Fig. 6.
Functional consequences for membrane nanotubes formed by NK cells. (A) Representative example of time-lapse microscopy of NKL/mem-YFP (green) and P815/MICA connected by a membrane nanotube, where the target cell is moved back into close contact with the NK cell (n > 60). (B) Speed of target cell movement was compared for target cells (P815/MICA or P815/MICA-YFP) freely migrating unidirectionally or moving via a nanotube. (C) Orientation of target cell polarity in the direction of movement was scored for target cells (P815/MICA or P815/MICA-YFP) moved toward NKL via nanotubes (n = 60). Each target cell was scored as either being (i) unpolarized, having its (ii) uropod or (iii) leading edge facing the direction of movement, or (iv) polarized such that neither the leading edge nor the uropod faced the direction of movement. (D) Representative time-lapse microscopy shows an example in which a 221/MICA-YFP target cell (green) is moved back along the nanotube path into close contact with NKL stained with DiD (red) and is subsequently lysed as evidenced by membrane blebbing (at 60 min). (E) Time-lapse microscopy of 221/MICA-YFP (green) target cells connected to NKL stained with DiD (red) in the presence of DNA dye (blue). Incorporation of the DNA dye was observed at later time points, indicating cell death (n = 30). (F) Time-lapse fluorescence micrographs of single optical slices show an example of how a nanotube was removed by moving the DiD-labeled (red) NK cell away using a 7-μm needle (starred and red attributable to autofluorescence). (G) Time-lapse micrographs of reconstructed z-stacks show how the target cell shown in F (221/MICA-YFP, green) was followed after nanotube removal to determine whether or not lysis occurred (i.e., whether or not it stained with a DNA dye, Sytox-blue). (H) Time required for DNA dye incorporation following initial intercellular contact was determined for three different processes: (i) cell death occurring at a conventional (i.e., large) cytolytic synapse, (ii) cell death occurring while the target cell was tethered to a distant NK cell, and (iii) cell death occurring after the target cell has come back to the NK cell body via a nanotube. (I) Relative frequency of different processes that led to death of 221/MICA target cells was assessed by analysis of a series of 2-h long movies (n > 400). Events were scored as death (detected by DNA dye incorporation) occurring (i) at a tight cell/cell contact (i.e., via a conventional immune synapse), (ii) when target cells were connected to NK cells by nanotubes and had moved back to reform a tight contact, (iii) when target cells were connected to a distant NK cell via a membrane nanotube, (iv) when target cells had previously been in contact with an NK cell and subsequently moved apart without remaining connected by a nanotube, or (v) when cells spontaneously died without interaction with an NK cell. (Scale bars: 10 μm.)