Figure 2
From: TMEM16F activation by Ca2+ triggers plasma membrane expansion and directs PD-1 trafficking
Plasma membrane internalization in TMEM16F-null Jurkat T cells treated with ionomycin. (A) TMEM16F-null Jurkat T cells were incubated with the cytoplasmic calcium indicator Fluo4-AM, then treated with 5 μM ionomycin for 15 minutes at 37 °C. Cells were then chilled on ice and stained with Annexin V (Anx V) to detect surface phosphatidylserine. Flow cytometry analysis shows intracellular calcium plotted against surface phosphatidylserine. (B) Single TMEM16F-null Jurkat T cells were patched with a glass micropipette loaded with cytoplasmic solution (see Materials and Methods) and incubated at 37 °C in Ringer’s solution. Cells were then treated with 5 μM ionomycin for the period shown. Measurements were made of total capacitance, Cm, which reflects plasma membrane area, and transmembrane conductance, Gm. The red trace shows the change in capacitance (∆Cm) compared to t = 0, while the blue trace shows Gm. A typical ∆Cm trace is shown, the error bar represents standard error of the mean (SEM) at 150 s post ionomycin addition (n = 10). In addition, the dye FM4-64, which binds reversibly to membranes, was added to the same patched cell and removed as shown. The solid trace shows total FM4-64 fluorescence of the cell, measured by a confocal microscope, relative to t = 0. Below are images of FM4-64 fluorescence taken using confocal microscopy at the time points shown (scale bar is 5 μm).
