Actin dynamics provides membrane tension to merge fusing vesicles into the plasma membrane.

Primary bovine chromaffin cell culture

Bovine chromaffin cells were prepared as described previously 11 . Briefly, fresh adrenal glands were obtained from a local slaughterhouse on the day of culture. After excess fat was trimmed off the glands (two were used per culture), glands were then perfused with cold 1 × Lock's buffer containing (in mM): NaCl, 145; KCl, 5.4; Na 2 HPO 4 , 2.2; NaH 2 PO 4 , 0.9; glucose, 5.6; and HEPES, 10 pH 7.3, to remove any residual blood. Each gland was then injected through the portal vein with ∼2 ml of filtered Lock's buffer containing collagenase P (1.5 mg ml −1 , Roche), trypsin inhibitor (0.325 mg ml −1 , Sigma) and BSA (5 mg ml −1 , Sigma), and incubated at 37 ° C for 20 min in a water bath. The glands were then cut open longitudinally, to expose the digested medulla. The medulla were carefully removed, minced in Lock's buffer and filtered through a nylon mesh. The filtrate was centrifuged at 500 r.p.m. for 4–5 min, to obtain the cell pellet. The supernatant was then removed and the pellets resuspended in Lock's buffer. The process was repeated one to two times until the supernatant was clear. Final cell pellet was resuspended in pre-warmed DMEM low glucose medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and plated onto poly- D -lysine and laminin I-coated 25 mm-diameter glass coverslips (Neuvitro Corp., USA). The plated cells were incubated at 37 ° C, 8% humidified CO 2 and used within 4 days after culturing. In some experiments, cells were transfected with 2 μg of Lifeact-tagGFP2 (Ibidi, Germany) by electroporation using a basic neuron nucleofector kit (Lonza, Program O-005) according to the manufacturer's instruction.

Electrophysiology

Whole-cell voltage-clamp and capacitance recordings were performed with an EPC-10 amplifier together with the lock-in software (PULSE, HEKA, Lambrecht, Germany) 56 . All experiments were carried out at room temperature (21 °C −24 °C) with the cells immersed in bathing solution containing (in mM): 125 NaCl, 10 glucose, 10 HEPES, 5 CaCl 2 , 1 MgCl 2 , 4.5 KCl, 0.001 tetrodotoxin and 20 tetraethylammonium ion, pH 7.3. The osmolarity of the bath solution was 305–310 mOsm. In some experiments, we increased the osmolarity to 640–650 mOsm by adding 295 mM sucrose to the bath solution. In some experiments, we decreased the osmolarity to 160–221 mOsm by reducing NaCl to 55–70 mM. The pipette (3–6 MΩ) solution contained (in mM): 130 glutamate, 0.5 EGTA, 12 NaCl, 30 HEPES, 1 MgCl 2 , 2 ATP and 0.5 GTP pH 7.2 adjusted with CsOH. The osmolarity was ∼308 mOsm. In some experiments, ATP (2 mM) in the pipette solution was replaced with a non-hydrolysable ATPγS (2 mM, Sigma) and MgCl 2 was raised to 3 mM to facilitate endogenous ATP turnover 57 . For intracellular Ca 2+ dialysis experiments, the pipette solution contained (in mM): 110 glutamate, 10 EGTA, 12 NaCl, 30 HEPES, 1 MgCl 2 , 2 ATP 0.5 GTP and 9 CaCl 2 pH 7.2 adjusted with CsOH. The free Ca 2+ concentration was ∼1.5 μM, which was calculated based on the Max-Chelator programme (Stanford University, Stanford, CA), in which the calcium dissociation constant of EGTA is 0.15 μM 58 59 . The holding potential was −80 mV. For Train 2Hz stimulation, a Train of 50 ms depolarization from −80 to +10 mV was given at 2 Hz; each 50 ms depolarization was preceded by a 50 ms pre-pulse from −80 to +120 mV, to facilitate Ca 2+ channel current 60 61 62 . Cell membrane capacitance was simultaneously recorded 15 s before, during and 30 s after depolarization. The sample interval for current recordings was 50 μs. The frequency of the sinusoidal stimulus was 1,000–1,500 Hz with a peak-to-peak voltage ≤50 mV. Confocal imaging Alexa 647 (A647; 20–30 μM in bathing solution, Invitrogen) and Alexa 488 dyes (A488; 20–30 μM in bathing solution, Invitrogen) were excited by a solid-state 638 nm (30 mW output) and 488 nm lasers (20 mW output) with an inverted confocal microscope (TCS SP8, Leica, Germany; original magnification, × 63/1.40 oil objective). Unless mentioned otherwise, the 638 nm laser was set at 20–25% of the maximum power and the 488 nm laser was set at 0.5–2% of the maximum power. A647 fluorescence was collected with a photomultiplier at 639–767 nm, whereas A488 was collected with a hybrid GaAsP spectral detector at 489–596 nm. For time-lapse A647/A488 imaging, images were collected with 40 ms inter-frame interval at 45–70 nm per pixel in an imaging area of ∼160–320 μm 2 . In some experiments, cells were pre-treated with 3 μM latrunculin A (Enzo Life Sciences), 4 μM Cyto D (Enzo Life Sciences), wiskostatin (10 μM, Tocris Bioscience) and SMIFH2 (25 μM, Tocris Bioscience) for 20 min in the bath solution, or whole-cell dialysis of phalloidin–FITC (1.3 μM, Invitrogen) for 2–3 min before imaging.

STED imaging

The inverted STED microscopes used in this study (TCS SP5 STED, TCS SP8 STED 3X, Leica) have a resolution of ∼60–90 nm. A488 (60 μM) was excited with an Argon laser at 488 nm at 20% of the maximum power (maximum power: 25 mW) and depleted with a continuous wave fibre laser at 592 nm using the maximum power (1.5 W). The fluorescence was acquired by GaAsP hybrid detection system at 498–580 nm. At 20% of the maximum power, 488 nm laser caused A488 bleaching after pore closure with a time course similar to that of A647 under the confocal setting 11 . Lifeact (TagGFP2) was excited by a tunable white light laser at 470 nm (7% of maximum power) with the STED depletion laser at 592 nm (25% of the maximum power) and its fluorescence at 480–560 nm was collected using time-gated detection (1.5–6.5 ns). mCherry was excited by the tunable white light laser at 570 nm (6% of maximum power) with the STED depletion laser at 660 nm (60% of the maximum power) and its fluorescence between 575 and 650 nm was collected using time-gated detection (0.5–6.5 ns). PH–mPapaya was excited by the tunable white light laser at 530 nm (14% of the maximum power) with the STED depletion laser at 660 nm (10% of the maximum power) and its fluorescence between 541 and 620 nm was collected using time-gated detection (1.5–6.5 ns). When NPY–mCherry or PH–mPapaya and Lifeact (TagGFP2) were imaged at the STED microscope, NPY–mCherry (or PH–mPapaya) was imaged first, to avoid bleaching of mCherry or mPapaya fluorescence by the 592 nm STED depletion laser. For XZ scanning, we used 70–80% of STED depletion laser power in the Z -direction to improve the z axis resolution.

Image analysis A647 or A488 spots

(Ω-profiles filled with A647 or A488) were identified during and