© 2020. Published by The Company of Biologists Ltd | Journal of Cell Science (2020) 133, jcs242875. doi:10.1242/jcs.242875

RESEARCH ARTICLE The DISC1–Girdin complex – a missing link in signaling to the T cell Nicholas Maskalenko1, Shubhankar Nath2, Adarsh Ramakrishnan1, Nadia Anikeeva3, Yuri Sykulev3 and Martin Poenie1,*

ABSTRACT alpha-L). Finally, there is the outermost zone known as the distal In this study, using Jurkat cells, we show that DISC1 (disrupted in supramolecular activation cluster (dSMAC), which is enriched in 1) and Girdin (girders of filament) are essential for lamellipodial actin (Freiberg et al., 2002). typical actin accumulation at the immunological . Furthermore, One of the prominent features of the immunological synapse is the DISC1, Girdin and are bound in a complex. Although this accumulation of actin, which is seen as a ring-like structure that forms complex initially forms as a central patch at the synapse, it relocates to a at the edges of the cell lamellae as they spread over the target cell. The peripheral ring corresponding to the peripheral supramolecular most obvious actin assembly at the synapse is thought to be triggered activation cluster (pSMAC). In the absence of DISC1, the classic by formation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3), actin ring does not form, cell spreading is blocked, and the dynein which in turn leads to activation of the WASP-family verprolin complex fails to relocate to the pSMAC. A similar effect is seen when homologous WAVE2 and the actin-related protein (ARP) 2/3 ’ Girdin is deleted. When cells are treated with inhibitors of actin structure (Le Floc h et al., 2013; Chen et al., 2017; Basu et al., 2016). polymerization, the dynein–NDE1 complex is lost from the synapse and However, recent studies have revealed a remarkable complexity to the the -organizing center fails to translocate, suggesting that regulation of actin, involving multiple actin regulators and nucleators ’ actin and dynein might be linked. Upon stimulation of T cell receptors, with a variety of effects on T cell functions (Le Floc hetal.,2013; DISC1 becomes associated with talin, which likely explains why the Kumari et al., 2015; Jankowska et al., 2018; Janssen et al., 2016; dynein complex colocalizes with the pSMAC. These results show that Comrie et al., 2015). Defects in proper actin assembly have been the DISC1–Girdin complex regulates actin accumulation, cell linked generally to immunodeficiency and autoimmune dysfunctions spreading and distribution of the dynein complex at the synapse. (Wickramarachchi et al., 2010). More specifically, actin dynamics have been linked to sustained T cell signaling, cell spreading, calcium This article has an associated First Person interview with the first author entry, formation of stable adhesions and target cell stimulated of the paper. secretion in cytotoxic T lymphocytes (CTLs) (Babich et al., 2012; Carisey et al., 2018; Nolz et al., 2006). KEY WORDS: DISC1, Girdin, Immunological synapse, Dynein, In this study, we introduce new players in the T cell actin schema, Actin dynamics Girdin (girders of actin filaments) and DISC1 (disrupted in schizophrenia 1). DISC1 is a scaffolding protein that, in addition INTRODUCTION to dynein, interacts with over 100 different including those When T cells engage antigen-presenting cells they form a associated with centromeres, the cytoskeleton, cell signaling and specialized contact site known as the immunological synapse. neuronal (Camargo et al., 2007; Chubb et al., 2008). We Classically, the synapse can be described in terms of concentric previously showed that DISC1 forms a complex with dynein, NDE1 zones of receptors, adhesion proteins and cytoskeletal elements (neurodevelopment protein 1, also known as nuclear distribution (Monks et al., 1998; Bunnell et al., 2001; Kupfer et al., 1987; protein nudE homolog 1) and LIS1 (lissencephaly 1) in Jurkat cells Grakoui et al., 1999). The central region, known as the central (Nath et al., 2016) but its function was not explored. Girdin (also supramolecular activation cluster (cSMAC), is characterized by the known as GIV, G α-interacting vesicle-associated protein) exhibits a accumulation of T cell receptors (TCR) and protein kinase C-theta variety of signaling functions and is involved in cytoskeletal (PKC-θ) (Monks et al., 1998). Surrounding the cSMAC is an outer reorganization and integrin signaling (Leyme et al., 2016, 2015; zone known as the peripheral supramolecular activation cluster Aznar et al., 2016; Weng et al., 2010). (pSMAC), which is characterized by clusters of the lymphocyte We initially showed there are two DISC1 isoforms expressed in function-associated protein 1 (LFA-1, also known as integrin Jurkat cells, the full-length L isoform (DISC1L) and the Lv splice variant (DISC1Lv) (Nakata et al., 2009). We show that the L isoform accumulates at the immunological synapse, whereas DISC1Lv is 1Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712, USA. associated with mitochondria. When DISC1 was deleted using 2Cellink LLC, 100 Franklin St., Boston, MA 02110, USA. 3Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson CRISPR/Cas9, actin accumulation at the immunological synapse was University, Philadelphia, PA 19107, USA. greatly reduced and members of the dynein complex (dynein, NDE1 and LIS1), which colocalizes with ADAP (FYN-binding protein 1) at *Author for correspondence ([email protected]) the pSMAC, remain clustered together near the center of the synapse N.M., 0000-0001-7690-5234; S.N., 0000-0001-5778-7615; A.R., 0000-0002- (Combs et al., 2006; Nath et al., 2016). We also found that DISC1 2922-5778; N.A., 0000-0003-1494-6270; Y.S., 0000-0002-9685-0223; M.P., 0000- forms a complex with Girdin and that deletion of Girdin gave a 0003-2524-4039 phenotype similar to that of DISC1 knockout (DISC1-KO) cells. Handling Editor: Daniel Billadeau When DISC1L or Girdin were reintroduced as cDNAs into their

Received 14 December 2019; Accepted 26 May 2020 respective deletion mutant cell lines, the DISC1–dynein complex was Journal of Cell Science

1 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs242875. doi:10.1242/jcs.242875 again localized at the pSMAC and actin accumulation at the synapse expression was only partially reduced and that cells quickly was restored to levels seen in wild-type (WT) Jurkat cells. recovered. As an alternative, a CRISPR/Cas9 construct targeted to The loss of actin accumulation and failure of the dynein complex DISC1 was used to disrupt the . Chemical selection and cell to locate at the pSMAC suggested that there was a connection sorting were then used to obtain a pure DISC1 deletion cell line between actin assembly and localization of the NDE1–dynein (DISC1-KO). Absence of DISC1 expression in this cell line was complex. To explore this further, we treated Jurkat cells with verified by immunoblotting and an absence of DISC1 cytochalasin B (CytB) or latrunculin B (LatB) to disrupt actin immunofluorescence at the synapse (Fig. S2). polymerization prior to formation of conjugates with staphylococcal One of the most obvious effects of DISC1 deletion was the loss of enterotoxin E (SEE)-coated Raji cells. These treatments resulted in actin accumulation at the immunological synapse, as detected by loss of the dynein complex from the synapse and a failure of staining with phalloidin-TRITC (Fig. 2A,B). Actin accumulation microtubule organizing center (MTOC) translocation to the was analyzed by plotting average phalloidin-TRITC fluorescence immunological synapse. Moreover, we showed that after Jurkat for pixels within segments spanning the width of the synapse; 30 cells are activated by anti-TCR Ig, DISC1 co-immunoprecipitates wild-type (WT) and 30 DISC1-KO cells were analyzed (Fig. 2C). with talin. Because talin binds to LFA-1, this finding may explain For the five segments closest to the immunological synapse there how the dynein complex becomes associated with the pSMAC was significant difference in phalloidin-TRITC fluorescence (Klapholz and Brown, 2017). (P<0.001). Additionally, to determine whether disruption of DISC1 had an effect on LFA-1 recruitment to the synapse, we RESULTS immunostained for the LFA-1 adapter talin in WT and DISC1-KO DISC1 isoform L promotes actin polymerization at the cells (Fig. 2A,B). The results show that talin was recruited to the immunological synapse immunological synapse in the absence of DISC1. Initial immunostaining studies showed that DISC was concentrated To verify that the loss of actin at the immunological synapse was around the MTOC in unstimulated Jurkat cells but accumulated at specific to deletion of DISC1, we introduced DISC1-eGFP the synapse after conjugation with SEE-coated Raji cells (Fig. 1A). constructs for L and Lv isoforms into DISC1-KO cells. However, These results were not unique to Jurkat cells as DISC1 also neither of the DISC1-eGFP isoforms restored actin accumulation at accumulates at the immunological synapse in NK-92-Daudi cell the synapse (Fig. S3). We then repeated the experiment using pairs and in mouse OT-1 CTLs engaged with peptide-pulsed EL4 DISC1 constructs without the fused eGFP. In this case, only cells (Fig. S1). expression of the L isoform was able to restore visible actin staining We then proceeded to clone DISC1 from a Jurkat cDNA library. at the immunological synapse (Fig. 2D,E). Finally, to determine Sequencing of the clones revealed two previously identified whether detectable actin remained at the immunological synapse in isoforms, L and Lv (Nakata et al., 2009). We used these two the DISC1-KO cell line, we used confocal microscopy together clones to produce DISC1-eGFP chimeras, which were expressed in with phalloidin-TRITC staining to compare WT Jurkat cells Jurkat cells. We found that DISC1 isoform Lv localized to and DISC1-KO cells that were either untreated or treated with that accumulate near the synapse after stimulation with LatB to disrupt actin (Fig. 2F–H). The results show that treatment of SEE-coated Raji cells (Fig. 1B). These organelles were DISC1-KO cells with LatB visibly reduced actin staining below the subsequently identified as mitochondria, which are known to levels seen in untreated DISC1-KO cells. We found that the accumulate at the immunological synapse in a microtubule- fluorescent intensity around the synapse was reduced by 42% in dependent manner (He et al., 2019; Maccari et al., 2016; DISC1-KO cells compared with the WT, subtracting for background Quintana et al., 2007). Isoform L concentrated around the MTOC and using LatB-treated cells as a baseline for 0% (n=10; P<0.05). in unstimulated Jurkat cells but also became localized to the synapse This suggests that some polymerized actin remains in the after stimulation with SEE-coated Raji cells (Fig. 1C). DISC1-KO cells. To explore the function of DISC1, we initially introduced siRNA to reduce DISC1 expression. The results showed that DISC1 Actin accumulation at the immunological synapse requires Girdin In searching for a functional connection between DISC1 and actin at the synapse, immunoprecipitation experiments revealed that Girdin was part of the DISC1 complex (Fig. 3A). Like DISC1, Girdin also accumulated at the synapse of Jurkat cells activated by SEE-coated Raji cells (Fig. S4A,B). To determine whether Girdin was needed for actin accumulation at the synapse, a CRISPR/Cas9 construct targeted to Girdin was used to disrupt the gene. Chemical selection and cell sorting were then used to obtain a pure Girdin knockout cell line (Girdin- KO). Absence of Girdin expression was verified by immunoblotting and by absence of any accumulated immunofluorescence at the synapse (Fig. S4C–F). When Girdin-KO cells were paired with SEE-coated Raji cells, Fig. 1. Cellular distribution of DISC1. (A) Fluorescent images were obtained fixed and stained with phalloidin-TRITC, the normally obvious for WT Jurkat cells that were either unpaired or paired with SEE-coated Raji actin ring at the immunological synapse was not detected, similar to cells and immunostained for DISC1. (B) WT Jurkat cells expressing the observations for DISC1-KO cells (Fig. 3B,C). When Girdin was N-terminal DISC1Lv-eGFP construct that were either unpaired or paired with reintroduced into Girdin-KO cells as an eGFP-fusion protein, a SEE-coated Raji cells. (C) WT Jurkat cells expressing the N-terminal DISC1L- eGFP construct that were either unpaired or paired with SEE-coated Raji cells. repeat of the phalloidin-TRITC staining experiment showed that In all cell pairs, the presence of DISC1 is marked with a white arrow. R, Raji cell. actin was clearly present at the synapse (Fig. 3D). Plots of average

Scale bars: 10 μm. phalloidin-TRITC fluorescence for segments across the width of the Journal of Cell Science

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Fig. 2. DISC1 knockout and restoration. (A,B) WT Jurkat (A) or DISC1-KO (B) cells paired with SEE-coated Raji cells were fixed and stained for actin using phalloidin-TRITC and immunostained for talin using an FITC-conjugated secondary Ig. (C) Fluorescence intensities of phalloidin-TRITC-stained WT and DISC1-KO cell pairs plotted as mean±s.e.m. for strips 5 pixels (0.33 μm) wide starting at the contact site and moving to the rear of the Jurkat cell (n=30). A one-tailed Student’s t-test showed that the signal intensity of the first five bands was significantly different between WT and DISC1-KO cells (P<0.001). (D,E) DISC1-cloned cDNAs for isoforms L or Lv were introduced into the Jurkat DISC1-KO line. Subsequently, Jurkat-SEE-Raji pairs from cells expressing the L (D) or Lv (E) isoforms were stained with phalloidin-TRITC and immunostained for talin. (F–H) Confocal fluorescence micrographs showing the phalloidin-TRITC staining at the synapse for unpaired and paired WT Jurkat cells (F), DISC1-KO cells (G), and WT Jurkat cells treated with LatB (H). The arrow in G indicates residual actin at the synapse. Experiments A–E were performed three times, whereas experiments F–H were performed twice. R, Raji cell. Scale bars: 10 μm. synapse were generated from 30 WT cells, Girdin-KO cells or (Fig. 4C). When DISC1Lv was expressed in DISC1-KO cells, the Girdin-KO cells expressing Girdin-eGFP (Fig. 3E). Comparisons of distribution of dynein and its associated proteins remained in the the first five segments showed a significant difference between WT center (Fig. 4D). In Girdin-KO cells, dynein was confined to a and Girdin-KO cells (P<0.001) and no significant difference central position, resembling the distribution in DISC1-KO cells between WT cells and Girdin-KO cells expressing Girdin-eGFP (Fig. 4E). (P>0.05). We repeated the experiment, but co-immunostained for NDE1 and LIS1 (Fig. 4F–J). We found that NDE1–LIS1 similarly formed Localization of dynein, NDE1 and LIS1 at the pSMAC requires a peripheral ring at the synapse in WT cells, which was not seen in DISC1 DISC1-KO and Girdin-KO cell lines. This ring formation was Previous studies have shown that dynein complexes similarly recovered by reintroducing the DISC1L-eGFP construct immunoprecipitated from Jurkat cells contained NDE1 and LIS1 but not the DISC1Lv-eGFP construct. Taking 30 cells of each type, and that all three proteins form a ring-like pattern corresponding to we grouped cells based on whether they formed a ring, a central spot the pSMAC (Nath et al., 2016). To determine whether DISC1 was or a miscellaneous pattern (Fig. 4K) and compared the results with a needed to recruit the NDE1–LIS1 complex to the immunological chi-squared test. This showed that WT cells were significantly synapse, WT and DISC1-KO Jurkat cells were immunostained for different in actin ring formation compared with DISC1-KO cells, χ2 NDE1 and LIS1 (Fig. S5A,B). Images showed that both NDE1 and (1, N=30)=28.1, P<0.001; DISC1-KO cells expressing DISC1Lv- LIS1 accumulated at the synapse in the absence of DISC1. When the eGFP, χ2 (1, N=30)=28.8, P<0.001; and Girdin-KO cells, χ2 average NDE1 fluorescence was plotted for segments across the (1, N=30)=11.6, P<0.01. Interestingly, in Girdin-KO cells, loss of cell, the average NDE1 fluorescence at the Jurkat–Raji junction was the ring-like distribution of NDE1 and LIS1 was not as severe as that slightly but significantly different across the first five segments from seen for DISC1-KO cells. WT cells showed no significant the synapse (Fig. S5C; P<0.05). difference in ring distribution compared with DISC1-KO cells To investigate further how these dynein-associated proteins were expressing DISC1L-eGFP, χ2 (1, N=30)=0.33, P>0.05. distributed at the synapse, Jurkat cells were settled on anti-TCR- We next looked at the distribution of the dynein–NDE1 complex coated coverslips, fixed and immunostained for the dynein using talin as a marker for the pSMAC. We also used interference intermediate chain (DIC). In WT Jurkat cells, dynein showed a reflection microscopy (IRM) to image the total adhesion area ring-like pattern (Fig. 4A). In the DISC1-KO cells, dynein was between the cell and the coverslip. In WT cells, we found that NDE1 confined to the central region of the synapse (Fig. 4B). When and talin colocalize at the pSMAC (Fig. 4L). In DISC1-KO cells, an DISC1L-eGFP was expressed in DISC1-KO cells, the distribution apparent pSMAC still formed, as evidenced by the ring of talin, but of dynein was essentially identical to that seen for WT Jurkat cells NDE1 remained at the center of the synapse (Fig. 4M). Journal of Cell Science

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DISC1 forms a complex with talin in Jurkat cells upon TCR stimulation The data in Fig. 4 show that the dynein complex ultimately formed a ring at the pSMAC when Jurkat cells were bound to TCR-coated coverslips. However, the dynein complex failed to locate at the pSMAC in the absence of DISC1. Given that the pSMAC is often defined in terms of LFA-1 clustering, we looked for a direct link between DISC1 and LFA-1. Unfortunately, data from cell sorting, western blots and attempts to visualize ICAM (intercellular cell adhesion molecule) clustering on supported lipid bilayers all indicated that LFA-1 was expressed at very low levels on Jurkat cells. As such, we were not successful at immunostaining for LFA-1 or detecting LFA-1 in DISC1 immunoprecipitates. However, interference reflection microscopy showed that the contact site formed on supported lipid bilayers was substantially smaller in DISC1-KO cells than in WT Jurkat cells (Fig. S7). Furthermore, to the extent that it could be discerned, ICAM clustering in DISC1-KO cells was confined to a smaller ring than seen for WT Jurkat cells or DISC1-KO cells expressing isoform L. Although we could not show that DISC1 binds to LFA-1, we were able to show an association between DISC1 and talin, which is known to bind LFA-1 (Tadokoro et al., 2003; Simonson et al., 2006). We immunostained Jurkat–Raji pairs for both DISC1 and talin and found that they colocalized at the synapse (Fig. 5A). We also found that DISC1 and talin co-immunoprecipitated when Jurkat cells were first treated with anti-TCR Ig (Fig. 5B). To determine which DISC1 isoform bound to talin, we used anti-GFP antibody to pull down each DISC1 isoform and then probed for talin on blots (Fig. 5C). The results showed that talin is primarily associated with isoform L.

Actin inhibitors block the recruitment of dynein and MTOC translocation to the synapse The loss of actin and failure of the dynein complex to localize at the pSMAC suggested that DISC1 might be linked to actin, perhaps through Girdin. To test this idea, we treated Jurkat cells with either CytB or LatB to disrupt actin assembly and then immunostained Fig. 3. Girdin in Jurkat cells. (A) DISC1 immunoprecipitates (IP) were probed – on blots for Girdin and DISC1. Beads lacking DISC1 antibody (Neg) showed Jurkat Raji pairs for members of the dynein complex. The results no obvious nonspecific binding. Analysis of the supernatant (Sup) from the IP show that treatment with either drug results in the loss of all showed that some Girdin still remained but DISC1 was not detected. Both members of the dynein complex (dynein, NDE1, LIS1) from the DISC1 and Girdin were present in the supernatant of lysates treated with beads synapse (Fig. 6A,B). These results were confirmed by comparing only. (B–D) WT Jurkat cells (B), Girdin-KO cells (C) and Girdin-KO cells average NDE1 fluorescence plotted from 30 treated and 30 transfected with an Girdin-eGFP construct (D) were paired with SEE-coated untreated cells (Fig. 6C). From the segment-by-segment analysis Raji cells and fixed. Each cell type was then stained for actin with phalloidin- of the plots, we found that fluorescence was significantly TRITC and immunostained for Girdin using an FITC secondary Ig. Experiment A was performed twice and experiments B–D were performed three times. diminished in the first five segments of both CytB- and LatB- (E) The fluorescence intensity of phalloidin-TRITC staining was plotted treated cells compared with WT cells (P<0.001). as the mean±s.e.m. (n=30). A one-tailed Student’s t-test showed that the To test how disrupting actin polymerization affects MTOC signal intensity of the first five bands was significantly different between translocation, we immunostained Jurkat–Raji conjugates for tubulin WT and Girdin-KO cells (P<0.001) but not between WT and Girdin-KO cells to compare MTOC position in the presence or absence of actin- μ expressing Girdin-eGFP (P>0.05). R, Raji cell. Scale bars: 10 m. disrupting drugs (Fig. 7A–D). The distance of the MTOC was recorded for 33 cells of WT and DISC1-KO cell types, as well as for We next sought to track the temporal movements of the DISC1 and CytB- and LatB-treated cells (Fig. 7E). In WT cells, the distance of the dynein complexes. For these studies, DISC1L-eGFP was expressed the MTOC was an average of 1.03±0.47 µm away from the synapse. in WT and DISC1-KO cell lines (Fig. S6). Because NDE1 is In the absence of DISC1, the MTOC was an average of 2.05± complexed to and colocalizes with dynein, we used the NDE1- 1.22 µm away from the synapse, a small but significant difference mCherry chimera to follow the dynein complex in WT, DISC1-KO, or compared with the distance in WT cells (P<0.001). However, for DISC1-KO cells expressing the eGFP fusions of either DISC1L or Lv. cells treated with either CytB or LatB, the MTOC remained much In WT Jurkat cells settled on anti-TCR coated coverslips, we found that further away from the synapse than in WT (3.62±1.86 µm for CytB DISC1L-eGFP and NDE1-mCherry initially accumulated at the center and 4.34±1.80 µm for LatB) indicating that little or no MTOC of the synapse before spreading out into a ring corresponding to the translocation had taken place (P<0.001). Finally, we saw no pSMAC. In the absence of DISC1, NDE1-mCherry failed to spread significant difference in MTOC position between CytB and LatB into a ring and remained at the center of the immunological synapse. treatments (P>0.05). Journal of Cell Science

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Fig. 4. Jurkat cells on anti-TCR-coated coverslips. (A–J) To monitor the movements of NDE1, LIS1, and dynein during Jurkat cell activation, cells were settled on coverslips coated with anti-TCR Ig. After 15 min incubation, cells were fixed and then some cells were immunostained for DIC using a FITC secondary Ig (A–E) whereas others were immunostained for NDE1 using an AlexaFluor 594 secondary Ig and LIS1 using an FITC secondary Ig (F–J). Each set of immunostaining procedures for either DIC or NDE1 and LIS1 was repeated for WT Jurkat cells (A,F), DISC1-KO cells (B,G), DISC1-KO cells expressing isoform L-eGFP (C,H), DISC1-KO cells expressing DISC1Lv-eGFP (D,I) and Girdin-KO cells (E,J). Merged composite images show NDE1 in red and LIS1 in green. (K) Fluorescence images were classified as having a ring-like staining pattern (Ring), a central patch (Spot) or neither pattern (Misc) from the interface in NDE1-stained cells (n=30). Results were averaged from multiple experiments and plotted as mean±s.e.m. Chi-squared tests were used to show the significant difference in ring formation between WT and DISC1-KO cells (P<0.001), WT and DISC1Lv-eGFP- expressing DISC1-KO cells (P<0.001), or WT and Girdin-KO cells (P<0.001). WT and L-eGFP-expressing DISC1-KO cells were not found to be significantly different (P>0.05). (L,M) WT Jurkat (L) and DISC1-KO (M) cells were settled onto Vβ8-coated coverslips, immunostained for NDE1 and talin, and imaged using confocal microscopy. Experiments A–J were performed four times and experiment L,M was performed twice. Scale bars: 10 μm.

DISCUSSION activation of Jurkat cells. The Lv isoform accumulates around Previous studies have shown that polarization of the microtubule mitochondria, where it is known to bind the mitochondrial proteins cytoskeleton begins with a group of projecting to the Trak and Miro (James et al., 2004; Norkett et al., 2016). As we will central region of the interface between a T cell and a target (Kuhn detail in a separate study, DISC1Lv is required for accumulation of and Poenie, 2002). These microtubules then fan out to form a mitochondria near the immunological synapse. hollow cone that projects from the MTOC to the pSMAC. It was Our results show that when Jurkat cells are activated by SEE- noted that microtubules exhibit sharp bends in the zone where coated Raji cells, DISC1L, Girdin and members of the dynein LFA-1 is clustered, and we speculated that these bends might be due complex first accumulate at or near the central region of the nascent to dynein. Tracking of the MTOC showed that, initially, it moves immunological synapse and then become associated with the linearly towards the center of the contact site but begins oscillating pSMAC. CRISPR-mediated deletion of either DISC1 or Girdin laterally as it nears the immunological synapse. Subsequently, results in a loss of bulk actin accumulation at the immunological dynein forms a ring that colocalizes with ADAP, a protein that synapse and failure of the dynein complex to form a peripheral ring together with SKAP55 plays an important role in formation of the corresponding to the pSMAC. pSMAC (Combs et al., 2006). This ADAP complex colocalizes To examine the specificity of the CRISPR-mediated deletion of with the pSMAC by activating integrin clustering and binding to Girdin, a Girdin-eGFP construct was introduced into the Girdin-KO integrin complexes (Kliche et al., 2006; Wang et al., 2009; Burbach line and this restored the accumulation of actin at the synapse. et al., 2011). Finally, our previous work showed that dynein at the Adding back DISC1Lv-eGFP did not restore actin at the synapse nor immunological synapse is found in a complex with NDE1, LIS1 and association of the DISC1–dynein complex with the pSMAC. DISC1 (Nath et al., 2016). We also showed that incompletely Expression of DISC1L-eGFP showed that this isoform could bind to functional NDE1 could localize to the synapse in the absence of talin and restore accumulation of the NDE1–dynein complex at the dynein, but that fully functional NDE1 is required to bind and pSMAC, but does not support the polymerization of actin. recruit dynein. Expression of the DISC1L isoform without the fused eGFP was The present study explores the function of DISC1 in more detail. able to restore actin accumulation at the immunological synapse. We We began by showing that there are two isoforms of DISC1 suspect that eGFP placed at the N terminus of DISC1, near where expressed in Jurkat cells (L and Lv). We focused primarily on the L Girdin binds, interferes with the binding or function of Girdin isoform that accumulates at the immunological synapse upon (Enomoto et al., 2009). It should also be noted that we tested a Journal of Cell Science

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The dramatic decrease in actin in the absence of DISC1L or Girdin, as well as the failure of the dynein complex to relocate to the pSMAC initially, suggests that these two events are linked. This idea was supported by data showing that CytB or LatB can reduce synaptic phalloidin staining below that seen for the DISC1-KO or Girdin-KO lines and that these treatments appear to eliminate the dynein complex from the synapse. One potentially attractive model is that the early DISC1–dynein complex is linked to a small patch of actin at the center of the immunological synapse and progresses to the periphery on a wave of actin polymerization. This appears unlikely given what is known about actin polymerization at the synapse, where formation of the actin network begins at the periphery instead of spreading outward. Although there is some evidence suggesting that actin is needed to recruit dynein to the synapse, at present we do not know how the initial dynein complex is anchored to the central region of the synapse. The results obtained when DISC1L-eGFP was expressed in the DISC1-KO cells show that dynein relocates to the pSMAC in the absence of the actin ring. Thus DISC1, if linked to actin, is not linked to the same set of actin filaments that Girdin generates. In principle, DISC1 could bind to actin through LIS1. LIS1 interacts with actin filaments through the actin-binding protein IQGAP and also interacts with the actin guanine-nucleotide exchange factors (GEFs) Cdc42, Rac and RhoA. Additionally, formins are required for MTOC translocation, so formin-generated actin filaments might link actin to the DISC1 complex (Gomez et al., 2007). Formins are needed to generate the concentric arcs of actin seen in the pSMAC. Fig. 5. DISC1 interaction with talin. (A) WT Jurkat cells were paired with SEE- Because these actin arcs are linked to LFA1, and DISC1 is bound to coated Raji cells, fixed, and immunostained for DISC1 using an AlexaFluor 594 talin, this seems to be a plausible connection (Murugesan et al., secondary Ig and for talin using an FITC secondary Ig. (B) DISC1 was 2016). However, we note that formins are also involved in immunoprecipitated (IP) from WT Jurkat cells that were either treated with anti- microtubule acetylation, which in turn can affect MTOC TCR Ig (+) or left untreated (−). The immunoprecipitates were then probed on blots for talin and DISC1. (C) eGFP-tagged DISC1 was immunoprecipitated translocation, so this issue needs to be resolved (Thurston et al., from lysates of WT cells, DISC1-KO cells or DISC1-KO cells (DISC1L-eGFP or 2012). DISC1Lv-eGFP). All cells were treated with anti-TCR Ig prior to lysis. These From our studies, the single biggest defect we see in this loss of immunoprecipitates were then probed on blots for talin and DISC1. As a actin is a failure of the cell to spread, as seen from interference control, the beads used for the immunoprecipitations were used without the reflection microscopy on supported lipid bilayers. The use of ICAM anti-eGFP antibody. These experiments were all performed twice. R, Raji cell. as a marker on these lipid bilayers shows that the diameter of the Scale bars: 10 μm. LFA-1 ring, insofar as one can see it, is also reduced. This is construct in which eGFP was placed at the C terminus of DISC1. consistent with earlier studies showing that polymerization of actin This construct failed to accumulate at the synapse. It is known that drives spreading of the contact site (Bunnell et al., 2001; Barda- the C terminus of DISC1 contains binding regions for NDE1 and Saad et al., 2005). More recently, actin polymerization at the LIS1 (Burdick et al., 2008; Sanchez-Pulido and Ponting, 2011), so synapse has been shown to depend on WAVE2 and associated in this case the eGFP tag may interfere with DISC1 binding to the proteins (Nolz et al., 2006; Zipfel et al., 2006). Blocking the ability dynein complex. of the WAVE2 complex to activate ARP2/3 also blocks spreading. In both the DISC1 and Girdin KO cell lines, the NDE1–dynein This spreading defect could be one possible explanation for the complex remains in an approximately central location at the failure of the NDE1–dynein complex to form a peripheral ring in the synapse. This dynein complex is sufficient to draw the MTOC close absence of DISC1. In this case, the failure to spread could be a but not all the way to the immunological synapse. Indeed, the physical barrier that prevents outward relocation of the dynein difference in MTOC position seen in WT Jurkat and DISC1-KO complex to the pSMAC. To examine this question, we used cells is subtle and was initially missed. However, the MTOC–cell coverslips coated with anti-TCR Ig and poly-L-lysine where Jurkat edge measurements on at least 30 cells of each category show a cells bind and form a larger contact area (Bunnell et al., 2001). significant difference. When Jurkat cells were treated with either We also immunostained these preparations for talin to identify CytB or LatB and then paired with SEE-coated Raji cells, the dynein the putative pSMAC. The results show that in the absence of DISC1 complex was absent from the synapse and the MTOC failed to the NDE1–dynein complex remains confined to the central region translocate. These results support the idea that the NDE1–dynein of the synapse, even though a peripheral ring of talin is evident. complex might be directly or indirectly linked to actin, and explains Thus, spreading as a physical barrier might be necessary for the why dynein-dependent translocation of the MTOC to the synapse is peripheral spread of the dynein complex, but that alone is not blocked by treatment with these drugs (Filbert et al., 2012; Orange sufficient. et al., 2003; Wulfing et al., 2003). However, although a small At present, it is also not clear how the DISC1–Girdin complex amount of actin in the center of the immunological synapse has been regulates actin polymerization. Girdin is essential for cytoskeletal reported, we were not able to detect it by phalloidin staining reorganization in a number of systems (Enomoto et al., 2005; Wang

(Sanchez et al., 2019; Murugesan et al., 2016). et al., 2018; Gu et al., 2014). Furthermore, there are several reports Journal of Cell Science

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Fig. 6. Effects of actin inhibitors on the dynein complex at the immunological synapse. (A,B) WT Jurkat cells were treated with DMSO, CytB or LatB and paired with SEE-coated Raji cells. Cells were then fixed and stained with phalloidin-TRITC (actin) and immunostained for either NDE1 (A) or DIC using a FITC secondary Ig (B). (C) The fluorescence intensity of NDE1 immunostaining was analyzed from images of CytB-treated, LatB-treated and DMSO-treated cell pairs, plotted as the mean±s.e.m. (n=30). A one-tailed Student’s t-test showed that the signal intensity of the first five bands was significantly different between DMSO- treated and CytB- or LatB-treated cells (P<0.001). This experiment was performed twice. R, Raji cell. Scale bars: 10 μm.

showing that reduced Girdin expression leads to a reduction in The results obtained here complement those of Kuhn and polymerized actin (Gu et al., 2014; Wu et al., 2016; Enomoto et al., Poenie (2002). In that study, microtubules were seen to initially 2005). There are also studies showing that depletion of DISC1 is concentrate at the center of the synapse and then fan out to form a associated with a reduction of actin, although this might also be a hollow cone that projects to the pSMAC. In tracking MTOC result of the link between DISC1 and Girdin (Steinecke et al., 2014). translocation to the immunological synapse, they found that the There are several possible ways that Girdin could be involved in MTOC initially moved linearly toward the synapse. Then, as pathways that stimulate actin polymerization. Girdin is a non- the MTOC came near the immunological synapse, it began to receptor GEF for the Gαi group of trimeric G proteins and there is oscillate laterally. Here we expand on those results and propose a evidence that it links activation of receptor tyrosine kinases to revised model for translocation of the MTOC to the immunological signaling through these G proteins (Lin et al., 2014). T cells are synapse (Fig. 8). We have shown that the NDE1–dynein complex known to express Gαi, which is important for T cell development, first accumulates at the center of the immunological synapse. so this signaling pathway could be in play (Hwang et al., 2017; Dynein at this central location would tend to pull the MTOC Leyme et al., 2016). Another possible signaling pathway is through straight towards the immunological synapse. Then, the dynein the binding of DISC1 to the Ras effector RASSF7, which could play complex moves peripherally to associate with the pSMAC. At this a role in activating the Ras signaling pathways (Wang et al., 2016). It point, the MTOC would be at the center of the dynein ring and should be noted that Girdin was first identified as an enhancer of opposing dynein forces would act to pull the MTOC laterally in an AKT signaling (Enomoto et al., 2005; Anai et al., 2005) and might oscillating manner. However, in the absence of DISC1, the dynein be part of a FAK-dependent positive feedback loop whereby complex is unable to move peripherally and the MTOC is not integrin stimulation leads to activation of phosphoinositide 3 (PI-3) brought directly adjacent to the center of the synapse by opposing kinase (Leyme et al., 2016). Regardless of how it takes place, it is dynein forces. clear that Girdin serves as a hub for amplifying signaling through A final point that should be mentioned concerns the role of the PI-3 kinase/AKT pathway (Lin et al., 2014, 2011; Enomoto DISC1 in schizophrenia. Up to this point, almost all studies of et al., 2005; Ni et al., 2015; Wu et al., 2016). DISC1 have focused on its role in the brain and in Of the various signaling pathways in which Girdin participates, neurons. However, the connection between schizophrenia and the ones that appears most closely related to actin signaling are the immune system has recently come to the fore. As Debnath signaling through the Ras pathway and the PI-3 kinase pathway (Le stated in a review, “immunopathogenesis has emerged as one of Floc’h et al., 2013; Hammer et al., 2019). Interestingly, Le Floc’h the most compelling etiological models of schizophrenia” and colleagues suggest that the Ras and PI-3 kinase pathways are (Debnath, 2015). Although models relating immune effects to early signaling steps that lead to actin polymerization (Le Floc’h schizophrenia are largely correlative, this study provides a et al., 2013). Clearly, more work needs to be done to understand the concrete link between a known genetic risk factor for role of the DISC1–Girdin complex in regulating actin. schizophrenia and T cells. Journal of Cell Science

7 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs242875. doi:10.1242/jcs.242875

Fig. 8. Model for the role of DISC1 in MTOC translocation. (A) The DISC1 and the dynein complexes become concentrated at a central location at the nascent immunological synapse where they are possibly linked to actin. At this stage, dynein can draw the MTOC linearly towards the synapse. (B) Subsequently, congruous with cell spreading, the DISC1–dynein complex becomes linked to talin and LFA-1 at the pSMAC. (C) In the absence of DISC1, dynein still concentrates at a central region and can draw the MTOC towards the synapse. (D) In the absence of DISC1, cells do not spread and the dynein complex remains concentrated at a central location at the synapse. In this case, compared to WT cells, the MTOC does not come all the way to the synapse.

Fig. 7. Effects of actin inhibitors on MTOC polarization. (A–D) Jurkat cells were treated with CytB, LatB or DMSO alone for 30 min and paired with Cell lines, reagents and antibodies SEE-coated Raji cells. Cells were then fixed and stained with phalloidin-TRITC for actin and immunostained for β-tubulin (B-tub) using a FITC secondary Ig to The Jurkat (E6.1), Raji and Daudi cell lines were obtained from the track the MTOC. The results are shown for WT cells (A), DISC1-KO cells American Type Central Collection. The EL-4 cell line was obtained from treated with DMSO (B), WT cells treated with CytB (C), and WT cells treated Dr Anne-Marie Schmitt-Verhulst (Centre d'Immunologie de Marseille- with LatB (D). (E) The position of the MTOC relative to the immunological Luminy, Marseille, France). OT-1 splenocytes were obtained from synapse was measured for each cell type (n=33), as described in the Materials Dr Lauren Ehrlich (Molecular Biosciences, University of Texas at and Methods, and arranged in groups by its distance from the immunological Austin). These C57BL/6-Tg(TCRaTCRb)1100Mjb/J (OT-I) mice were synapse (in micrometers). A one-tailed Student’s t-test showed that the sourced from Jackson Laboratories and bred in house. All strains were bred distance between the synapse and the MTOC was significantly different and maintained under specific pathogen-free conditions in the University of between WT, DISC1-KO and CytB- or LatB-treated cells (P<0.001). This Texas at Austin animal facility. Experiments were performed using mice 1- experiment was performed three times. R, Raji cell. Scale bars: 10 μm. 3 months of age of mixed sex. Mouse maintenance and experimental procedures were carried out with approval from the Institutional Animal Care and Use at the University of Texas at Austin. MATERIALS AND METHODS Opti-MEM cell media was obtained from Gibco Thermo-Fisher (Cat Design # 31985062). Heat-inactivated fetal bovine serum (FBS) was obtained from The goal of this study was to characterize the role of DISC1 in the Atlas Biologicals (Cat # F-0500-D). The 4 mm gap transfection cuvettes formation of the immunological synapse. Images in each experiment were were obtained from Fisher Scientific (Cat # FB104). Goat serum (Cat taken under the same conditions and brightness/contrast was adjusted to # G2093), poly-L-lysine (Cat # P2636) and cytochalasin B (Cat # C6762) the same degree and in the same manner for all representative images of the were obtained from Sigma-Aldrich. SuperSignal West Pico same experiment. Cell selections were imaged randomly in all chemiluminescent substrate solution (Cat # 34580) and X-ray film (Cat experiments. Measurements of cell pairs were conducted blind of the # 34090) were obtained from Thermo Scientific. G418 Sulfate was experimental groups being measured. For all experiments, a minimum of purchased from Gold Biotechnology (Cat # G-418-5). All restriction 30 cells or cell pairs were used per experimental group. The minimum enzymes were obtained from New England Biolabs. Mini Plasmid and Midi number of cell pairs needed to avoid a type II (nonrejection of a false Fast Ion Plasmid Kits were obtained from IBI Scientific (Cat # IB47111 and hypothesis) error was established as 30 through power analysis IB47111). Xfect transfection reagent was obtained from Clonetech (Cat calculations made using the R programming environment. For each # 631318). ProLong Gold Anti-Fade Mounting Reagent was obtained from experiment, a value for power of at least 80% could be expected. Life Technologies (Cat # P36930). The Cas9, DISC1 and Girdin sgRNA Journal of Cell Science

8 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs242875. doi:10.1242/jcs.242875 plasmids were obtained from Genecopoeia (Cat # CP-LvC9NU-02-B, standard PCR protocol to insert several additional restriction sites into the HCP268459-LvSG03-1-B and HCP259879-LvSG03-1-B). Partially multicloning site. purified staphylococcal enterotoxin E (SEE) was obtained from Toxin All DNA constructs were transformed from frozen aliquots of competent Technologies (Cat # ET404). bacteria suspended in CaCl2 solution. These aliquots were thawed and DISC1 rabbit polyclonal antibody (Cat # PA2023) and Talin mouse mixed with DNA constructs, then put through heat shock at 43°C. monoclonal antibody (Cat # MA1092) were obtained from Boster Biological. Depending on the concentrations needed, DNA constructs were isolated CCDC88A (Girdin) rabbit polyclonal antibody (Cat # A16132) was obtained using the Mini Plasmid or Midi Fast Ion Plasmid Kits and plasmid from Abclonal. LIS1 mouse monoclonal antibody (Cat # L7391), eGFP sequences were verified by Sanger sequencing. DNA was introduced into polyclonal rabbit antibody (Cat # G1544), Dynein (intermediate chain) mouse Jurkat cells through electroporation. For the transformation, Jurkat cells antibody (Cat # D5167) and β-tubulin mouse monoclonal antibody (Cat # were washed and resuspended in Opti-MEM reduced serum medium at a T8328) were obtained from Sigma-Aldrich. The TCR Vβ8 mouse monoclonal concentration of 2×107 cells/ml and incubated with 10 µg of plasmid DNA antibody was obtained from BD Biosciences (Cat # 555604). Rabbit anti- for 15 min at 37°C. Cells were placed in 4 mm gap transfection cuvettes NDE1 antibody was obtained from Proteintech Group (Cat # 10233-1-AP). and pulsed at 250 V (950 µF) using the Gene Pulser Electroporation Goat anti-rabbit AlexaFluor 594-conjugated antibody (Cat # A11037) and System (Bio-Rad). After electroporation, cells were resuspended in fresh goat anti-mouse FIT-conjugated antibody (Cat # F2012) were obtained from ACC growth media. Cells containing DISC1 constructs were grown under Invitrogen. Goat anti-mouse IgM FITC-conjugated antibody (Cat # F2959), selection with 1 mg/ml G418, whereas cells containing NDE1 constructs goat anti-mouse horse radish peroxidase (HRP)-conjugated antibody (Cat # were grown under selection with 2 µg/ml puromycin. Selection began 24 h A9917) and goat anti-rabbit HRP-conjugated antibody (Cat # A0545) were post-transfection and continued for two weeks. Afterwards, cells were obtained from Sigma-Aldrich. Cell Tracker Blue (Cat # C2110) was obtained sorted for the expression of fluorescent proteins using the FACSAria cell from Invitrogen. TRITC-conjugated phalloidin (Cat # P-1951) was obtained sorter. from Sigma-Aldrich. CRISPR/Cas9 gene knockouts Cell culture A DISC1 or Girdin sgRNA plasmid and a Cas9 plasmid were transfected Jurkat cells and Raji cells were grown in RPMI 1640 supplemented with into the Gryphon viral packaging cell line using the Xfect transfection 24 mM sodium bicarbonate, 1 mM sodium pyruvate, 2 mM L-glutamine, reagent. Fresh growth medium was added 4 h post-transfection. At 48 h after 50 µM β-mercaptoethanol, 10,000 U/ml penicillin, 10 mg/ml streptomycin transfection, supernatants containing the viral particles were collected. For and 10% (v/v) FBS (ACC growth media). Gryphon cells were grown in transduction, 2×106 Jurkat cells in six-well plates were spinfected at 500×g DMEM supplemented with 44 mM sodium bicarbonate, 1 mM sodium and 30°C for 1 h with medium containing viral particles and 8 µg/ml pyruvate, 2 mM L-glutamine, 50 µM β-mercaptoethanol, 10,000 U/ml polybrene. Spinfection through centrifugation was repeated every 12 h for penicillin, 10 mg/ml streptomycin, and 10% (v/v) FBS. All cells were 36 h. After the final centrifugation, the medium was replaced with fresh cultured at 37°C in 5% CO2. growth medium. Successful transduction was confirmed through For expansion and stimulation of OT-1 cells, EL-4 cells were treated with observation of eGFP and mCherry fluorescent proteins expressed by the 50 µg/ml mitomycin C for 2 h, washed thoroughly and then treated with Cas9 and sgRNA plasmids. Complete knockout of DISC1 in culture was 1 µM Ova peptide. These EL-4 cells were then mixed with OT-1 achieved through FACSAria sorting of eGFP- and mCherry-expressing splenocytes or activated OT-1 CTLs, or used as targets in immunostaining cells, followed by verification through DISC1 western blotting of the experiments. OT-1 CTLs were maintained in ACC growth media resultant cells. Cells were grown under selection with 1 mg/ml G418 sulfate supplemented with 20 U/ml IL-2. and 2 µg/ml puromycin. Selection began 36 h post-transduction and continued for two weeks, until sorting was conducted with a FACSAria DNA constructs cell sorter. After sorting, selection was stopped. Cells were finally used for We began identifying possible DISC1 isoforms expressed in Jurkat cells by immunostaining or transfected with a new construct, after the loss of eGFP adapting an RT-PCR method used by Nakata et al. (2009). First, total Jurkat and mCherry fluorescence had been observed. mRNA was isolated using the RNeasy Midi Kit. This mRNA was converted into a cDNA library using a MMLV reverse transcriptase kit. Using eight Preparation of cell conjugates for staining different sets of primers, DISC1 exon fragments were identified from this To prepare coverslips for cell staining and fluorescence microscopy, cDNA library through PCR. Through this method, DISC1L and Lv isoforms coverslips were first cleaned with a 9:1 mixture of ethanol and 1 M KOH for were identified and verified through Sanger sequencing on an Applied 1 h. They were then washed in dH2O and coated with an aqueous solution of Biosystems 3730 DNA Analyzer (UT ICMB Core Facilities). 0.1 µg/ml 30,000-70,000 kDa poly-L-lysine. These were rinsed again in Full-sized DNA fragments for DISC1 isoforms L and Lv were dH2O and left to dry for 30 min at room temperature. synthesized through PCR of Jurkat cDNA. A tagless DISC1 construct To prepare Jurkat–Raji cell conjugates, Raji cells were suspended at a was made by making DNA fragments of DISC1 isoforms L and Lv concentration of 1×106 cells/ml in serum-free1 RPMI 1640 and treated containing the XhoI and XmaI restriction sites. These fragments were with SEE at 1 µg/ml for 1 h at 37°C. They were subsequently stained inserted into a peGFP-C1 vector to generate DISC1-eGFP constructs, or into with 10 µM Cell Tracker Blue for 15 min at 37°C in order to identify Raji a peGFP-N1 vector that contained a premature stop codon at the start of the cells from Jurkat cells during imaging. In experiments using CytB or eGFP sequence, created through site-directed mutagenesis. LatB, Jurkat cells were treated with 20 μg/ml CytB or 10 μMLatBfor Full-sized Girdin DNA fragments were derived from our Jurkat cDNA 30 min. Both Jurkat and Raji cells were washed with ACC media, paired library using PCR. A C-terminal eGFP-tagged Girdin construct was made at a ratio of 3:2 Jurkat to Raji cells and centrifuged at a light speed by first adding XhoI and XmaI restriction sites to the ends of Girdin DNA (500×g) for 5 min. Cells were then washed again with ACC and settled fragments. These fragments were then inserted into a peGFP-N1 plasmid. on poly-L-lysine-coated coverslips at a total concentration of Both peGFP-C1 and -N1 plasmids contain a monomeric eGFP sequence. 1×106 cells/ml for 15 min. PCR point editing was used to change amino acid 206 of the eGFP sequence To prepare TCR antibody-coated coverslips, a 10 µg/ml solution of from alanine to lysine. mouse Vβ8 anti-TCR antibody was coated over dried poly-L-lysine-treated The NDE1-mCherry construct was made by creating DNA fragments of coverslips for 3 h. Coverslips were then washed in three 10 min intervals the NDE1 coding sequence through PCR amplification of a pmeGFP-N1- with phosphate-buffered saline (PBS) and then either used directly or stored NDE1 template, as described by Nath et al. (2016). DNA fragments for up to 24 h at 4°C. Jurkat cells in ACC growth medium at a cellular contained AgeI and XbaI restriction sites on their 5′ and 3′ ends, which were concentration of 1×106 cells/ml were settled onto antibody-coated used to insert the sequence into the pIRES-PURO3-mCherry vector. The coverslips and used for imaging studies. pIRES-PURO3-mCherry vector was obtained from Dr Roger Tsien For immunostaining, cells settled on coverslips were fixed with PBS

(University of California San Diego), and subsequently modified using a containing 1% paraformaldehyde for 30 min before being washed with Journal of Cell Science

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PBS. Cells were then permeabilized with a 1:1 solution of ice-cold methanol Then, we measured the distance of the MTOC to the edge of the Raji cell, as and acetone for 15 min. Cells were washed again with PBS and blocked with determined through Cell Tracker Blue staining. This distance was recorded PBS containing 5% goat serum and 0.5% Tween-20 for 30 min. Cells were as micrometers converted from pixels (5 pixels=0.33 μm). A two-tailed then incubated with a 1:50 solution of primary antibody in blocking solution t-test with independent variance was then performed comparing the MTOC for 1 h. After primary antibody staining, the cells were then washed again polarization of 33 cell pairs from each cell type. Cell pairs of each cell type with PBS and incubated with a 1:100 solution of secondary antibody or were grouped and plotted on a graph depending on the degree of MTOC phalloidin-TRITC in blocking solution for another hour. After a final wash polarization. with PBS, the coverslips were mounted onto slides with ProLong Gold Cross-sectional imaging of the immunological synapse was taken with a antifade reagent overnight at room temperature before being stored long- Zeiss LSM 710 confocal microscope from the University of Texas ICMB term at −20°C. core facilities. Image processing was carried out by reslicing z-stacks of images using ImageJ. Immunoprecipitation and western blotting Jurkat cells meant to be stimulated prior to lysis and immunoprecipitation Acknowledgements were pelleted at 750×g and resuspended in RPMI at a cellular concentration We thank Dr Jeffrey Kuhn (currently Scientific Director, Microscopy Core Facility, of 1×106 cells/ml. Cells were treated with 500 ng/ml Vβ8 anti-TCR Koch Institute, Massachusetts Institute of Technology) for consultation and feedback given during the course of this project. We would also like to thank Dr antibody and incubated at 37°C for 30 min. After that, cells were pelleted at Lauren Ehrlich (Molecular Biosciences, University of Texas at Austin) for providing 750×g and resuspended in lysis buffer containing 200 mM NaCl, 50 mM OT-1 splenocytes, as well as Dr Jessica Lancaster (currently of the Mayo Clinic, Tris pH 8, 2 mM EDTA, 2 mM NaVO4, 20 mM NaF, 3 mM PMSF, 2 mM Phoenix, AZ) for extracting splenocytes and providing consultation for cell culture imidazole, 1 mM Na-β-glycerophosphate and 1% Triton X-100. The methods. suspension was then passed through a 21-gage needle repeatedly to homogenize it and then clarified at 16,000×g and 4°C for 10 min to remove Competing interests cell debris. The authors declare no competing or financial interests. To prepare beads for immunoprecipitation, 5 µg antibody was added to 300 µl PBS and 80 µl of a 50% slurry of Protein A agarose beads. This Author contributions solution was mixed gently on a rotator overnight at 4°C. The next day, beads Conceptualization: N.M., S.N., N.A., Y.S., M.P.; Methodology: N.M., S.N., N.A., Y.S., were washed with cell lysis buffer for 15 min and added to cell lysate made M.P.; Validation: N.M., A.R., M.P.; Formal analysis: N.M., M.P.; Investigation: N.M., from 1×107 Jurkat cells as previously described. Cells were then incubated S.N., A.R.; Resources: Y.S., M.P.; Data curation: M.P.; Writing - original draft: N.M., M.P.; Writing - review & editing: N.M., S.N., Y.S., M.P.; Visualization: N.M., M.P.; on a rotator at 4°C for 2 h. Afterwards, the beads were washed four times Supervision: N.M., S.N., N.A.; Project administration: M.P.; Funding acquisition: with PBS, diluted with SDS-PAGE loading buffer and boiled for 5 min. The M.P. lysate was then adjusted with SDS-PAGE loading buffer to a final β concentration of 2% (w/v) SDS and 5% (v/v) -mercaptoethanol. Funding Samples prepared in SDS-PAGE sample buffer were run through SDS- This work was supported in part by R01AI118694 National Institutes of Health grant PAGE and transferred to nitrocellulose paper for western blotting. Samples to Michael R. Betts, which includes sub-award 566950 to Y.S. Deposited in PMC for were blocked in a blocking solution of Tris-buffered saline with 0.1% Tween release after 12 months. and 5% BSA. Primary antibody was then added, diluted to a concentration of 1 µg/ml in blocking solution. Primary antibodies were tagged with HRP using Supplementary information a goat anti-rabbit or goat anti-mouse HRP-conjugated secondary antibody and Supplementary information available online at treated with SuperSignal West Pico chemiluminescent substrate solution https://jcs.biologists.org/lookup/doi/10.1242/jcs.242875.supplemental before being exposed to X-ray film. References Anai, M., Shojima, N., Katagiri, H., Ogihara, T., Sakoda, H., Onishi, Y., Ono, H., Preparation of supported lipid bilayers Fujishiro, M., Fukushima, Y., Horike, N. et al. (2005). A novel protein kinase B + Primary CD4 T cells were derived from peripheral blood mononuclear cells (PKB)/AKT-binding protein enhances PKB kinase activity and regulates DNA and monitored on lipid bilayers, as described by Steblyanko et al. (2018). synthesis. J. Biol. Chem. 280, 18525-18535. doi:10.1074/jbc.M500586200 The procedure was adjusted by preparing bilayers with Cy5-ICAM1-His6 at Aznar, N., Kalogriopoulos, N., Midde, K. K. and Ghosh, P. (2016). Heterotrimeric 1 µg/ml in order to eliminate the high background fluorescence of Cy5 G protein signaling via GIV/Girdin: breaking the rules of engagement, space, and caused by the relatively low levels of LFA-1 expressed on the surface of time. BioEssays 38, 379-393. doi:10.1002/bies.201500133 Babich, A., Li, S., O’connor, R. S., Milone, M. C., Freedman, B. D. and Jurkat cells. Burkhardt, J. K. (2012). F-actin polymerization and retrograde flow drive sustained PLCγ1 signaling during T cell activation. J. Cell Biol. 197, 775-787. Imaging and data processing doi:10.1083/jcb.201201018 Images were viewed using a Nikon inverted microscope and captured using Barda-Saad, M., Braiman, A., Titerence, R., Bunnell, S. C., Barr, V. A. and a CMOS camera (Andor). The images were processed and analyzed using Samelson, L. E. (2005). Dynamic molecular interactions linking the T cell antigen the ImageJ processing software. To determine protein accumulation at the receptor to the actin cytoskeleton. Nat. Immunol. 6, 80-89. doi:10.1038/ni1143 Basu, R., Whitlock, B. M., Husson, J., Le Floc’h, A., Jin, W., Oyler-Yaniv, A., synapse, a line of length 230 pixels and width 70 pixels was drawn on the – Dotiwala, F., Giannone, G., Hivroz, C., Biais, N. et al. (2016). Cytotoxic T cells Jurkat Raji cell pairs such that the mid-point of the line (pixel 115) was on use mechanical force to potentiate target cell killing. Cell 165, 100-110. doi:10. the synapse. Background intensity was obtained from a region outside the 1016/j.cell.2016.01.021 cell pairs and subtracted from the fluorescence intensity. The fluorescence Bunnell, S. C., Kapoor, V., Trible, R. P., Zhang, W. and Samelson, L. E. (2001). was normalized by dividing all the pixel measurements by the average Dynamic actin polymerization drives T cell receptor–induced spreading: a role for intensity of the row furthest away from the synapse. Beginning at the the adaptor LAT. Immunity 14, 315-329. doi:10.1016/S1074- synapse (row 115) and moving toward the opposite edge of the Jurkat cell 7613(01)00112-1 Burbach, B. J., Srivastava, R., Ingram, M. A., Mitchell, J. S. and Shimizu, Y. (row 1), intensity values for 5 pixel groups were treated as one increment (2011). The Pleckstrin homology domain in the SKAP55 adapter protein defines and used for statistical analysis (mean±s.e.m.). The compound mean and the ability of the adapter protein ADAP to regulate integrin function and NF-κB standard error for the increments was plotted against the mean intensity of activation. J. Immunol. 186, 6227-6237. doi:10.4049/jimmunol.1002950 fluorescence. A one-tailed t-test with independent variance was performed Burdick, K. E., Kamiya, A., Hodgkinson, C. A., Lencz, T., Derosse, P., Ishizuka, for the first five increments starting from the synapse and moving to the back K., Elashvili, S., Arai, H., Goldman, D., Sawa, A. et al. (2008). Elucidating the of the Jurkat cell. relationship between DISC1, NDEL1 and NDE1 and the risk for schizophrenia: evidence of epistasis and competitive binding. Hum. Mol. Genet. 17, 2462-2473. 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