This protocol describes a confocal imaging technique to detect three fusion modes in bovine adrenal chromaffin cells. These fusion modes include 1) close-fusion (also called kiss-and-run), involving fusion pore opening and closure, 2) stay-fusion, involving fusion pore opening and maintaining the opened pore, and 3) shrink-fusion, involving fused vesicle shrinkage.
Dynamic fusion pore opening and closure mediate exocytosis and endocytosis and determine their kinetics. Here, it is demonstrated in detail how confocal microscopy was used in combination with patch-clamp recording to detect three fusion modes in primary culture bovine adrenal chromaffin cells. The three fusion modes include 1) close-fusion (also called kiss-and-run), involving fusion pore opening and closure, 2) stay-fusion, involving fusion pore opening and maintaining the opened pore, and 3) shrink-fusion, involving shrinkage of the fusion-generated Ω-shape profile until it merges completely at the plasma membrane.
To detect these fusion modes, the plasma membrane was labeled by overexpressing mNeonGreen attached with the PH domain of phospholipase C δ (PH-mNG), which binds to phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) at the cytosol-facing leaflet of the plasma membrane; vesicles were loaded with the fluorescent false neurotransmitter FFN511 to detect vesicular content release; and Atto 655 was included in the bath solution to detect fusion pore closure. These three fluorescent probes were imaged simultaneously at ~20-90 ms per frame in live chromaffin cells to detect fusion pore opening, content release, fusion pore closure, and fusing vesicle size changes. The analysis method is described to distinguish three fusion modes from these fluorescence measurements. The method described here can, in principle, apply to many secretory cells beyond chromaffin cells.
Membrane fusion mediates many biological functions, including synaptic transmission, blood glucose homeostasis, immune response, and viral entry1,2,3. Exocytosis, involving vesicle fusion at the plasma membrane, releases neurotransmitters and hormones to achieve many important functions, such as neuronal network activities. Fusion opens a pore to release vesicular contents, after which the pore may close to retrieve the fusing vesicle, which is termed kiss-and-run1,4. Both irreversible and reversible fusion pore opening can be measured with cell-attached capacitance recordings combined with fusion pore conductance recordings of single vesicle fusion.
This is often interpreted as reflecting full-collapse fusion, involving dilation of the fusion until flattening of the fusing vesicle, and kiss-and-run, involving fusion pore opening and closure, respectively5,6,7,8,9,10,11,12,13. Recent confocal and stimulated emission depletion (STED) imaging studies in chromaffin cells directly observed fusion pore opening and closure (kiss-and-run, also called close-fusion), fusion pore opening that maintains an Ω-shape with an open pore for a long time, termed stay-fusion, and shrinking of the fusing vesicle until it complete merges with the plasma membrane, which replaces full-collapse fusion for merging fusing vesicles with the plasma membrane4,8,14,15,16,17.
In neurons, fusion pore opening and closure have been detected with imaging showing the release of quantum dots preloaded in vesicles that are larger than the fusion pore and with fusion pore conductance measurements at the release face of nerve terminals5,18,19. Adrenal chromaffin cells are widely used as a model for the study of exo- and endocytosis20,21. Although chromaffin cells contain large dense-core vesicles, whereas synapses contain small synaptic vesicles, the exocytosis and endocytosis proteins in chromaffin cells and synapses are quite analogous10,11,12,20,21,22,23.
Here, a method is described to measure these three fusion modes using a confocal imaging method combined with electrophysiology in bovine adrenal chromaffin cells (Figure 1). This method involves loading of fluorescent false neurotransmitters (FFN511) into vesicles to detect exocytosis; addition of Atto 655 (A655) in the bath solution to fill the fusion-generated Ω-shape profile, and labeling of the plasma membrane with the PH domain of phospholipase C δ (PH), which binds to PtdIns(4,5)P2 at the plasma membrane8,15,24. Fusion pore dynamics can be detected through changes in different fluorescent intensities. Although described for chromaffin cells, the principle of this method described here can be applied widely to many secretory cells well beyond chromaffin cells.
NOTE: The animal use procedure followed NIH guidelines and was approved by the NIH Animal Care and Use Committee.
1. Bovine chromaffin cell culture
2. Transfection with electroporation
3. Preparation for patch-clamp recording and confocal imaging
NOTE: This protocol was performed with a laser scanning confocal microscope and patch-clamp amplifier with voltage-clamp recording together with a lock-in amplifier for capacitance recording. An XY plane confocal imaging at a fixed Z-plane (XY/Zfixed scanning) was used to image all three fluorescent signals simultaneously. The Z-plane was focused at the cell bottom where the plasma membrane was adhering to the coverslips.
4. Patch-clamp recording and confocal imaging
5. Patch-clamp data analysis
6. Confocal imaging data analysis
Following the experimental procedures shown in Figure 1 and Figure 2, chromaffin cells from bovine adrenal glands were transfected with PH-mNG to label the plasma membrane; A655 was added to the bath solution to detect fusion pore closure; and fluorescent false neurotransmitter FFN511 was loaded in vesicles for detection of release. Next, XY-plane confocal timelapse imaging of FFN511, PH-mNG, and A655 was performed every 20-90 ms at the cell bottom (Z-focal plane ~100-200 nm above the cell membrane). Whole-cell patch-clamp recording and application of a 1 s depolarization from -80 to +10 mV was performed to evoke exo- and endocytosis (Figure 3A–C). This depolarization induced an inward calcium current, a capacitance jump that indicates exocytosis, and a capacitance decay after the jump that indicates endocytosis (Figure 3D, E).
With timelapse XY/Zfixed imaging at the cell bottom, many individual fusion events were observed8,24 after depolarization (Figure 4A), whereas rare fusion events were observed before depolarization. Fusion events induced by the 1 s depolarization protocol were identified as FFN511 spot fluorescence (FFFN) decrease reflecting FFN511 release, accompanied by FPH and A655 spot fluorescence (F655) increase reflecting PH-mNG and A655 diffusion from the plasma membrane and the bath solution into the fusing vesicle (the fusion-generated Ω-profile)15.
After fusion, the Ω-profile generated by vesicle fusion with the plasma membrane may 1) close its pore, termed close-fusion, 2) maintain an open fusion pore, termed stay-fusion, or 3) shrink to merge into the plasma membrane, termed shrink-fusion. Close-fusion was identified as F655 dimming while FPH sustained or decayed with a delay (Figure 4B). Stay-fusion was detected as the sustained presence of both PH-mNG and A655 spots (Figure 4C). Shrink-fusion was detected as parallel FPH and F655 decay accompanied with a parallel size reduction of the PH-mNG spot and the A655 spot (Figure 4D)8,15,16,24.
Figure 1: Schematic representation of the experimental protocol. (A, B) Bovine adrenal glands are trimmed with scissors to remove fat tissue (A), flushed with Locke's solution, and digested through adrenal vein (B). (C) The interior of an adrenal gland without digestion (left) or after proper digestion (right). (D) After being washed and digested, the medullae are isolated and minced into small pieces, and chromaffin cells are separated from minced medullae after filtering and centrifuging. (E) Chromaffin cells are electroporated and plated on coverslips for incubation. (F) On days 2-3, check the chromaffin cells under a microscope before experimentation. (G) The cell sample is embedded in the chamber for patch-clamp recording and confocal imaging. Calcium current and capacitance changes are recorded, amplified, and displayed on the monitor. Fluorescence changes upon stimulation are detected and displayed on the monitor. Scale bars = 40 mm (A), 20 mm (B-D), 20 µm (F). Please click here to view a larger version of this figure.
Figure 2: Patch-clamp and confocal setup. (A) On the day of experimentation, the chromaffin cells grown on coverslips are incubated with FFN511 for 20 min. The dye A655 is added to the bath solution. (B) Chromaffin cells on the coverslip are transferred to a recording chamber, and the bath solution with A655 is added to the chamber. (C) After adding a drop of oil on the 100x oil immersion objective, the chamber is mounted onto the stage of an inverted confocal microscope. The tip of the ground wire is immersed in bath solution. The pipette is brought into position after loading with pipette solution and held by a pipette holder, which is attached to a headstage. The headstage is controlled by a motorized micromanipulator. (D) After finding a good cell, move the pipette tip into the bath solution with the micromanipulator to start whole-cell patch-clamp recording and confocal imaging. Scale bars = 10 mm (A), 5 mm (B). Please click here to view a larger version of this figure.
Figure 3: Whole-cell voltage-clamp recordings of calcium currents and capacitance changes induced by depolarization. (A) Setup drawing of chromaffin cells during whole-cell voltage-clamp recording. The chromaffin cell is immersed in A655-containing bath solution (red), and the cell membrane and vesicles are labeled with PH-mNG (green) and FFN511 (cyan), respectively. This image has been modified with permission from 24. (B) A representative image of a patch-clamped chromaffin cell observed by brightfield. (C) Representative images of PH-mNG (green), A655 (red), and FFN511 (cyan) in a cell with good focus at the cell footprint. (D) An example of calcium current and capacitance changes induced by 1 s depolarization from -80 to +10 mV. (E) The averaged traces of calcium currents (ICa) and capacitance (Cm) changes collected from 20 chromaffin cells. This image has been modified with permission from 26. Scale bars = 5 µm (B, C). Abbreviations: ICa = calcium current; Cm = capacitance; Depol = depolarization. Please click here to view a larger version of this figure.
Figure 4: Visualization of fusion events under the confocal microscope. (A) Many fusion spots can be detected with confocal XY-plane imaging of PH-mNG (green), A655 (red), and FFN511 (cyan) at the cell bottom. Fusion was evoked by a 1 s depolarization from -80 to +10 mV (depol1s). FFN spots underwent release and close-fusion (circles). Images before (-1 s) and after (+1 s and +10 s) depolarization are shown. (B) An example of close-fusion. (C) An example of stay-fusion. (D) An example of shrink-fusion. This image has been modified with permission from 24. Scale bars = 1 µm (A), 0.5 µm (B–D). Abbreviations: Depol = depolarization; F = fluorescent intensity. Please click here to view a larger version of this figure.
Medium/Solution | Description | ||
Locke's solution | 145 mM NaCl, 5.4 mM KCl, 2.2 mM Na2HPO4, 0.9 mM NaH2PO4, 5.6 mM glucose, and 10 mM HEPES, pH 7.3, adjusted with NaOH | ||
Enzyme solution | 1.5 mg/mL collagenase P, 0.325 mg/mL trypsin inhibitor, and 5 mg/mL bovine serum albumin in Locke's solution | ||
Culture medium | DMEM medium supplemented with 10% fetal bovine serum | ||
Internal solution | 130 mM Cs-glutamate, 0.5 mM Cs-EGTA, 12 mM NaCl, 30 mM HEPES, 1 mM MgCl2, 2 mM ATP, and 0.5 mM GTP, pH 7.2, adjusted with CsOH | ||
Bath solution | 125 mM NaCl, 10 mM glucose, 10 mM HEPES, 5 mM CaCl2, 1 mM MgCl2, 4.5 mM KCl, and 20 mM TEA, pH 7.3, adjusted with NaOH |
Table 1: Details regarding the composition of culture medium and solutions.
A confocal microscopic imaging method is described to detect the dynamics of fusion pore and transmitter release, as well as three fusion modes, close-fusion, stay-fusion, and shrink-fusion in bovine adrenal chromaffin cells4,24. An electrophysiological method to depolarize the cell and thereby evoke exo- and endocytosis is described. Systematic confocal image processing provides information about different modes of pore behaviors for fusion and fission events.
Simultaneous monitoring of calcium current and capacitance changes at the same cell with the whole-cell configuration provides additional information about exo- and endocytosis, implying the ratio of pore opening and closure at a whole-cell level at any given time. These methods are, in principle, applicable to other excitable cells and nonelectrically excitable cells in primary culture or in cell lines containing vesicles of ~200-1,600 nm15. In addition to chromaffin cells, the method described here has been applied to a rat pancreatic beta-cell line, INS-1 cells4.
In principle, the method can also be applied to secretory cells containing vesicles smaller than 200 nm. However, as the vesicle size is decreased, the signal-to-noise-ratio may be too low for reliable detection. Super-resolution imaging instead of confocal imaging may become necessary. So far, the methods described here have not been applied to synapses containing ~40 nm synaptic vesicles.
Successful implementation of the method described here depends critically on several general steps, such as the culture cell quality, plasmid transfection efficiency, and electrophysiological recording success rate. First, healthy cells are vital for both patch-clamp recording and confocal imaging. It is not advised to use antibacterial or antifungal agents during culture because these chemicals may affect the excitability of chromaffin cells. Thus, diligent exercise of sterile technique and use of freshly prepared media are important. Although primary culture chromaffin cells can survive in dishes for at least one week25, it is important for new users to use cells on days 2 to 3 for the experiments. As fibroblasts grow gradually, it becomes more difficult to establish the whole-cell patch-clamp configuration. Second, the electroporation efficiency of plasmids into chromaffin cells is approximately 20-30%. The electroporation efficiency needs to be optimized if it is too low or PH-mNG fluorescence is too dim. Third, cells were imaged at their bottoms with a 100x oil objective to obtain substantial events indicated by changes in three fluorescent probes: FFN511, PH-mNG, and A655. Although DIC provides a clearer view than bright field or naked-eye imaging when establishing the whole-cell configuration, any visualization technique that allows the user to establish the configuration is sufficient.
Appropriate laser power strength is vital for confocal imaging in live cells. As FFN511 and mNG may be bleached by high laser power, it is better to look for a good cell with epifluorescence before visualization with confocal lasers. For A655, high-power laser excitation is needed to bleach the dye inside the vesicles. In addition, the most common reason this experiment is unsuccessful is a failure of patch-clamp recording. A clean and polished pipette tip of the proper size and practice generating the whole-cell configuration on chromaffin cells are key factors. Although it may take some time and effort to succeed, once established, these experiments provide substantial data regarding both fusion and fission events, indicating membrane pore opening and closure at a single event level.
The protocol described here can be used with some modifications to further different experimental goals. However, regardless of experimental goals, it is advisable to first use high potassium solution (such as 70 mM KCl) as a stimulus to see the changes the membrane undergoes upon being depolarized, ensuring the intensity changes in these three dyes, FFN, PH, and A655, can be visualized. The plasma membrane can be labeled with other membrane binding fluorophores, such as mCLING (membrane-binding fluorophore-cysteine-lysine-palmitoyl group)4,27 or CAAX (a protein motif that targets cytosol-faced plasma membrane via cysteine residue isoprenylation)4,28. A655 can be replaced with other dyes with different spectra, such as Atto 532 (A532), depending on the combination of fluorescent molecules used in certain experiments15. Neuropeptide Y can be used instead of fluorescent false neurotransmitters16.
FFN511 was excited at 458 nm instead of 405 nm in this experiment even though the peak excitation spectrum of FFN511 is about 405 nm. Study with capacitance recording showed no significant difference in chromaffin cells with or without PH-mNG, indicating that overexpression of PH-mNG does not affect exocytosis and endocytosis15,24. Similar percentages for fusion events were verified in chromaffin cells labeled with PH-mNG and A532 only or with FFN511, demonstrating that loading of FFN511 into chromaffin vesicles does not affect the content release and different fusion modes16. The 1 s depolarization stimulation can be replaced by high potassium solution or other pattens of depolarization. These modifications can be used to adapt to different experimental aims.
Despite the advantages of this powerful method, it has some limitations. The biggest limitation is related to the diffraction-limited resolution (~230 nm) of confocal microscopy, which can be overcome by more advanced super-resolution microscopy techniques29. The granule diameter in bovine chromaffin cells is ~300 nm6, making it possible to observe membrane pore opening and closure within the spatial resolution of confocal microscopy. However, synaptic vesicles are ~30-60 nm30, which is beyond confocal resolution. Extending these live-cell imaging measurements to small synaptic vesicles proves to be difficult with confocal imaging.
Live-cell imaging techniques with better temporal and/or spatial resolution, such as STED microscopy, stochastic optical reconstruction microscopy (STORM), photoactivated localization microscopy (PALM), total internal reflection fluorescence (TIRF) microscopy, and minimal photon fluxes (MINFLUX) nanoscopy have been developed and may be applied to this method31,32,33,34. Indeed, STED microscopy has been used to reveal fusion pore dynamics in chromaffin cells. The different modes of fusion events and preformed Ω-profile closure can be further verified using super-resolution microscopy such as STED microscopy24. Other limitations include photobleaching and cytotoxicity of fluorescent proteins and dyes.
This combination of electrophysiology and confocal microscopy can be applied widely to many secretory cells. The combination of super-resolution microscopy with the methods described here would be a valuable tool for measuring fusion pore dynamics in neural circuits in the future, provided that super-resolution microscopy will develop to such an extent that it can resolve the fusion pore at nerve terminals.
The authors have nothing to disclose.
We thank the NINDS Intramural Research Programs (ZIA NS003009-13 and ZIA NS003105-08) for supporting this work.
Adenosine 5'-triphosphate magnesium salt | Sigma | A9187-500MG | ATP for preparing internal solution |
Atto 655 | ATTO-TEC GmbH | AD 655-21 | Atto dye to label bath solution |
Basic Nucleofector for Primary Neurons | Lonza | VSPI-1003 | Electroporation transfection buffer along with kit |
Boroscilicate capillary glass pipette | Warner Instruments | 64-0795 | Standard wall with filament OD=2.0 mm ID=1.16 mm Length=7.5 cm |
Bovine serum albumin | Sigma | A2153-50G | Reagent for gland digestion |
Calcium Chloride 2 M | Quality Biological | 351-130-721 | Reagent for preparing bath solution |
Cell Strainers, 100 µm | Falcon | 352360 | Material for filtering chromaffin cell suspension |
Cesium hydroxide solution | Sigma | 232041 | Reagent for preparing internal solution and Cs-glutamate/Cs-EGTA stock buffer |
Collagenase P | Sigma | 1.1214E+10 | Enzyme for gland digestion |
Coverslip | Neuvitro | GG-14-Laminin | GG-14-Laminin, 14 mm dia.#1 thick 60 pieces Laminin coated German coverslips |
D-(+)-Glucose | Sigma | G8270-1KG | Reagent for preparing Locke’s solution and bath solution |
DMEM | ThermoFisher Scientific | 11885092 | Reagent for preparing culture medium |
EGTA | Sigma | 324626-25GM | Reagent for preparing Cs-EGTA stock buffer for bath solution |
Electroporation and Nucleofector | Amaxa Biosystems | Nucleofector II | Transfect plasmids into cells |
Fetal bovine serum | ThermoFisher Scientific | 10082147 | Reagent for preparing culture medium |
FFN511 | Abcam | ab120331 | Fluorescent false neurotransmitter to label vesicles |
Guanosine 5'-triphosphate sodium salt hydrate | Sigma | G8877-250MG | GTP for preparing internal solution |
HEPES | Sigma | H3375-500G | Reagent for preparing Locke’s solution |
Igor Pro | WaveMetrics | Igor pro | Software for patch-clamp analysis and imaging data presentation |
Leica Application Suite X software | Leica | LAS X software | Confocal software for imaging data collection and analysis |
Leica TCS SP5 Confocal Laser Scanning Microscope | Leica | Leica TCS SP5 | Confocal microscope for imaging data collection |
L-Glutamic acid | Sigma | 49449-100G | Reagent for preparing Cs-glutamate stock buffer for bath solution |
Lock-in amplifier | Heka | Lock-in | Software for capacitance recording |
Magnesium Chloride 1 M | Quality Biological | 351-033-721EA | Reagent for preparing internal solution and bath solution |
Metallized Hemacytometer Hausser Bright-Line | Hausser Scientific | 3120 | Counting chamber |
Microforge | Narishige | MF-830 | Polish pipettes to enhance the formation and stability of giga-ohm seals |
Millex-GP Syringe Filter Unit, 0.22 µm | Millipore | SLGPR33RB | Material for glands wash and digestion |
mNG(mNeonGreen) | Allele Biotechnology | ABP-FP-MNEONSB | Template for PH-mNeonGreen construction |
Nylon mesh filtering screen 100 micron | EIKO filtering co | 03-100/32 | Material for filtering medulla suspension |
Patch clamp EPC-10 | Heka | EPC-10 | Amplifier for patch-clamp data collection |
PH-EGFP | Addgene | Plasmid #51407 | Backbone for PH-mNeonGreen construction |
Pipette puller | Sutter Instrument | P-97 | Make pipettes for patch-clamp recording |
Potassium Chloride | Sigma | P5404-500G | Reagent for preparing Locke’s solution and bath solution |
Pulse software | Heka | Pulse | Software for patch-clamp data collection |
Recording chamber | Warner Instruments | 64-1943/QR-40LP | coverslip chamber, apply patch-clamp pipette on live cells |
Sodium chloride | Sigma | S7653-1KG | Reagent for preparing Locke’s solution, bath solution and internal solution |
Sodium hydroxide | Sigma | S5881-500G | Reagent for preparing Locke’s solution |
Sodium phosphate dibasic | Sigma | S0876-500G | Reagent for preparing Locke’s solution |
Sodium phosphate monobasic | Sigma | S8282-500G | Reagent for preparing Locke’s solution |
Stirring hot plate | Barnsted/Thermolyne | type 10100 | Heater for pipette coating with wax |
Syringe, 30 mL | Becton Dickinson | 302832 | Material for glands wash and digestion |
Tetraethylammonium chloride | Sigma | T2265-100G | TEA for preparing bath solution |
Trypsin inhibitor | Sigma | T9253-5G | Reagent for gland digestion |
Type F Immersion liquid | Leica | 195371-10-9 | Leica confocal mounting oil |