Figure 3
From: TMEM16F activation by Ca2+ triggers plasma membrane expansion and directs PD-1 trafficking
Simultaneous surface phosphatidylserine exposure and plasma membrane expansion is followed by membrane vesicle shedding. (A) Jurkat T cells were loaded with the cytoplasmic calcium indicator Fluo4-AM, then treated with 5 μM ionomycin for 400 s at 37 °C in the presence of polylysine-rhodamine (K7r) which binds rapidly to exposed phosphatidylserine. Confocal microscope images of Fluo4-AM and K7r fluorescence in a single field of cells are shown (scale bar is 10 μm). (B) A single TMEM16F-null Jurkat T cells was patched with a glass micropipette loaded with cytoplasmic solution (see Materials and Methods), incubated at 37 °C in Ringer’s solution, then treated with 5 μM ionomycin at the time shown. Total capacitance, Cm, was measured and the red trace shows the change in capacitance (∆Cm) compared to t = 0. In addition, K7r was added to the same patched cell and removed as shown. The green trace shows total K7r fluorescence of the cell, measured by a confocal microscope, relative to t = 0. Below are images of K7r fluorescence taken using confocal microscopy at the time points shown (scale bar is 5 μm). (C) Wild-type or TMEM16F-null Jurkat T cells were treated with 5 μM ionomycin for 15 minutes in the presence of eFluor780, a plasma membrane impermeable dye. The cell preparations were analyzed by FACS. The events shown were first gated on subcellular sized particles using forward scatter and side scatter. Staining of these subcellular particles is displayed versus side scatter, with unstained events representing dye impermeable membrane vesicles rather than apoptotic bodies. The numbers shown represent the percentage of total events.
