Fig. 2. Ca²⁺ gradients are formed on the melanoma cell side of the lytic synapse.

Conjugate formation was analyzed using spinning disk time-lapse microscopy at a time resolution of one image every 60 ms. D10 cells and CTLs were previously loaded with Fluo-4-AM and Tubulin Tracker Green, respectively. (A and B) Snapshots illustrating [Ca²⁺]_i in melanoma cells and MTOC in CTL (pseudocolor) after cell-cell contact. In (B), the three white arrows point at Ca²⁺ “hotspot” microdomains in a melanoma cell. (C) Scheme of Fluo-4-AM fluorescence intensity quantification strategy in synaptic versus distal area. (D) Measurement of Fluo-4-AM fluorescence intensity over time in the synaptic and distal areas in the cell-cell conjugate shown in (A). The time required to achieve half-maximum Fluo-4-AM intensity in D10 cells in the synaptic area and the distal area was quantified and used to calculate the Δtime. (E) The Δtime of [Ca²⁺]_i increase in synaptic versus distal area in melanoma cells was quantified using 22 conjugates from four independent experiments. Data are expressed as means ± SEM. See also movies S1 and S2. Of note, the threshold for the pseudocolor scale in these experiments has been set to minimize signal at baseline and provide optimal sensitivity for the presence and localization of the gradient that emerges upon synaptic attack. Images with baseline staining visible are shown in fig. S2.