Integration of the movement of signaling microclusters with cellular motility in immunological synapses.

Mice CD8 + OT1 +

TCR-transgenic mice, which recognize the SIINFEKL peptide of ovalbumin bound to H-2K(b) 38 , were obtained from Taconic, and then bred in-house. The floxed-MyH9 mice, described previously 30 , were crossed with OT1 + mice. Mice were housed and bred at UCSF according to Laboratory Animal Resource Center guidelines. Protocols were approved by the Institutional Animal Care and Use Committee of the University of California. Cells OT1 + T cell blasts were prepared from the lymph nodes of OT1 + mice. Cells were retrovirally transduced with CD3ζ-GFP or Lifeact-GFP Phoenix cell supernatants as described previously 39 . Conditional myosin II-deficient OT1 + T cell blasts were generated by transducing proliferating OT1 + MyH9 flox/flox T cells with a Cre-GFP retroviral supernatant 30 . Blasts were used 4–6 days after stimulation. On the day of imaging, live T cells were collected, stained as required for experiments, washed and then held on ice in complete RPMI without phenol red indicator until used for imaging. To label surface TCRs, 2×10 6 cells were stained for 30 min on ice with 1 μg Alexa Fluor 568-labeled H57-597 anti-TCRβ in 0.1 ml complete RPMI without indicator. To inhibit actin depolymerization, stained, washed cells on ice were treated with 25–100 μM jasplakinolide. After 15 min, cells were transferred to a pre-warmed bilayer well containing jasplakinolide at the same concentration used to treat the cells. To inhibit actin polymerization, cells were incubated with 10 μM cytochalasin D for 30 min at 37 C. To inhibit myosin II activity, 50 μM (−)-blebbistatin was added to cells for 30 min at 22 C before addition to a well pre-loaded with blebbistatin. Blebbistatin was loaded as a 100 μM racemic mixture of (+, inactive) and (−, active) enantiomers. DMSO vehicle was used as a control for drug treatments.

Reagents

H57-597 anti-TCRβ was purchased from Bio-X-Cell and conjugated to Alexa Fluor 568 at the UCSF hybridoma facility. The dodecahistidine-tagged extracellular domain of ICAM1 (his-ICAM) was purified from the supernatant of High Five cells transfected using a baculovirus expression system. The protein was purified using nickel-affinity resin, followed by MonoQ, then Superdex FPLC. His-ICAM was labeled with Alexa Fluor 488 using Alexa Fluor 488 succinimidyl ester. Biotinylated H-2K(b) loaded with SIINFEKL was purchased from Beckman Coulter, or obtained from the NIH Tetramer Facility, and used in monomeric form. Lipid Bilayers Phospholipid mixtures consisting of 96.5% POPC, 2% DGS-NTA (Ni), 1% Biotinyl-Cap-PE and 0.5% PEG5,000-PE were mixed in a round bottom flask and dried. The following day, liposomes were prepared from the rehydrated lipid cakes by extrusion through 100 nm pore-size filters using a LiposoFast extruder (Avestin). To setup lipid bilayers, liposomes dilutions were applied to a rigourously cleaned LabTek II chambered coverglass (Nalge Nunc). Excess liposomes were rinsed away, and bilayers then blocked in 1% bovine serum albumin in PBS (PBS-BSA). Streptavidin was loaded in PBS-BSA, and then the excess streptavidin washed away. His-ICAM and biotinylated pMHC were added to bilayers at loading concentrations of 2.5×10 2 –2.5×10 7 fg/ml biotinylated pMHC and 62.5–500 ng/ml his-ICAM. After loading proteins, bilayers were rinsed, inhibitors added if needed, and pre-warmed before applying cells. To generate standardized bilayers, we measured the concentrations of his-ICAM and biotinylated pMHC on bilayers relative to cultured BMDCs pulsed with 100 ng/ml SIINFEKL peptide. Lipid bilayers were setup on silica microspheres (Bangs Labs) by the same procedure used for coverslip supported bilayers. Microsphere bilayer standards and BMDCs were stained for ICAM (YN1/1.7.4) and SIINFEKL:H-2K(b) (25-D1.16), and analyzed by flow cytometry. Except where indicated, stimulating bilayers refers to a bilayer loaded with his-ICAM and pMHC under conditions that generate similar protein presentation as compared to peptide-pulsed BMDCs.

Microscopy A Zeiss Axiovert 200-M equipped with Laser

TIRF slider was used to perform all imaging (Zeiss). For TIRF microscopy, a 1.45 NA, 100x Plan-Fluar or a 1.46 NA, 100x PlanApo objective lens were used. A DG-4 (Sutter Instruments) was used to provide epifluorescent illumination for fura-2 ratiometric imaging and CellTracker Orange imaging. A 1.3 NA, 40x PlanFluar objective was used for imaging cell movements in widefield. In two-color TIRF imaging, a DV2, two-channel simultaneous imaging system (Photometrics) with a 560 nm long pass dichroic filter and 525/50 nm and 605/70 nm bandpass emission filters was used to split the camera field into two image channels for simultaneous GFP and Alexa Fluor 568 imaging. Images were collected by one of two cameras: an Evolve emCCD (Photometrics) or a Stanford Photonics XR-10MegaZ iCCD camera (Stanford Photonics). For TIRF image sequences acquired using the iCCD camera, QED InVivo (Media Cybernetics) software was used. For TIRF, FRAP and Fura ratiometric images acquired by the emCCD camera, Metamorph software was used (Universal Imaging). To image OT1 + T cells interacting with the bilayers, 10 5 cells in 0.1 ml of complete RPMI without Phenol Red indicator were added to the 0.5 ml PBS volume overlaying the bilayer. For Fura ratiometric image time-lapse sequences, acquisition was started as soon as the first cells tethered to the bilayer (typically within 1 minute of addition of cells). Fura-2 component images, consisting of 340/10 nm or 380/10 nm excitation with emission recorded at 520/20 nm, were collected with 33–66 msec exposures at 15 s intervals for 20 min. For TIRF image time-lapses, cells undergoing initial spreading onto bilayers were located, and TIRF images acquired at 1 or 2 s intervals using 33–100 ms exposure lengths for 3–5 min. Cells were imaged until all cells had bound to the bilayers (typically 10–15 min) or, when imaging jasplakinolide or blebbistatin treated cells, for 5 min after delivering cells into wells.

Image Analysis

All image arithmetic operations, for example: filtering, background subtraction, masking, and division, were performed in MATLAB (The Mathworks). TCR microcluster identification was perfomed using the polynomial fitting with Gaussian weight method 40 . Assignment of identified microclusters to tracks was performed in Imaris (Andor) by transferring the microcluster data using the ImarisXT MATLAB interface. Further track analyses, such as categorization of tracks by their time of formation or calculation of movement vectors, was performed after transferring the assembled tracks to data structures in MATLAB. Cell tracking was performed in Imaris using Fura-2 ratiometric images calculated and masked in MATLAB. Conversion of fura-2 ratiometric intensities to calcium concentrations was performed as described previously 41 . Detailed descriptions of the image processing routines can be found at the Nature Protocol Exchange. Statistical analyses were performed in Prism (GraphPad Software). The Mann-Whitney U test or Student’s t test, where appropriate, were used to compare samples. For comparing multiple groups, 1-way ANOVA (α = 0.05) was used with Dunnett’s post-test. The tests applied, resulting p-values, and sample numbers are reported in figure legends.

Supplementary Material Movie 1 Supplementary Figure 1. Generation of high mobility standardized lipid bilayers. The cushioned bilayer system generated consistent bilayers that supported high lateral mobility of ligated proteins. ( a ) Time-lapse widefield FRAP images of Alexa Fluor 488–his-ICAM (top) and TRITC-streptavidin (SA, bottom) ligated to a PEG-cushioned lipid bilayer. Numbers below images are time (s). The bleach pulse occurred just before acquisition of the 6 s image. ( b ) FRAP profiles for bilayer-bound Alexa Fluor 488–his-ICAM (top) or TRITC–streptavidin (bottom). Data represents the mean of ten randomly selected regions of a bilayer. Error bars represent the standard deviation. In ( a,b ), unlabeled, biotinylated pMHC was present as a non-visualized streptavidin ligand. ( c,d ) Flow cytometric analysis of lipid bilayer standards formed on 5 μm silica microspheres and loaded with a series of concentrations of biotinylated pMHC and his-ICAM protein. Top: microsphere bilayer standards and BMDCs (loaded with 100 ng/ml SIINFEKL peptide) stained with YN1/1.7.4 anti–ICAM ( c ) and 25D1.16 anti–pMHC ( d ). Bottom: plots of input protein concentration (log scale) versus the median fluorescence intensities (from the graphs at top) for the bilayer standards and reference BMDCs. ( e ) Normalized fura-2 ratiometric intensities and displacements of OT1 + T cell blasts on bilayers loaded with pMHC at concentrations of 2.5×10 4 –2.5×10 6 fg/ml. ICAM was loaded at 250 ng/ml. ( f ) Normalized fura-2 ratiometric intensities and displacements of OT1 + T cell blasts on bilayers loaded with pMHC at 2.5×10 7 fg/ml and the indicated concentrations of ICAM (ng/ml). FRAP analyses and ligand standardization measurements were performed whenever new bilayer reagents were acquired. Supplementary Figure 2. Depletion of myosin II in OT1 + T cell blasts. Myosin II depletion by Cre-GFP expression in T cells with ‘floxed’ MyH9 alleles. ( a ) Western blotting analysis and ( b ) FACS analysis of myosin II protein levels in MyH9 flox/flox /OT1 + T cell blasts transduced with Cre-GFP compared to GFP-transduced control cells. Cells were sorted using GFP fluorescence, then analyzed. Gray profile represents the IgG isotype control. Loss of >70–80% of myosin II appeared to compromise long-term survival of cells. ( c ) Forward scatter histograms of T cells subjected to Cre-GFP mediated knockdown of myosin II and control (GFP transduced) cells. To image TCR dynamics in GFP + cells, cells were stained with a non-blocking TCR antibody (Alexa Fluor 568–labeled H57-597). ( d ) Calcium fluxes, measured by Fura-2 ratiometric imaging, of antibody-labeled and unlabeled OT1 + T cell blasts presented with stimulating bilayers. ( e ) The temporally aligned displacements for antibody labeled and control cells on stimulating bilayers. Time 0 represents T cell binding to the stimulating bilayers. ( f ) The proliferation of H57-597 stained and non-labeled OT1 + T cells in response to pMHC and anti–CD28 coated surfaces. Data in ( d,e ) were pooled from two separate experiments with 24 control cells or 24 antibody labeled cells. Data in ( f ) is from a single experiment with each data point representing the mean of a triplicate. Error bars represent the s.e.m. Supplementary Figure 3. Actin depolymerization associated edge collapse and microcluster centralization. ( a )Time-lapse Lifeact-GFP TIRF images during formation of a protrusion in a spread synapse. At bottom, images are overlaid with the grid used for calculating local F-actin density in the edge regions. Gridded regions are color-coded according to the protrusion-contraction regions (red), the flanking non-protruding-contracting regions (blue) and outer regions (magenta). Scale bar is 5 μm. ( b ) The Lifeact-GFP intensity temporal derivative (solid lines) and distance (dashed lines, relative to the center of the synapse) of the grid regions over time. Plot colors correspond to the grid border colors in ( a ). The correlation between increasing Lifeact-GFP intensity and edge growth is representative of three cells in which protrusions were analyzed. The black arrow at left indicates the initiation of the protrusion; the gray arrow indicates the start of local edge collapse. ( c ) The linkage between microcluster and footprint edge movements is illustrated. Left: the initial position of the microcluster is shown. Middle: The path followed by the microcluster (black) along with the position of the footprint edge at the time the microcluster appeared (green), the end of spreading (magenta) and following contraction and microcluster centralization (cyan). The line from the microcluster starting position to the centroid of the cSMAC is shown in orange. Right: the radial displacement over time for the microcluster and the proximal edge region. Positive values indicate inward displacements. Spreading associated outward movement of edge microclusters was observed in all cells where the spreading process was imaged. ( d ) TIRF images of TCRs (labeled with Alexa Fluor 568–H57-597) and Lifeact-GFP at the end of spreading. The F-actin dense band and F-actin poor sink are visible. ( e ) Expanded view of the boxed region in ( d ) over the 16 s period after spreading. The location of a gap in the high F-actin density band is indicated by the arrow. The paths followed by microclusters that formed in the edge region in the vicinity of the growing gap are overlaid at lower-right. Sub-panels are 6.4 μm×6.4 μm. Numbers below images in ( a,e ) indicate the time (s) since the start of spreading. Scale bars in ( a,d ) are 5 μm. Scale bars in ( c ) are 2 μm. Supplementary Movie 1. Microcluster flow couples with cell motility. TCR microcluster flows and cSMAC movement adapted to cell movement. Microcluster flows were directed ahead of the cSMAC, which positioned to the rear of the synapse. As cell motility underwent directional changes, microcluster and cSMAC movements re-aligned to the direction of cell motility. Movie: time-lapse TIRF microscopy images of a CD3ζ-GFP transduced OT1 + T cell blast generating microcluster flow and cSMAC movement during motile synapse formation. The borders of the cSMAC regions are outlined in magenta. Microclusters (in particular, at bottom and bottom-right at 2.8–4.7 s through the movie) can be seen centralizing ahead of the cSMAC, which eventually reaches and merges with the centralized microclusters. The image sequence consists of 0.1 s exposures acquired at 2 s intervals. Images are 19.2 μm by 19.2 μm. Movie 2 Supplementary Movie 2. Synapse motility and cSMAC formation are independent In motile synapses, the aggregation of TCR microclusters into cSMACs was simultaneous with polarization and the initiation of cell motility. Contraction of the rear of the cell positioned the cSMAC to the trailing edge of the crawling cell. Movie: a CD3ζ-GFP transducted OT1 + T cell blast forming a motile synapse on a stimulating bilayer and imaged by time-lapse TIRF microscopy. After coalescing, the cSMAC cotranslates with the cell footprint. The border of the cell is highlighted in light-blue, while the borders of the TCR accumulations marking the cSMAC are highlighted in magenta. The timelapse images were acquired at 2 s intervals using 0.1 s exposures. The video measures 17 μm by 23.6 μm. Movie 3 Supplementary Movie 3. Blebbistatin does not impair motile synapse formation under non-detrimental experimental conditions OT1 + T cells form centralize TCRs and form motile synapses when myosin II activity is blocked by blebbistatin, but illumination of blebbistatin treated cells with ~491 nm light must be avoided. Exposure of blebbistatin treated cells to 491 nm light inhibited the ability of cells to generate a spreading response, form microclusters and centralize TCRs. Movie: Time-lapse TIRF microscopy images of OT1 + T cell blasts treated with blebbistatin, labeled with Alexa Fluor 568-H57-597 anti-TCRβ and subjected to various illumination conditions during image acquisition. Images of labeled TCRs were acquired with 0.1 s exposures at 1 s intervals. In the bottom two panels, TCR image 0.1 s exposures to the 491 nm TIRF illumination laser were interleaved with the TCR imaging exposures. Top: a blebbistatin treated T cell spreading onto a bilayer generated a robust clustering response and centralized TCRs within a motile synapse. Inhibition of myosin II activity with blebbistatin did not prevent cells from forming cSMACs and motile synapses. Middle: a blebbistatin treated OT1 + T cell was allowed to spread onto a bilayer and generate TCR microclusters before exposures to the 491 nm TIRF field began. The cell was able to generate TCR microclusters and centralization was initiated but was significantly reduced, and the cell failed to form a cSMAC. In the bottom panel, a blebbistatin treated T cell was exposed to the 491 nm TIRF field as it contacted the bilayer but before it was allowed to spread onto the bilayer. This blocked spreading onto the bilayer, and significantly reduced microcluster formation. For the bottom two panels, 0.1 s exposures to the 491 nm TIRF laser were interleaved after each TIRF image was acquired. The video panel is 18 μm wide. Movie 4 Supplementary Movie 4. Synapse formation by control, blebbistatin treated and conditional myosin II knockout cells Inhibition of myosin II activity by blebbistatin or Cre-GFP mediated excision of myosin II did not alter TCR centralization, cSMAC formation or synapse dynamics. Movie: Time-lapse TIRF microscopy images of OT1 + T cell blasts interacting with stimulating bilayers. Cell treatments (from left to right): DMSO vehicle control, 50 μM (−)-blebbistatin, or retroviral transduction with Cre-GFP to eliminate myosin II expression. Cells were labeled with Alexa Fluor 568–H57-597 anti-TCRβ on ice prior to imaging to visualize TCRs. Images were acquired using 0.1 s exposures at 2 s intervals. Each sub-panel of the video is 21.76 μm by 21.76 μm. Movie 5 Supplementary Movie 5.

Cell footprint contraction coordinated TCR microcluster flows

TCR microcluster flows were associated with morphological changes in the cell footprint. Movie: a time-lapse TIRF microscopy image sequences of OT1 + T cell blasts spreading and contracting on stimulating bilayers. T cells were retrovirally transduced with CD3ζ-GFP to visualize TCRs. During spreading, microclusters tended to move outward, especially when located near the edge of expanding footprints. As spreading ended, footprints contracted and microclusters began moving inward with the collapsing footprint edges. The image sequences consist of 0.1 s exposures acquired at 2 s intervals. The images measure 22 μm by 22 μm. Movie 6 Supplementary Movie 6. TCR microclusters flow into regions of actin depolymerization The appearance of a gap in the dense F-actin band correlated with the flow of edge TCRs toward the center of the synapse. Movie: TIRF images of Lifeact-GFP (left), TCRs (middle, labeled with Alexa Fluor 568–H57-597) and overlaid Lifeact/TCR images (right) as expansion ended and edge TCRs began to move inward. Images are taken from the upper-left region of the cell presented in Figure 6 . Video sub-panels are 9.6 μm by 9.6 μm. Movie 7 Supplementary Movie 7. TCR microclusters and cSMAC movement are directed to an F-actin-poor sink region in motile synapses In motile synapses, TCR microclusers and the cSMAC move toward the actin-poor interior sink. Movie: a mature motile synapse formed by a CD3ζ-GFP (left) and Lifeact-RFP (middle) transfected OT1 + T cell. The overlay of the two image sequences is shown at right. Lifeact-RFP revealed the presence of an F-actin poor interior region in the motile synapse. The cSMAC was positioned to the rear of this region. The timelapse TIRF image sequence consists of 0.1 s GFP and RFP exposures acquired at 2 s intervals. The video panels are 20.48 μm by 20.48 μm.