GTPases. We have shown that the modulation of RhoA activity by the recently described Fam65b protein interferes with T cell changes in morphology and their capacity to migrate in a Transwell assay6. As polarization is a prerequisite step for cellular motility, it is thus crucial to be able to quantify it as a main readout of chemokine responses. Cell shape alterations were previously measured manually8. However, this kind of quantification is very time-consuming, so that usually only a few tens of cells are taken into account.
Here, a new method is presented to rapidly quantify the degree of shape alterations of T lymphocytes exposed to chemokine stimulation. An imaging flow cytometry technology (see Table of Specific Reagents/Equipment) is used, which combines the advantages of a flow cytometer and a microscope9 to quantify efficiently the amount of polarized cells in different conditions of chemokine stimulation. In addition to the quantification of the morphological alterations that one can measure robustly with this technology, it is also possible to evaluate changes in the subcellular localization of some proteins upon chemokine signaling.

<table><tbody><tr><td>RPMI</td><td>Gibco</td><td>61870-010</td><td></td></tr><tr><td>Human serum AB</td><td>PAA</td><td>C11-021</td><td>Pre-heat to inactivate the complement.</td></tr><tr><td>Fetal calf serum</td><td>PAN Biotech</td><td>P30-3300</td><td>Pre-heat to inactivate the complement.</td></tr><tr><td>Human T cell nucleofactor kit</td><td>Lonza</td><td>VCA-1002</td><td></td></tr><tr><td>Murine IL7</td><td>Peprotech</td><td>217-17</td><td></td></tr><tr><td>HBSS</td><td>Gibco</td><td>14025-050</td><td>Warm in 37 &amp;deg;C water bath before use.</td></tr><tr><td>Hepes</td><td>Gibco</td><td>15630-056</td><td></td></tr><tr><td>Murine CCL19</td><td>Peprotech</td><td>250-27B</td><td>Aliquots are thawed on ice before adding the chemokine to the cells.</td></tr><tr><td>Human CCL19</td><td>Peprotech</td><td>300-29B</td><td>Aliquots are thawed on ice before adding the chemokine to the cells.</td></tr><tr><td>Human CXCL12</td><td>Peprotech</td><td>300-28A</td><td>Aliquots are thawed on ice before adding the chemokine to the cells. This chemokine can also be used on mouse T cells.</td></tr><tr><td>PFA</td><td>Electron Microscopy Sciences</td><td>157-8-100</td><td>This is a 8% PFA solution in water. Mix volume to volume with 2x PBS to obtain a 4% PFA solution in PBS.</td></tr><tr><td>BSA</td><td>Sigma</td><td>A3059</td><td></td></tr><tr><td>Saponin</td><td>Fluka</td><td>84510</td><td></td></tr><tr><td>Alexa Fluor 594 phalloidin</td><td>Invitrogen</td><td>A12381</td><td></td></tr><tr><td>FITC-anti-HLA-ABC antibody</td><td>Beckman Coulter</td><td>IM1838</td><td>clone B9.12.1</td></tr><tr><td>Anti-P-ERM antibody</td><td>Cell Signaling Technology</td><td>3149P</td><td></td></tr><tr><td>ImageStream&lt;sup&gt;x&lt;/sup&gt; Mark II</td><td>Amnis</td><td></td><td></td></tr></tbody></table>

rapid and robust analysis of cellular and molecular polarization induced by chemokine signaling

Cells respond to chemokine stimulation by losing their round shape in a process called polarization, and by altering the subcellular localization of many proteins. Classic imaging techniques have been used to study these phenomena. However, they required the manual acquisition of many cells followed by time consuming quantification of the morphology and the co-localization of the staining of tens of cells. Here, a rapid and powerful method is described to study these phenomena on samples consisting of several thousands of cells using an imaging flow cytometry technology that combines the advantages of a microscope with those of a cytometer. Using T lymphocytes stimulated with CCL19 and staining for MHC Class I molecules and filamentous actin, a gating strategy is presented to measure simultaneously the degree of shape alterations and the extent of co-localization of markers that are affected by CCL19 signaling. Moreover, this gating strategy allowed us to observe the segregation of filamentous actin (at the front) and phosphorylated Ezrin-Radixin-Moesin (phospho-ERM) proteins (at the rear) in polarized T cells after CXCL12 stimulation. This technique was also useful to observe the blocking effect on polarization of two different elements: inhibition of actin polymerization by a pharmacological inhibitor and expression of mutants of the Par6/atypical PKC signaling pathway. Thus, evidence is shown that this technique is useful to analyze both morphological alterations and protein redistributions.

The first example chosen here concerns the use of human primary T lymphocytes stimulated with CCL19. However, the same strategy can be used with primary mouse T cells, T cell lines or any other cell type responsive to chemokine stimulation. The gating strategy presented here includes a series of windows that select the focused events, then the single cells. Finally the range of fluorescence intensity is followed here as an example on two markers: the MHC Class I HLA-ABC and filamentous actin for resting (Figure 1) or CCL19-stimulated (Figure 2) T lymphocytes.
First, the Circularity index is measured in untreated (Figure 1) or CCL19-stimulated (Figure 2) T lymphocytes. The fact that resting cells have a major single peak with a high Circularity index value indicates that most cells are close to round. However, it is clear that CCL19 stimulation elicits the appearance of a second subpopulation of cells with a low Circularity index. This subpopulation encompasses cells that have adopted a polarized morphology. Also, when representing the Bright Detail Similarity index between HLA-ABC and F-actin staining, one can observe that some cells exhibit a drop in the value of this index upon CCL19 treatment, indicating that both markers show a lower degree of co-localization. Interestingly, from the dot-plots presented previously, one can position the curser on any particular dot to get a picture of the cell consisting of a brightfield image and an image for each of the stainings performed. Figure 3 thus presents examples of CCL19 stimulated T lymphocytes that fall in the gate of non polarized (Figure 3A) or polarized (Figure 3B) cells. One can clearly visualize the differences in morphology between both subpopulations. From the statistics tables below the Circularity histograms from Figures 1 and 2, one can plot the percentage of cells that fall in the polarized gate in the absence or presence of CCL19 (Figure 3C). As expected, it is clear that chemokine stimulation increases the percentage of polarized T cells (from 11.2% to 50.6%). One can also plot the mean Circularity index of the whole cell population obtained in both conditions (Figure 3D). CCL19 stimulation elicits a drop in this index. However, as only half the cells are polarized, the change in the mean Circularity index observed upon CCL19 stimulation is small.
Similarly, Figure 4 represents examples of CCL19-stimulated cells, in which both the HLA-ABC and the F-actin stainings do (Figure 4A) or do not colocalize (Figure 4B). From the statistics tables below the Bright Detail Similarity histograms shown in Figures 1 and 2, one can obtain the proportion of cells that exhibit an index value below 1.5, representing the cells that exhibit a very low degree of co-localization between HLA-ABC and F-actin. Figure 4C shows that the fraction of cells that falls in this gate increases upon CCL19 stimulation (from 3.4% to 13.9%). Collecting the values of the index for each individual cell, one can also plot the mean Bright Detail Similarity index of the whole cell population obtained in both conditions (Figure 4D). CCL19 elicits a drop in the index, however, for the same reasons as previously, changes are small. By looking at the images of the corresponding cells, it appears that this drop in HLA-ABC &#8211; F-actin co-localization is mainly due to the clustering of F-actin in one pole of the cell. Conversely, the subcellular distribution of HLA-ABC MHC Class I molecules does not seem to be grossly affected by CCL19 stimulation.
That is the reason why we next decided to test further the methodology by analyzing two markers (F-actin and phospho-ERM) known to segregate in opposing poles of chemokine-stimulated T cells. Human T cells stimulated with CXCL12 polarized strongly, as quantified in Figure 5A with the same gating strategy than before (from 5.95% to 79.10% polarized cells after 8 min of stimulation with 50 ng/mL CXCL12). In these conditions, there is a strong decrease in the mean Bright Detail Similarity index as shown in Figures 5B and 5D, meaning that F-actin and phospho-ERM colocalize less after CXCL12 stimulation. As expected, the percentage of cells exhibiting a segregation of the markers chosen here is higher (48%, Figure 5C) compared to the ones previously studied (14%, Figure 4C). Accordingly, the drop in the mean Similarity index of each condition is also more obvious (Figure 5D). Therefore, the imaging flow cytometry technique presented here is suitable to quantify the degree of co-localization of two stainings in different settings.
Finally, we decided to illustrate to what extent the perturbation of some cytoskeletal element or signaling pathway known to affect changes in T cell morphology upon chemokine stimulation, could be measured by imaging flow cytometry. Control T cells were first compared with some pretreated with the actin-disrupting drug latrunculin A (Figure 6A). Cells were stimulated with CXCL12, stained with fluorescent phalloidin and analyzed by imaging flow cytometry. Histograms showing the Raw Max Pixel intensity of the F-actin staining are shown for both cell populations (Figure 6A, top). As expected from the severing activity of latrunculin A towards actin filaments, the intensity of the phalloidin staining is lower in these cells (right panel) compared to control T lymphocytes treated with the vehicle DMSO (left panel). Moreover, latrunculin A-treated T cells exhibit a larger Circularity index indicating that they remain rounder than control cells (Figure 6A, bottom). This can be quantified by a gate set on the histograms to measure the percentage of cells that exhibit morphological alterations. Although 32% of control cells get polarized in this condition, only 10% of latrunculin A &#8211;treated T lymphocytes exhibit any shape changes (Figure 6B). Therefore, it is shown here that the imaging flow cytometry technology is suitable to measure differences in morphological modifications when one studies the potential effect of some cytoskeletal elements in this phenomenon.
Secondly, the acquisition of a large number of cells with this method also opens the possibility to analyze rapidly small subsets of cells present as a minority. In Figure 7, an example is presented where our analysis is specifically restricted to the behavior of T cells co-transfected with GFP together with plasmids encoding for dominant negative mutants of the polarity complex Par6/PKC&#950;. The histogram corresponding to the non-transfected cells has allowed us to gate specifically on the relevant GFP+ T cells (Figure 7A, left). The intensity of GFP measured as histograms that quantify the distribution of the fluorescence Intensity of the channel 2 indeed shows that only a small fraction of the whole cell population has been transfected (Figure 7A). The degree of morphological polarization of these transfected cells was then quantified by measuring only the Circularity index of the GFP+ cells (Figure 7B). An additional gate for T cells exhibiting low values of Circularity allowed estimation of the percentage of polarized cells in each condition (Figure 7C). The results show that disruption of the function of the Par6/PKC&#950; signaling pathway inhibited T cell polarization induced by both CCL19 and CXCL12 chemokines, in line with our previous findings7.
<img alt="Figure 1" src="/files/ftp_upload/52140/52140fig1.jpg" width="650" /> 
Figure 1. Representative non-stimulated staining results on primary human T cells. The upper left panel shows the histogram repartition of the Gradient RMS calculated with the M01 mask on the bright field images (Channel 1). The upper right panel shows the Area of the M01 mask eroded of 2 pixels, based on the brightfield images (Channel 1) of the In Focus cells gated from the previous plot. The middle panels show the Raw Max Pixel values of both the HLA-ABC and Phalloidin stainings (Channels 2 and 4, respectively) either as a dot plot (left panel) or as histograms (middle and right panels) for the individual, in-focus cells selected at the previous step. From this population of individual, in-focus cells with correct staining, is calculated the Circularity feature on the M02 mask eroded of 2 pixels of the HLA-ABC staining (lower left panel) and the Bright Detail Similarity R3 feature (lower right panel) showing the degree of similarity between the HLA-ABC and the Phalloidin stainings. For these two latest plots, statistics indicating the repartition of the population (% gated, mean of the feature, S.D.) are indicated in a table below. <a href="https://www.jove.com/files/ftp_upload/52140/52140fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52140/52140fig2.jpg" width="650" /> 
Figure 2: Representative staining results on primary human T cells stimulated with 200 ng/ml CCL19 for 8 min. The panel layout and gating strategy are identical to Figure 1. <a href="https://www.jove.com/files/ftp_upload/52140/52140fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52140/52140fig3highres.jpg" /> 
Figure 3: Quantification of the morphological status of T lymphocytes stimulated by CCL19. (A,B) Examples of cells from Figure 2 outside or in the &#8220;polarized&#8221; gate, respectively. (C) Histogram showing the percentage of polarized T cells obtained in the absence of CCL19 (Figure 1, -) or with 200 ng/ml CCL19 (Figure 2, +). (D) Histogram representing the mean values of the circularity index &#177; SE in the individual, in-focus cell population in the absence (-) or presence (+) of CCL19. *** p &#60; 0.001. <a href="https://www.jove.com/files/ftp_upload/52140/52140fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52140/52140fig4highres.jpg" /> 
Figure 4. Quantification of the degree of co-localization between the HLA-ABC and phalloidin stainings. (A,B) Examples of cells from Figure 2 outside or in the &#8220;not colocalized&#8221; gate from the Bright Detail Similarity histogram, respectively. (C) Histogram showing the percentage of T cells exhibiting no co-localization between both markers in un-stimulated (-) or CCL19-stimulated (+) conditions. (D) Histogram representing the mean values of the Similarity index &#177; SE in the individual, in-focus cell population in the absence (-) or presence (+) of CCL19. *** p &#60; 0.001. <a href="https://www.jove.com/files/ftp_upload/52140/52140fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52140/52140fig5highres.jpg" /> 
Figure 5. Analysis of F-actin &#8211; phospho-ERM exclusion upon CXCL12 stimulation of human primary T cells. (A) Percentage of polarized T cells in the absence or presence of CXCL12 (10 or 50 ng/mL). (B) Imaging flow cytometry histograms showing the repartition of the individual, in-focus cell population according to the Bright Detail Similarity index that compares the extent of co-localization between the phospho-ERM and phalloidin stainings, in the absence of CXCL12 or with 10 ng/ml or 50 ng/ml CXCL12. (C) Quantification of the proportion of cells exhibiting no co-localization of the pERM and the phalloidin stainings in T cells stimulated or not with CXCL12. (D) Quantification of the mean values of the Similarity index &#177; SE in each condition. *** p &#60; 0.001. <a href="https://www.jove.com/files/ftp_upload/52140/52140fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/52140/52140fig6highres.jpg" /> 
Figure 6. Effect of actin polymerization on T cell polarization. (A) F-actin staining intensity (upper panels) and Circularity (lower panels) of primary human T cells treated with Latrunculin A (500 nM, 30 min) or DMSO and then stimulated with 100 ng/ml CXCL12 for 8 min. (B) Histogram showing the percentage of polarized T cells treated with DMSO or Latrunculin A after 8 min of stimulation with CXCL12. <a href="https://www.jove.com/files/ftp_upload/52140/52140fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/52140/52140fig7.jpg" width="650" /> 
Figure 7. Expression of the PKC&#950; kinase-dead mutant or the N-terminal mutant of Par6 blocks T cell polarization after CCL19 or CXCL12 treatment. (A) Imaging flow cytometry histograms showing the expression intensity of GFP, in non-transfected primary T cells or primary T cells co-transfected with GFP and an empty vector (Control) or the kinase-dead mutant of PKC&#950; or the N-terminal part of Par6. (B) Imaging flow cytometry histograms representing the Circularity index of the GFP-positive cell population expressing the different constructs at the basal level (NS: No Stimulation) or after CCL19 or CXCL12 stimulation (100 ng/ml for 8 min). (C) Quantification of the percentage of morphologically polarized T cells expressing the different constructs at the basal level or after CCL19 or CXCL12 stimulation. <a href="https://www.jove.com/files/ftp_upload/52140/52140fig7highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Rapid and Robust Analysis of Cellular and Molecular Polarization Induced by Chemokine Signaling at JoVE.com

Rapid and Robust Analysis of Cellular and Molecular Polarization Induced by Chemokine Signaling

Chemokine signaling elicits marked alterations of cellular morphology and some important redistributions of intracellular proteins. Here, a rapid and detailed protocol is provided to study these events.

Cells respond to chemokine stimulation by losing their round shape in a process called polarization, and by altering the subcellular localization of many ...

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Unit 1

Cell Polarization and Migration

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8.1K Views.  Institut Cochin. Chemokine signaling elicits marked alterations of cellular morphology and some important redistributions of intracellular proteins. Here, a rapid and detailed protocol is provided to study these events.

Video: Rapid and Robust Analysis of Cellular and Molecular Polarization Induced by Chemokine Signaling

Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. The various functions of these cells are related to their activation states, which is also called polarization. The precise molecular description of these various polarizations is a priority in the field of macrophage biology. It is currently acknowledged that a multidimensional approach is necessary to describe how polarization is controlled by environmental signals. In this report, we describe a protocol designed to obtain the proteomic signature of various polarizations in human macrophages. This protocol is based on a label-free quantification of macrophage protein expression obtained from in-gel fractionated and Lys C/trypsin-digested cellular lysis content. We also provide a protocol based on in-solution digestion and isoelectric focusing fractionation to use as an alternative. Because oxygen concentration is a relevant environmental parameter in tissues, we use this protocol to explore how atmospheric composition or a low oxygen environment affects the classification of macrophage polarization.

We present a protocol to obtain proteomic signatures of human macrophages and apply this to determination of the impact of a low oxygen environment on macrophage polarization.

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. ...

Biochemistry

Three-dimensional Confocal Analysis of Microglia/macrophage Markers of Polarization in Experimental Brain Injury

After brain stroke microglia/macrophages (M/M) undergo rapid activation with dramatic morphological and phenotypic changes that include expression of novel surface antigens and production of mediators that build up and maintain the inflammatory response. Emerging evidence indicates that M/M are highly plastic cells that can assume classic pro-inflammatory (M1) or alternative anti-inflammatory (M2) activation after acute brain injury. However a complete characterization of M/M phenotype marker expression, their colocalization and temporal evolution in the injured brain is still missing.	
	Immunofluorescence protocols specifically staining relevant markers of M/M activation can be performed in the ischemic brain. Here we present immunofluorescence-based protocols followed by three-dimensional confocal analysis as a powerful approach to investigate the pattern of localization and co-expression of M/M phenotype markers such as CD11b, CD68, Ym1, in mouse model of focal ischemia induced by permanent occlusion of the middle cerebral artery (pMCAO). Two-dimensional analysis of the stained area reveals that each marker is associated to a defined M/M morphology and has a given localization in the ischemic lesion. Patterns of M/M phenotype marker co-expression can be assessed by three-dimensional confocal imaging in the ischemic area. Images can be acquired over a defined volume (10 &mu;m z-axis and a 0.23 &mu;m step size, corresponding to a 180 x 135 x 10 &mu;m volume) with a sequential scanning mode to minimize bleed-through effects and avoid wavelength overlapping. Images are then processed to obtain three-dimensional renderings by means of Imaris software. Solid view of three dimensional renderings allows the definition of marker expression in clusters of cells. We show that M/M have the ability to differentiate towards a multitude of phenotypes, depending on the location in the lesion site and time after injury.

A way to gain new insights into the complexity of the brain inflammatory response is presented. We describe immunofluorescence-based protocols followed by three-dimensional confocal analysis to investigate the pattern of co-expression of microglia/macrophage phenotype markers in a mouse model of focal ischemia.

After brain stroke microglia/macrophages (M/M) undergo  rapid activation with dramatic morphological and phenotypic changes that  include expression of ...

Neuroscience

An Ectopic Chemokine Expression Model for Testing Macrophage Recruitment In Vivo

Zebrafish are widely used in basic and biomedical research. Many zebrafish transgenic lines are currently available to label various types of cells. Owing to the transparent embryonic body of zebrafish, it is convenient for us to study the effect of one chemokine on the behavior of a certain type of cells in vivo. Here we provided a workflow to investigate the function of a chemokine on macrophage migration in vivo. We constructed a tissue-specific overexpression plasmid to overexpress IL-34 and injected the plasmid into one-cell stage transgenic fish embryos whose macrophages were specifically labeled by a fluorescent protein. We then used whole mount fluorescent in situ hybridization and immunostaining to detect the pattern of the chemokine expression and the number or location of macrophages. The injected WT embryos were raised to generate a stable transgenic line. Finally, we used confocal live imaging to directly observe macrophage behavior in the stable transgenic fish to study the function of IL-34 on macrophages in vivo.

To test the effect of a chemokine on macrophage recruitment in vivo, the whole mount in situ hybridization was used to detect the ectopic expression of the chemokine, and immunostaining was used to label macrophages. Live imaging was used for real-time observation of macrophage migration.

Zebrafish are widely used in basic and biomedical research. Many zebrafish transgenic lines are currently available to label various types of cells. Owing ...

Developmental Biology

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

An All-on-chip Method for Rapid Neutrophil Chemotaxis Analysis Directly from a Drop of Blood

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil migration and chemotaxis owing to their advantages in real-time visualization, precise control of chemical concentration gradient generation, and reduced reagent and sample consumption. Recently, a growing effort has been made by the microfluidic researchers toward developing integrated and easily operated microfluidic chemotaxis analysis systems, directly from whole blood. In this direction, the first all-on-chip method was developed for integrating the magnetic negative purification of neutrophils and the chemotaxis assay from small blood volume samples. This new method permits a rapid sample-to-result neutrophil chemotaxis test in 25 min. In this paper, we provide detailed construction, operation and data analysis method for this all-on-chip chemotaxis assay with a discussion on troubleshooting strategies, limitations and future directions. Representative results of the neutrophil chemotaxis assay testing a defined chemoattractant, N-Formyl-Met-Leu-Phe (fMLP), and sputum from a chronic obstructive pulmonary disease (COPD) patient, using this all-on-chip method are shown. This method is applicable to many cell migration-related investigations and clinical applications.

This article provides the detailed method of performing a rapid neutrophil chemotaxis assay by integrating the on-chip neutrophil isolation from whole blood and the chemotaxis test on a single microfluidic chip.

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil ...

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and chemical complexity of living tissues and mimic in vivo tissue functions. Recent advances in microfabrication technologies have facilitated the development of 3D in vitro environments in which cells can be integrated into a well-defined extracellular matrix (ECM) and a defined set of soluble or matrix associated biomolecules. However, technological barriers have limited their widespread use in research laboratories. Here, we describe a method to construct simple devices for 3D culture and experimentation with cells and multicellular organoids in 3D microenvironments with a defined chemoattractant gradient. We illustrate the use of this platform for analysis of the response of epithelial cells and organoids to gradients of growth factors, such as epidermal growth factor (EGF). EGF gradients were stable in the devices for several days leading to directed branch formation in breast organoids. This analysis allowed us to conclude that collective gradient sensing by groups of cells is more sensitive vs. single cells. We also describe the fabrication method, which does not require photolithography facilities nor advanced soft lithography techniques. This method will be helpful to study 3D cellular behaviors in the context of the analysis of development and pathological states, including cancer.

We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs.

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and ...

Bioengineering

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1. This guided cell migration is referred as chemotaxis, which is essential for various cells to carry out their functions such as trafficking of immune cells and patterning of neuronal cells 2, 3. A large family of G-protein coupled receptors (GPCRs) detects variable small peptides, known as chemokines, to direct cell migration in vivo 4. The final goal of chemotaxis research is to understand how a GPCR machinery senses chemokine gradients and controls signaling events leading to chemotaxis. To this end, we use imaging techniques to monitor, in real time, spatiotemporal concentrations of chemoattractants, cell movement in a gradient of chemoattractant, GPCR mediated activation of heterotrimeric G-protein, and intracellular signaling events involved in chemotaxis of eukaryotic cells 5-8. The simple eukaryotic organism, Dictyostelium discoideum, displays chemotaxic behaviors that are similar to those of leukocytes, and D. discoideum is a key model system for studying eukaryotic chemotaxis. As free-living amoebae, D. discoideum cells divide in rich medium. Upon starvation, cells enter a developmental program in which they aggregate through cAMP-mediated chemotaxis to form multicullular structures. Many components involved in chemotaxis to cAMP have been identified in D. discoideum. The binding of cAMP to a GPCR (cAR1) induces dissociation of heterotrimeric G-proteins into G&gamma; and G&beta;&gamma; subunits 7, 9, 10. G&beta;&gamma; subunits activate Ras, which in turn activates PI3K, converting PIP2 into PIP3 on the cell membrane 11-13. PIP3 serve as binding sites for proteins with pleckstrin Homology (PH) domains, thus recruiting these proteins to the membrane 14, 15. Activation of cAR1 receptors also controls the membrane associations of PTEN, which dephosphorylates PIP3 to PIP2 16, 17. The molecular mechanisms are evolutionarily conserved in chemokine GPCR-mediated chemotaxis of human cells such as neutrophils 18. We present following methods for studying chemotaxis of D. discoideum cells. 1. Preparation of chemotactic component cells. 2. Imaging chemotaxis of cells in a cAMP gradient. 3. Monitoring a GPCR induced activation of heterotrimeric G-protein in single live cells. 4. Imaging chemoattractant-triggered dynamic PIP3 responses in single live cells in real time. Our developed imaging methods can be applied to study chemotaxis of human leukocytes.

Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Here, we describe detailed live cell imaging methods for investigating chemotaxis. We present fluorescence microscopic methods to monitor spatiotemporal dynamics of signaling events in migrating cells. Measurement of signaling events permits us to further understand how a GPCR-signaling network achieves gradient sensing of chemoattractants and controls directional migration of eukaryotic cells.

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1.  This guided cell migration is referred as ...

Biology

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many ...

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium discoideum revealed that a complex signal transduction network of parallel pathways amplifies the response to chemoattractants, and leads to biased actin polymerization and protrusion of a pseudopod in the direction of a gradient. In contrast, molecular mechanisms driving other types of directed migration, for example, due to exposure to shear flow or electric fields, are not known. Many regulators of chemotaxis exhibit localization at the leading or lagging edge of a migrating cell, as well as show transient changes in localization or activation following global stimulation with a chemoattractant. To understand the molecular mechanisms of other types of directed migration we developed a method that allows examination of cellular response to acute mechanical stimulation based on brief (2 - 5 s) exposure to shear flow. This stimulation can be delivered in a channel while imaging cells expressing fluorescently-labeled biosensors to examine individual cell behavior. Additionally, cell population can be stimulated in a plate, lysed, and immunoblotted using antibodies that recognize active versions of proteins of interest. By combining both assays, one can examine a wide array of molecules activated by changes in subcellular localization and/or phosphorylation. Using this method we determined that acute mechanical stimulation triggers activation of the chemotactic signal transduction and actin cytoskeleton networks. The ability to examine cellular responses to acute mechanical stimulation is important for understanding the initiating events necessary for shear flow-induced motility. This approach also provides a tool for studying the chemotactic signal transduction network without the confounding influence of the chemoattractant receptor.

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Here we describe methods for assessing cellular response to acute mechanical stimulation. In the microscopy-based assay, we examine localization of fluorescently-labeled biosensors following brief stimulation with shear flow. We also test activation of various proteins of interest in response to acute mechanical stimulation biochemically.

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...

Rapid and Robust Analysis of Cellular and Molecular Polarization Induced by Chemokine Signaling

Podsumowanie

Przeglądaj więcej filmów

Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment

Three-dimensional Confocal Analysis of Microglia/macrophage Markers of Polarization in Experimental Brain Injury

An Ectopic Chemokine Expression Model for Testing Macrophage Recruitment In Vivo

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

An All-on-chip Method for Rapid Neutrophil Chemotaxis Analysis Directly from a Drop of Blood

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation