Name: rapid and robust analysis of cellular and molecular polarization induced by chemokine signaling
Uploaded: 2014-12-12T14:00:00
Description: Watch this Scientific Journal Video about Rapid and Robust Analysis of Cellular and Molecular Polarization Induced by Chemokine Signaling at JoVE.com

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 ...

<ol>
	<li>Rossi, D., Zlotnik, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+chemokines+and+their+receptors.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+chemokines+invite+leukocytes+to+dance.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GTPases:+masters+of+T+lymphocyte+migration+and+activation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+polarization+in+cell+migration+and+immune+interactions.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocyte+growth+factor+and+macrophage+inflammatory+protein+1+beta:+structurally+distinct+cytokines+that+induce+rapid+cytoskeletal+changes+and+subset-preferential+migration+in+T+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fam65b+is+a+new+transcriptional+target+of+FOXO1+that+regulates+RhoA+signaling+for+T+lymphocyte+migration.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+Atypical+PKCs+regulate+T+lymphocyte+polarity+and+scanning+behavior.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarity+of+T+cell+shape,+motility,+and+sensitivity+to+antigen.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ImageStream+System:+a+key+step+to+a+new+era+in+imaging.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+CD4++T+cells+from+mouse+lymph+nodes+using+Miltenyi+MACS+purification.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+antigen+specific+CD4++T+cells+using+MHC+class+II+tetramers.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L-plastin+phosphorylation:+a+novel+target+for+the+immunosuppressive+drug+dexamethasone+in+primary+human+T+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L-plastin+regulates+polarization+and+migration+in+chemokine-stimulated+human+T+lymphocytes.">L-plastin regulates polarization and migration in chemokine-stimulated human T lymphocytes.</a> J Immunol. 188 (12), 6357-6370 (2012).</li></ol>

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...

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 border="0" cellpadding="0" cellspacing="0" width="569">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">Name of the material/Equipment</td>
			<td style="width:95px;">Company</td>
			<td style="width:80px;">Catalog number</td>
			<td style="width:255px;">Comments/Description (optional)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">RPMI</td>
			<td style="width:95px;">Gibco</td>
			<td style="width:80px;">61870-010</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:140px;">Human serum AB</td>
			<td style="width:95px;">PAA</td>
			<td style="width:80px;">C11-021</td>
			<td style="width:255px;">Pre-heat to inactivate the complement.</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:140px;">Fetal calf serum</td>
			<td style="width:95px;">PAN Biotech</td>
			<td style="width:80px;">P30-3300</td>
			<td style="width:255px;">Pre-heat to inactivate the complement.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:140px;">Human T cell nucleofactor kit</td>
			<td style="width:95px;">Lonza</td>
			<td style="width:80px;">VCA-1002</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Murine IL7</td>
			<td style="width:95px;">Peprotech</td>
			<td style="width:80px;">217-17</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:140px;">HBSS</td>
			<td style="width:95px;">Gibco</td>
			<td style="width:80px;">14025-050</td>
			<td style="width:255px;">Warm in 37 &#176;C water bath before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Hepes</td>
			<td style="width:95px;">Gibco</td>
			<td style="width:80px;">15630-056</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:140px;">Murine CCL19</td>
			<td style="width:95px;">Peprotech</td>
			<td style="width:80px;">250-27B</td>
			<td style="width:255px;">Aliquots are thawed on ice before adding the chemokine to the cells.</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:140px;">Human CCL19</td>
			<td style="width:95px;">Peprotech</td>
			<td style="width:80px;">300-29B</td>
			<td style="width:255px;">Aliquots are thawed on ice before adding the chemokine to the cells.</td>
		</tr>
		<tr height="88">
			<td height="88" style="height:88px;width:140px;">Human CXCL12</td>
			<td style="width:95px;">Peprotech</td>
			<td style="width:80px;">300-28A</td>
			<td style="width:255px;">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 height="65">
			<td height="65" style="height:65px;width:140px;">PFA</td>
			<td style="width:95px;">Electron Microscopy Sciences</td>
			<td style="width:80px;">157-8-100</td>
			<td style="width:255px;">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 height="21">
			<td height="21" style="height:21px;width:140px;">&#160;BSA</td>
			<td style="width:95px;">Sigma</td>
			<td style="width:80px;">A3059</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Saponin</td>
			<td style="width:95px;">Fluka</td>
			<td style="width:80px;">84510</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:140px;">Alexa Fluor 594 phalloidin</td>
			<td style="width:95px;">Invitrogen</td>
			<td style="width:80px;">A12381</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:140px;">FITC-anti-HLA-ABC antibody</td>
			<td style="width:95px;">Beckman Coulter</td>
			<td style="width:80px;">IM1838</td>
			<td style="width:255px;">clone B9.12.1</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:140px;">Anti-P-ERM antibody</td>
			<td style="width:95px;">Cell Signaling Technology</td>
			<td style="width:80px;">3149P</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:140px;">ImageStreamx Mark II</td>
			<td style="width:95px;">Amnis</td>
			<td style="width:80px;"></td>
			<td style="width:255px;"></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...

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 ...

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Immunology and Infection

Unit 1

Cell Polarization and Migration

SCIENTISTS IN ACTION

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &alpha;-actinin by Confocal Imaging

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &#945;-actinin by Confocal Imaging

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an ...

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1  Bacterial biofilms are encased within a matrix and attached ...

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

Rapid Quantification of Mitogen-induced Blastogenesis in T Lymphocytes for Identifying Immunomodulatory Drugs

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., ...

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...