We thank the UBC Life Sciences Institute (LSI) Imaging Facility for supporting and maintaining the STED microscope. This work was funded by grant #68865 from the Canadian Institutes of Health Research (to M.R.G.). We thank Dr. Kozo Kaibuchi (Nagoya University, Nagoya, Japan) for the CLIP-170-GFP plasmid.

All animal procedures were approved by the University of British Columbia Animal Care Committee.
1. Expressing GFP-fusion proteins in A20 B-lymphoma cells
<ol>
	<li>Culture A20 cells in complete RPMI medium (RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 50 &#181;M 2-mercaptoethanol, 2 mM glutamine, 1 mM pyruvate, 50 U/mL penicillin, and 50 &#181;g/mL streptomycin) at 37 &#176;C in a tissue culture incubator with 5% CO2.</li>
	<li>Prepare the transfection reagent (see the Table of Materials) according to the manufacturer&#39;s instructions. 
	NOTE: This protocol has been optimized for the specific reagents and transfection protocol described in the Table of Materials. Other transfection reagents that we have tried have not yielded sufficient transfection efficiency.</li>
	<li>Count the cells using a hemocytometer. Centrifuge 2.5 x 106 A20 cells (for each transfection) for 5 min at 525 x g. Resuspend the cells in 100 &#181;L of transfection reagent containing 1-2.5 &#181;g of plasmid DNA. For DNA encoding CLIP-170-GFP23, use 2.5 &#181;g per transfection. Mix gently.</li>
	<li>Transfer the cells to a well of a 6-well plate, and adjust the volume to 2 mL with pre-warmed complete RPMI medium. Culture the cells for 18 h at 37 &#176;C to allow protein expression.</li>
</ol>
2. Isolating Primary Mouse B Cells and Activating Them with LPS
<ol>
	<li>Use sterilized surgical tools to remove the spleen from a mouse, following protocols approved by the institution&#39;s Animal Care Committee.</li>
	<li>In the tissue culture hood, place a sterile 70-&#181;m cell strainer into a 35-mm tissue culture dish containing 3 mL of room temperature sterile PBS. Use the rubber part of the plunger from a 5-ml syringe to crush the spleen through the cell strainer.</li>
	<li>Pipet the single cell suspension of splenoctyes into a sterile 15-mL tube and centrifuge at 525 x g for 5 min.</li>
	<li>Use a magnetic bead-based B cell isolation kit (see Table of Materials) to obtain a highly enriched population of B cells.</li>
	<li>Resuspend the B cells to 3 x 106/mL in complete RPMI medium supplemented with 2.5 &#181;g/mL LPS plus 5 ng/mL B cell activating factor (BAFF; a survival cytokine).</li>
	<li>Culture the cells for 6 hr at 37 &#176;C.</li>
</ol>
3. Coating Glass Coverslips with Anti-Ig Antibodies
<ol>
	<li>Prepare the antibody solution for coating the coverslips. Dilute the stock solution of goat-anti-mouse Ig antibodies into room temperature sterile PBS to make a 12.5 &#181;g/mL solution; 400 &#181;L of antibody solution is required for each coverslip. 
	NOTE: Use goat anti-mouse IgG for A20 cells; use goat anti-mouse IgM for primary B cells. Goat IgG antibodies do not bind well to mouse Fc receptors. However, to avoid any potential Fc receptor binding, one can use F(ab) or F(ab&#39;)2 fragments of anti-mouse IgG or anti-mouse IgM antibodies.</li>
	<li>Dip a #1.5 18-mm round glass coverslip in 100% methanol and let it dry completely (~10 min).</li>
	<li>Using forceps, place the dried coverslip into a 12-well tissue culture plate and pipet 400 &#181;L of the 12.5 &#181;g/mL antibody solution (5 &#181;g total; 2 &#181;g/cm2) onto the coverslip so that it forms a bubble at the center of the coverslip and spreads to the edges.</li>
	<li>Be careful to cover the entire coverslip, but do not allow the antibody solution to extend past the edge of the glass coverslip and onto the tissue culture plastic. Incubate at room temperature for 30 min.</li>
	<li>Wash the coverslips by pipetting 1 mL of sterile PBS into the well and subsequently aspirating out the solution. Repeat two more times to remove unbound antibodies.</li>
	<li>Add 1 mL of sterile PBS to the well containing the antibody-coated coverslip until cells are ready to be added. 
	NOTE: The coverslips can be kept at room temperature, covered with PBS, for use on the same day.</li>
</ol>
4. B Cell Spreading on Anti-Ig-Coated Coverslips
<ol>
	<li>Prepare modified HEPES-buffered saline (mHBS): 25&#8197;mM HEPES, pH 7.2, 125&#8197;mM NaCl, 5&#8197;mM KCl, 1&#8197;mM CaCl2, 1&#8197;mM Na2HPO4, 0.5&#8197;mM MgSO4, 1&#8197;mg/mL dextrose, 2&#8197;mM glutamine, 1&#8197;mM sodium pyruvate, and 50&#8197;&#181;M 2-mercaptoethanol). Filter sterilize this solution and store at 4 &#176;C.</li>
	<li>On the day of the experiment, make 50 mL of mHBS with 2% FBS (mHBS-FBS) by adding 1 mL of heat-inactivated FBS to 50 mL mHBS. Warm the mHBS-FBS to 37 &#176;C before use.</li>
	<li>Centrifuge the transfected A20 cells or the primary B cells that had been cultured with BAFF plus LPS at 525 x g for 5 min.</li>
	<li>Resuspend the cells in 1 mL of mHBS-FBS, count the cells using a hemocytometer, and dilute the cells to 2 x 105 cells/mL in mHBS-FBS.</li>
	<li>Using forceps, transfer the coverslip from the tissue culture plate to a piece of paraffin film.</li>
	<li>Add 250 &#181;L of B cells (5 x 104 cells) to each coverslip.</li>
	<li>Incubate the coverslips at 37 &#176;C for 15 min in the dark to allow the B cells to spread over the anti-IgG-coated surface.</li>
</ol>
5. Fixing and Immunostaining the Cells
<ol>
	<li>Prepare the fixation solution by diluting the 16% paraformaldehyde stock solution and the 50% glutaraldehyde stock solution in room-temperature distilled water to yield final concentrations of 3% paraformaldehyde and 0.1% glutaraldehyde. Prepare the fixation solution on the day it is to be used and discard any excess. 
	CAUTION: The paraformaldehyde and glutaraldehyde stock solutions should only be used in a chemical fume hood. Follow the instructions on the material safety data sheet (MSDS).</li>
	<li>Prepare the permeabilization/blocking buffer (3% BSA and 0.1% Triton X-100 diluted in PBS). Filter sterilize this solution and store at 4 &#176;C. On the day of the experiment, warm the required amount to room temperature.</li>
	<li>Prepare the staining buffer (1% BSA and 0.1% saponin in PBS). Sterilize this solution using a 0.2 &#181;m filter and store at 4 &#176;C. On the day of the experiment, warm the required amount to room temperature.</li>
	<li>Pipet 350 &#181;L of the fixation solution onto each coverslip, ensuring that the surface is completely covered. Incubate at room temperature for 10 min in the dark.</li>
	<li>Remove the fixation solution from the coverslip by carefully aspirating from the edge of the coverslip using a micropipet. 
	CAUTION: The fixation solution should be discarded as hazardous chemical waste.</li>
	<li>Wash the coverslips once with 500 &#181;L of permeabilization/blocking buffer. Pipet off the liquid.</li>
	<li>Permeabilize and block the cells by adding 250 &#181;L of permeabilization/blocking buffer to each coverslip, and incubating for 10 min at room temperature in the dark.</li>
	<li>Dilute the rabbit anti-tubulin antibody 1:100 in staining buffer.</li>
	<li>Aspirate off the permeabilization/blocking buffer from the coverslips, and add 50 &#181;L of the diluted anti-tubulin antibody solution to each coverslip. Incubate at room temperature for 30 min in the dark.</li>
	<li>Prepare the secondary antibody/actin stain solution by diluting both the Alexa Fluor 532-conjugated goat anti-rabbit IgG secondary antibody and Alexa Fluor 568-conjugated phalloidin 1:100 in staining buffer.</li>
	<li>Wash the coverslips 3 times to remove excess primary antibody by adding 500 &#181;L of staining buffer and then aspirating it.</li>
	<li>Add 50 &#181;L of the secondary antibody/actin stain solution to each coverslip, and incubate for 30 min at room temperature in the dark.</li>
	<li>Wash the coverslips 3 times to remove excess secondary antibody by adding 500 &#181;L of staining buffer and then aspirating it.</li>
	<li>Add 10 &#181;L of mounting reagent to a microscope slide. Mount the coverslip onto the microscope slide with the cell side down. 
	NOTE: An &#34;anti-fade&#34; mounting medium (see Table of Materials) is strongly recommended to preserve the fluorescence intensity of GFP fusion proteins. Avoid the use of mounting reagents containing DAPI, which can interfere with the imaging of the fluorophores when using the STED laser.</li>
	<li>Allow the slides to dry overnight in the dark. Image the slides the next day. 
	NOTE: Imaging the slides as soon as the coverslip is dry is strongly recommended, as the GFP fluorescence will decrease over time.</li>
</ol>
6. Imaging Using the STED Microscope
NOTE: Please note that all software steps described below are specific to the microscope and software we used (see the Table of Materials). The steps and settings will need to be adjusted if imaging is performed using a different microscope/software.
<ol>
	<li>Turn on the STED microscope, activate the lasers and epifluorescence illumination lamp, and start up the microscope software.</li>
	<li>Set the parameters for the STED depletion lasers. 
	NOTE: This will depend on the specific microscope being used and the lasers that it is equipped with. Some recommended settings are white light (WLL) 70%; 592 nm laser 80%; 660 nm laser 80%.</li>
	<li>Activate the STED settings and align the STED laser beams using the 100X objective.</li>
	<li>Using the eyepiece, focus the sample and select a cell with moderate GFP expression. 
	NOTE: Low GFP expression will not be visible because STED reduces the emission intensity. Conversely, high expression of CLIP-170-GFP induces the spontaneous formation of large clusters, which does not reflect the normal microtubule plus-end protein complex morphology and distribution. Selecting a cell with moderate GFP expression is essential for accurately imaging the cytoskeletal structures at the antigen contact site.</li>
	<li>Zoom into the selected cell and choose the region of interest to be imaged. Adjust the focus so that the x-y plane of the B cell that is closest to the coverslip is in focus.
	<ol>
		<li>To do this, move the objective progressively closer to the coverslip so that the B cell cytoskeletal structures come into focus and then go out of focus. Then gradually move the objective in the opposite direction until the B cell cytoskeletal structures first come back into focus.</li>
	</ol>
	</li>
	<li>Optimize the settings including the laser power, excitation beam, and detector range. Start with the settings recommended by the manufacturer&#39;s instructions. Then make adjustments as needed to optimize the signal for the samples. Image all samples to be compared to each other with the same settings.</li>
	<li>Under the &#34;acquire&#34; tab of the software, set the image acquisition to sequential frame acquisition in the lower left &#34;Sequential Scan&#34; panel by checking the option &#34;between frames&#34;.</li>
	<li>Set the acquisition sequence. 
	Note: We acquire the GFP fluorescence first in order to avoid degradation of the GFP signal. 
	Depending on the combination of fluorophores that are used in addition to GFP, imaging the longer wavelength fluorescence signal first may yield better results. However, the order in which the fluorophores or fluorescent proteins are subjected to stimulated emission depletion and imaged should be optimized to obtain the strongest fluorescence signals and best resolution.</li>
	<li>Select and set the STED laser power for each fluorophore under the &#34;Acquire&#34; tab of the software, and use the slider bar for either the 592-nm or the 660-nm STED laser to adjust the laser power. Adjust the power for the 592-nm depletion laser for GFP and for Alexa Fluor 532. Adjust the power for the 660-nm depletion laser for Alexa Fluor 568.</li>
	<li>To increase the resolution and reduce the background signal, go to the &#34;Acquisition&#34; settings under the &#34;Acquire&#34; tab of the software, increase the line and/or frame averaging by selecting a value greater than 1 using the drop-down menu for each option.</li>
	<li>Also, acquire the fluorescent signal using time-gated STED by checking the gating option for the fluorophore under the &#34;Acquire&#34; tab, and specifying values for time-gating (see note below). Increase the STED laser power to enhance the resolution, though this will also increase photobleaching of the fluorophores. 
	NOTE: The gating time will need to be optimized for the microscope, sample, and fluorophores being used. A time gating of 0.3 to 6 ns is recommended. After fixation, GFP is particularly sensitive to high laser power. To reduce the photobleaching of GFP, time gating should be applied and the STED laser power should be reduced.</li>
	<li>To capture 3D STED images, use the slider to change the point spread function (PSF) to &#34;3D STED&#34;.</li>
	<li>Manually acquire multiple images to ensure reproducibility. Deconvolve the images (see Figure 1) using a deconvolution software package41(see Table of Materials).</li>
</ol>

<ol>
	<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=The+cytoskeleton+coordinates+the+early+events+of+B-cell+activation.">The cytoskeleton coordinates the early events of B-cell activation.</a> Cold Spring Harb Perspect Biol. 3 (2), a002360 (2011).</li><li>Batista, F. D., Treanor, B., 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=Visualizing+a+role+for+the+actin+cytoskeleton+in+the+regulation+of+B-cell+activation.">Visualizing a role for the actin cytoskeleton in the regulation of B-cell activation.</a> Immunol Rev. 237 (1), 191-204 (2010).</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.">Early events in B cell activation.</a> Annu Rev Immunol. 28, 185-210 (2010).</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> Nat Rev Immunol. 9 (1), 15-27 (2009).</li><li>Kumari, 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=T+cell+antigen+receptor+activation+and+actin+cytoskeleton+remodeling.">T cell antigen receptor activation and actin cytoskeleton remodeling.</a> Biochim Biophys Acta. 1838 (2), 546-556 (2014).</li><li>Dustin, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+Cell-Cell+Interaction+Contacts:+Synapses+and+Kinapses.">Visualization of Cell-Cell Interaction Contacts: Synapses and Kinapses.</a> Self Nonself. 2 (2), 85-97 (2011).</li><li>Dustin, M. L., Chakraborty, A. K., Shaw, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+Structure+and+Function+of+the+Immunological+Synapse.">Understanding the Structure and Function of the Immunological Synapse.</a> Cold Spring Harb Perspect Biol. 2 (10), a002311 (2010).</li><li>Dustin, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modular+design+of+immunological+synapses+and+kinapses.">Modular design of immunological synapses and kinapses.</a> Cold Spring Harb Perspect Biol. 1 (1), a002873 (2009).</li><li>Dustin, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+cell-cell+interaction+contacts-synapses+and+kinapses.">Visualization of cell-cell interaction contacts-synapses and kinapses.</a> Adv Exp Med Biol. 640, 164-182 (2008).</li><li>Dustin, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hunter+to+gatherer+and+back:+immunological+synapses+and+kinapses+as+variations+on+the+theme+of+amoeboid+locomotion.">Hunter to gatherer and back: immunological synapses and kinapses as variations on the theme of amoeboid locomotion.</a> Annu Rev Cell Dev Biol. 24, 577-596 (2008).</li><li>Dustin, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-cell+activation+through+immunological+synapses+and+kinapses.">T-cell activation through immunological synapses and kinapses.</a> Immunol Rev. 221, 77-89 (2008).</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. 285 (5425), 221-227 (1999).</li><li>Monks, C. R., 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=Three-dimensional+segregation+of+supramolecular+activation+clusters+in+T+cells.">Three-dimensional segregation of supramolecular activation clusters in T cells.</a> Nature. 395 (6697), 82-86 (1998).</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=How+B+cells+capture,+process+and+present+antigens:+a+crucial+role+for+cell+polarity.">How B cells capture, process and present antigens: a crucial role for cell polarity.</a> Nat Rev Immunol. 13 (7), 475-486 (2013).</li><li>Mattila, P. K., Batista, F. D., Treanor, B. <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+the+actin+cytoskeleton+mediates+receptor+cross+talk:+An+emerging+concept+in+tuning+receptor+signaling.">Dynamics of the actin cytoskeleton mediates receptor cross talk: An emerging concept in tuning receptor signaling.</a> J Cell Biol. 212 (3), 267-280 (2016).</li><li>Kuhn, J. R., Poenie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+polarization+of+the+microtubule+cytoskeleton+during+CTL-mediated+killing.">Dynamic polarization of the microtubule cytoskeleton during CTL-mediated killing.</a> Immunity. 16 (1), 111-121 (2002).</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-cofilin-1+pathway+coordinates+actin+reorganization+and+MTOC+polarization+at+the+B+cell+immune+synapse.">The Rap1-cofilin-1 pathway coordinates actin reorganization and MTOC polarization at the B cell immune synapse.</a> J Cell Sci. 130 (6), 1094-1109 (2017).</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. 34 (6), 905-918 (2011).</li><li>Nath, 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=Dynein+Separately+Partners+with+NDE1+and+Dynactin+To+Orchestrate+T+Cell+Focused+Secretion.">Dynein Separately Partners with NDE1 and Dynactin To Orchestrate T Cell Focused Secretion.</a> J Immunol. 197 (6), 2090-2101 (2016).</li><li>Combs, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recruitment+of+dynein+to+the+Jurkat+immunological+synapse.">Recruitment of dynein to the Jurkat immunological synapse.</a> Proc Natl Acad Sci U S A. 103 (40), 14883-14888 (2006).</li><li>Akhmanova, A., Steinmetz, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+microtubule+organization+and+dynamics:+two+ends+in+the+limelight.">Control of microtubule organization and dynamics: two ends in the limelight.</a> Nat Rev Mol Cell Biol. 16 (12), 711-726 (2015).</li><li>Akhmanova, A., Steinmetz, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule++TIPs+at+a+glance.">Microtubule +TIPs at a glance.</a> J Cell Sci. 123 (20), 3415-3419 (2010).</li><li>Fukata, 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=Rac1+and+Cdc42+capture+microtubules+through+IQGAP1+and+CLIP-170.">Rac1 and Cdc42 capture microtubules through IQGAP1 and CLIP-170.</a> Cell. 109 (7), 873-885 (2002).</li><li>Wegel, E., 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=Imaging+cellular+structures+in+super-resolution+with+SIM,+STED+and+Localisation+Microscopy:+A+practical+comparison.">Imaging cellular structures in super-resolution with SIM, STED and Localisation Microscopy: A practical comparison.</a> Sci Rep. 6, 27290 (2016).</li><li>Huang, B., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+fluorescence+microscopy.">Super-resolution fluorescence microscopy.</a> Annu Rev Biochem. 78, 993-1016 (2009).</li><li>Mace, E. M., Orange, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+channel+STED+nanoscopy+of+lytic+granules+on+actin+filaments+in+natural+killer+cells.">Dual channel STED nanoscopy of lytic granules on actin filaments in natural killer cells.</a> Commun Integr Biol. 5 (2), 184-186 (2012).</li><li>Ritter, A. 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=Actin+depletion+initiates+events+leading+to+granule+secretion+at+the+immunological+synapse.">Actin depletion initiates events leading to granule secretion at the immunological synapse.</a> Immunity. 42 (5), 864-876 (2015).</li><li>Tamarit, B., 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=Membrane+microdomains+and+cytoskeleton+organization+shape+and+regulate+the+IL-7+receptor+signalosome+in+human+CD4+T-cells.">Membrane microdomains and cytoskeleton organization shape and regulate the IL-7 receptor signalosome in human CD4 T-cells.</a> J Biol Chem. 288 (12), 8691-8701 (2013).</li><li>Brown, A. 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=Super-resolution+imaging+of+remodeled+synaptic+actin+reveals+different+synergies+between+NK+cell+receptors+and+integrins.">Super-resolution imaging of remodeled synaptic actin reveals different synergies between NK cell receptors and integrins.</a> Blood. 120 (18), 3729-3740 (2012).</li><li>Dupre, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+Lymphocyte+Migration:+An+Action+Movie+Starring+the+Actin+and+Associated+Actors.">T Lymphocyte Migration: An Action Movie Starring the Actin and Associated Actors.</a> Front Immunol. 6, 586 (2015).</li><li>Rak, G. 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=Natural+killer+cell+lytic+granule+secretion+occurs+through+a+pervasive+actin+network+at+the+immune+synapse.">Natural killer cell lytic granule secretion occurs through a pervasive actin network at the immune synapse.</a> PLoS Biol. 9 (9), e1001151 (2011).</li><li>Lin, K. B., 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+Rap+GTPases+regulate+the+migration,+invasiveness+and+in+vivo+dissemination+of+B-cell+lymphomas.">The Rap GTPases regulate the migration, invasiveness and in vivo dissemination of B-cell lymphomas.</a> Oncogene. 29 (4), 608-615 (2010).</li><li>Lin, K. B., 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+rap+GTPases+regulate+B+cell+morphology,+immune-synapse+formation,+and+signaling+by+particulate+B+cell+receptor+ligands.">The rap GTPases regulate B cell morphology, immune-synapse formation, and signaling by particulate B cell receptor ligands.</a> Immunity. 28 (1), 75-87 (2008).</li><li>McLeod, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Rap+GTPases+regulate+integrin-mediated+adhesion,+cell+spreading,+actin+polymerization,+and+Pyk2+tyrosine+phosphorylation+in+B+lymphocytes.">The Rap GTPases regulate integrin-mediated adhesion, cell spreading, actin polymerization, and Pyk2 tyrosine phosphorylation in B lymphocytes.</a> J. Biol. Chem. 279 (13), 12009-12019 (2004).</li><li>Christian, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+Rap+GTPases+in+B+lymphocytes+modulates+B+cell+antigen+receptor-induced+activation+of+Akt+but+has+no+effect+on+MAPK+activation.">Activation of the Rap GTPases in B lymphocytes modulates B cell antigen receptor-induced activation of Akt but has no effect on MAPK activation.</a> J Biol Chem. 278 (43), 41756-41767 (2003).</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>Treanor, B., 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+cortical+actin+remodeling+by+ERM+proteins+controls+BCR+microcluster+organization+and+integrity.">Dynamic cortical actin remodeling by ERM proteins controls BCR microcluster organization and integrity.</a> J Exp Med. 208 (5), 1055-1068 (2011).</li><li>Mattila, P. K., 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+and+tetraspanin+networks+organize+receptor+nanoclusters+to+regulate+B+cell+receptor-mediated+signaling.">The actin and tetraspanin networks organize receptor nanoclusters to regulate B cell receptor-mediated signaling.</a> Immunity. 38 (3), 461-474 (2013).</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>Mace, E. M., Orange, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+the+immunological+synapse+by+dual+color+time-gated+stimulated+emission+depletion+(STED)+nanoscopy.">Visualization of the immunological synapse by dual color time-gated stimulated emission depletion (STED) nanoscopy.</a> J Vis Exp. (85), (2014).</li><li>Russ, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Image Processing Handbook, Sixth Edition. , (2011).</li><li>Stinchcombe, 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=Centrosome+polarization+delivers+secretory+granules+to+the+immunological+synapse.">Centrosome polarization delivers secretory granules to the immunological synapse.</a> Nature. 443 (7110), 462-465 (2006).</li><li>Vicidomini, 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=Sharper+low-power+STED+nanoscopy+by+time+gating.">Sharper low-power STED nanoscopy by time gating.</a> Nat Methods. 8 (7), 571-573 (2011).</li></ol>

The authors have nothing to disclose.

Detailed images of cytoskeletal structures can be obtained using STED super-resolution microscopy, which can theoretically achieve a resolution of 50 nm, compared to conventional confocal microscopy, for which the diffraction-limited resolution is ~200 nm24. The ability to resolve finer structures is further enhanced by using deconvolution software to calculate the most likely position of the original light source from the observed &#34;blurred&#34; fluorescence signal. This protocol describes met...

When B cells bind to polarized arrays of antigens (e.g., displayed on the surface of antigen-presenting cells (APCs)), the resulting B-cell receptor (BCR) signaling drives the formation of a classic immune synapse structure, which was first described in T cells1,2,3,4,5,6,7,8,9,10,11,12,13. Initially, microclusters of antigen-bound BCRs form at the periphery of the B cell:APC contact site. These microclusters then move towards the center of the antigen contact site, where they coalesce into a central supramolecular activation cluster (cSMAC) that forms the core of the immune synapse. Immune synapse formation optimizes BCR signaling and facilitates BCR-mediated antigen extraction from the APC membrane14. This antigen acquisition, which is followed by BCR-mediated antigen internalization and subsequent antigen processing, allows B cells to present peptide:MHC II complexes to T cells and elicit T cell help14. Because immune synapse formation promotes B cell activation, elucidating the mechanisms that establish this functional pattern of receptor organization can provide new insights into how humoral immune responses are initiated and regulated.
Reorganization of both the actin and microtubule cytoskeletons is essential for immune synapse formation. Localized BCR signaling stimulated by a spatially-polarized array of antigens induces rapid and dramatic remodeling of the actin cytoskeleton1,15. The formation of dendritic actin structures at the periphery of the B cell exerts pushing forces on the plasma membrane and promotes B cell spreading. This allows the B cell to scan a greater area of the antigen-bearing surface, and increases the number of BCRs that bind antigen and activate BCR signaling pathways. At the same time, the MTOC and the microtubule network are reoriented towards the site of antigen contact. As the MTOC approaches the antigen contact site, microtubules emanating from the MTOC extend along the inner face of the plasma membrane at the interface between the B cell and the antigen-bearing surface16,17. These juxtamembrane microtubules can then act as tracks for the dynein-mediated centripetal movement of antigen-bound BCR microclusters18, leading to the formation of a cSMAC.
The reorientation and polarization of the MTOC towards the immune synapse requires intact actin and microtubule cytoskeletons, and often depends on interactions between the cortical actin network and microtubules16,17,19,20. Cortical actin-binding proteins, such as IQGAP1, can capture microtubules by interacting with protein complexes that decorate the microtubule plus-ends21. These dynamic complexes of plus-end binding proteins include EB1 and CLIP-170, which are collectively referred to as microtubule plus-end tracking proteins (+TIPs)21,22. +TIPs at the ends of microtubules can bind to proteins that are associated with either the plasma membrane or the cortical actin cytoskeleton. This allows force-generating mechanisms (e.g., the minus-end directed movement of cortically-anchored dynein along microtubules) to exert pulling forces on microtubules, and thereby reposition the MTOC. CLIP-170 can bind to the actin-associated scaffolding protein IQGAP123, and we have shown that both of these proteins are required for BCR-induced MTOC polarization towards the immune synapse17. This IQGAP1-CLIP-170 interaction may play a key role in coordinating the remodeling of the actin cytoskeleton with the repositioning of the microtubule network at the B-cell immune synapse.
Conventional fluorescence microscopy has revealed the dramatic reorganization of the actin and microtubule cytoskeletons during B-cell immune synapse formation2. However, this approach cannot resolve small cellular structures in detail due to the diffraction limit of light, which, according to Abbe&#39;s law, is dependent on the wavelength of light used to illuminate the sample and the aperture of the objective24. This diffraction limit constrains the resolution of conventional light microscopes to 200-300 nm in the lateral direction and 500-700 nm in the axial direction25. Therefore, smaller subcellular structures, as well as the fine details of the actin and microtubule cytoskeletons, could only be observed using electron microscopy. Electron microscopy imaging of the cytoskeleton is time consuming, requires harsh sample fixation and preparation protocols that can alter biological structures, and is limited to antibody-mediated detection. The ability to immunostain and simultaneously image multiple proteins or cellular structures is a substantial advantage of fluorescence microscopy. Moreover, expressing fluorescent fusion proteins in cells enables real-time imaging and is useful when effective antibodies for immunostaining the protein of interest are not available.
Recent technological advances in super-resolution microscopy have overcome the diffraction limits of light and allowed the visualization of nanoscale cellular structures24. One such super-resolution microscopy technique is called stimulated emission depletion (STED) microscopy. STED employs two lasers, where one laser excites the fluorophore and a second laser with a donut-shaped pattern selectively suppresses the fluorescence emission around the fluorophore. This reduces the point-spread function (apparent area) of a single fluorescent particle and provides a sub-diffraction limit fluorescent image25,26. Ground-state depletion microscopy also employs fluorescence-based techniques to acquire super-resolution images. However, the image acquisition and reconstruction times are long, there are only a limited number of fluorophores that can be used, and the simultaneous high-resolution imaging of multiple cytoskeletal components is technically challenging because maintaining actin and microtubule structures requires different fixation procedures. Therefore, STED has multiple advantages over electron microscopy and other super-resolution microscopy approaches in that it offers rapid image acquisition, has minimal post-processing requirements, and employs the same fluorophores and staining techniques that are used for conventional fluorescence microscopy of fixed samples26.
Super-resolution microscopy has now been used to visualize actin structures at the immune synapse in natural killer (NK) cells and T cells26,27,28,29,30,31. However, super-resolution imaging of the microtubule cytoskeleton, as well as the coordinated reorganization of the actin and microtubule cytoskeletons during immune synapse formation, has only recently been reported17. We used STED microscopy to image B cells that had been allowed to spread on coverslips coated with anti-immunoglobulin (anti-Ig) antibodies, which stimulate BCR signaling and initiate cytoskeleton reorganization. When plated on immobilized anti-Ig antibodies, B cells undergo dramatic actin-dependent spreading, which recapitulates the initial events during immune synapse formation. Importantly, STED microscopy revealed the fine details of the dendritic ring of F-actin that forms at the periphery of the immune synapse and showed that the MTOC, as well as the microtubules attached to it, had moved close to the antigen contact site17. These microtubules extended outward towards the peripheral ring of F-actin. Moreover, multi-color STED imaging of various combinations of F-actin, tubulin, IQGAP1, and GFP-tagged CLIP-170 +TIPs showed that microtubule plus-ends marked by CLIP-170-GFP were closely associated with the peripheral actin meshwork and with IQGAP1, a cortical capture protein17.
Here, we present a detailed protocol for imaging the actin and microtubule cytoskeletons at the immune synapse using STED microscopy. These methods have been optimized using the A20 murine B cell line, which has been widely employed for studying BCR signaling and immune synapse formation17,32,33,34,35,36,37,38,39. Because commercial antibodies to CLIP-170 did not work well for immunostaining in previous experiments, we describe in detail the expression of GFP-tagged CLIP-170 in A20 cells, along with staining protocols for simultaneously visualizing up to three cytoskeletal components or cytoskeleton-associated proteins. Methods for using STED microscopy to image actin at NK cell immune synapses have been described previously40. Here, we extend this to acquiring multi-color super-resolution images of both the actin and microtubule cytoskeletons in B cells.
A critical consideration for super-resolution microscopy is using the appropriate fixation procedures for maintaining cellular structures and preventing damage to fluorescent proteins. The fixation and staining methods presented herein have been optimized to retain GFP fluorescence and provide high-resolution imaging of the actin and microtubule networks. When expressing fluorescent proteins, it should be noted that B cells are usually difficult to transfect. Using this protocol, 20-50% of A20 cells typically express the transfected GFP fusion protein, and among this population the levels of protein expression are variable. Nevertheless, super-resolution imaging of actin and microtubules using the procedures we describe is quite robust and high-quality images are readily obtained. Despite their small size relative to A20 cells, we show that these procedures can also be used to image the microtubule network in primary B cells that have been briefly activated with lipopolysaccharide (LPS). We have shown that LPS-activated primary B cells can be transfected with siRNAs at relatively high efficiency (i.e., such that protein depletion can be detected by immunoblotting), making them a good alternative to the use of B cell lines for some studies17.

<table><tbody><tr><td>&lt;strong&gt;Cell culture&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>A20 mouse B-lymphoma cells</td><td>ATCC</td><td>TIB-208</td><td>Murine B-cell lymphoma of Balb/c origin that expresses an IgG-containing BCR on its surface</td></tr><tr><td>RPMI-1640</td><td>Thermo Fisher Scientific</td><td>&amp;nbsp;R0883</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Thermo Fisher Scientific</td><td>12483020</td><td>Heat inactivate at 56 &lt;sup&gt;o&lt;/sup&gt;C for 30 min</td></tr><tr><td>2-mercaptoethanol</td><td>Millipore Sigma</td><td>M3148</td><td></td></tr><tr><td>Glutamine</td><td>Millipore Sigma</td><td>G5763</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Millipore Sigma</td><td>P5280</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Thermo Fisher Scientific</td><td>15140122</td><td>Liquid, 10,000 units</td></tr><tr><td>&lt;strong&gt;Additional materials for primary B cells&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>70-&amp;micro;m cell strainer</td><td>Corning</td><td>352350</td><td></td></tr><tr><td>Magnetic bead-based B cell isolation kit</td><td>Stemcell Technologies</td><td>19854</td><td>EasySep Mouse B cell Isolation kit</td></tr><tr><td>Lipopolysaccharide (LPS)</td><td>Millipore Sigma</td><td>L4391</td><td>LPS from &lt;em&gt;E. coli &lt;/em&gt;0111:B4</td></tr><tr><td>B cell-activating factor (BAFF)</td><td>R&amp;amp;D Systems</td><td>2106-BF-010</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Transfection&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Plasmid encoding CLIP-170-GFP</td><td></td><td></td><td>Gift from Dr. Kozo Kaibuchi (Nagoya Univ., Nagoya, Japan); described in Fukata &lt;em&gt;et al.&lt;/em&gt; &lt;strong&gt;Cell&lt;/strong&gt; 109:873-885, 2002</td></tr><tr><td>&lt;strong&gt;Recommended transfection reagents and equipment &lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Amaxa Nucleofection kit V</td><td>Lonza</td><td>VCA-1003</td><td>Follow the manufacturer&#039;s directions for mixing the transfection reagents with the DNA</td></tr><tr><td>Amaxa Nucleofector model 2b</td><td>Lonza</td><td>AAB-1001</td><td>Program L-013 used</td></tr><tr><td>Falcon 6-well plates, TC treated, sterile</td><td>Corning</td><td>353046</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Coating coverslips&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>18-mm diameter round #1.5 cover glasses</td><td>Thomas Scientific</td><td>1217N81</td><td>Similar product: Marienfield-Superior catalogue #117580</td></tr><tr><td>Forceps</td><td>Fisher Scientific</td><td>1381242</td><td>Dissecting extra fine splinter forceps</td></tr><tr><td>Falcon 12-well sterile tissue polystyrene tissue culture plate</td><td>Corning</td><td>353043</td><td></td></tr><tr><td>100% methanol</td><td>Fisher Scientific</td><td>A412-4</td><td></td></tr><tr><td>Sterile phosphate buffered saline (PBS) without calcium or magnesium</td><td>Thermo Fisher Scientific</td><td>10010049</td><td></td></tr><tr><td>Goat-anti-mouse IgG antibody</td><td>Jackson ImmunoResearch</td><td>115-005-008</td><td>For A20 cells</td></tr><tr><td>Goat-anti-mouse IgM antibody</td><td>Jackson ImmunoResearch</td><td>115-005-020</td><td>For primary mouse B cells</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Staining&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Paraformaldehyde (16% stock solution)</td><td>Electron Microscopy Sciences</td><td>15710</td><td>Dilute with PBS to working concentration</td></tr><tr><td>Glutaraldehyde (50% stock solution)</td><td>Millipore Sigma</td><td>340855</td><td>Dilute with PBS to working concentration</td></tr><tr><td>Triton X-100</td><td>Fisher Scientific</td><td>BP151-500</td><td></td></tr><tr><td>Saponin</td><td>Millipore Sigma</td><td>S2149</td><td></td></tr><tr><td>Bovine serum albumin (BSA), Fraction V</td><td>Millipore Sigma</td><td>10735094001</td><td></td></tr><tr><td>Rabbit anti-tubulin antibody</td><td>Abcam</td><td>ab52866</td><td>1:250 dilution recommended but should be optimized</td></tr><tr><td>Alexa Fluor 532-conjugated goat anti-rabbit IgG</td><td>Thermo Fisher Scientific</td><td>A11009</td><td>1:100 dilution recommended but should be optimized</td></tr><tr><td>Alexa Fluor 568-conjugated phalloidin</td><td>Thermo Fisher Scientific</td><td>A12380</td><td>1:100 dilution recommneded but should be optimized</td></tr><tr><td>Prolong Diamond Antifade Mountant</td><td>Thermo Fisher Scientific</td><td>P36970</td><td>Without DAPI</td></tr><tr><td>Fisherbrand Superfrost Plus Microscope Slides</td><td>Fisher Scientific</td><td>12-550-15</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Parafilm</td><td>VWR</td><td>P1150-2</td><td></td></tr><tr><td>HEPES</td><td>Millipore Sigma</td><td>H3375</td><td></td></tr><tr><td>NaCl</td><td>Fisher Scientific</td><td>BP358</td><td></td></tr><tr><td>KCl</td><td>Millipore Sigma</td><td>P9333</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Millipore Sigma</td><td>C1016</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific</td><td>S374-500</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific</td><td>M63</td><td></td></tr><tr><td>Dextrose</td><td>Fisher Scientific</td><td>D16-500</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Microscopy&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Leica SP8 TCS STED microscope</td><td>Leica</td><td></td><td></td></tr><tr><td>Huygens deconvolution software</td><td>Scientific Voume Imaging</td><td></td><td>See https://svi.nl/HuygensProducts</td></tr></tbody></table>

visualizing the actin and microtubule cytoskeletons at the b-cell immune synapse using stimulated emission depletion (sted) microscopy

B cells that bind to membrane-bound antigens (e.g., on the surface of an antigen-presenting cell) form an immune synapse, a specialized cellular structure that optimizes B-cell receptor (BCR) signaling and BCR-mediated antigen acquisition. Both the remodeling of the actin cytoskeleton and the reorientation of the microtubule network towards the antigen contact site are essential for immune synapse formation. Remodeling of the actin cytoskeleton into a dense peripheral ring of F-actin is accompanied by polarization of the microtubule-organizing center towards the immune synapse. Microtubule plus-end binding proteins, as well as cortical plus-end capture proteins mediate physical interactions between the actin and microtubule cytoskeletons, which allow them to be reorganized in a coordinated manner. Elucidating the mechanisms that control this cytoskeletal reorganization, as well as understanding how these cytoskeletal structures shape immune synapse formation and BCR signaling, can provide new insights into B cell activation. This has been aided by the development of super-resolution microscopy approaches that reveal new details of cytoskeletal network organization. We describe here a method for using stimulated emission depletion (STED) microscopy to simultaneously image actin structures, microtubules, and transfected GFP-tagged microtubule plus-end binding proteins in B cells. To model the early events in immune synapse formation, we allow B cells to spread on coverslips coated with anti-immunoglobulin (anti-Ig) antibodies, which initiate BCR signaling and cytoskeleton remodeling. We provide step-by-step protocols for expressing GFP fusion proteins in A20 B-lymphoma cells, for anti-Ig-induced cell spreading, and for subsequent cell fixation, Immunostaining, image acquisition, and image deconvolution steps. The high-resolution images obtained using these procedures allow one to simultaneously visualize actin structures, microtubules, and the microtubule plus-end binding proteins that may link these two cytoskeletal networks.

For B cells spreading on immobilized anti-Ig, STED microscopy used in conjunction with deconvolution software provides higher resolution images of cytoskeletal structures than confocal microscopy. This is evident in Figure 1, where the F-actin network was visualized using the protocol described above. A comparison of confocal and STED super-resolution images of the same sample shows that the STED images are of higher resolution and reveal more detailed struct...

Watch this Scientific Journal Video about Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion STED Microscopy at JoVE.com

Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion STED Microscopy

We present a protocol for using STED microscopy to simultaneously image actin structures, microtubules, and microtubule plus-end binding proteins in B cells that have spread on coverslips coated with antibodies to the B-cell receptor, a model for the initial phase of immune synapse formation.

Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion (STED) Microscopy

B cells that bind to membrane-bound antigens (e.g., on the surface of an antigen-presenting cell) form an immune synapse, a specialized cellular structure ...

visualizing-actin-microtubule-cytoskeletons-at-b-cell-immune-synapse

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

13.6K Views.  University of British Columbia. We present a protocol for using STED microscopy to simultaneously image actin structures, microtubules, and microtubule plus-end binding proteins in B cells that have spread on coverslips coated with antibodies to the B-cell receptor, a model for the initial phase of immune synapse formation.

Video: Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion STED Microscopy

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion (STED) Nanoscopy

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic actin reorganization is critical to the function of NK cells and the formation of the IS. Imaging of F-actin at the synapse has traditionally utilized confocal microscopy, however the diffraction limit of light restricts resolution of fluorescence microscopy, including confocal, to approximately 200 nm. Recent advances in imaging technology have enabled the development of subdiffraction limited super-resolution imaging. In order to visualize F-actin architecture at the IS we recapitulate the NK cell cytotoxic synapse by adhering NK cells to activating receptor on glass. We then image proteins of interest using two-color stimulated emission depletion microscopy (STED). This results in &#60;80 nm resolution at the synapse. Herein we describe the steps of sample preparation and the acquisition of images using dual color STED nanoscopy to visualize F-actin at the NK IS. We also illustrate optimization of sample acquisition using Leica SP8 software and time-gated STED. Finally, we utilize Huygens software for post-processing deconvolution of images.

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion STED Nanoscopy

Here we illustrate the protocol for imaging by two-color STED nanoscopy the cytotoxic immune synapse of NK cells recapitulated on glass. Using this method we obtain sub-100 nm resolution of synapse proteins and the cytoskeleton.

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic ...

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological synapses&#39;. Within these intimate cell-cell interfaces discrete sub-cellular clusters of MHC/Ag-TCR, F-actin, adhesion and signaling molecules form and remodel rapidly. These dynamics are thought to be critical determinants of both the efficiency and quality of the immune responses that develop and therefore of protective versus pathologic immunity. Current understanding of immunological synapses with physiologic APCs is limited by the inadequacy of the obtainable imaging resolution. Though artificial substrate models (e.g., planar lipid bilayers) offer excellent resolution and have been extremely valuable tools, they are inherently non-physiologic and oversimplified. Vascular and lymphatic endothelial cells have emerged as an important peripheral tissue (or stromal) compartment of &#39;semi-professional APCs&#39;. These APCs (which express most of the molecular machinery of professional APCs) have the unique feature of forming virtually planar cell surface and are readily transfectable (e.g., with fluorescent protein reporters). Herein a basic approach to implement endothelial cells as a novel and physiologic &#39;planar cellular APC model&#39; for improved imaging and interrogation of fundamental antigenic signaling processes will be described.

Adaptive immunity is controlled by dynamic 'immunological synapses' formed between T cells and antigen presenting cells. This protocol describes methods for investigating endothelial cells both as understudied physiologic APCs and as a novel type of 'planar cellular APC model'.

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological ...

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell interaction known as the immunological synapse. F-actin is also speculated to play a role in regulating molecular distributions at the membrane of cells including sub-membranous vesicle dynamics and protein clustering. While standard light microscope techniques allow generalized and diffraction-limited observations to be made, many cellular and molecular events including clustering and molecular flow occur in populations at length-scales far below the resolving power of standard light microscopy. By combining total internal reflection fluorescence with the super resolution imaging method structured illumination microscopy, the two-dimensional molecular flow of F-actin at the immune synapse of T cells was recorded. Spatio-temporal image correlation spectroscopy (STICS) was then applied, which generates quantifiable results in the form of velocity histograms and vector maps representing flow directionality and magnitude. This protocol describes the combination of super-resolution imaging and STICS techniques to generate flow vectors at sub-diffraction levels of detail. This technique was used to confirm an actin flow that is symmetrically retrograde and centripetal throughout the periphery of T cells upon synapse formation.

To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy.

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell ...

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) Microscopy

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and regulated by different suites of binding proteins unique for each polymer. Many studies now demonstrate that the dynamics of both cytoskeletal polymers are intertwined and that this crosstalk is required for most cellular behaviors. A number of proteins involved in actin-microtubule interactions have already been identified (i.e., Tau, MACF, GAS, formins, and more) and are well characterized with regard to either actin or microtubules alone. However, relatively few studies showed assays of actin-microtubule coordination with dynamic versions of both polymers. This may occlude emergent linking mechanisms between actin and microtubules. Here, a total internal reflection fluorescence (TIRF) microscopy-based in vitro reconstitution technique permits the visualization of both actin and microtubule dynamics from the one biochemical reaction. This technique preserves the polymerization dynamics of either actin filament or microtubules individually or in the presence of the other polymer. Commercially available Tau protein is used to demonstrate how actin-microtubule behaviors change in the presence of a classic cytoskeletal crosslinking protein. This method can provide reliable functional and mechanistic insights into how individual regulatory proteins coordinate actin-microtubule dynamics at a resolution of single filaments or higher-order complexes.

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence TIRF Microscopy

This protocol is a guide for visualizing dynamic actin and microtubules using an in vitro total internal fluorescence (TIRF) microscopy assay.

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and ...

Biochemistry

Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins towards the immune synapse to assure a stable binding and signal exchange. Classical confocal, TIRF, or super-resolution microscopy have been used to study the immune synapse. Since these methods require manual image acquisition and time-consuming quantification, the imaging of rare events is challenging. Here, we describe a workflow that enables the morphological analysis of tens of thousands of cells. Immune synapses are induced between primary human T cells in pan-leukocyte preparations and Staphylococcus aureus enterotoxin B (SEB)-loaded Raji cells as APCs. Image acquisition is performed with imaging flow cytometry, also called In-Flow microscopy, which combines features of a flow cytometer and a fluorescence microscope. A complete gating strategy for identifying T cell/APC couples and analyzing the immune synapses is provided. As this workflow allows the analysis of immune synapses in unpurified pan-leukocyte preparations and hence requires only a small volume of blood (i.e., 1 mL), it can be applied to samples from patients. Importantly, several samples can be prepared, measured, and analyzed in parallel.

Here, we describe a complete workflow for the qualitative and quantitative analysis of immune synapses between primary human T cells and antigen-presenting cells. The method is based on imaging flow cytometry, which allows the acquisition and evaluation of several thousand cell images within a relatively short period of time.

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins ...

Traction Force Microscopy to Study B Lymphocyte Activation

Traction force microscopy (TFM) enables the measurement of forces produced by a cell on a substrate. This technique infers traction force measurements from an experimentally observed displacement field produced by a cell pulling on an elastic substrate. Here, we adapted TFM to investigate the spatial and temporal structure of the force field exerted by B cells when activated by antigen engagement of the B cell receptor. Gel rigidity, bead density, and protein functionalization must be optimized for the study of relatively small cells (~ 6 &#181;m) that interact with, and respond specifically to ligands for cell surface receptors.

Here we present a protocol used to perform traction force microscopy experiments on B cells. We describe the preparation of soft polyacrylamide gels and their functionalization, as well as data acquisition at the microscope and a summary of data analysis.

Traction force microscopy (TFM) enables the measurement of forces produced by a cell on a substrate. This technique infers traction force measurements ...

Bioengineering

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.

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 ...

Imaging the Human Immunological Synapse

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) and an effector helper T lymphocyte (Th) cell, and to record the images corresponding to the first stages of the IS formation and the subsequent trafficking events (occurring both in the APC and in the Th cell). These events will eventually lead to polarized secretion at the IS. In this protocol, Jurkat cells challenged with Staphylococcus enterotoxin E (SEE)-pulsed Raji cells as a cell synapse model was used, because of the closeness of this experimental system to the biological reality (Th cell-APC synaptic conjugates). The approach presented here involves cell-to-cell conjugation, time-lapse acquisition, wide-field fluorescence microscopy (WFFM) followed by image processing (post-acquisition deconvolution). This improves the signal-to-noise ratio (SNR) of the images, enhances the temporal resolution, allows the synchronized acquisition of several fluorochromes in emerging synaptic conjugates and decreases fluorescence bleaching. In addition, the protocol is well matched with the end point cell fixation protocols (paraformaldehyde, acetone or methanol), which would allow further immunofluorescence staining and analyses. This protocol is also compatible with laser scanning confocal microscopy (LSCM) and other state-of-the-art microscopy techniques. As a main caveat, only those T cell-APC boundaries (called IS interfaces) that were at the right 90&#176; angle to the focus plane along the Z-axis could be properly imaged and analyzed. Other experimental models exist that simplify imaging in the Z dimension and the following image analyses, but these approaches do not emulate the complex, irregular surface of an APC, and may promote non-physiological interactions in the IS. Thus, the experimental approach used here is suitable to reproduce and to confront some biological complexities occurring at the IS.

This protocol images both the immunological synapse formation and the subsequent polarized secretory traffic towards the immunological synapse. Cellular conjugates were formed between a superantigen-pulsed Raji cell (acting as an antigen-presenting cell) and a Jurkat clone (acting as an effector helper T lymphocyte).

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) ...

Assessing Immunological Synapse Topology through Live-Cell Imaging

Source: Martinelli, R. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/53288">An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics.</a>&#160;J. Vis. Exp. (2015)
This video demonstrates live-cell imaging of immunological synapse topology, investigating the interactions between T lymphocytes and epithelial cells carrying fluorescent antigens. Fluorescence microscopy reveals red cytoplasm displacement and yellow membrane rings in endothelial cells, indicating the formation of immunological synapses.

Ocena topologii synaps immunologicznych za pomocą obrazowania żywych komórek

1. Preparing Human CD4+&#160;Th1 Effector/Memory T Cells

	Apply a tourniquet to the arm of the donor, wipe the vein with alcohol, and insert a needle. ...