n of myosin leads to slower TCR transport and diminishes signaling. The bigger question is whether force from myosin plays a direct role in the modulation of TCR signaling. Although actin, microtubule, and some molecular motors have all been shown to play important mechanical roles in T cell signaling, they are unlikely to directly transduce mechanical PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22180813 forces into biochemical AG-221 site signaling cascades. Our observation of a decrease in CasL phosphorylation in response to myosin inhibition suggests that CasL may be involved in a mechanical signal transduction process in T cells. While working out details of the possible regulatory pathways is well beyond the scope of this paper, CasL is clearly a candidate for relating myosin to TCR signaling pathways. Studies have shown that CasL may be a substrate for Fyn and Lck, two key tyrosine kinases in initiating TCR activation. Phosphorylated CasL can also bind to Src homology domains of signaling proteins, such as Crk, Cbl, and nucleotide exchange protein C3G, to regulate T cell Myosin IIA in Immunological Synapse Formation signaling. We observed the phosphorylation of CasL upon TCR ligation and its association with discrete TCR microclusters at the IS. Our results of calcium influx, ZAP-70 phosphorylation, and TCR microcluster formation all suggest that myosin is more important for sustained signaling than initiation. CasL might be involved in a feedback loop between myosin and multiple signaling pathways. While much remains to be uncovered concerning the nature of mechanical influences on TCR activation, our observation of differential CasL phosphorylation with myosin inhibition clearly pinpoints a starting point to look into. DNA constructs A plasmid containing enhanced green fluorescent protein fused to the calponin homology domain of utrophin was a gift of Dr. William Bement, University of Wisconsin, Madison, WI. The EGFP-UtrCH coding sequence was amplified using PCR and subcloned into a murine stem cell virus plasmid. A plasmid containing EGFP fused to the heavy chain of human non-muscle myosin IIA was provided by Dr. Robert Adelstein, National Institutes of Health, Bethesda, MD through Addgene.org , and the EGFP-NMHCIIA coding sequence was subcloned into pMSCVPuro plasmid. Materials and Methods Ethics statement All animal work was conducted with prior approval by Lawrence Berkeley National Laboratory Animal Welfare and Research Committee and performed under the approved protocol 17702. Reagents Histidine-tagged ICAM-1 and MHC Class II I-EK were expressed and purified as previously described. Briefly, secreted ICAM-1 with a decahistidine tag at its C terminus was expressed using the baculovirus expression system in High Five cells and purified using a Ni2+-NTA-agarose column. Secreted MHC with a hexahistidine tag at the C terminus of both a and b chains was similarly expressed and purified from S2 cells. Blebbistatin, ML-7 and jasplakinolide were purchased from EMD Chemicals. ZAP-70 antibody and p130Cas antibody were purchased from Cell Signaling. Animals AND X B10.BR transgenic mice, of both genders and of age between 616 weeks, were used as CD4+ cell donors. Mice were housed in a facility certified by AWRC, under continuous veterinary animal care with adequate water, food and comfort. Only AWRC veterinary certified researchers, who 
have passed specific animal handling tests for the procedure, were allowed to handle the mice. Retroviral transfection T cells were retrovirally transduced using sup
