In this study, using Jurkat cells, we show that DISC1 (Disrupted in Schizophrenia 1), and Girdin (Girders of actin filaments) are essential for typical actin accumulation at the immunological synapse. Furthermore, DISC1, Girdin, and dynein are bound in a complex. While initially this complex is seen as a central patch at the synapse, it relocates to a peripheral ring corresponding to the pSMAC. In the absence of DISC1, actin accumulation at the synapse is disrupted while dynein and the dynein-binding protein NDE1 fail to reorganize to the pSMAC. A similar effect is seen when Girdin is deleted. When cells are treated with inhibitors of actin polymerization, the dynein-NDE1 complex is lost from the synapse and the MTOC fails to translocate, suggesting that actin and dynein may be linked. Upon TCR stimulation, DISC1 becomes associated with talin which likely explains why the dynein complex colocalizes with the pSMAC. These results show that DISC1-Girdin regulates actin accumulation, cell spreading, and the distribution of the dynein complex at the synapse.

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DISC1 (Disrupted in Schizophrenia-1) is a dynein-binding scaffold protein that interacts with many other proteins. In neurons, DISC1 is important for proliferation, migration, and signaling. We found that DISC1 is expressed in Jurkat cells and human NK cells and that it coimmunoprecipitates with dynein in Jurkat cells. Immunostaining for DISC1 in unstimulated Jurkat cells showed that it was generally located around the MTOC. When Jurkat cells were stimulated with SEE-coated Raji cells, depending on the antibody used, we found DISC1 either clustered around mitochondria or colocalized with dynein and NDE1 at the immunological synapse (IS). Biochemical studies showed that after Jurkat cells are activated (SEE-coated Raji cells or PMA), DISC1 coimmunoprecipitates with talin and paxillin. Sequence analysis of cloned DISC1 cDNAs from Jurkat cells revealed that at least two isoforms (L and Lv) are expressed. When GFP-DISC1 isoforms were individually expressed in Jurkat cells, we found that the Lv isoform concentrated around mitochondria whereas the L isoform colocalized with dynein at the synapse as seen in the immunostaining data. While RNAi was relatively ineffective at reducing DISC1 expression, we could abolish expression by using a DISC1-targeted CAS9/CRISPR construct. In these DISC1-disrupted cells, we found that mitochondria failed to accumulate at the immunological synapse and the MTOC did not appear to move all the way to the contact site (perhaps suggesting a defect in docking). When GFP-DISC1 Lv was expressed in the DISC1 CAS9/CRISPR cells mitochondria accumulation at the IS was restored. Similarly, expression of the GFP-DISC1 L restored the normal positioning of the MTOC upon T cell activation.

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