Remodelling of cortical actin where lytic granules dock at natural killer cell immune synapses revealed by super-resolution microscopy

Abstract

Natural Killer (NK) cells are innate immune cells that secrete lytic granules to directly kill virus-infected or transformed cells across an immune synapse. However, a major gap in understanding this process is in establishing how lytic granules pass through the mesh of cortical actin known to underlie the NK cell membrane. Research has been hampered by the resolution of conventional light microscopy, which is too low to resolve cortical actin during lytic granule secretion. Here we use two high-resolution imaging techniques to probe the synaptic organisation of NK cell receptors and filamentous (F)-actin. A combination of optical tweezers and live cell confocal microscopy reveals that microclusters of NKG2D assemble into a ring-shaped structure at the centre of intercellular synapses, where Vav1 and Grb2 also accumulate. Within this ring-shaped organisation of NK cell proteins, lytic granules accumulate for secretion. Using 3D-structured illumination microscopy (3D-SIM) to gain super-resolution of ~100 nm, cortical actin was detected in a central region of the NK cell synapse irrespective of whether activating or inhibitory signals dominate. Strikingly, the periodicity of the cortical actin mesh increased in specific domains at the synapse when the NK cell was activated. Two-colour super-resolution imaging revealed that lytic granules docked precisely in these domains which were also proximal to where the microtubule-organising centre (MTOC) polarised. Together, these data demonstrate that remodelling of the cortical actin mesh occurs at the central region of the cytolytic NK cell immune synapse. This is likely to occur for other types of cell secretion and also emphasises the importance of emerging super-resolution imaging technology for revealing new biology.

Figure 1. Organisation of NKG2D, Vav1, and Grb2 at the intercellular cytolytic NK cell synapse.

(A) A schematic to show how optical tweezers were used to place a target cell into contact with an NK cell in order that the immune synapse could be imaged with higher resolution and at high speed. The dotted line shows the imaging plane. (B) Time-lapse imaging (1 fps) of NKG2D microcluster formation and supramolecular reorganisation into a ring-shaped structure between live conjugates of Daudi/MICA and NKL/NKG2D-GFP (representative of 67% of synapses, n = 45). (C) Tracks showing the movement of the individual NKG2D-GFP microclusters (labelled 1–5 in (C) between t = 0 and t = 150 s (the scale on the graph is in µm)). (D) Representative live cell image of a conjugate between Daudi/MICA and NKL transfected to express both Vav1-GFP and Grb2-mCherry, at the moment of cell-cell contact. (E) Representative live cell image of a conjugate between Daudi/MICA and NKL/Vav1-GFP/Grb2-mCherry. Upper panels show the conjugate immediately prior to reorientation using the optical tweezers (∼170 s after conjugate formation) and lower panels the same conjugate with the synapse orientated in the imaging plane. Vav1 and Grb2 co-localise, with Pearson's correlation coefficient R_r = 0.7, and organise into a central ring. (F) The diameter at the inner and outer borders of fluorescent protein rings imaged in NKL cells expressing either NKG2D-GFP, Vav1-GFP/Grb2-mCherry, or actin-YFP; graph shows mean ± SD (n = 10 cells). (G) Representative live cell image of a conjugate between Daudi/MICA and NKL expressing actin-YFP. (H) Scaled schematic representing the relative size and organisation of NKG2D-GFP and actin-YFP rings detected in NKL–Daudi/MICA conjugates. All scale bars = 10 µm.

Figure 2. Lytic granules are delivered within the ring-shaped organisation of NKG2D at the synapse centre.

(A) Time-lapse microscopy of live cell conjugates of NKL/NKG2D-GFP loaded with Lysotracker Red and Daudi/MICA. Lysotracker was absent from the early immune synapse when NKG2D microclusters initially formed (top, t) but appeared within the ring-shaped organisation of NKG2D at later time points (bottom, t+240 s) (n = 7). (B) Conjugates between NKL/NKG2D-GFP and Daudi/MICA were fixed and stained for perforin. Perforin (red) is detected within the central ring of NKG2D (green). (C) Live cell imaging shows vesicles marked with Lysotracker (red) move from the NKL to the target cell within regions in which NKG2D-GFP is less dense (green). Lower panels show the synapse between the NKL and target cell enlarged. (D) Quantification of the extent of Lysotracker staining localised within the inner border of the NKG2D ring, co-localising with the NKG2D ring or outside the outer border of the NKG2D ring. Graph shows mean ± SEM for granules from n = 10 cells analysed for 2 min post-NKG2D-GFP ring formation. (E) The distribution of the frequency normalised by area at a given radius from the NKG2D-GFP ring centroid of Lysotracker particles at the NK synapse. All scale bars = 5 µm.

Figure 3. Structured Illumination microscopy permits super-resolution imaging of F-actin at the NK cell synapse.

(A) Widefield (top) and SI images (bottom) of 40 nm beads. Bar = 1 µm. (B) Plot profiles from the region marked by red bars in (A) show that two beads approximately 200 nm apart can only be resolved following SI reconstruction. Fitting of the curve with two Gaussians gives full width half maximums (FWHMs, red bars on the graph) of 99 nm and 105 nm for each bead, respectively. (C) Confocal (left), widefield (middle), and SI image (right) of human primary NK (pNK) cells activated on a surface coated with anti-NKG2D mAb. Bars = 5 µm. (D) Regions at the centre of the synapses in (C) are enlarged to demonstrate the increased level of detail in cortical F-actin structure labelled using AlexaFluor488-conjugated phalloidin that can be observed when SI microscopy is used. Bars = 1 µm. (E) Plot profiles to directly compare the widefield and SI reconstructed image for the region indicated by the red bar shown in panel D. Estimation of the FWHM (red bar on the graph) from the Gaussian fit of the SI data gives a resolution of ∼115 nm.

Figure 4. Organisation of cortical actin at the NK cell immune synapse.

(A) The proportion of pNK cells (top) or NKL cells (bottom) that form a F-actin ring when stimulated on surfaces coated with control protein (poly-L-lysine, Isotype-matched mAb), inhibitory (αNKG2A, αNKG2A+ICAM-1), or activating mAb/ligand (αNKG2D, αNKG2D+ICAM-1, MICA-Fc, MICA-Fc+ICAM-1). Graph shows the mean ± SEM, n>200, *** p<0.001. (B) The intensity of F-actin distribution in SI microscopy images across pNK cells stimulated on control or inhibitory surfaces (top) or activating surfaces (bottom). Graphs show the mean, n = 10–75 cells per condition. (C) Representative images obtained by SI microscopy of cortical F-actin at the interface between pNK cells and coverslips coated with inhibitory or control ligands. Bar = 5 µm. The central synaptic region is enlarged in the lower panels. Bar = 1 µm. (D) SI images of cortical F-actin structure at the interface between NKL cells and surfaces coated as in (C). Bar = 5 µm. The centre of the synapse is enlarged in the lower panels. Bar = 1 µm. (E) SI images of cortical F-actin structure at the interface between pNK cells and surfaces coated with activating antibody or ligand. Bar = 5 µm. The central region of the synapse is enlarged in the lower panels. Bar = 1 µm. (F) SI images of cortical F-actin structure at the interface between NKL cells and surfaces coated as in (E). Bar = 5 µm. The central region of the synapse is enlarged in the lower panels, bar = 1 µm.

Figure 5. Increased periodicity of the cortical actin mesh at the centre of the NK cell synapse.

(A) SI microscopy images of cortical F-actin in pNK cells stimulated on surfaces coated with control (poly-L-lysine, Isotype-matched mAb, ICAM-1) or inhibitory proteins (αNKG2A, αNKG2A + ICAM-1). Actin mesh domains (or “holes”) are shown as heat maps related to hole area, with the smallest holes shown in blue set at the resolution limit of the microscope (0.01 µm²) and largest areas (>3.0 µm²) shown in red. (B) Heat mapped SI images of cortical F-actin in pNK cells activated on surfaces coated with activating mAb/ligand (αNKG2D, αNKG2D+ICAM-1, MICA-Fc, MICA-Fc+ICAM-1). All scale bars = 5 µm. (C) Quantification of gaps or “holes” in the F-actin mesh in the synapse centre for pNK cells (top) or NKL cells (bottom) stimulated on control, inhibitory, or activating surfaces. The periodicity (and “hole area”) of the actin mesh significantly increases when cells are activated, with or without LFA-1 engagement. Graph shows mean ± SEM (n = 10–75 cells, *** p<0.001).

Figure 6. Predicted regions of lytic granule penetration across the NK cell immune synapse.

(A) SI microscopy of lytic granules stained for perforin in pNK cells stimulated on control (poly-L-lysine, left) or activating (MICA-Fc, middle, or MICA-Fc + ICAM-1, right) surfaces. Bar = 1 µm. (B) Distributions of granule diameters above the measured 100 nm SI microscope resolution limit for pNK cells stimulated as in (A) (n = 10 cells per condition). (C) Average granule diameter for pNK cells stimulated as in (A) (mean ± SEM, n>400). (D) The fraction of the synapse predicted to be readily penetrable by a granule with a diameter of 250 nm quantified for pNK cells (left) or NKL cells (right) stimulated on control, inhibitory, or activating surfaces. Graphs show mean ± SEM, n = 10–75 cells. (E) Quantification of the proportion of the immune synapse in pNK on control surfaces or activated with MICA-Fc + ICAM-1 predicted to be penetrable by granules of diameters ranging from 100 nm to 500 nm. Graph shows the mean ± SEM (n = 10 cells). (F) Regions within the cortical F-actin mesh through which a lytic granule of diameter 200 nm (blue) to 800 nm (red) may penetrate were mapped on images of pNK cells stimulated on control, inhibitory, and activating surfaces. (G) The average distance of the predicted granule penetrable areas from the synapse centre was measured and graphs show mean ± SEM (n = 10 cells). Bar = 5 µm.

Figure 7. Cortical F-actin rearrangements can be stimulated through engagement of CD16.

(A) Images from SI microscopy of cortical F-actin in pNK cells stimulated on surfaces coated with mAb to CD16 with or without ICAM-1. The central panel shows actin mesh domains as a heat map related to hole area with smallest holes shown in blue (0.01 µm²) and largest in red (>3.0 µm²). The right panel shows the regions within the cortical F-actin mesh through which a lytic granule of diameter 200 nm (blue) to 800 nm (red) may penetrate. Bar = 5 µm. (B) The proportion of pNK cells that form an F-actin ring when stimulated on surfaces coated with αNKG2D or αCD16 with or without ICAM-1. (C) The mean hole area within the central region of the pNK cell synapse for cells stimulated as in (B). (D) The proportion of the NK cell synapse predicted to be penetrable by a granule with a diameter of 250 nm for cells stimulated as in (B).

Figure 8. Polarised lytic granules in activated pNK cells preferentially localise to predicted granule penetrative domains.

(A) Two-colour SI images of F-actin (red) and perforin (green) (left panels) in the immune synapse of a representative pNK cell activated with MICA-Fc. Right panels show the predicted region of lytic granule penetration for granules with diameter 200–800 nm. Bar = 5 µm. Lower panels show the centre of the synapse enlarged. Bar = 1 µm. (B) As for (A), SI images of F-actin (red) and perforin (green) (left panels) and predicted regions of lytic granule penetration (right panels) for a representative pNK cell activated with MICA-Fc and ICAM-1. Bar = 5 µm. (C) The centroid (position) of each lytic granule was mapped in 3D to a depth of 1 µm above the coverslip, in pNK cells stimulated with MICA-Fc. Left panels show granule positions overlaid onto an en face 2D map of the cell-slide contact. This map shows the cell border (cyan), the dense F-actin ring (green), and the regions calculated to be penetrable by granules 200–800 nm in diameter (red). Percentages on right panels represent the proportion of granules which fall within regions 0–250 nm, 250–500 nm, and 500–750 nm of the surface. (D) The centroid for each lytic granule was mapped in 3D as for (C), for pNK cells stimulated with MICA-Fc and ICAM-1. Percentages are shown as in (C). (E) Lateral distances (mean ± SEM) from predicted granule penetrable areas are plotted for granules, within the central synapse, at positions 0–1 µm above the surface. Graph compares pNK cells stimulated with MICA-Fc or MICA-Fc + ICAM-1 (n = 10). (F) Calculation of the Odds Ratio for granules to land on granule penetrative areas at axial distances ranging from 0–900 nm above the slide surface that has been coated with MICA-Fc or MICA-Fc + ICAM-1.

Figure 9. MTOC polarisation towards Granule Penetrable Areas.

(A) Two-colour 3D-SI image of F-actin (green) and the MTOC (red) in a pNK cell stimulated on an ICAM-1 coated surface. Upper and left panels show orthogonal XZ and YZ slices, respectively, taken from the super-resolved Z-stack. (B) Two-colour 3D-SI image of F-actin (green) and the MTOC (red) in a pNK cell stimulated on a surface coated with MICA-Fc and ICAM-1. Upper and left panels show orthogonal XZ and YZ views of the super-resolved Z-stack, respectively. (C) The average axial distance or “height” of the MTOC above the cell-surface interface for pNK cells on poly lysine, ICAM-1, or MICA-FC + ICAM-1 coated surfaces. Graph shows mean ± SD (n = 10 cells per condition (*** p<0.001). (D) Left and upper right panels show the regions of the cortical F-actin mesh through which a lytic granule diameter of 200–800 nm in diameter would be predicted to pass. Lower right panel shows the presence of the MTOC at the cell surface interface, in close proximity to the granule penetrable areas in a pNK cell stimulated on MICA-Fc + ICAM-1. The distance of the MTOC from granule penetrable areas for n = 7 cells is 0.25±0.17 µm (mean ± SD). Scale bars = 5 µm except right panels in (D) where scale bars = 1 µm.