Download Powerpoint Fig. 6 Peripheral F-actin ring formation is impaired in the IS of mDia1/3-deficient naïve CD8 T cells. ( A ) Representative TIRF images of TCRβ and mDia1 in the IS of a naïve CD8 OT-I T cell fixed at 3 min after stimulation on an SLB. Scale bar, 5 μm. ( B ) Representative TIRF images of TCR microclusters together with EGFP-mDia1 in a CD8 OT-I T blast stimulated on an SLB. Time is relative to initial cell-SLB contact. Scale bar, 5 μm. ( C ) Scheme of the experimental design for the cell conjugation assay for control OT-I or mDia1/3 cDKO naïve CD8 OT-I T cells cocultured with SL8 peptide–loaded splenic B cells. Naïve CD8 OT-I T cells from control mice or tamoxifen-induced mDia1/3 cDKO mice and SL8 peptide–loaded splenic B cells were mixed at a one-to-one ratio, centrifuged, and then incubated for 15 min. After fixation, conjugated cells were stained with phalloidin. Half of the stained cells were then analyzed by FACS, while the remaining cells were subjected to F-actin staining and imaging by confocal microscopy. ( D ) Percentage of cell conjugates from coculture assays, as determined by FACS. Data are from three independent experiments. Bars represent means ± SEM. n.s. = not significant (one-way ANOVA with post hoc test). ( E ) Representative confocal image stacks of cell conjugates from control (upper) and mDia1/3 cDKO (lower) naïve CD8 OT-I T cells cultured with SL8 peptide–pulsed splenic B cells. Cells were labeled with anti-B220 antibody (red) and phalloidin (green) and imaged with a confocal microscope. White arrows indicate the cell conjugates used for the reconstructed en face views shown in (F). B, B cell. Scale bar, 10 μm. ( F ) Reconstructed en face views of F-actin at the IS in control and mDia1/3 cDKO naïve CD8 OT-I T cells (left) and surface plots of the corresponding en face view (right). Quantification of IS F-actin intensity ( G ) and IS diameter ( H ) in conjugates of control and mDia1/3 cDKO naïve CD8 OT-I T cells. n = 11 for control cells and n = 13 for mDia1/3 cDKO cells. Bars represent means ± SEM. *** P < 0.001 (Student’s t test). Next, we assessed the impact of mDia1/3 loss on IS formation between naïve CD8 OT-I T cells and SL8-pulsed splenic B cells using a conjugation assay ( Fig. 6C ). Although fluorescence-activated cell sorting (FACS) analysis revealed that the formation of cell conjugates was not significantly affected ( Fig. 6D ), confocal imaging analysis revealed that the IS of mDia1/3 cDKO cells was smaller and that F-actin staining at the IS interface was reduced when compared with controls ( Fig. 6E ). Confocal image stacks and the reconstructed en face view of the IS revealed a strong signal for polymerized F-actin at the periphery and an F-actin–devoid space at the center in control cells ( Fig. 6F , top), a finding that was consistent with previous reports ( 31 , 32 ). Conversely, the polymerized F-actin signal was weaker in the IS of mDia1/3 cDKO cells ( Fig. 6F , bottom left). Impaired peripheral F-actin ring formation in the IS of mDia1/3 cDKO naïve CD8 OT-I T cells was further confirmed by surface plot analysis of the en face view ( Fig. 6F , bottom right). Quantification of image data confirmed the significant reduction in F-actin intensity at the IS interface of cell conjugates formed using mDia1/3 cDKO naïve CD8 OT-I T cells ( Fig. 6G ) and also an associated decrease in IS diameter ( Fig. 6H ). Together, these results suggest that mDia1/3 is indispensable for both IS peripheral F-actin ring formation and IS spreading. Notably, live-cell imaging of TCR microcluster dynamics in mDia1/3 cDKO naïve CD8 OT-I T cells stimulated on SLBs revealed that loss of mDia1/3 impairs IS spreading, TCR microcluster centralization, and cSMAC formation (fig. S4 and movies S14 and S15) in a manner similar to that observed for SMIFH2-treated cells ( Fig. 2B and movies S1 and S2). mDia1/3 are indispensable for the positive selection of T cells To explore the physiological significance of mDia1/3 in T cell function, we investigated the phenotype of conventional mDia1/3 double-KO (DKO) mice ( 26 ). We found that these mice exhibited T cell lymphopenia (fig. S5, A to D). Histological analysis of the thymus revealed that the cortex, the region where CD4 and CD8 double-positive (DP) thymocytes reside, was larger and the medulla, the region where CD4 single-positive (SP) and CD8 SP thymocytes reside, was smaller in mDia1/3 DKO mice when compared with control WT mice (fig. S5E). We therefore performed FACS analysis of CD4 and CD8 surface expression on thymocytes and found significantly higher proportions of DP and CD4 + CD8 int DP thymocytes and significantly lower proportions of CD4 SP and CD8 SP thymocytes in the thymus of mDia1/3 DKO mice when compared with WT controls (fig. S5, F and G). This phenotype was not observed in mDia1 KO mice (fig. S6, A and B) or mDia3 KO mice (fig. S6, C and D). These findings therefore suggested that, together, mDia1 and mDia3 are required for the positive selection of T cells in the thymus. The impaired positive selection phenotype associated with mDia1/3 DKO is intrinsic to developing T cells because bone marrow transfer of mDia1/3 DKO cells to irradiated WT mice (fig. S7) and lck-cre–driven conditional deletion of mDia1/3 mimicked the impaired positive selection phenotype (fig. S8). To exclude the possibility that the polyclonal TCR repertoire may complicate the interpretation of these results, we also assessed the effects of mDia1/3 deficiency on positive selection in a defined antigen-specific TCR background, OT-I and OT-II, and obtained similar results (fig. S9). These results demonstrate the critical and redundant role of mDia1 and mDia3 in the positive selection of T cells, a process dependent on TCR signaling ( 1 ). Last, we confirmed the impact of mDia1 and mDia3 double deficiency on IS spreading, TCR microcluster dynamics, and F-actin assembly in thymocytes by TIRF imaging of cells stimulated on SLBs. Similar to naïve CD8 OT-I T cells ( Fig. 2B , top, and fig. S4A, top), control OT-I thymocytes rapidly spread and that TCR microclusters continuously formed at the cell membrane edge (fig. S10A, top, black arrows). Subsequently, when IS spreading reached the maximum, TCR microclusters moved toward the cell center to form a cSMAC (fig. S10A, top, white arrow, and movie S16). On the other hand, in mDia1/3 cDKO (lck-cre) OT-I thymocytes, while the initial formation of TCR microclusters at the contact site was unaffected, their movement was significantly slower when compared with controls (fig. S10A, bottom, and movie S17). Moreover, rapid membrane spreading associated with IS maturation was largely abolished and TCR microclusters in mDia1/3 cDKO (lck-cre) OT-I thymocytes failed to coalesce to form a cSMAC (fig. S10A, bottom, and movie S17). Quantitative analysis of image data revealed that the TCR microcluster number and the speed of TCR microcluster movement during IS formation were significantly reduced in mDia1/3 cDKO (lck-cre) OT-I thymocytes when compared with controls (fig. S10B). Consistent with these findings, an analysis of fixed samples revealed that, after seeding on SLBs at 10 min, while 71.8 ± 3.7% of TCRβ-positive control OT-I thymocytes formed a cSMAC, cSMAC formation was seen in only 35.6 ± 11.2% of TCRβ-positive mDia1/3 cDKO (lck-cre) OT-I thymocytes at the same time point (fig. S10C). In addition, mDia1/3 cDKO (lck-cre) OT-I thymocytes fixed 3 min after stimulation on SLBs also showed reduced IS area (fig. S10D). Analysis of F-actin and TCRβ staining in cells fixed 10 min after contact with SLBs revealed a peripheral F-actin ring surrounding the cSMAC in control OT-I thymocytes (fig. S10E, top). In contrast, F-actin was localized throughout the IS and cSMAC formation was impaired in mDia1/3 cDKO (lck-cre) OT-I thymocytes (fig. S10E, bottom). Quantitative analysis of the F-actin intensity distribution within the IS confirmed a significant reduction in peripheral F-actin in the IS of mDia1/3 cDKO (lck-cre) OT-I thymocytes when compared with controls (fig. S10, F and G). Together, these results suggest that mDia1/3, through their regulation of F-actin, are critical for IS spreading, TCR microcluster dynamics, and cSMAC formation also in thymocytes. DISCUSSION Stimulation of T cells with cognate antigen rapidly induces phosphorylation of proximal components of the TCR signaling complex. In this study, we started by examining the effects of formin inhibition on the phosphorylation kinetics of the key proximal TCR signaling molecules Zap70, LAT, and SLP76 in naïve CD8 T cells TCR-stimulated in suspension. We found that, while Zap70 phosphorylation was relatively sustained, the phosphorylation of the Zap70 substrates LAT and SLP76 was transient and dependent upon the activity of formins. It has previously been shown that two distinct pools of pLAT exist in TCR-stimulated T cells, a plasma membrane pLAT pool and a vesicular pLAT pool ( 4 , 8 ). Recently, it has also been demonstrated that, while the plasma membrane pool of LAT is rapidly phosphorylated, the vesicular pool is phosphorylated at later time point following TCR activation ( 33 ). Because formin inhibition suppressed LAT phosphorylation from the earliest time points examined, we concluded that phosphorylation of the membrane pool of LAT occurs in a formin-dependent manner. However, formin activity is dispensable for Zap70 phosphorylation. Zap70 phosphorylation has previously been shown to be regulated by its catch and release from the TCR ( 34 ), and its TCR dwell time has been shown to determine its kinase activity independently of LAT ( 35 ). Our results therefore suggest that formin-dependent F-actin polymerization and subsequent F-actin remodeling are not involved in the interaction between Zap70 and the TCR. It should be noted that a previous study has reported that formin activity is critical for TCR signaling from the Zap70 level ( 20 ). Although we did not address the reason for this discrepancy experimentally, this may be because of technical difference between the studies, such as the cell types used; our study used naïve CD8 T cells, while the previous study used Jurkat T cells ( 20 ). It should also be noted that in this previous study, the authors examined the level of Zap70 and LAT phosphorylation in cell conjugate assays using APCs. Given that F-actin has previously been implicated in APC-mediated TCR signaling ( 36 ), it is possible that formin inhibition in APCs may also indirectly affect TCR signaling. In our study, we also found a correlation between Zap70-mediated LAT phosphorylation and formin-dependent F-actin polymerization in TCR-stimulated naïve CD8 T cells in suspension. Our results suggest the possibility that formin-dependent F-actin networks interact with pZap70 and pLAT upon TCR stimulation. We further dissected the role of formin-dependent F-actin in the regulation of pZap70 and pLAT in the context of the IS using an SLB system. Using this system, we observed that pZap70 is localized beneath the peripheral F-actin ring in the IS of stimulated control T cells. However, treatment with a formin inhibitor suppressed the formation of this peripheral F-actin ring and impaired pZap70 localization to the IS. Consistent with this finding, phosphorylated LAT, which also localizes to the IS periphery in control cells, was significantly diminished in the IS of formin inhibitor–treated T cells. Together, these results suggest that pZap70-dependent LAT phosphorylation at the IS is regulated by formin-dependent F-actin assembly. We speculate that the recently proposed mechanism whereby Lck served as a bridge between pZap70 and pLAT ( 9 ) may occur subsequently to the formin-mediated transient and physical coupling of pZap70 and LAT that we report here to further stabilize the complex and therefore promote efficient LAT phosphorylation. It should also be noted that we found formin activity to be critical for IS spreading and TCR microcluster formation during the early stages of IS formation in naïve CD8 T cells, but found it to be dispensable at later stages, after IS spreading or cSMAC formation. Given that IS spreading upon TCR stimulation occurs downstream of LAT phosphorylation ( 37 ), impaired IS spreading and the subsequent impaired TCR microcluster formation are likely to be secondary consequences of suppressed LAT phosphorylation upon formin inhibition. Although the TCR microcluster number was reduced, total cellular levels of Zap70 phosphorylation were not significantly affected. Our 3D image reconstruction analysis revealed that although pZap70 levels at the IS of SMIFH2-treated naïve CD8 T cells were reduced, this was not a consequence of impaired protein phosphorylation, but rather because the phosphorylated protein was mislocalized to the cytoplasm. Given the catch and release mechanisms for the regulation of phosphorylated Zap70 at the TCR ( 34 ), it is possible that pZap70 may be released into the cytoplasm in the absence of formin-dependent polymerized F-actin at the IS. A previous study into the role of the formin and Rho effector, mDia1, in TCR signaling and IS formation in Jurkat T cells concluded that this protein had no effect on TCR signaling and the TCR activation–dependent formation of F-actin–rich structures ( 19 ). Consistent with these findings, we found that naïve CD8 T cells from mDia1 KO mice did not exhibit an impaired proximal TCR signaling phenotype. However, given that previous studies from our group ( 26 – 29 ) and others ( 30 ) have demonstrated functionally redundant roles between mDia1 and mDia3 isoforms in the polymerization of F-actin, we further investigated potential T cell phenotypes associated with loss of both mDia1 and mDia3. We found that Zap70-dependent LAT phosphorylation was significantly impaired in TCR stimulated naïve CD8 T cells from mDia1/3 cDKO mice in suspension, a finding that was similar to that observed for SMIFH2-treated cells. Loss of mDia1/3 in naïve CD8 T cells strongly suppressed TCR stimulation–dependent IL-2 production and cell proliferation, suggesting a role of mDia1/3 in T cell activation. Using the SLB system, we have shown that mDia1 initially colocalizes with early TCR microclusters but then concentrates at the IS periphery, which is the site where the formin-dependent peripheral F-actin ring subsequently forms. Furthermore, using a cell conjugation assay, we demonstrated that loss of mDia1/3 in naïve CD8 T cells impaired peripheral F-actin ring formation and IS spreading in a similar manner to that observed in SMIFH2-treated cells. We also found that thymocytes from mice deficient in mDia1/3 showed impaired IS spreading and TCR microcluster centralization on SLBs. Notably, the positive selection of these cells in vivo was also impaired, suggesting that mDia1/3 is critical for TCR signaling in the physiological context. It has previously been shown that thymocytes expressing a constitutively active form of RhoA, an upstream regulator of mDia1/3 ( 38 ), demonstrate augmented positive selection ( 39 ). On the basis of this previous report and our findings, we speculate that RhoA may exert its effects on thymocyte positive selection through mDia1/3. The underlying molecular mechanism controlling mDia1/3 activation upon TCR stimulation warrants future investigation. In this study, we have combined pharmacological inhibition of formins, genetic manipulation of mDia1/3, an SLB system, and high-resolution imaging in conjunction with 3D image reconstruction to reveal an indispensable yet hitherto unrecognized role for the formins, mDia1 and mDia3, in Zap70-dependent LAT phosphorylation at the IS. Our study therefore advances a new conceptual model in which F-actin spatiotemporally regulates proximal TCR signaling, providing an important framework for further dissection of the role of F-actin in T cell development and activation. MATERIALS AND METHODS Study design The aim of this study was to reveal the function of formin-mediated F-actin assembly in proximal TCR signaling using mouse naïve CD8 T cells and mouse CD8 T blasts. All animal experiments were conducted in accordance with the U.S. National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of Kyoto University Graduate School of Medicine and University of California, San Francisco. WT mice were used as the control for experiments with mDia1 KO, mDia3 KO, and mDia1/3 DKO mice. mDia1/3 double-floxed mice were used as the control for experiments with mDia1/3 double-floxed × UBC-creERT2 mice (mDia1/3 cDKO mice). mDia1/3 double-floxed × OT-I mice were used as the control for experiments with mDia1/3 double-floxed × UBC-creERT2 × OT-I mice. Lck-cre transgenic mice were used as the control for experiments with mDia1/3 double-floxed × lck-cre mice [mDia1/3 cDKO (lck-cre) mice]. mDia1/3 double-floxed × OT-I mice and mDia1/3 double-floxed × OT-II mice were used as the control for experiments with mDia1/3 double-floxed × lck-cre × OT-I mice and mDia1/3 double-floxed × lck-cre × OT-II mice, respectively. Age- and gender-matched mice of the same strain were used in each experiment. Experimental replicates were indicated in the figure legends. Randomization was not performed in the animal experiments of this study. Mice mDia1 KO mice, mDia3 KO mice, and mDia1/3 DKO mice have been described previously ( 26 ). In brief, mDia1 KO mice and mDia3 KO mice (each lacking exon 1 of the WT gene) were generated by homologous recombination and backcrossed for more than 10 generations to C57BL/6N mice. mDia1/3 DKO mice were generated as follows: mDia1 heterozygous males were crossed with mDia3 heterozygous females. Among the offspring, mDia1 heterozygous/mDia3 hemizygous male mice were then mated with mDia3 heterozygous female mice, and the resultant male and female mice of mDia1 heterozygous/mDia3 homozygous genotype were then intercrossed to generate mDia1/3 DKO mice. mDia1/3 double-floxed mice were generated by crossing mDia1 floxed mice with mDia3 floxed mice ( 28 ) backcrossed to C57BL/6N mice for more than 10 generations. To delete mDia1 and mDia3 in the T cell–lineage, mDia1/3 double-floxed mice were mated with transgenic mice expressing Cre recombinase under the control of lck proximal promoter. Mice with transgenic expression of lck-cre, UBC-creERT2, OT-I, and OT-II were from the Jackson Laboratory. CD45.1-congenic C57BL/6J mice for bone marrow transplantation experiments were also obtained from the Jackson Laboratory. Tamoxifen (T-5648, Sigma) was dissolved in ethanol and then diluted in corn oil (C8267, Sigma) to a final concentration of 20 mg/ml. For tamoxifen-induced deletion of mDia1/3, tamoxifen was intraperitoneally injected into mDia1/3 double-floxed × UBC-creERT2 mice or mDia1/3 double floxed × UBC-creERT2 × OT-I mice at a dose of 3.2 mg/day for five consecutive days. Cell preparation and stimulation Thymocytes from thymus and lymphocytes from spleen and lymph nodes were isolated by mechanical disruption between the frosted ends of glass slides (S2215, Matsunami) and filtrated through a 70-μm nylon cell strainer (352350, BD Falcon). Naïve CD8 T cells from control or tamoxifen-induced mDia1/3 conditional DKO mice were isolated using a negative selection MACS isolation kit (130-096-543, Miltenyi Biotec). For the stimulation of naïve CD8 T cells in suspension, cells were incubated at 37°C under 5% CO 2 for 2 hours in RPMI 1640 medium (R-8758, Sigma) containing 10% fetal calf serum (12483-020, Gibco), resuspended in phosphate-buffered saline (PBS), incubated with biotin-conjugated anti-mouse CD3ε antibody (553059, BD Pharmingen) and anti-mouse CD28 antibody (102102, BioLegend) on ice for 15 min, washed, and then stimulated by cross-linking with streptavidin (43-4301, Invitrogen) at 37°C. Immunocytochemistry (in suspension) For immunocytochemistry of naïve CD8 T cells in suspension, cells were fixed with 4% paraformaldehyde (PFA) in phosphate buffer for 15 min, washed with PBS, and allowed to settle for 45 min onto poly- l -lysine (P6282, Sigma)–coated coverslips (Matsunami) at 37°C. Cells were then washed with PBS, permeabilized with 0.3% Triton-X100 in PBS for 10 min, and blocked with 1% bovine serum albumin in PBS for 45 min at room temperature. The cells were then incubated with indicated primary antibodies at 4°C overnight, washed, and stained with secondary antibodies for 45 min at room temperature. For F-actin staining, cells were incubated with fluorescent dye–labeled phalloidin diluted in 0.3% Triton-X100/PBS for 20 min at room temperature. Cells were finally washed with PBS and mounted onto slides with ProLong Diamond Antifade reagent (P36970, Life Technologies). Fluorescence images were acquired with an SD-OSR IX83 inverted microscope (Olympus) equipped with a 100× numerical aperture (NA) 1.4, UPLSAPO oil immersion objective (Olympus) and Yokogawa W1 spinning disk unit (Yokogawa) controlled by MetaMorph software (Universal Imaging). Quantification of F-actin, pZap70 [Y319], and pLAT [Y171] staining intensity was performed using ImageJ software. Coefficient of variant of F-actin staining intensity was calculated as the ratio of the SD and average staining intensity. Imaging of the IS on SLBs TIRF imaging was performed as previously described ( 23 ). An Olympus IX-81 invert microscope equipped with a laser TIRF slider (Olympus) was used for imaging of naïve CD8 OT-I T cells and CD8 OT-I T blasts. For TIRF microscopy, a 100× NA 1.4 PlanApo objective lens was used. For two-color TIRF imaging, a G-base, two-channel simultaneous imaging system (G-Angstrom) with a 560-nm longpass dichroic filter and 525-nm/50-nm and 605-nm/70-nm bandpass emission filters was used to split the camera field into two image channels for simultaneous imaging of EGFP and Alexa Fluor 568 fluorescence. Images were acquired with an iXon Ultra 888 EMCCD (Andor) and MetaMorph software (Universal Imaging). For imaging of OT-I thymocytes, a Zeiss Axiovert 200M equipped with a laser TIRF slider and a 100× NA 1.45 Plan-Fluar objective lens was used. Images were acquired with an Evolve EMCCD (Photometrics) and MetaMorph software (Universal Imaging). For labeling of surface TCRs, 2 × 10 6 OT-I thymocytes, naïve CD8 OT-I T cells, or CD8 OT-I T blasts were stained for 30 min on ice with 1 μg of Alexa Fluor 568–labeled H57-597 anti-TCRβ antibody (Bio X Cell) in 0.05 ml of complete phenol red–free RPMI medium. For imaging of cells stimulated on bilayers, 1 × 10 5 cells in 0.1 ml of complete phenol red–free RPMI medium were added to the PBS (0.25 ml) overlaying the bilayer. In TIRF time-lapse imaging experiments, cells undergoing initial spreading on bilayers were located, and TIRF images were acquired for 2 to 5 min at intervals of 1 or 2 s for naïve CD8 OT-I T cells and CD8 OT-I T blasts and for 2 to 5 min at intervals of 0.25 or 0.5 s for OT-I thymocytes. The exposure length was 100 to 200 ms. Imaris software was used for tracking and quantitative analysis of TCR microcluster dynamics. In immunocytochemistry of experiment of cells stimulated on SLBs, cells were fixed with 4% PFA and then blocked and stained according to standard protocols. TIRF images of fixed cells were acquired on the same microscope used for live-cell imaging. Confocal images of fixed cells on SLBs were acquired with a laser scanning confocal imaging system (Zeiss LSM710) using a 63×, NA 1.4 oil immersion objective lens. Line scanning analysis of F-actin staining within a single confocal plane corresponding to the IS was conducted using ImageJ software (NIH). 3D reconstruction of confocal image stacks was performed using Imaris software (Bitplane). Quantification of the percentage of cSMAC was performed by visual inspection of the TIRF images. Quantification of IS area and staining intensity in the images of each confocal plane was conducted using ImageJ software (NIH). mRNA electroporation For electroporation, 1 μg of LifeAct-EGFP mRNA (TriLink), 1 μg of EGFP-mDia1 mRNA (TriLink), or 1 μg of EGFP-mDia3 mRNA (TriLink) was mixed with 5 × 10 5 CD8 OT-I T blasts (TCR-stimulated with SL8 peptide–loaded splenocytes for 48 hours) in 10 μl of T buffer (Thermo Fisher Scientific). Cells were then loaded in 10-μl tips and electroporated with the Neon Transfection System (Invitrogen) according to the manufacturer’s protocol with three pulses of 1400 V for 10 ms. Conjugation assay Naïve CD8 T cells were isolated from control or mDia1/3 cDKO OT-I mice using a negative selection MACS purification kit (130-096-543, Miltenyi Biotec). B cells from WT mice were isolated using MACS beads (18954, Stemcell Technologies), incubated with SL8 peptide (1 μg/ml) (Sigma) for 30 min at 37°C, and then surface-stained with phycoerythrin (PE)–conjugated anti-B220 antibody (12-0452-83, eBioscience). For cell conjugation assays, equal volumes of naïve CD8 OT-I T cells and SL8 peptide–pulsed B cells were mixed and incubated at 37°C for 15 min. Conjugated cells were then fixed with 0.5% PFA for FACS analysis using the LSR Fortessa System (BD Biosciences). The percentage of conjugates within the culture was analyzed using FlowJo software (Tree Star Inc.). For imaging of the F-actin ring, cell conjugates were fixed with 4% PFA, permeabilized, stained with Alexa Fluor 488-conjugated phalloidin, and then imaged with a Leica SP5 laser confocal microscope equipped with 100× NA 1.4 HCX PL APO CS objective lens (Leica). 3D reconstruction of stacked confocal images and en face view images were generated by Volocity software (PerkinElmer). Surface plots of 3D reconstructed images were generated using ImageJ software (NIH). Quantification of IS F-actin intensity and IS diameter was performed using ImageJ software (NIH). Statistical analyses Quantitative data were reported as means ± SEM or means ± SD. Excel (Microsoft) and Prism (GraphPad Software) were used for statistical analyses. The Student’s t test or one-way ANOVA with post hoc test was used for the evaluation of significance as specified in the figure legends. The sample number ( N ) refers to the number of biological replicates. n.s. = not significant ( P > 0.05), * P < 0.05, ** P < 0.01, and *** P < 0.001. SUPPLEMENTARY MATERIALS Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/1/eaay2432/DC1 Supplementary Materials and Methods Fig. S1. Effects of formin inhibition after initial TCR microcluster formation in naïve CD8 OT-I T cells stimulated on SLBs. Fig. S2. TCR signaling in naïve CD8 T cells from mDia1 KO or mDia3 KO mice. Fig. S3. Dynamic localization of EGFP-mDia3 in the IS. Fig. S4. IS spreading and TCR microcluster centralization are impaired in mDia1/3 cDKO naïve CD8 OT-I T cells. Fig. S5. T cell lymphopenia results from impaired positive selection in mDia1/3 DKO mice. Fig. S6. T cell development in mDia1 KO mice and mDia3 KO mice. Fig. S7. Impaired T cell development phenotypes in mDia1/3 DKO are cell intrinsic. Fig. S8. Conditional deletion of mDia1 and mDia3 in the T cell lineage. Fig. S9. mDia1/3 deficiency impaired thymocyte-positive selection in OT-I and OT-II transgenic mice. Fig. S10. Impaired TCR microcluster centralization and peripheral F-actin ring formation following loss of mDia1/3 in OT-I thymocytes. Movie S1. TIRF live imaging of TCR microcluster dynamics in a control naïve CD8 OT-I T cell stimulated on a SLB (2 s per frame; 60 frames). Movie S2. TIRF live imaging of TCR microcluster dynamics in a 10 μM SMIFH2–treated naïve CD8 OT-I T cell stimulated on an SLB (2 s per frame; 60 frames). Movie S3. TIRF live imaging of TCR microcluster dynamics in a naïve CD8 OT-I T cell stimulated on an SLB and treated with 15 μM SMIFH2 at 10 s after initial microcluster formation (2 s per frame; 75 frames). Movie S4. TIRF live imaging of TCR microcluster dynamics in a naïve CD8 OT-I T cell stimulated on an SLB and treated with 15 μM SMIFH2 at 20 s after initial microcluster formation (2 s per frame; 75 frames). Movie S5. TIRF live imaging of cSMAC dynamics in a naïve CD8 OT-I T cell stimulated on an SLB for 10 min and treated with 15 μM SMIFH2 (2 s per frame; 160 frames). Movie S6. TIRF live imaging of TCR microcluster and LifeAct-EGFP dynamics in a control CD8 OT-I T blast stimulated on an SLB (2 s per frame; 60 frames). Movie S7. TIRF live imaging of TCR microcluster and LifeAct-EGFP dynamics in a 10 μM SMIFH2–treated CD8 OT-I T blast stimulated on an SLB (2 s per frame; 60 frames). Movie S8. 3D reconstruction of a confocal image stack of pZap70 and F-actin staining in a control naïve CD8 OT-I T cell stimulated on an SLB for 1.5 min. Movie S9. 3D reconstruction of a confocal image stack of pZap70 and F-actin staining in a 10 μM SMIFH2–treated naïve CD8 OT-I T cell stimulated on an SLB for 1.5 min. Movie S10. 3D reconstruction of a confocal image stack of pLAT and F-actin staining in a control naïve CD8 OT-I T cell stimulated on an SLB for 1.5 min. Movie S11. 3D reconstruction of a confocal image stack of pLAT and F-actin staining in a 10 μM SMIFH2–treated naïve CD8 OT-I T cell stimulated on an SLB for 1.5 min. Movie S12. TIRF live imaging of TCR microcluster and EGFP-mDia1 dynamics in a CD8 OT-I T blast stimulated on an SLB (2 s per frame; 60 frames). Movie S13. TIRF live imaging of TCR microcluster and EGFP-mDia3 dynamics in a CD8 OT-I T blast stimulated on an SLB (2 s per frame; 60 frames). Movie S14. TIRF live imaging of TCR microcluster dynamics in a control naïve CD8 OT-I T cell stimulated on an SLB (2 s per frame; 60 frames). Movie S15. TIRF live imaging of TCR microcluster dynamics in an mDia1/3 cDKO naïve CD8 OT-I T cell stimulated on an SLB (2 s per frame; 60 frames). Movie S16. TIRF live imaging of TCR microcluster dynamics in a control OT-I thymocyte stimulated on an SLB (0.5 s per frame; 240 frames). Movie S17. TIRF live imaging of TCR microcluster dynamics in an mDia1/3 cDKO (lck-cre) OT-I thymocyte stimulated on an SLB (0.5 s per frame; 240 frames). View/request a protocol for this paper from Bio-protocol . This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license , which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited. REFERENCES AND NOTES
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