PS, but not PIP2, levels are reduced in microclusters during early synapse formation. B-A8 T cells coexpressing Lact-C2-mRFP and PH-PLC-eGFP were injected into flow cells with lipid bilayers displaying HA-DR4 and ICAM-1. Synapse formation was imaged by TIRFM at 10-s intervals (3 min). (A) Images show early synapse formation (time after T cell contact is indicated in seconds). To simplify visualization, probe signals are also shown in grayscale. Images representative of n = 3 experiments. Bars 10 µm. (B) Pearson’s correlation coefficient calculated for each zoomed image using all fluorophore combinations to examine spatial relationships between distribution of HA-DR4 microclusters, Lact-C2-mRFP, and PH-PLC-eGFP (r, correlation factor). (C) Spatial relationships between HA-DR4 (blue), Lact-C2-mRFP (red), and PH-PLC-eGFP (green) fluorescence were examined by connecting several microclusters (dotted line in A) with line tool.
