Question 1: (4 points) A commonly used assay to measure Ca 2+ influx in response

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Question 1: (4 points) A commonly used assay to measure Ca2+ influx in response to TCR stimulation involves loading T cells with a Ca2+–sensitive dye, and then stimulating the TCR using anti-CD3 antibody coupled to biotin, followed by cross-linking with Streptavidin (S-Av). As the antibody and then S-Av are added, the cells are run on the flow cytometer to examine the fluorescence of the Ca2+-sensitive dye. After several minutes of analysis, the cells are stimulated with ionomycin (Iono), to induce Ca2+ influx; this is used as a positive control to ensure that the cells are loaded with the dye. In the figure below, the characteristic pattern of Ca2+ influx is shown in the red line (wild-type; WT). In this you line you can see where TCR stimulation causes a sharp rise in cytoplasmic Ca2+, followed by a slow decline over hours. Also as shown below, cytoplasmic Ca2+ concentrations do not normally return to baseline for the timecourse of this experiment. A mutant mouse is identified with a defect in T cell activation in response to TCR stimulation. The calcium response of T cells from the mutant mouse is shown in the blue line.

a) Given these data, name three T cell signaling proteins that could be defective in the mutant T cells. Then name three T cell signaling proteins that could not be responsible for this defect, even if mutated. (1.5 points)

aCD3-biotin Streptavidin lonomycin T cells 1 minute 00o 8 minutes ??? Cytoplasmic 5 T calcium concentration4 Wild-type Mutant 200 600 Time 400 Streptavidin lonomycin

Explanation / Answer

a) the three T cell signaling proteins taht coud be defective in the mutant T cell are: phospholipase C?1 (PLC?1), which it is implicated in the the hydrolysis of phosphatidylinositol-3,4-bisphospahte (PIP2) into inositol-1,4,5-trisphosphate (IP3). Other coul be the inositol triphosphate receptor (IP3R) a defective receptor will not interact with the specific antigen. And finally the calcium release activated Ca2+ channels (CRAC) which allows the relase of the calcium ion from the endoplasmic reticulum (ER). On the other hand, the proteins that are not responsable for this defect are: the Calmodulin (CaM), the Calcineurin and the STIAM1 and STIAM2.

The proteins in bold are related with the process of calcium releasement from the endoplasmic retoculum while the underline proteins interact with the calcium once in the cytosol.

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