The author(s) have made the following declarations about their contributions: Conceived and designed the experiments: GDR TS JSO. Performed the experiments: GDR EMM PPB. Analyzed the data: GDR EMM PPB JSO. Contributed reagents/materials/analysis tools: TS. Wrote the paper: GDR JSO.
The authors have declared that no competing interests exist.
Super-resolution imaging provides a new look at how the lytic granules in natural killer cells penetrate the filamentous actin network of the immunological synapse.
Accumulation of filamentous actin (F-actin) at the immunological synapse (IS) is a prerequisite for the cytotoxic function of natural killer (NK) cells. Subsequent to reorganization of the actin network, lytic granules polarize to the IS where their contents are secreted directly toward a target cell, providing critical access to host defense. There has been limited investigation into the relationship between the actin network and degranulation. Thus, we have evaluated the actin network and secretion using microscopy techniques that provide unprecedented resolution and/or functional insight. We show that the actin network extends throughout the IS and that degranulation occurs in areas where there is actin, albeit in sub-micron relatively hypodense regions. Therefore we propose that granules reach the plasma membrane in clearances in the network that are appropriately sized to minimally accommodate a granule and allow it to interact with the filaments. Our data support a model whereby lytic granules and the actin network are intimately associated during the secretion process and broadly suggest a mechanism for the secretion of large organelles in the context of a cortical actin barrier.
The immune system's natural killer cells eliminate diseased cells in the body. They do so by secreting toxic molecules directly towards the diseased cells, so causing their death. This process is essential for the host organism to defend itself against infectious diseases. The interface between the natural killer cell and its target—the lytic immunological synapse—forms by close apposition of the surface membranes of the two cells. It is characterized by coordinated rearrangement of proteins to allow lytic granules, which contain the toxic molecules, to fuse with the cell surface at the synapse. Given the large size of the granules, one challenge the natural killer cell faces is how to contend with network of actin filaments just under the cell surface, which potentially could pose a barrier to secretion. The current model proposes large-scale clearing of actin filaments from the center of the immunological synapse to provide granules access to the synaptic membrane. By using very high-resolution imaging techniques, we now demonstrate that actin filaments are present throughout the synapse and that natural killer cells overcome the actin barrier not by wholesale clearing but by making minimally sufficient conduits in the actin network. This suggests a model in which granules access the surface membrane by means of specific and facilitated contact with the actin cytoskeleton.
Natural killer (NK) cells are lymphocytes of the innate immune system that function in clearance of tumor and virally infected cells
F-actin accumulation at the synapse is the first major cytoskeletal reorganization event and is critical to subsequent steps and function of the IS
One question that arises from the creation of a dense polarized network at the IS is how secretion of lytic granules occurs through a potential barrier. The traditional view of granule delivery through the actin network holds that granules reach the synaptic membrane through a void of actin in the center of the network. This model is based on the observation from 3-D confocal microscopy that actin forms a dense peripheral ring around the IS
Here we use microscopy techniques that provide enhanced sensitivity and resolution over those used previously to investigate the NK cell IS. We show that F-actin is present throughout the synapse and that lytic granules likely navigate and are secreted through the filamentous network by accessing minimally sufficiently sized clearances. These data demonstrate a previously unappreciated distribution of F-actin at the NK cell IS and redefine granule access to the synaptic membrane and functional secretion.
Visualization of the synaptic actin network has relied on 3-D reconstructions of confocal slices
(A) 3-D projection of NK-92 GFP-actin expressing cell (green) conjugated to mel1190 target cell (yellow). Scale bar = 5 m. (B) X–Y projection of a synapse taken from a conjugate similar to (A). (C,D) X–Y projections of a representative NK-92 (left) or ex vivo (right) cell that had been activated for 30 min at 37°C on immobilized antibody to NKp30 and CD18, fixed, stained with 568 phalloidin (red), and imaged by TIRF microscopy. Above each X–Y projection is a plot of the mean fluorescent intensity (MFI) of concentric circles at a given distance beginning at the periphery of the cell and moving inward to the center (radial intensity profiles). (E,F) Plot of radial intensity profiles from 70 cells from 3 experiments for each cell type. Mean value is shown in black.
To more directly image the cortical region of the NK cell immunologic synapse, we used total internal reflection fluorescence microscopy (TIRFm), which has the benefit of an improved signal to noise ratio over confocal microscopy and is limited to visualization within the first membrane proximal 100 nm
To evaluate the kinetics of actin accumulation at the activated synapse, NK-92 cells expressing GFP-actin were imaged using TIRFm after contacting an activating surface. Actin accumulated quickly, within 5 min, and was sustained over the period of observation (50 min) (
Because there was abundant actin present at the synapse, we wanted to determine if lytic granules might utilize relative clearances in the actin network to access the synaptic membrane. To address this, GFP-actin expressing cells were loaded with LysoTracker Red dye, which enables tracking of lytic granules and definition of their position relative to actin, and followed in real time after activation. Numerous granules were identified in the synaptic actin network using two-color TIRFm. Although some relative hypodensities were apparent in the synaptic actin network (
(A) NK-92 cells expressing GFP-actin (green) were loaded with LysoTracker Red (red) and activated by immobilized antibody to NKp30 and CD18. Cells were imaged at 1 frame per minute for 60 min using TIRFm. Images are shown at 10 min intervals starting at 10 min following contact. Images for the GFP channel (top), LysoTracker Red channel (middle), and a merge of both (bottom) are shown. (B–E) NK-92 cells were activated on glass, fixed, stained, and imaged by both confocal and TIRF microscopy using a 100× objective. (B) Images show pericentrin (blue), perforin (yellow), actin (green), and indicated merges at the plane of contact with the glass. (C) Full merge from (B) is shown to the right of a Y–Z projection of the cell. (D) TIRFm image of the cell from (B, C). (E) Distances of granules to the MTOC were measured and averaged on a per cell basis for 30 cells over 2 experiments. The red dashed line denotes the mean of all cells. Scale bars = 5 µm.
The MTOC is known to deliver lytic granules to the immunological synapse in NK cells
Because there was variability in colocalization between synaptic actin and granules (
Lysosomal-associated membrane protein 1 (LAMP1, CD107a), which is sorted to lytic granules
(A) Histogram demonstrating green fluorescence measured by flow cytometry of NK-92 cells expressing pHluorin-LAMP1. Cells were untreated or treated with PMA/Ionomycin or CMA. (B) NK-92 cells expressing pHluorin-LAMP1 (green) were loaded with LysoTracker Red (red) and imaged by TIRF under activating conditions. Frames were acquired at a rate of 2 frames per min following 10 min of activation and the image sequence depicts a cropped section showing a single lytic granule over time. (C) NK-92 cells expressing pHluorin-LAMP1 and mCherry-actin were activated by immobilized antibody to NKp30 and CD18 and imaged by TIRFm. Images shown were taken at 10-s intervals at indicated time of activation. Scale bar = 5 µm. (D) Ratio measurements of the MFI of mCherry-actin at the site of degranulation, or adjacent points, to the MFI of the whole cell footprint were calculated and represent 52 events; means = 0.965 and 1.013, respectively. (***
We next used pHluorin-LAMP1 expressing NK-92 cells to address whether granule approximation results in degranulation. LysoTracker Red loaded, pHluorin-LAMP1 expressing cells were imaged over time using TIRFm (
To directly investigate where degranulation occurs relative to the synaptic actin network, we stably coexpressed pHluorin-LAMP1 and mCherry-actin in cells and imaged them following activation using two-color TIRFm. Timelapse imaging demonstrated that degranulation events occurred in areas of at least some actin fluorescence, similar to that which was seen with granule approximations (
To further characterize the local actin network at the point of degranulation in consideration of focal hypodense regions, we quantitatively evaluated actin fluorescence in the entire immediate vicinity of degranulation events. Measurements of actin fluorescence were made along sequential pixel radii emanating from the centroid of individual degranulations extending approximately 1 µm outwards (
Since granules are in contact with at least some actin during degranulation, we next investigated the role of actin dynamics in degranulation. We inhibited actin polymerization and dynamics with drugs that prevent F-actin assembly (latrunculin A and cytochalasin D) or disassembly (jasplakinolide). Inhibitor addition at the time of activation almost completely inhibited degranulation (
NK-92 cells were activated by immobilized antibody to NKp30 and CD18 and incubated at 37°C. (A) Indicated inhibitors were added to samples at 0 min, 10 min, or 20 min following activation. Supernatants were harvested after 60 min of total incubation and assayed for Granzyme A activity. All values are statistically significantly different from respective DMSO controls (range:
To further evaluate the effects of the inhibitors on NK cells, we imaged F-actin at the synapse following inhibitor treatment. Jasplakinolide had no effect on F-actin presence or distribution; latrunculin A completely depleted the actin network; cytochalasin D had variable effects on cells, with some cells appearing unaffected while others showing a relative depletion of some filaments (
To determine if the synaptic actin network was dynamic at the time corresponding to degranulation, GFP-actin expressing cells were imaged by TIRFm and evaluated at both early and late timepoints of activation. Subtle but consistent changes in actin intensity were visualized over the course of imaging (
The resolution of fluorescence microscopy is diffraction limited to around 200 nm
Using CW-STED microscopy, we first imaged Citrine-actin expressing NK-92 cells conjugated to mel1190 cells (
(A) The synapse between a Citrine-actin expressing NK-92 and mel1190 target was imaged in the X–Y plane. (B, C) NK-92 cells were stimulated for 30 min on glass coated with either antibody to CD18 (B) or to both CD18 and NKp30 (C) and stained for F-actin with phalloidin. (D–F) Measurements of the number of clearances at the synapses of 30 cells from 2 experiments activated as in (B,C). (***
We next asked whether there was an activation-dependent change in the synaptic actin architecture. To this end, we imaged F-actin in cells that had been stimulated with antibody to CD18, which does not induce degranulation, or in combination with antibody to NKp30, which robustly induces degranulation (
Having defined the presence of clearances in the synaptic actin network upon activation, we next sought to identify granule localization relative to the network. Thus we simultaneously imaged the actin network by STED microscopy and granules by laser scanning confocal microscopy. Granules within activated cells displayed a range of interaction with the actin network and all that were present at the synapse had at least some (
To obtain unprecedented, nanometer resolution of actin filaments at the synapse, we used platinum replica electron microscopy. In order to expose the inner surface of cells, corresponding to the synapse, for metal coating, cells were “unroofed” by mechanical removal of the bulk of the cell body with the nucleus. Images of platinum replicas of the activated synapse confirmed our earlier light microscopy data that the F-actin network exists throughout the synapse and contains small granule-sized clearances (
NK-92 cells activated by immobilized antibodies to NKp30 and CD18 for 10 min or 30 min and then sheared apart by sonication were viewed by platinum replica electron microscopy. (A) Activated cortical synapse at 30 min of activation. (B) Enlargement of central boxed region in (A). Scale bars = 1 µm. (C–E) Measurements and comparison of the number of clearances of specified sizes: 250–499 nm (C), 500–749 nm (D), and greater than 750 nm (E).
In order to determine whether the abundance or distribution of clearances changes over time, the filamentous network at the synapse was evaluated after 10 and 30 min of activation. Quantitative assessment of the actin network in multiple cells defined a similar total contact area and total area occupied by filaments (filament density) between the two timepoints (
Our TIRF data suggested, and our STED data demonstrated, that granules approximate the synapse in close association with the actin network. To confirm this observation on the nanometer level we used platinum replica electron microscopy to image “unroofed,” membrane-intact NK cells. Granule-sized organelles could be identified on the intracellular face of the actin network in approximation with filaments as well as intercalated within filament clearances (
(A,B) 20,000× images taken using platinum replica electron microscopy following activation of NK-92 cells by immobilized antibody to NKp30 and CD18 and then “unroofing” with nitrocellulose membrane (top) (scale bar = 1 µm). Pseudocolored images are shown (bottom) highlighting filaments (blue), granules (yellow), and plasma membrane (green). (C) Traditional (left) and proposed (right) model of granule delivery through the immunologic synapse.
Actin accumulation defines an early stage in the maturation of the NK cell IS and is required for subsequent cytolytic function. Without proper reorganization of the actin cytoskeleton, lytic granules fail to polarize to the synapse and NK cells display inadequate cytotoxicity. Upon polarization, lytic granules require myosin IIA function to approximate the plasma membrane and have their contents directly secreted. The dependence on an actin motor protein for the secretory process suggests a requirement for the actin network itself. We have defined the distribution of actin at the IS using techniques that provide unprecedented sensitivity and resolution. We have done so both by live cells conjugated to target cells as well as a more flexible model system. We have also shown that lytic granules reach the plasma membrane and are secreted in areas of actin. Furthermore, our data suggest that secretion events likely occur in minimally yet sufficiently sized clearances in the actin network.
While our studies relied upon activating an NK cell line using immobilized antibody we were able to obtain physiologically relevant supporting data. Firstly, NK-92 cells activated in this manner released contents of lytic granules (
The F-actin network in secretory cells was first believed to be a barrier to exocytosis
Investigations with F-actin and granule secretion at the IS in cytotoxic lymphocytes have thus far been limited to T cells. In cytotoxic T lymphocytes, it has been suggested that actin is not a barrier to secretion
Our data support a role for F-actin as a facilitator of secretion rather than a barrier. Inhibiting actin polymerization with cytochalasin D or latrunculin A after activation resulted in diminished secretion of lytic granule contents rather than an increase. This suggested that actin was not a barrier to secretion, but that its dynamics were required. The latter hypothesis is supported by the results showing a similar inhibitory effect when cells were treated with jasplakinolide. Interestingly, the effect of the actin inhibitors was most pronounced at 10 min following activation and less so at 20 min following activation, and thus defined a critical window of actin reorganization that facilitates degranulation (
Our work defines an actin network that is more pervasive at the NK cell IS than previously thought. Although this could serve as a potential barrier, we have identified abundant granule-sized clearances that could function as sufficient access points to the plasma membrane. These could provide functionality by allowing granules to pass between filaments and to simultaneously interact with them, whereby myosin IIA could exert force in squeezing granules between filaments or in post-fusion expulsion of granule contents. This latter possibility has been suggested in chromaffin cells where myosin II function was required for appropriate release of catecholamines
NK-92 and GFP-actin expressing NK-92 cell lines were a kind gift from K. Campbell and were maintained in Myelocult (StemCell) media supplemented with 100 U/mL penicillin and streptomycin (Gibco) and 100 U/mL IL-2 (Hoffman-La Roche). mCherry-actin, Citrine-actin, and pHluorin-LAMP1 expressing cells were generated by retroviral transduction of NK-92 cells as described
The pHluorin-LAMP1 retroviral plasmid was generated by BioMeans, Inc. by inserting the sequence for pHluorin (a kind gift from G. Miesenböck) between the signal sequence and the transmembrane domain of IL-2Rα linked to the cytoplasmic tail of LAMP1 (a kind gift from M. Marks). A flexible GS linker was added between pHluorin and the transmembrane domain sequences. The entire construct was subsequently cloned into the MIGR1-puromycin vector.
The mCherry-actin retroviral plasmid was generated by PCR amplifying mCherry-actin from a pmCherry plasmid with 5′ BglII and 3′ EcoRI restriction site overhangs. The PCR product was digested and ligated into the pMSCV-Hygromycin plasmid (a kind gift from W. Pear), which had an EcoRI site in the Hygromycin resistance gene sequence eliminated by site-directed mutagenesis. The Citrine-actin retroviral plasmid was generated by amplifying the Citrine sequence from the pRSET-b Citrine plasmid (a kind gift from R. Tsien) with 5′ and 3′ BglII overhangs. The product was digested and ligated into a similarly digested mCherry-actin retroviral plasmid, effectively removing the mCherry sequence and inserting the Citrine sequence. Proper orientation of insert was verified by DNA sequencing by the Children's Hospital of Philadelphia Research Institute sequencing core facility.
Flow cytometry was performed to verify pHluorin-LAMP1 expression. Cells were untreated or treated with phorbol myristate acetate (PMA, 100 ng/mL, Sigma) and Ionomycin (1 µg/mL, Sigma) for 30 min or Concanamycin A (CMA, 100 nM, Sigma) for 90 min and samples were run on a BD FACSCalibur.
Cells were washed and resuspended in supplemented Myelocult prior to use. For imaging of lytic granules, cells were incubated with 100 nM LysoTracker Red DND-99 (Molecular Probes) for 30 min at 37°C, washed once, and resuspended in supplemented Myelocult. ΔT dishes (Bioptechs) were coated with 5 µg/mL anti-NKp30 (Beckman-Coulter) and 5 µg/mL anti-CD18 (Clone IB4) for 1 h at 37°C, washed with PBS, and prewarmed prior to imaging with 1 mL dye free R10 (dye free RPMI 1640 (Gibco), 10% fetal bovine serum (Atlanta Biologicals), 10 mM HEPES (Gibco), 100 U/mL penicillin and streptomycin, 100 µM MEM nonessential amino acids (Gibco), 1 mM sodium pyruvate (CellGro), and 2 mM L-glutamine (Gibco). 4×105 cells were added to the dishes, which were maintained at 37°C with a heated stage and lid (Bioptechs).
For live cell imaging of actin dynamics following inhibitor treatment, cells were activated as above for 10 min before addition of media containing DMSO or jasplakinolide (1 µM, Calbiochem). Following 5 min of incubation, media containing DMSO or latrunculin A (10 µM, Sigma) was added to the dish. After 5 min of further incubation, cells were imaged.
For fixed cell experiments, 1×105 cells were adhered to No. 1 glass coverslips coated with antibody as described above. Samples were fixed and stained with Alexa Fluor 488 phalloidin or 568 phalloidin (Molecular Probes) as described
Samples were imaged through a 1.49 NA, oil immersion, 60×, APO N TIRFm objective or a 1.45 NA, oil immersion, 100×, PlanApo TIRFm objective (Olympus) when noted. 488 nm (Spectra-Physics) and 561 nm (Cobalt) diode lasers were launched through a two-line combiner of an LMM5 (Spectral Applied Research) into a rear mounted TIRF illuminator (Olympus) on an Olympus IX-81. Lasers were aligned for total internal reflection prior to each experiment. Images were captured using Volocity (PerkinElmer) to control a C9100 EM-CCD camera (Hamamatsu).
Mel1190 cells were plated into ΔT dishes 1 d prior to use. Cells were stained with CellMask Deep Red (Invitrogen) according to manufacturer's instructions just prior to imaging. GFP-actin expressing NK-92 cells were added to the dishes, which were maintained at 37°C, and imaged for up to 1 h. Cells were imaged through a 63×1.4 NA Plan-APOCHROMAT objective (Zeiss) on a Zeiss Observer.Z1 using a C10600 ORCA-R2 camera (Hamamatsu). The microscope was equipped with a CSU10 spinning disk system (Yokogawa). 491 nm (Cobalt) and 655 nm (CrystaLaser) diode lasers were launched through an LMM5 (Spectral Applied Research).
NK-92 cells were activated on antibody coated glass coverslips as described above for 30 min and then fixed and stained with rabbit anti-human Pericentrin (Abcam), Alexa Fluor 488-phalloidin, and anti-Perforin-Alexa Fluor 647 (Biolegend). The secondary antibody to anti-Pericentrin was a goat anti-mouse Pacific Blue (Molecular Probes). Cells were imaged in three dimensions on a spinning disk confocal Olympus DSU IX-81 microscope.
Mel1190 cells were grown in a monolayer overnight on No. 1.5 glass coverslips. Citrine-actin expressing NK-92 cells (106) were resuspended in media and incubated on Mel1190 targets for 30′ at 37°C. Cells were fixed with 2% paraformaldehyde and mounted with ProLong antifade reagent (Invitrogen). Cells were imaged at the plane of the interface between NK and target cells. Separately, for imaging of granules, Citrine-actin expressing NK-92 cells were immobilized to bound antibody as described above, then fixed, permeabilized, and stained with anti-perforin Alexa Fluor 488 (Biolegend).
For visualization of actin and perforin in NK-92 cells, cells were immobilized to bound antibody as described above, then fixed, permeabilized, stained with Alexa Fluor 488 phalloidin and anti-perforin Alexa Fluor 647, and imaged at the plane of the glass or 0.5–1 µm above it.
All samples were mounted with Prolong anti-fade reagent (Invitrogen). Cells were imaged through a 100×1.4 NA HCX APO objective on a Leica TCS STED CW system controlled by Leica AS AF software. Alexa Fluor 488 and Citrine were excited using a 488 nm Argon laser and STED depletion was achieved using a 592 nm continuous wave fiber laser. Alexa Flour 647 was excited using a HeNe 633 laser and imaged using the laser scanning confocal modality of the system. Fluorescence was detected with HyD detectors (Leica).
Cells were washed, resuspended in supplemented Myelocult, and allowed to adhere to 0.15 glass coverslips coated with antibody as described above. For imaging of the actin network alone, samples were prepared following a modified protocol described in
Immulon 4HBX 96 well flat bottom plates (Thermo) were coated with murine IgG (BD), anti-human NKp30, anti-human CD18, or both anti-NKp30 and anti-CD18 at 5 µg/mL in PBS overnight at 4°C. Plates were washed twice with PBS and blocked for 1 h at room temperature with R10 media. 1×105 cells were added to wells and plates were spun at 1,000 rpm for 2 min before incubation at 37°C. For the timecourse degranulation assay, supernatants from spontaneous and activated wells were harvested at indicated times. For inhibitor treatments, media containing inhibitor or vehicle (DMSO) was added at indicated times. Thapsigargin (Calbiochem) was used at 1 µM. Supernatants were harvested after 60 min. For total release, cells were lysed in 0.5% Nonidet P40 (Accurate Chemical and Scientific).
Supernatants were assayed by mixing 20 µL supernatant with 200 µL of a solution containing PBS, 9.8 mM HEPES (Gibco), 196 µM Z-L-Lys-SBzl hydrochloride (BLT, Sigma), and 218 µM 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB, Sigma). Samples were incubated for 30 min at 37°C and absorbance was measured immediately at 405 nm. Percent total release was measured by subtracting the spontaneous release value from activated release values (A–S) and the total release value (T–S), and then dividing (A–S) by (T–S).
Images and timelapse series were analyzed using either Volocity or the FIJI package of ImageJ (
Distance of granules from the MTOC was determined as described
FIJI was used to generate radial intensity profiles using the radial profile plugin (
To measure changes in the actin network over time, sequential images were imported into FIJI, a line was drawn across the center of the cell, and line intensity profile data were generated for each time point at the same location within the cell. Data were exported to Excel and standard deviations calculated. Surface plots were generated using the surface plot function in FIJI.
Images of granules taken using STED microscopy were analyzed in Volocity and diameters measured by drawing lines across the center of the granules. STED microscopy images of the actin network were imported into FIJI and processed before analysis. Background was subtracted using the Rolling Ball Subtraction algorithm with the radius set to 150 pixels and then pixel intensities were squared twice. An ROI was drawn around the interior of the cell and clearances were identified using the default autothreshold with “dark background” unchecked. Clearance areas were sorted, grouped, and counted in Excel based on size. Dividing the number of clearances per cell by the area measured normalized the values. Area cutoffs were implemented by using granule diameter as a reference. The smallest two sizes calculated assume that granules are uniformly spherical and require a clearance that has an area that would accommodate the equatorial area of the granule. The larger size categorizes all clearances that are larger than most granules.
Colocalization analysis was performed in Volocity using the “Find objects” tool to identify granules and a fixed intensity threshold, which was adjusted to generate interfilament or intercellular black space on a per field basis, to identify the actin network. The colocalized area of granule and actin network staining was divided by the area of the granule to yield a value denoting the percent of the granule area colocalized with actin. Five granules from 10 cells over 2 experiments were measured (
Images from platinum replica electron microscopy were inverted and linearly contrast enhanced using Photoshop (Adobe) and imported into FIJI for processing and analysis. Background was subtracted from each image using the Rolling Ball Subtraction algorithm with the radius set to 25 pixels. Pixel intensities were subsequently squared. Cells were identified using the default autothreshold and cell contact area and cell centroid were measured with “include holes” checked. ROIs were drawn around the interior of the cell to more accurately identify filaments and to avoid debris. Filaments were identified using the default automatic threshold with “dark background” checked. Clearances in the filamentous network were identified as mentioned above. Distance from the cell centroid was determined by inputting the coordinates of the cell centroid and a clearance centroid into the Pythagorean equation as described above for granule to MTOC distance.
All data were plotted using Prism (Graphpad).
Statistical significance was determined using Prism to perform one- or two-sample, unpaired or paired, two-tailed Student's
Timecourse of degranulation of activated NK-92 cells. NK-92 cells were activated by immobilized antibody to NKp30 and CD18 and incubated at 37°C. (A) Supernatants were harvested at indicated times and assayed for Granzyme A activity using the BLT esterase assay and results are shown as a percent of total potential release. Single antibody control supernatants were harvested following 60 min of activation. Values shown represent the mean + SD of three independent experiments.
(TIF)
Kinetics and sustenance of actin accumulation at the activated IS. (A) GFP-actin (green) expressing NK-92 cells were activated on immobilized antibody to NKp30 and CD18 and imaged by TIRF microscopy. Images were acquired over 50 min at a rate of 1 frame per minute. Images of a representative cell are shown at 5-min intervals beginning following 2.5 min of contact. Scale bar = 5 µm. (B) Area and mean fluorescence intensity (MFI) for 6 cells plotted over time (error bars, ± SD). Data are representative of three independent experiments.
(TIF)
Actin hypodensities present within the NK cell synaptic cortex. NK-92 cells expressing GFP-actin were activated and imaged by TIRFm. (A) Image of GFP-actin at 30 min post-activation. Scale bar = 5 µm. (B) Magnification of boxed region from (A). (C) Line profile of intensity from dotted line in (B).
(TIF)
Quantitative analysis of granule approximation to the actin network. (A) The intensity of GFP-actin fluorescence at the point of granule approximation was determined by dividing the MFI of GFP-actin in the granule region by the MFI of the GFP-actin of the whole cell in the TIRF field. This yielded a ratio of MFI signals. Each point represents one granule. (B) Minimum (min) and maximum (max) possible values are plotted along with the mean. Min and max values were determined by using the minimum and maximum pixel values of the GFP-actin signal in the TIRF field and the equation described in (A). (C) GFP ratio values for 14 cells plotted as in (A).
(TIF)
Model and implementation of pHluorin-LAMP1 construct. (A) Model of the construct depicting relative locations of sequences: endoplasmic reticulum targeting signal sequence (SS), flexible glycine-serine linker (GS), transmembrane domain (TM). (B) Diagram depicting fluorescent state of pHluorin depending on intralumenal versus surface location.
(TIF)
Degranulation events are less abundant than granule approximations. (A) pHluorin-LAMP1 expressing cells were loaded with LysoTracker Red and imaged for approximately 60 min at a rate of 1 frame per minute. (B) To count events, all frames from the acquisition were merged into a single image. (C) Number of Lysotracker positive and pHluorin positive events for each cell are plotted (
(TIF)
Degranulation MFI actin ratios plotted relative to minimum and maximum potential ratios. MFI ratio of actin intensities at the point of degranulation to that of the respective footprints (black) is plotted relative to minimum (blue) and maximum (red) potential values for 52 events.
(TIF)
Radial intensity profile plots for synaptic actin following treatment with actin inhibitors. NK-92 cells were activated for 10 (A) or 20 min (B) before addition of DMSO or inhibitor. Following 5 min of incubation, cells were fixed, stained for actin with phalloidin, and imaged by TIRFm using a 100× objective. Radial intensity profiles were generated and averaged for 30 cells/condition over 3 experiments. For latrunculin A treated cells, DIC images were used for spatial reference since actin fluorescent signal was undetectable.
(TIF)
The actin network is dynamic at early and late timepoints of activation. GFP-actin expressing NK-92 cells were activated and imaged at a rate of 2 frames per minute after 10 min and 30 min of activation for 5 min. Scale bar = 5 µm. (A) Images from the first 2.5 min of the 10 to 15 min timeframe are shown. (B) Corresponding intensity surface plots from the timepoints shown in (A). Overlay of line profiles through the centroid of the cell contact from images taken between 10 and 15 min of activation (C), or 30–35 min (D) of activation. (E) To compare variation in multiple cells (
(TIF)
Diameters of granules imaged by STED microscopy and their relation to clearance area. (A) NK-92 cells were activated on glass, fixed, and stained for perforin. 104 granules were measured. (B) The mean clearance area for each cell (defined as any area large enough to accommodate a 250 nm in diameter granule) was divided by the mean granule equatorial area derived from (A) and plotted according to interval. The mean granule diameter of 333 nm corresponds to a mean equatorial area of 0.0871 µm2.
(TIF)
Branched networks at the activated IS. (A) High magnification image of filaments at the activated synapse using platinum replica electron microscopy. (B) Image from (A) with pseudocolored region indicating examples of branching filaments. Scale bar = 100 nm.
(TIF)
Additional analyses of cells imaged by platinum replica electron microscopy. (A–C) Comparative measurements of the synapse include: contact area (A); filament density (B); and distance from the cell centroid of individual clearances that would be greater than or equal in size to the equatorial area of a 250 nm granule (C) (*
(TIF)
Algorithm-based identification of clearances in the F-actin network. Colored regions indicate appropriately sized clearances that were identified.
(TIF)
Live cell imaging of activated GFP-actin expressing NK-92 cells. Cells were imaged at 2 frames per minute and are shown at 6 frames per second starting at time zero relative to activation.
(MOV)
Live cell imaging of activated GFP-actin expressing, LysoTracker Red loaded NK-92 cells. Cells were imaged at 1 frame per minute and are shown at 4 frames per second starting 2 min after activation.
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Live imaging of a Lysotracker Red loaded granule in a pHluorin-LAMP1 expressing cell undergoing degranulation and attaining pHluorin fluorescence. Images were acquired at 2 frames per minute and are shown at 6 frames per second.
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Live cell imaging of activated pHluorin-LAMP1 expressing, LysoTracker Red loaded NK-92 cells. Cells were imaged at 1 frame per minute and are shown at 4 frames per second starting at time zero relative to activation. LysoTracker Red events outnumber pHluorin events.
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Live cell imaging of activated pHluorin-LAMP1 and mCherry-actin expressing NK-92 cells. Cells were imaged at 6 frames per minute and are shown at 6 frames per second. Video begins 25 min after activation.
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Live cell imaging and intensity surface plots of activated GFP-actin expressing NK-92 cells. Cells were imaged at 2 frames per minute and are shown at 2 frames per second starting at 10 min after activation.
(AVI)
The authors thank P. Kumar, R. Pandey, and L. Monaco-Shawver for technical assistance; K. Campbell for gifts of cell lines; W. Pear, G. Miesenböck, R. Balice-Gordon, M. Marks, and R.Y. Tsien for gifts of reagents; K. Rak for valuable suggestions; and C. Yang and F. Korobova for technical training.
concanamycin A
continuous wave
filamentous-actin
immunological synapse
lysosomal-associated membrane protein 1
mean fluorescence intensity
microtubule organizing center
natural killer
phorbol myristate acetate
stimulated emission depletion
total internal reflection fluorescence microscopy
Wiskott-Aldrich Syndrome
Wiskott-Aldrich Syndrome protein