Tirf system

Yeast cells were imaged with a 60x TIRF objective (Nikon, MRD01691) on a Nikon TI-E equipped with a Nikon TIRF system and a SCMOS camera (Andor, Ixon Ultra 888). Protoplasts were imaged on a Nikon TI-2 equipped with a Diskovery Multi-modal imaging system from Andor and a SCMOS camera (Andor, Ixon Ultra 888) using a 60x TIRF objective (Nikon, MRD01691). Cells were imaged at 100 Hz, in TIRF, for 10 s with a 488 nm excitation laser.

VAMP2-pHluorin^{45 (link)} was transfected into INS-1 cells after siRNA knockdown treatment. Twenty four h later, cells were subjected to TIRF live cell imaging to quantify fusion events as described.^{46 (link)} On the day of the live cell imaging experiment, cells were preincubated with KRBH buffer containing 2 mM glucose in an incubator and then placed in a metal chamber mounted on a heated stage and kept at 37°C throughout the imaging experiments. Time lapse images were acquired by a Nikon TIRF system equipped with the PerfectFocus stability mechanism at 100 ms exposure and readout rate 540 MHz from an Andor Zyla 4.2 Megapixel sCMOS camera. To analyze fusion events, background normalized image sequences were first processed by Trackmate plugin in Fiji-ImageJ.^{47 (link)} The statistics of all identified spots were extracted. The data were then calculated by custom script in Python to generate time series intensity curves for each spot track including the preceding and following frames. The spot tracks were finally compiled for visualization within Fiji-Image J for manual confirmation. Individual fusion events were defined by the rapid appearance and disappearance of VAMP2-pHluorin fluoresence labeled vesicles. Kinetics of fusion decay curve for identified fusion events were extracted and normalized by the peak signal.

For measurement of docked insulin granules, dispersed islet cells from control or APT1 global deficient mice were fixed with 4% paraformaldehyde for 10 min, then monolayer cells were permeabilised using 0.1% Triton-X 100, and blocked with 5% goat serum in PBS at room temperature for 1 h. Then cells were stained for insulin (guinea pig polyclonal antibody from Abcam, Cat. No. ab7842) overnight at 4°C. TIRF images were then acquired by a Nikon TIRF system in the Cell and Tissue Imaging Core of the Washington Univesity Diabetes Research Center and granule density was counted by Fiji-ImageJ. For the assessment of cortical F-actin, Alexa Fluor 594 conjugated phalloidin (Thermofisher, Cat. No. A12381) was used to stain dispersed islet cells and images were acquired by a Nikon A1plus laser scan confocal microscope. Cell borders were delineated and fluoresence intensity of F-actin was quantitated after subtraction of background (Fiji-ImageJ).

For mouse islets, dispersed islet cells from control mice were fixed and stained for insulin and Scamp1 (Thermofisher, Cat. No. PA1-739) as described above. For INS-1 cells, the cells were co-transfected with GFP-Scamp1 and mCherry-NPY (Addgene, Cat. No. 67156). TIRF images were acquired by a Nikon TIRF system as described above.