Fig. 4.
Accumulation of activating NK cell protein MICA at nanotube junctions. (A) Bright-field image with the corresponding fluorescence of NKL (labeled with DiD, red) and P815/MICA-YFP, where MICA-YFP (green) has accumulated at nanotube junctions. This particular field of view is chosen to show several nanotubes, and boxed regions (A′, A′′) have been enlarged to allow better visualization of the accumulation of MICA-YFP. (B) Frequency of nanotubes wherein MICA accumulated for NKL (n = 14 independent experiments) and primary NK cells (n = 5 independent experiments) in coculture with P815/MICA-YFP. (C) Fold increase of MICA-YFP or mem-YFP at the nanotube junction was measured in comparison to the plasma membrane of the target cell body. Time-lapse microscopy of NKL (red) and P815/MICA-YFP (green) reveals that MICA accumulation can occur either before (D) or after (E) the formation of membrane nanotubes. Fold increase of MICA-YFP at the nanotube junction (F) vs. time for the nanotube shown in E, starting from when the nanotube first forms (t = 0). (G) Time-lapse microscopy shows MICA persists at nanotube junctions for long times. (H) Primary human NK cells were tested for lysis of P815 transfectants expressing different mean amounts of MICA at the surface, as quantified by flow cytometry. Percent specific lysis is shown plotted against the mean number of MICA proteins expressed by each target cell transfectant for E/T ratios of 2:1, 1:1, and 0.5:1 (red circles, blue squares, and green triangles, respectively). Error bars are SD of triplicates from a representative sample of 3 independent experiments. (I) Number of MICA proteins accumulated at individual nanotube junctions, estimated by correlating the distribution in fluorescence among cells observed by microscopy and flow cytometry (n = 41). [Scale bars: 10 μm (inserts: 2 μm).]