Supplementary Figure Legends:

Figure S1: Regulated degranulation elicited by PMA and ionomycin in NKL cells. (A) NKL cells on poly-L-lysine coated glass coverslips were stimulated with PMA and ionomycin and imaged by TIRF microscopy in the presence of 0.016 µM Alexa Fluor 647-conjugated CD107a F(ab). A stimulated NKL cell (upper panel) and an unstimulated NKL cell (bottom panel) were imaged by TIRF microscopy. Movie frames from the indicated time points are shown. Corresponding DIC images are shown on the left. The time point ‘0’ indicates the start time point at which TIRF images were acquired. Note the dynamic and dispersed distribution of exocytosed LAMP-1 (upper panel). Very little LAMP-1 is detected in the absence of PMA and ionomycin treatment (bottom panel). Note that 0.016 µM Alexa Fluor 647-conjugated CD107a F(ab) did not label NK cells non-specifically. The scale bars are 5 µm. (B) Maximum fluorescence intensity (FI) for individual NKL cells treated either with DMSO vehicle (Control) or with PMA and ionomycin. The average and standard deviation of the mean are indicated. Statistical significance was determined using the t-test. Data are representative of two independent experiments.

Figure S2: Complete fusion of a granzyme B-DsRed containing lytic granule in live NKL cell under TIRF microscopy.

Time-lapsed images taken at ~ 20 min from a fusion event after addition of PMA and ionomycin. Images were acquired by TIRF microscopy. The scale bar is 500 nm. The images are representative of at least 5 fusion events from 12 cells in two independent experiments.

Figure S3: Colocalization of granzyme B-DsRed with GFP-FasL in live NKL cells.

NKL cells were double transfected with granzyme B-DsRed and GFP-FasL. Images were acquired by Meta-510 Zeiss confocal microscopy. The scale bar is 5.0 µm. The images are representative of at least 50 cells in two independent experiments.

Figure S4: Colocalization of GFP-FasL with perforin containing LG in NKL cells.

Stably transfected NKL-GFP-FasL cells were fixed and permeabilized on Poly-L-Lysine coated coverslips. Perforin containing lytic granules were stained by anti-perforin monoclonal antibody followed by Alexa Fluor conjugated secondary antibody. The scale bar is 3.0 µm. The images are representative of 10 cells in two independent experiments.

Figure S5: Complete fusion of two DsRed-FasL-pHluorin positive lytic granules in live NKL cell under TIRF microscopy.

Time-lapsed images taken at ~ 38 min from two complete fusion events after addition of PMA and ionomycin. Sequential TIRF microscopy images of single LG in complete fusion event #1 and #2 are shown. Images were acquired by TIRF microscopy. The scale bars are 1.0 µm. The images are representative of at least 5 fusion events from 20 cells in two independent experiments. The weak DsRed signal in the stably transfected NKL cells may be due to proteolytic cleavage in the cytoplasmic tail of DsRed-FasL-pHluorin.

Figure S6: DsRed-FasL-pHluorin positive lytic granules in primary NK cells do not undergo spontaneous fusion.

Primary, unstimulated NK cells were transiently transfected with DsRed-FasL-pHluorin and attached onto poly-L-lysine coated coverslips. Dual color images were acquired simultaneously by TIRF microscopy equipped with GFP/DsRed dual-view microimager. The scale bar is 500 nm. The images are representative of 65 lytic granules from 13 cells in two independent experiments. Note that a DsRed signal was observed but without a pHluorin fluorescence signal.


Supplementary Movie Legends

Movie S1: Live images of regulated degranulation elicited by PMA and ionomycin.

Accumulation of Alexa Fluor 647-CD107a Fab over time under PMA and ionomycin stimulation (upper-left). In the upper-right panel, the updated mean fluorescence intensity of CD107a over time on the highlighted region by a purple square is shown over time. Note that the time interval between frames is 5 seconds. The scale bar is 5 µm. The images were converted into gray scale 8 in Image-pro-plus software. In contrast, very little CD107a staining was observed in NKL cells in the absence of PMA and ionomycin (bottom panel). The time point ‘0’ indicates the start time point at which TIRF images were acquired. The data is representative of NK cells that have been undergoing degranulation.

Movie S2: Complete fusion of LG visualized with GFP-FasL.

Single color TIRF imaging was carried out in NKL cell expressing GFP-FasL. The arrow indicates the LG that is undergoing complete fusion under TIRF microscopy. LG approached the PM, appeared to dock, and rapidly fused with the PM, releasing a bright fluorescence cloud that diffused at the PM, consistent with complete fusion. Scale bar is 3 µm.

Movie S3: Visualization of incomplete fusion by double labeling of LG membrane and cargo protein.

Two-color TIRF imaging was carried out in NKL cell co-expressing granzyme B-DsRed (LG cargo protein) and GFP-FasL (LG membrane protein). This movie was acquired by simultaneous illumination of pHluorin and DsRed using a GFP/DsRed dual-view microimager. The arrow indicates the LG that is undergoing incomplete fusion. Scale bar is 3 µm.

Movie S4: Addition of ammonium chloride (NH4Cl) to raise the pH in LG induced strong pHluorin fluorescence.

Two-color confocal imaging was carried out in NKL cell expressing DsRed-FasL-pHluorin after addition of 500 mM NH4Cl (pH 7.4) solution. Imaging was acquired at 20 seconds per frame. The movie was played at 5 frames per second. Scale bar is 10 µm. Please note the cell labeled with circle. The pHluorin fluorescent signals (Green) increased as the DsRed fluorescent signals (Red) are relatively constant.

Movie S5: Visualization of incomplete fusion event using DsRed-FasL-pHluorin probe.

Two-color TIRF imaging was carried out in NKL cell expressing DsRed-FasL-pHluorin. The DsRed and pHluorin fluorescent signals were acquired simultaneously by dual-view microimager under TIRF microscopy. The left panel is pHluorin signal. The right panel is DsRed signal. The circle indicates the LG that is undergoing incomplete fusion. Scale bar is 0.5 µm.

Movie S6: Visualization of complete fusion event using DsRed-FasL-pHluorin probe.

Two-color TIRF imaging was carried out in NKL cell expressing DsRed-FasL-pHluorin. The DsRed and pHluorin fluorescent signals were acquired simultaneously by dual-view microimager under TIRF microscopy. The left panel is pHluorin signal. The right panel is DsRed signal. The arrow indicates the LG that is undergoing complete fusion. Scale bar is 3.0 µm.

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