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. 2019 Apr 5;8:e42475. doi: 10.7554/eLife.42475

Figure 5. The opto-ligand-TCR can be switched ON and OFF.

(A) GFP-PIFS-TCR cells were labeled with Indo-1 and calcium influx measured by flow cytometry. The arrow marks the addition of the stimuli indicated, and the grey rectangle the absence of any light. Results show one experiment of n > 3. (B) Calcium influx into GFP-PIFS-TCR cells stimulated with anti-Vβ3, PhyBt(660), PhyBt(740) or PBS was measured as in (A). Results show one experiment of n > 3. (C) GFP-PIFS-TCR Jurkat cells were incubated with PhyBt bound to sepharose beads after a 30 s 660 nm or 740 nm light pulse for 6 hr. Expression of CD69 was quantified by flow cytometry using an APC-labeled anti-CD69 antibody. Data points depict two experiments. (D) Calcium influx was measured as in (A). PhyBt(660) induced calcium influx (blue and orange lines). After 2 min a 1 s short pulse of 100% intensity 740 nm light (red break in the grey bar) terminated the calcium response (blue line). Addition of PhyBt(740) did not induce calcium influx (red line). Results show one experiment of n > 3.

Figure 5.

Figure 5—figure supplement 1. Characterization of the optogenetic PhyBt - GFP-PIFS-TCR system.

Figure 5—figure supplement 1.

(A) Calcium influx was only seen in cells expressing GFP-PIFS-TCR and treated with PhyBt(660) (orange line). Cells lacking GFP-PIFS-TCR (parental Jurkat, grey and blue lines) or treatment with PhyBt(740) (red line) did not lead to intracellular calcium mobilization. Stimuli addition is marked by an arrow and the illumination procedure by a bar above the graph (grey = dark); One representative experiment is depicted of n = 2. (B) Treatment of GFP-PIFS-TCR cells with 660 or 740 nm light alone in the absence of PhyBt did not induce calcium influx (grey and blue lines). Addition of PhyBt(660) elicited a calcium response, whereas PhyBt(740) did not. Stimuli addition is marked by an arrow and the illumination procedures by bars above the graph (grey = dark, orange = 660 nm, red = 740 nm); One representative experiment is depicted of n = 3. (C) In contrast to PhyBt(660) (orange line) the PhyB(660) monomers (blue line) did not stimulate calcium influx in the GFP-PIFS-TCR cells. As controls, both PhyB forms illuminated with 740 light (red and grey lines) did not evoke any calcium influx; One representative experiment is depicted of n = 3. Panels A, B and C are from the same experiment.