Membrane nanotubes facilitate long-distance interactions between natural killer cells and target cells

Abstract

Membrane nanotubes are membranous tethers that physically link cell bodies over long distances. Here, we present evidence that nanotubes allow human natural killer (NK) cells to interact functionally with target cells over long distances. Nanotubes were formed when NK cells contacted target cells and moved apart. The frequency of nanotube formation was dependent on the number of receptor/ligand interactions and increased on NK cell activation. Most importantly, NK cell nanotubes contained a submicron scale junction where proteins accumulated, including DAP10, the signaling adaptor that associates with the activating receptor NKG2D, and MHC class I chain-related protein A (MICA), a cognate ligand for NKG2D, as occurs at close intercellular synapses between NK cells and target cells. Quantitative live-cell fluorescence imaging suggested that MICA accumulated at small nanotube synapses in sufficient numbers to trigger cell activation. In addition, tyrosine-phosphorylated proteins and Vav-1 accumulated at such junctions. Functionally, nanotubes could aid the lysis of distant target cells either directly or by moving target cells along the nanotube path into close contact for lysis via a conventional immune synapse. Target cells moving along the nanotube path were commonly polarized such that their uropods faced the direction of movement. This is the opposite polarization than for normal cell migration, implying that nanotubes can specifically drive target cell movement. Finally, target cells that remained connected to an NK cell by a nanotube were frequently lysed, whereas removing the nanotube using a micromanipulator reduced lysis of these target cells.

Fig. 1.

Human NK cells readily form membrane nanotubes. Primary cultured human NK cells labeled with membrane dye DiD (red) readily formed nanotubes with other primary NK cells labeled with 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO) (green, n > 50) (A) or with P815 transfectants expressing MICA-YFP (green, n > 100) (B). (C) After 45 min of coculture, the frequency of nanotubes formed by primary NK cells was assessed for cells incubated alone (none) or in the presence of different target cells as indicated (n > 1,500 cells). Bars show the frequency of nanotubes between NK cells (shaded) and the frequency of nanotubes connecting NK cells to target cells (white). (D) Length of nanotubes formed between primary NK cells and different target cells (n > 150). (Scale bars: 10 μm.)

Fig. 2.

Characteristics of human NK cell membrane nanotubes. (A) Time-lapse microscopy reveals the formation of a nanotube between NKL labeled with membrane dye DiD (red) and P815/MICA-YFP (green, n > 100). Images acquired by time-lapse microscopy of primary NK cells (n = 109) (B) and NKL cells (n = 110) (C) coincubated with P815/MICA-YFP were analyzed to record the length of time of contact between cells and then whether or not membrane nanotubes formed as cells departed. Cocultures of NKL (labeled NK) and P815/MICA-YFP (labeled T for target cell) were fixed and stained with phalloidin-Alexa633, which marks f-actin (white, n > 100) (D) or anti-α-tubulin (E), followed by secondary mAb conjugated to Alexa633 (white, n = 78). Thin panels show an enlarged view of the nanotube. (Scale bars: 10 μm.)

Fig. 3.

Frequency of membrane nanotube formation. The frequency at which NK cells formed nanotubes was assessed for cocultures of NKL (A) or primary NK (B) cells and untransfected P815 or transfectants of P815 expressing different levels of MICA-YFP. (C) Frequency at which NKL cells formed nanotubes with transfectants of P815 expressing human ICAM-1 in the presence or absence of manganese (denoted Mn⁺⁺ or none). (D) Untransfected NKL (□) and NKL transfected to express WT DAP10-GFP (△) or DAP10(Y85F)-GFP (○) were each tested for their ability to lyse P815 (blue) or P815/MICA-YFP (red) target cells at different E/T ratios. Data are representative of three independent experiments performed in triplicate. (E) These same NK cell lines and transfectants were assessed for the frequency at which they formed nanotubes with P815/MICA-YFP (n = 8 experiments). (F) Time-lapse microscopy of NKL/DAP10(Y85F)-GFP coincubated with P815/MICA-YFP was analyzed to record the length of time of contact between cells and whether or not membrane nanotubes formed. (G) Freshly isolated human NK cells were incubated with different cytokines as indicated and assessed for the frequency at which they made nanotubes with P815/MICA-YFP (n = 5 experiments). P values shown are in comparison with unstimulated cells.

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).]

Fig. 5.

Accumulation of DAP-10, Vav-1, and tyrosine-phosphorylated proteins at nanotube junctions. (A) Representative micrograph (n > 100) shows NKL transfected to express DAP10-GFP (green) labeled with membrane dye DiD (red), which is connected to THP-1 (Lower Left in bright-field image) via a nanotube. Panels show the bright-field image of cells and the corresponding fluorescence of DAP10-GFP (green) and DiD (red). Also evident in this image is a membrane connection between two THP-1 cells (shown in the bright-field image; not fluorescent). Using transfectants of NKL expressing DAP10-GFP or mem-YFP and THP-1, the fold increase at nanotube junctions of DAP10-GFP and DiD (n = 25) (B) or mem-YFP and DiD (n = 37) (C) was measured in comparison with that elsewhere along the nanotube. (D) NKL coincubated with P815/MICA-YFP was fixed and stained for phosphotyrosine (red). The boxed region is shown enlarged to allow better visualization of the accumulation of phosphotyrosine (red) along with MICA-YFP (green, n = 20). (E) Representative micrograph shows NKL transfected to express Vav-1 tagged with GFP, stained with DiD (red), and cocultured with P815/MICA. Panels show the bright-field image and corresponding fluorescence of Vav-1-GFP (green) and an overlay (Right, n = 60). The boxed regions are shown enlarged in lower panels to visualize the nanotube junction better. (F) Using transfectants of NKL expressing Vav-1-GFP and P815/MICA, the fold increase at nanotube junctions of Vav-1-GFP and DiD was measured in comparison with that elsewhere along the nanotube. [Scale bars: 10 μm (inserts: 2 μm).]

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.)