Name: studying organelle dynamics in b cells during immune synapse formation
Uploaded: 2019-06-01T14:30:00
Description: Watch this Scientific Journal Video about Studying Organelle Dynamics in B Cells During Immune Synapse Formation at JoVE.com

M.-I.Y. is supported by a research grant from FONDECYT #1180900. J.I., D.F. and J.L. were supported by fellowships from the Comisi&#243;n Nacional de Ciencia y Tecnolog&#237;a. We thank to David Osorio from Pontificia Universidad Cat&#243;lica de Chile for the video recording and editing.

&#160;&#160;&#160;&#160;&#160;NOTE: The following steps were performed using IIA1.6 B cells.
1. B cell activation with antigen-coated beads
<ol>
	<li>Preparation of antigen-coated beads
	<ol>
		<li>To activate B cells, use NH2-beads covalently coated with antigen (Ag-coated beads), which are prepared using 50 &#181;L (~20 x 106 beads) of 3 &#181;m NH2-beads with activating (BCR-ligand+) or non-activating (BCR-ligand-) antigens.</li>
		<li>For IIA1.6 B cells use a...

<ol>
	<li>Carrasco, Y. R., Batista, F. D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cells+Acquire+Particulate+Antigen+in+a+Macrophage-Rich+Area+at+the+Boundary+between+the+Follicle+and+the+Subcapsular+Sinus+of+the+Lymph+Node.">Cells Acquire Particulate Antigen in a Macrophage-Rich Area at the Boundary between the Follicle and the Subcapsular Sinus of the Lymph Node.</a> Immunity. 27 (1), 160-171 (2007).</li><li>Batista, F. D., Harwood, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+who,+how+and+where+of+antigen+presentation+to+B+cells.">The who, how and where of antigen presentation to B cells.</a> Nature Reviews Immunology. 9 (1), 15-27 (2009).</li><li>Grakoui, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immunological+synapse:+a+molecular+machine+controlling+T+cell+activation.">The immunological synapse: a molecular machine controlling T cell activation.</a> Science (New York, N.Y.). 285 (5425), 221-227 (1999).</li><li>Batista, F. D., Iber, D., Neuberger, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B+cells+acquire+antigen+from+target+cells+after+synapse+formation.">B cells acquire antigen from target cells after synapse formation.</a> Nature. 411 (6836), 489-494 (2001).</li><li>Obino, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+nucleation+at+the+centrosome+controls+lymphocyte+polarity.">Actin nucleation at the centrosome controls lymphocyte polarity.</a> Nature Communications. 7, (2016).</li><li>Yuseff, M. I., 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=Polarized+Secretion+of+Lysosomes+at+the+B+Cell+Synapse+Couples+Antigen+Extraction+to+Processing+and+Presentation.">Polarized Secretion of Lysosomes at the B Cell Synapse Couples Antigen Extraction to Processing and Presentation.</a> Immunity. 35 (3), 361-374 (2011).</li><li>Spillane, K. M., Tolar, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanics+of+antigen+extraction+in+the+B+cell+synapse.">Mechanics of antigen extraction in the B cell synapse.</a> Molecular Immunology. 101, 319-328 (2018).</li><li>Schnyder, 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=B+Cell+Receptor-Mediated+Antigen+Gathering+Requires+Ubiquitin+Ligase+Cbl+and+Adaptors+Grb2+and+Dok-3+to+Recruit+Dynein+to+the+Signaling+Microcluster.">B Cell Receptor-Mediated Antigen Gathering Requires Ubiquitin Ligase Cbl and Adaptors Grb2 and Dok-3 to Recruit Dynein to the Signaling Microcluster.</a> Immunity. , (2011).</li><li>Wang, J. C., 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=The+Rap1+&#8211;+cofilin-1+pathway+coordinates+actin+reorganization+and+MTOC+polarization+at+the+B+cell+immune+synapse.">The Rap1 &#8211; cofilin-1 pathway coordinates actin reorganization and MTOC polarization at the B cell immune synapse.</a> Journal of Cell Science. , 1094-1109 (2017).</li><li>Fleire, S. J., Goldman, J. P., Carrasco, Y. R., Weber, M., Bray, D., Batista, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B+cell+ligand+discrimination+through+a+spreading+and+contraction+response.">B cell ligand discrimination through a spreading and contraction response.</a> Science. 312 (5774), 738-741 (2006).</li><li>Vascotto, F., 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=The+actin-based+motor+protein+myosin+II+regulates+MHC+class+II+trafficking+and+BCR-driven+antigen+presentation.">The actin-based motor protein myosin II regulates MHC class II trafficking and BCR-driven antigen presentation.</a> Journal of Cell Biology. 176 (7), 1007-1019 (2007).</li><li>Reversat, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarity+protein+Par3+controls+B-cell+receptor+dynamics+and+antigen+extraction+at+the+immune+synapse.">Polarity protein Par3 controls B-cell receptor dynamics and antigen extraction at the immune synapse.</a> Molecular biology of the cell. 26 (7), 1273-1285 (2015).</li><li>Lankar, D., 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=Dynamics+of+Major+Histocompatibility+Complex+Class+II+Compartments+during+B+Cell+Receptor&#8211;mediated+Cell+Activation.">Dynamics of Major Histocompatibility Complex Class II Compartments during B Cell Receptor&#8211;mediated Cell Activation.</a> The Journal of Experimental Medicine. 195 (4), 461-472 (2002).</li><li>Duchez, 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=Reciprocal+Polarization+of+T+and+B+Cells+at+the+Immunological+Synapse.">Reciprocal Polarization of T and B Cells at the Immunological Synapse.</a> The Journal of Immunology. 187 (9), 4571-4580 (2011).</li><li>P&#233;rez-Montesinos, G., 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=Dynamic+changes+in+the+intracellular+association+of+selected+rab+small+GTPases+with+MHC+class+II+and+DM+during+dendritic+cell+maturation.">Dynamic changes in the intracellular association of selected rab small GTPases with MHC class II and DM during dendritic cell maturation.</a> Frontiers in Immunology. 8 (MAR), 1-15 (2017).</li><li>Le Roux, D., Lankar, D., Yuseff, M. I., Vascotto, F., Yokozeki, T., Faure-Andre&#180;, G., Mougneau, E., Glaichenhaus, N., Manoury, B., Bonnerot, C., Lennon-Dume&#180;nil, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Syk-dependent+Actin+Dynamics+Regulate+Endocytic+Trafficking+and+Processing+of+Antigens+Internalized+through+the+B-Cell+Receptor.">Syk-dependent Actin Dynamics Regulate Endocytic Trafficking and Processing of Antigens Internalized through the B-Cell Receptor.</a> Molecular biology of the cell. 18 (September), 3451-3462 (2007).</li><li>Reber, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+8+Isolation+of+Centrosomes+from+Cultured+Cells.">Chapter 8 Isolation of Centrosomes from Cultured Cells.</a> Methods in Molecular Biology. 777 (2), 107-116 (2011).</li><li>Gogendeau, D., Guichard, P., Tassin, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+centrosomes+from+mammalian+cell+lines.">Purification of centrosomes from mammalian cell lines.</a> Methods in Cell Biology. 129, (2015).</li><li>Obino, D., 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=Vamp-7&#8211;dependent+secretion+at+the+immune+synapse+regulates+antigen+extraction+and+presentation+in+B-lymphocytes.">Vamp-7&#8211;dependent secretion at the immune synapse regulates antigen extraction and presentation in B-lymphocytes.</a> Molecular Biology of the Cell. 28 (7), 890-897 (2017).</li><li>Harwood, N. E., Batista, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+Events+in+B+Cell+Activation+BCR:+B+cell+receptor.">Early Events in B Cell Activation BCR: B cell receptor.</a> Annual Review of Immunology. , (2010).</li><li>Yuseff, M. I., Lennon-Dum&#233;nil, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B+cells+use+conserved+polarity+cues+to+regulate+their+antigen+processing+and+presentation+functions.">B cells use conserved polarity cues to regulate their antigen processing and presentation functions.</a> Frontiers in Immunology. 6, 1-7 (2015).</li></ol>

We describe a comprehensive method to study how B lymphocytes re-organize their intracellular architecture to promote the formation of an IS. This study includes the use of imaging techniques to quantify the intracellular distribution of organelles, such as centrosome, Golgi apparatus and lysosomes during B cell activation, and how they polarize to the IS. Additionally, we describe a biochemical approach to study changes in the centrosome composition upon B cell activation.
To promote the form...

B lymphocytes are an essential part of the adaptive immune system responsible for producing antibodies against different threats and invading pathogens. The efficiency of antibody production is determined by the ability of B cells to acquire, process and present antigens encountered either in a soluble or surface-tethered form1,2. Recognition of antigens attached to the surface of a presenting cell, by the BCR, leads to the formation of a close intercellular contact termed IS3,4. Within this dynamic platform both BCR-dependent downstream signaling and internalization of antigens into endo-lysosome compartments takes place. Uptaken antigens are processed and assembled onto MHC-II molecules and subsequently presented to T lymphocytes. Productive B-T interactions, termed B-T cell cooperation, allow B lymphocytes to receive the appropriate signals, which promote their differentiation into antibody-producing plasma cells or memory cells8.
Two mechanisms have been involved in antigen extraction by B cells. The first one relies on the secretion of proteases originating from lysosomes which undergo recruitment and fusion at the synaptic cleft5,6. The second one, depends on Myosin IIA-mediated pulling forces that triggers the invagination of antigen containing membranes which are internalized into clathrin-coated pits7. The mode of antigen extraction relies on the physical properties of the membrane in which antigens are found. Nevertheless, in both cases, B cells undergo two major remodeling events: actin cytoskeleton reorganization and polarization of organelles to the IS. Actin cytoskeleton remodeling involves an initial spreading stage, where actin-dependent protrusions at the synaptic membrane increase the surface in contact with the antigen. This is followed by a contraction phase, where BCRs coupled with antigens are concentrated at the center of the IS by the concerted action of molecular motors and actin cytoskeleton remodeling8,9,10,11. Polarization of organelles also relies on the remodeling of actin cytoskeleton. For instance, the centrosome becomes uncoupled from the nucleus, by local depolymerization of associated actin, which allows the repositioning of this organelle to the IS5,12. In B cells, repositioning of the centrosome to one cell pole (IS) guides lysosome recruitment to the synaptic membrane, which upon secretion can facilitate the extraction and/or processing of surface-tethered antigens6. Lysosomes recruited at the IS are enriched with MHC-II, which favors the formation of peptide-MHC-II complexes in endosomal compartments to be presented to T cells13. Additionally, the Golgi apparatus has also been observed to be closely recruited to the IS14, suggesting that Golgi-derived vesicles from the secretory pathway could be involved in antigen extraction and/or processing.
Altogether, intracellular organelle and cytoskeleton rearrangements in B cells&#160;during synapse formation are the key steps that allow efficient antigen acquisition and processing required for their further activation. In this work we introduce detailed protocols on how to perform imaging and biochemical analysis in B cells to study the intracellular remodeling of organelles associated with the formation of an IS. These techniques include: (i) Immunofluorescence and image analysis of B cells activated with antigen-coated beads and on antigen-coated coverslips, which allows visualization and quantification of intracellular components that are mobilized to the IS and (ii) isolation of centrosome-enriched fractions in B cells by ultracentrifugation on sucrose gradients, which allows the identification of proteins associated with the centrosome, potentially involved in regulating&#160;cell polarity.

<table border="0" cellpadding="0" cellspacing="0" style="width:591px;" width="589">
	<tbody>
		<tr height="19">
			<td height="19" style="height:19px;width:223px;">IIA1.6 (A20 variant) mouse B-lymphoma cells</td>
			<td style="width:74px;">ATCC</td>
			<td style="width:123px;">TIB-208</td>
			<td style="width:172px;">Murine B-cell lymphoma of Balb/c origin that expresses an IgG-containing BCR on its surface without Fc&#947;IIR</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">100% methanol</td>
			<td>Fisher Scientific</td>
			<td>A412-4</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">10-mm diameter cover glasses thickness No. 1 circular</td>
			<td>Marienfield-Superior</td>
			<td align="right">111500</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">2-mercaptoethanol</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right">21985023</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Alexa 488 fluor- donkey ant-rabbit IgG&#160;</td>
			<td>LifeTech&#160;</td>
			<td>A21206</td>
			<td>1:500 dilution recomended but should be optimized</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Alexa Fluor 546 goat anti-Rabbit &#160;IgG</td>
			<td>Thermo Fisher Scientific</td>
			<td>&#160;A-11071</td>
			<td>1:500 dilution recomended but should be optimized</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Alexa Fluor 647-conjugated phalloidin</td>
			<td>Thermo Fisher Scientific</td>
			<td>&#160;A21238</td>
			<td>1:500 dilution recomended but should be optimized</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Amaxa Nucleofection kit V</td>
			<td>Lonza</td>
			<td>VCA-1003</td>
			<td>Follow the manufacturer&#39;s directions for mixing the transfection reagents with the DNA</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Amaxa Nucleofector model 2b</td>
			<td>Lonza</td>
			<td>AAB-1001</td>
			<td>Program L-013 used</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Amino- Dynabeads</td>
			<td>ThermoFisher</td>
			<td>14307D</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Anti-pericentrin</td>
			<td>&#160;Abcam</td>
			<td>ab4448&#160;</td>
			<td>1:200 dilution recomended but should be optimized</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Anti-rab6</td>
			<td>Abcam</td>
			<td>ab95954</td>
			<td>1:200 dilution recomended but should be optimized</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Anti-sec61</td>
			<td>Abcam</td>
			<td>ab15575</td>
			<td>1:200 dilution recomended but should be optimized</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">BSA</td>
			<td>&#160;Winkler</td>
			<td>&#160;BM-0150</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">CaCl2</td>
			<td>Winkler</td>
			<td>CA-0520</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Culture plate T25</td>
			<td>BD</td>
			<td align="right">353014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Fiji Software</td>
			<td style="width:74px;">Fiji col.</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Fluoromount G</td>
			<td>Electron Microscopy Science</td>
			<td>17984-25</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Glutamine</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right">35050061</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Glutaraldehyde</td>
			<td>Sigma&#160;</td>
			<td>G7651&#160;</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Glycine</td>
			<td>Winkler&#160;</td>
			<td>BM-0820</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Goat-anti-mouse IgG antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>315-005-003</td>
			<td>IIA1.6 positive ligand</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Goat-anti-mouse IgM antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right">31186</td>
			<td>IIA1.6 negative ligand</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">HyClone Fetal bovine serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>SH30071.03</td>
			<td>Heat inactivate at 56 oC for 30 min</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">KCl</td>
			<td>Winkler</td>
			<td>PO-1260</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Leica SP8 TCS microscope</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">NaCl</td>
			<td>Winkler</td>
			<td>SO-1455</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Nikon Eclipse Ti-E epifluorescence microscope</td>
			<td>&#160;Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Parafilm</td>
			<td>M</td>
			<td>P1150-2</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Paraformaldehyde</td>
			<td>Merck</td>
			<td>30525-89-4</td>
			<td>Dilute to 4% with PBS in a safety cabinet, use at the moment</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Penicillin-Streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right">15140122</td>
			<td>Liquid</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Polybead Amino Microspheres 3.00&#956;m</td>
			<td>Polyscience</td>
			<td>17145-5</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Poly-L-Lysine</td>
			<td>Sigma</td>
			<td>P8920</td>
			<td>Dilute with sterile water</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Rabbit anti- alpha tubulin antibody</td>
			<td>Abcam</td>
			<td>ab6160</td>
			<td>1:1000 dilution recommended but should be optimized</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Rabbit anti mouse lamp1 antibody</td>
			<td>Cell signaling</td>
			<td align="right">3243</td>
			<td>1:200 dilution recomended but should be optimized</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Rabbit anti-cep55&#160;</td>
			<td>Abcam</td>
			<td>ab170414</td>
			<td>1:500 dilution recomended but should be optimized</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Rabbit Anti-gamma Tubulin antibody</td>
			<td>&#160;Abcam</td>
			<td>ab16504</td>
			<td>&#160;1:1000 for Western Blot</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">RPMI-1640</td>
			<td>Biological Industries</td>
			<td>01-104-1A</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Saponin</td>
			<td>&#160;Merck</td>
			<td align="right">558255</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Sodium pyruvate</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right">11360070</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Sucrose</td>
			<td>Winkler&#160;</td>
			<td>SA-1390&#160;</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Triton X-100</td>
			<td>&#160;Merck</td>
			<td>9036-19-5</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Tube 50 ml</td>
			<td>Corning</td>
			<td align="right">353043</td>
			<td></td>
		</tr>
	</tbody>
</table>

studying organelle dynamics in b cells during immune synapse formation

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both signaling&#160;and antigen uptake are coordinated. IS formation involves dynamic actin remodeling accompanied by the polarized recruitment to the synaptic membrane of the centrosome and associated intracellular organelles such as lysosomes and the Golgi apparatus. Initial stages of actin remodeling allow B cells to increase their cell surface and maximize the quantity of antigen-BCR complexes gathered at the synapse. Under certain conditions, when B cells recognize antigens associated to rigid surfaces, this process is coupled to the local recruitment and secretion of lysosomes, which can facilitate antigen extraction. Uptaken antigens&#160;are internalized into specialized endo-lysosome compartments for processing into peptides, which are loaded onto major histocompatibility complex II (MHC-II) molecules for further presentation to T helper cells. Therefore, studying organelle dynamics associated with the formation of an IS is crucial to understanding how B cells are activated. In the present article we will discuss both imaging and a biochemical technique used to study changes in intracellular organelle positioning and cytoskeleton rearrangements that are associated with the formation of an IS in B cells.

The present article shows how B cells can be activated using immobilized antigen on beads or coverslips to induce the formation of an IS. We provide information on how to identify and quantify the polarization of different organelles by immunofluorescence and how to characterize proteins that undergo dynamic changes in their association to the centrosome, which polarizes to the IS, using a biochemical approach.
The imaging of B ...

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Studying Organelle Dynamics in B Cells During Immune Synapse Formation

Herein we describe two approaches to characterize cell polarization events in B lymphocytes during the formation of an IS. The first, involves quantification of organelle recruitment and cytoskeleton rearrangements at the synaptic membrane. The second is a biochemical approach, to characterize changes in composition of the centrosome, which undergoes polarization to the immune synapse.

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both ...

Research

JoVE Journal

Immunology and Infection

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