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1 SUPPLEMENTAL FIGURES FIGURE S1. Detection of MCs. A, Schematic representation of T cells stimulated on anti- CD3 coated cover slips indicating stimulatory contact site, F-actin polymerization and microclusters. B, Side projection of wide field z-stack of Jurkat T lymphocyte expressing TCR.GFP stimulated on anti-cd3 coated cover slip. Stimulatory contact site chosen for imaging microclusters is depicted. Jurkat T lymphocyte expressing TCR.GFP were stimulated on anti-cd3 coated cover glasses for 5 min, fixed and stained for F-actin with TRITC conjugated phalloidin. Representative micrographs of F-actin (C) and GFP (D). Scale bar = 1μm. Cell boundaries were manually defined based on the F-actin fluorescence in most cases and regions of interest were marked using the polygon ROI tool in Fiji (as shown in red lines in A). TCR MCs (as depicted in B) were analyzed using the approach described in the main text. The MCs were enhanced using difference of Gaussian filters (E) and the spots were detected after applying a threshold 6x higher than the estimated noise standard deviation and using Fiji s Find Maxima command to identify watershed-separated regions (F). A binary image showing the detected regions (G). The lower panel indicates a zoomed image from the respective square boxes in upper panel. Scale bar = 2μm FIGURE S2. Representative micrographs of cells analyzed for incorporation of the indicated proteins into MCs following stimulation on anti-cd3 coated cover glasses. At the indicated time points post stimulation, cells were fixed and analyzed. A, Jurkat T lymphocytes, TCR.GFP. B, J14.SLP-76.YFP. C, Jurkat T lymphocytes, phospho-lat. 1

2 FIGURE S3. HIV does not disrupt formation of pzap7 containing MCs but reduces MC recruitment of pzap7. A, 24 hours post transfection, J14 SLP-76.YFP cells transiently expressing the indicated proteins were stimulated on anti-cd3 coated cover glasses for 5 min, fixed and stained for pzap7. Upper and lower panel depict original and binary images, respectively. Grey lines indicate cell boundaries. Scale bar = 1μm. B, Frequency of cells shown in A with pronounced pzap7 MCs at stimulatory contact sites. Depicted are mean values from 3 independent experiments ± SD with at least 1 cells analyzed per condition. Cells were scored positive for pzap7 MCs if they exhibited MCs comparable to those of untransfected neighboring cells. p values were determined using students t-test. Single cell quantification indicating pzap7 MC density (C), mean MC size per cell (D) and magnitude of recruitment into MC in the contact plane (E) are shown. Depicted are values from more than 25 cells analyzed per condition where each symbol designates the value for an individual cell. Grey bars indicate the mean values of all cells analyzed. p values were determined using the Mann Whitney U test. (F), Representative micrographs of the subcellular localization of SLP-76.YFP in J14SLP-76.YFP cells transiently expressing the indicated proteins on poly lysine coated cover glasses. FIGURE S4. Colocalization of plat and SLP-76 in MCs is impaired in expressing cells. 24 hours post transfection, J14 SLP-76.GFP cells transiently expressing the indicated proteins were stimulated on anti-cd3 coated cover glasses for 5 min, fixed and stained for phospho-lat. A, Representative binary images were generated for each channel using identical settings and the degree of overlap of MC pixels were determined to compute Manders coefficients M1 and M2. Scale bar = 1μm. Single cell quantification indicating the 2

3 percentage of overlap of p-lat with SLP-76 MCs (M1) (B) and the percentage of overlap of SLP-76 with plat MCs (M2) (C). Depicted are values from more than 25 cells analyzed per condition where each symbol designates the value for an individual cell. Red bars indicate the mean values of all cells analyzed. p values were determined using the Mann Whitney U test. D, Flow cytomeric analysis of the expression levels of TCR proximal signaling proteins in individual cells. Jurkat T cells expressing the indicated fusion proteins were transfected with expression plasmids coding for or.. 24 hours post transfection, /. positive cells were analyzed to determine the expression levels of various signaling molecules (GFP/YFP) using BD FACSCanto II. Shown below are anti- immunoblots (with numbers indicating the percentage of -positive cells) from the same samples. Anti-tubulin serves as loading control. Note that the presence of does not affect expression levels of the TCR proximal signaling proteins analyzed. E, F, G, Single cell quantification of cells shown in Fig. 8D. Depicted are values from more than 25 cells analyzed per condition where each symbol designates the value for an individual cell. E, SLP-76 MC density. F, mean MC size per cell. G, magnitude of recruitment into MC in the contact plane. Red bars indicate the mean values of all cells analyzed. p values were determined using the Mann Whitney U test. 3

4 SUPPLEMENTAL MOVIES Movie S1. Anti-CD3 induced cell spreading and SLP-76 MC formation in -expressing J14SLP-76.YFP cells. Five minute time-lapse of cell spreading and SLP-76 MC formation of a -expressing J14SLP-76.YFP cell. Stills of this movie are shown in Fig. 4A, upper panel. Shown is the sequence of the YFP signal from SLP-76 MCs (left) and the corresponding detected MCs (right). Scale bar = 5 μm. Movie S2. Anti-CD3 induced cell spreading and SLP-76 MC formation in.expressing J14SLP-76.YFP cells. Five minute time-lapse of cell spreading and SLP-76 MC formation of a.-expressing J14SLP-76.YFP cell. Stills of this movie are shown in Fig. 4A, middle panel. Shown is the sequence of the YFP signal from SLP-76 MCs (left) and the corresponding detected MCs (right). Scale bar = 5 μm. Movie S3. Anti-CD3 induced cell spreading and SLP-76 MC formation in F195A.expressing J14SLP-76.YFP cells. Five minute time-lapse of cell spreading and SLP-76 MC formation of a F195A.-expressing J14SLP-76.YFP cell. Stills of this movie are shown in Fig. 4A, bottom panel. Shown is the sequence of the YFP signal from SLP-76 MCs (left) and the corresponding detected MCs (right). Scale bar = 5 μm. 4

5 A F-actin polymerization Nucleus Microclusters Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y B Stimulatory Contact Site Top Z Bottom C D E F G Actin TCR.GFP DoG Filtered Image Detected Spots Binary Image Supplemental Fig. 1

6 Supplemental Fig. 2 A TCR.GFP B t [min] F195I.. SLP-76 C F195I.. F195I.. plat

7 A pzap7 Microcluster Density [number/μm 2 ]. F195I. pzap7 Binary Image C D E * n.s. F195I. Mean pzap7 Microcluster Size per Cell [μm 2 ] F195I. B Frequency of Cells With pzap7 Microclusters at Contact Site [%] Magnitude of pzap7 MC Recruitment [% Signal MCs/Total Signal at Contact Site] F195I.. F195I. F. F195I. Palm. SLP-76 Supplemental Fig. 3

8 A plat SLP-76.YFP Merge M1=33.8%, M2=81.%. M1=2.8%, M2=89.8% F195I. B M1=2.7%, M2=92.% % plat Signal in SLP-76 MCs (M1) at Contact Site. F195I. C n.s n.s % SLP-76 Signal in plat MCs (M2) at Contact Site. F195I. Untransfected. 1 TCR.GFP ZAP7.GFP SLP-76.YFP LAT.GFP E n.s [number/μm 2 ] F195I F195I Unc119. F * Unc119. G n.s Mean SLP-76 Microcluster Size per Cell [μm 2 ] Magnitude of SLP-76 MC Recruitment [% Signal MCs/Total Signal at Contact Site] Percentage of max % 26% 31% 22% F195I F195I F195I F195I Supplemental Fig. 4 Unc119.

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