We thank the NINDS Intramural Research Programs (ZIA NS003009-13 and ZIA NS003105-08) for supporting this work.

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The authors have no conflicts of interest to disclose.

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 &#937;-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.

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 &#937;-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 &#937;-shape profile, and labeling of the plasma membrane with the PH domain of phospholipase C &#948; (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.

<table><tbody><tr><td>Adenosine 5&#039;-triphosphate magnesium salt</td><td>Sigma</td><td>A9187-500MG</td><td>ATP for preparing internal solution</td></tr><tr><td>Atto 655</td><td>ATTO-TEC GmbH</td><td>AD 655-21</td><td>Atto dye to label bath solution</td></tr><tr><td>Basic Nucleofector for Primary Neurons</td><td>Lonza</td><td>VSPI-1003</td><td>Electroporation transfection buffer along with kit</td></tr><tr><td>Boroscilicate capillary glass pipette</td><td>Warner Instruments</td><td>64-0795</td><td>Standard wall with filament OD=2.0 mm ID=1.16 mm Length=7.5 cm</td></tr><tr><td>Bovine serum albumin</td><td>Sigma</td><td>A2153-50G</td><td>Reagent for gland digestion</td></tr><tr><td>Calcium Chloride 2 M</td><td>Quality Biological</td><td>351-130-721</td><td>Reagent for preparing bath solution</td></tr><tr><td>Cell Strainers, 100 &amp;micro;m</td><td>Falcon</td><td>352360</td><td>Material for filtering chromaffin cell suspension</td></tr><tr><td>Cesium hydroxide solution</td><td>Sigma</td><td>232041</td><td>Reagent for preparing internal solution and Cs-glutamate/Cs-EGTA stock buffer</td></tr><tr><td>Collagenase P</td><td>Sigma</td><td>11213873001</td><td>Enzyme for gland digestion</td></tr><tr><td>Coverslip</td><td>Neuvitro</td><td>GG-14-Laminin</td><td>GG-14-Laminin, 14 mm dia.#1 thick 60 pieces Laminin coated German coverslips</td></tr><tr><td>D-(+)-Glucose</td><td>Sigma</td><td>G8270-1KG</td><td>Reagent for preparing Locke&amp;rsquo;s solution and bath solution</td></tr><tr><td>DMEM</td><td>ThermoFisher Scientific</td><td>11885092</td><td>Reagent for preparing culture medium</td></tr><tr><td>EGTA</td><td>Sigma</td><td>324626-25GM</td><td>Reagent for preparing Cs-EGTA stock buffer for bath solution</td></tr><tr><td>Electroporation and Nucleofector</td><td>Amaxa Biosystems</td><td>Nucleofector II</td><td>Transfect plasmids into cells</td></tr><tr><td>Fetal bovine serum</td><td>ThermoFisher Scientific</td><td>10082147</td><td>Reagent for preparing culture medium</td></tr><tr><td>FFN511</td><td>Abcam</td><td>ab120331</td><td>Fluorescent false neurotransmitter to label vesicles</td></tr><tr><td>Guanosine 5&#039;-triphosphate sodium salt hydrate</td><td>Sigma</td><td>G8877-250MG</td><td>GTP for preparing internal solution</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375-500G</td><td>Reagent for preparing Locke&amp;rsquo;s solution</td></tr><tr><td>Igor Pro</td><td>WaveMetrics</td><td>Igor pro</td><td>Software for patch-clamp analysis and imaging data presentation</td></tr><tr><td>Leica Application Suite X software</td><td>Leica</td><td>LAS X software</td><td>Confocal software for imaging data collection and analysis</td></tr><tr><td>Leica TCS SP5 Confocal Laser Scanning Microscope</td><td>Leica</td><td>Leica TCS SP5</td><td>Confocal microscope for imaging data collection</td></tr><tr><td>L-Glutamic acid</td><td>Sigma</td><td>49449-100G</td><td>Reagent for preparing Cs-glutamate stock buffer for bath solution</td></tr><tr><td>Lock-in amplifier</td><td>Heka</td><td>Lock-in</td><td>Software for capacitance recording</td></tr><tr><td>Magnesium Chloride 1 M</td><td>Quality Biological</td><td>351-033-721EA</td><td>Reagent for preparing internal solution and bath solution</td></tr><tr><td>Metallized Hemacytometer Hausser Bright-Line</td><td>Hausser Scientific</td><td>3120</td><td>Counting chamber</td></tr><tr><td>Microforge</td><td>Narishige</td><td>MF-830</td><td>Polish pipettes to enhance the formation and stability of giga-ohm seals</td></tr><tr><td>Millex-GP Syringe Filter Unit, 0.22 &amp;micro;m</td><td>Millipore</td><td>SLGPR33RB</td><td>Material for glands wash and digestion</td></tr><tr><td>mNG(mNeonGreen)</td><td>Allele Biotechnology</td><td>ABP-FP-MNEONSB</td><td>Template for PH-mNeonGreen construction</td></tr><tr><td>Nylon mesh filtering screen 100 micron</td><td>EIKO filtering co</td><td>03-100/32</td><td>Material for filtering medulla suspension</td></tr><tr><td>Patch clamp EPC-10</td><td>Heka</td><td>EPC-10</td><td>Amplifier for patch-clamp data collection</td></tr><tr><td>PH-EGFP</td><td>Addgene</td><td>Plasmid #51407</td><td>Backbone for PH-mNeonGreen construction</td></tr><tr><td>Pipette puller</td><td>Sutter Instrument</td><td>P-97</td><td>Make pipettes for patch-clamp recording</td></tr><tr><td>Potassium Chloride</td><td>Sigma</td><td>P5404-500G</td><td>Reagent for preparing Locke&amp;rsquo;s solution and bath solution</td></tr><tr><td>Pulse software</td><td>Heka</td><td>Pulse</td><td>Software for patch-clamp data collection</td></tr><tr><td>Recording chamber</td><td>Warner Instruments</td><td>64-1943/QR-40LP</td><td>coverslip chamber, apply patch-clamp pipette on live cells</td></tr><tr><td>Sodium chloride</td><td>Sigma</td><td>S7653-1KG</td><td>Reagent for preparing Locke&amp;rsquo;s solution, bath solution and internal solution</td></tr><tr><td>Sodium hydroxide</td><td>Sigma</td><td>S5881-500G</td><td>Reagent for preparing Locke&amp;rsquo;s solution</td></tr><tr><td>Sodium phosphate dibasic</td><td>Sigma</td><td>S0876-500G</td><td>Reagent for preparing Locke&amp;rsquo;s solution</td></tr><tr><td>Sodium phosphate monobasic</td><td>Sigma</td><td>S8282-500G</td><td>Reagent for preparing Locke&amp;rsquo;s solution</td></tr><tr><td>Stirring hot plate</td><td>Barnsted/Thermolyne</td><td>type 10100</td><td>Heater for pipette coating with wax</td></tr><tr><td>Syringe, 30 mL</td><td>Becton Dickinson</td><td>302832</td><td>Material for glands wash and digestion</td></tr><tr><td>Tetraethylammonium chloride</td><td>Sigma</td><td>T2265-100G</td><td>TEA for preparing bath solution</td></tr><tr><td>Trypsin inhibitor</td><td>Sigma</td><td>T9253-5G</td><td>Reagent for gland digestion</td></tr><tr><td>Type F Immersion liquid</td><td>Leica</td><td>195371-10-9</td><td>Leica confocal mounting oil</td></tr></tbody></table>

confocal microscopy to measure three modes of fusion pore dynamics in adrenal chromaffin cells

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 &#937;-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 &#948; (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.

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 &#937;-profile)15.
After fusion, the &#937;-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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63569/63569fig01.jpg" /> 
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&#39;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 &#181;m (F). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63569/63569fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63569/63569fig02.jpg" /> 
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). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63569/63569fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63569/63569fig03.jpg" /> 
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 &#181;m (B, C). Abbreviations: ICa = calcium current; Cm = capacitance; Depol = depolarization. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63569/63569fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63569/63569fig04.jpg" /> 
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 &#181;m (A), 0.5 &#181;m (B-D). Abbreviations: Depol = depolarization; F = fluorescent intensity. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63569/63569fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Medium/Solution</td>
			<td colspan="3">Description</td>
		</tr>
		<tr>
			<td>Locke&#39;s solution</td>
			<td colspan="3">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</td>
		</tr>
		<tr>
			<td>Enzyme solution</td>
			<td colspan="3">1.5 mg/mL collagenase P, 0.325 mg/mL trypsin inhibitor, and 5 mg/mL bovine serum albumin in Locke&#39;s solution</td>
		</tr>
		<tr>
			<td>Culture medium</td>
			<td colspan="3">DMEM medium supplemented with 10% fetal bovine serum</td>
		</tr>
		<tr>
			<td>Internal solution</td>
			<td colspan="3">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</td>
		</tr>
		<tr>
			<td>Bath solution</td>
			<td colspan="3">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</td>
		</tr>
	</tbody>
</table>
Table 1: Details regarding the composition of culture medium and solutions.

Watch this Scientific Journal Video about Confocal Microscopy to Measure Three Modes of Fusion Pore Dynamics in Adrenal Chromaffin Cells at JoVE.com

Confocal Microscopy to Measure Three Modes of Fusion Pore Dynamics in Adrenal Chromaffin Cells

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 ...

confocal-microscopy-to-measure-three-modes-fusion-pore-dynamics

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

2.1K Views.  National Institute of Neurological Disorders and Stroke. 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. 

Video: Confocal Microscopy to Measure Three Modes of Fusion Pore Dynamics in Adrenal Chromaffin Cells

Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion known as exocytosis that mediates synaptic transmission. Subsequent retrieval of synaptic vesicles is necessary to generate new neurotransmitter-filled vesicles in a process identified as endocytosis. During exocytosis, fusing vesicle membranes will result in an increase in surface area and subsequent endocytosis results in a decrease in the surface area. Here, our lab demonstrates a basic introduction to cell-attached capacitance recordings of single endocytic events in the mouse adrenal chromaffin cell. This type of electrical recording is useful for high-resolution recordings of exocytosis and endocytosis at the single vesicle level. While this technique can detect both vesicle exocytosis and endocytosis, the focus of our lab is vesicle endocytosis. Moreover, this technique allows us to analyze the kinetics of single endocytic events. Here the methods for mouse adrenal gland tissue dissection, chromaffin cell culture, basic cell-attached techniques, and subsequent examples of individual traces measuring singular endocytic event are described.

After exocytosis, fused plasma membrane is retrieved through a process known as endocytosis. This mechanism reforms new synaptic vesicles for the next round of release. Individual endocytic events are captured and analyzed through the use of the cell-attached capacitance recordings in mouse adrenal chromaffin cells.

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion ...

TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real time and dissecting the different steps of exo-endocytosis at the single-vesicle level are crucial for understanding synaptic functions in health and disease.
Genetically-encoded pH-sensitive probes directly targeted to synaptic vesicles and Total Internal Reflection Fluorescence Microscopy (TIRFM) provide the spatio-temporal resolution necessary to follow vesicle dynamics. The evanescent field generated by total internal reflection can only excite fluorophores placed in a thin layer (&#60;150 nm) above the glass cover on which cells adhere, exactly where the processes of exo-endocytosis take place. The resulting high-contrast images are ideally suited for vesicles tracking and quantitative analysis of fusion events.
In this protocol, SH-SY5Y human neuroblastoma cells are proposed as a valuable model for studying neurotransmitter release at the single-vesicle level by TIRFM, because of their flat surface and the presence of dispersed vesicles. The methods for growing SH-SY5Y as adherent cells and for transfecting them with synapto-pHluorin are provided, as well as the technique&#160;to perform TIRFM and imaging. Finally, a strategy aiming to select, count, and analyze fusion events at whole-cell and single-vesicle levels is presented.
To validate the imaging procedure and data analysis approach, the dynamics of pHluorin-tagged vesicles are&#160;analyzed under resting and stimulated (depolarizing potassium concentrations) conditions. Membrane depolarization increases the frequency of fusion events and causes a parallel raise of the net fluorescence signal recorded in whole cell. Single-vesicle analysis reveals modifications of fusion-event behavior (increased peak height and width). These data suggest that potassium depolarization not only induces a massive neurotransmitter release but also modifies the mechanism of vesicle fusion and recycling.
With the appropriate fluorescent probe, this technique can be employed in different cellular systems to dissect the mechanisms of constitutive and stimulated secretion.

This paper provides a method for investigating neurotransmitter vesicle dynamics in neuroblastoma cells, using a synaptobrevin2-pHluorin construct and Total Internal Reflection Fluorescence Microscopy. The strategy developed for image processing and data analysis is also reported.

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real ...

Isolation, Fixation, and Immunofluorescence Imaging of Mouse Adrenal Glands

Immunofluorescence is a well-established technique for detection of antigens in tissues with the employment of fluorochrome-conjugated antibodies and has a broad spectrum of applications. Detection of antigens allows for characterization and identification of multiple cell types. Located above the kidneys and encapsulated by a layer of mesenchymal cells, the adrenal gland is an endocrine organ composed by two different tissues with different embryological origins, the mesonephric intermediate mesoderm-derived outer cortex and the neural crest-derived inner medulla. The adrenal cortex secretes steroids (i.e., mineralocorticoids, glucocorticoids, sex hormones), whereas the adrenal medulla produces catecholamines (i.e., adrenaline, noradrenaline). While conducting adrenal research, it is important to be able to distinguish unique cells with different functions. Here we provide a protocol developed in our laboratory that describes a series of sequential steps required for obtaining immunofluorescence staining to characterize the cell types of the adrenal gland. We focus first on the dissection of the mouse adrenal glands, the microscopic removal of periadrenal fat followed by the fixation, processing and paraffin embedding of the tissue. We then describe sectioning of the tissue blocks with a rotary microtome. Lastly, we detail a protocol for immunofluorescent staining of adrenal glands that we have developed to minimize both non-specific antibody binding and autofluorescence in order to achieve an optimal signal.

Here we present a method to isolate adrenal glands from mice, fix the tissues, section them, and perform immunofluorescence staining.

Immunofluorescence is a well-established technique for detection of antigens in tissues with the employment of fluorochrome-conjugated antibodies and has ...

Immunology and Infection

Monitoring the Effect of Osmotic Stress on Secretory Vesicles and Exocytosis

Amperometry recording of cells subjected to osmotic shock show that secretory cells respond to this physical stress by reducing the exocytosis activity and the amount of neurotransmitter released from vesicles in single exocytosis events. It has been suggested that the reduction in neurotransmitters expelled is due to alterations in membrane biophysical properties when cells shrink in response to osmotic stress and with assumptions made that secretory vesicles in the cell cytoplasm are not affected by extracellular osmotic stress. Amperometry recording of exocytosis monitors what is released from cells the moment a vesicle fuses with the plasma membrane, but does not provide information on the vesicle content before the vesicle fusion is triggered. Therefore, by combining amperometry recording with other complementary analytical methods that are capable of characterizing the secretory vesicles before exocytosis at cells is triggered offers a broader overview for examining how secretory vesicles and the exocytosis process are affected by osmotic shock. We here describe how complementing amperometry recording with intracellular electrochemical cytometry and transmission electron microscopy (TEM) imaging can be used to characterize alterations in secretory vesicles size and neurotransmitter content at chromaffin cells in relation to exocytosis activity before and after exposure to osmotic stress. By linking the quantitative information gained from experiments using all three analytical methods, conclusions were previously made that secretory vesicles respond to extracellular osmotic stress by shrinking in size and reducing the vesicle quantal size to maintain a constant vesicle neurotransmitter concentration. Hence, this gives some clarification regarding why vesicles, in response to osmotic stress, reduce the amount neurotransmitters released during exocytosis release. The amperometric recordings here indicate this is a reversible process and that vesicle after osmotic shock are refilled with neurotransmitters when placed cells are reverted into an isotonic environment.

Osmotic stress affects exocytosis and the amount of neurotransmitter released during this process. We demonstrate how combining electrochemical methods together with transmission electron microscopy can be used to study the effect of extracellular osmotic pressure on exocytosis activity, vesicle quantal size, and the amount of neurotransmitter released during exocytosis.

Amperometry recording of cells subjected to osmotic shock show that secretory cells respond to this physical stress by reducing the exocytosis activity ...

A Faster, High Resolution, mtPA-GFP-based Mitochondrial Fusion Assay Acquiring Kinetic Data of Multiple Cells in Parallel Using Confocal Microscopy

Mitochondrial fusion plays an essential role in mitochondrial calcium homeostasis, bioenergetics, autophagy and quality control. Fusion is quantified in living cells by photo-conversion of matrix targeted photoactivatable GFP (mtPAGFP) in a subset of mitochondria. The rate at which the photoconverted molecules equilibrate across the entire mitochondrial population is used as a measure of fusion activity. Thus far measurements were performed using a single cell time lapse approach, quantifying the equilibration in one cell over an hour. Here, we scale up and automate a previously published live cell method based on using mtPAGFP and a low concentration of TMRE (15 nm). This method involves photoactivating a small portion of the mitochondrial network, collecting highly resolved stacks of confocal sections every 15 min for 1 hour, and quantifying the change in signal intensity. Depending on several factors such as ease of finding PAGFP expressing cells, and the signal of the photoactivated regions, it is possible to collect around 10 cells within the 15 min intervals. This provides a significant improvement in the time efficiency of this assay while maintaining the highly resolved subcellular quantification as well as the kinetic parameters necessary to capture the detail of mitochondrial behavior in its native cytoarchitectural environment.
Mitochondrial dynamics play a role in many cellular processes including respiration, calcium regulation, and apoptosis1,2,3,13. The structure of the mitochondrial network affects the function of mitochondria, and the way they interact with the rest of the cell. Undergoing constant division and fusion, mitochondrial networks attain various shapes ranging from highly fused networks, to being more fragmented. Interestingly, Alzheimer's disease, Parkinson's disease, Charcot Marie Tooth 2A, and dominant optic atrophy have been correlated with altered mitochondrial morphology, namely fragmented networks4,10,13. Often times, upon fragmentation, mitochondria become depolarized, and upon accumulation this leads to impaired cell function18. Mitochondrial fission has been shown to signal a cell to progress toward apoptosis. It can also provide a mechanism by which to separate depolarized and inactive mitochondria to keep the bulk of the network robust14. Fusion of mitochondria, on the other hand, leads to sharing of matrix proteins, solutes, mtDNA and the electrochemical gradient, and also seems to prevent progression to apoptosis9. How fission and fusion of mitochondria affects cell homeostasis and ultimately the functioning of the organism needs further understanding, and therefore the continuous development and optimization of how to gather information on these phenomena is necessary.
Existing mitochondrial fusion assays have revealed various insights into mitochondrial physiology, each having its own advantages. The hybrid PEG fusion assay7, mixes two populations of differently labeled cells (mtRFP and mtYFP), and analyzes the amount of mixing and colocalization of fluorophores in fused, multinucleated, cells. Although this method has yielded valuable information, not all cell types can fuse, and the conditions under which fusion is stimulated involves the use of toxic drugs that likely affect the normal fusion process. More recently, a cell free technique has been devised, using isolated mitochondria to observe fusion events based on a luciferase assay1,5. Two human cell lines are targeted with either the amino or a carboxy terminal part of Renilla luciferase along with a leucine zipper to ensure dimerization upon mixing. Mitochondria are isolated from each cell line, and fused. The fusion reaction can occur without the cytosol under physiological conditions in the presence of energy, appropriate temperature and inner mitochondrial membrane potential. Interestingly, the cytosol was found to modulate the extent of fusion, demonstrating that cell signaling regulates the fusion process 4,5. This assay will be very useful for high throughput screening to identify components of the fusion machinery and also pharmacological compounds that may affect mitochondrial dynamics. However, more detailed whole cell mitochondrial assays will be needed to complement this in vitro assay to observe these events within a cellular environment.
A technique for monitoring whole-cell mitochondrial dynamics has been in use for some time and is based on a mitochondrially-targeted photoactivatable GFP (mtPAGFP)6,11. Upon expression of the mtPAGFP, a small portion of the mitochondrial network is photoactivated (10-20%), and the spread of the signal to the rest of the mitochondrial network is recorded every 15 minutes for 1 hour using time lapse confocal imaging. Each fusion event leads to a dilution of signal intensity, enabling quantification of the fusion rate. Although fusion and fission are continuously occurring in cells, this technique only monitors fusion as fission does not lead to a dilution of the PAGFP signal6. Co-labeling with low levels of TMRE (7-15 nM in INS1 cells) allows quantification of the membrane potential of mitochondria. When mitochondria are hyperpolarized they uptake more TMRE, and when they depolarize they lose the TMRE dye. Mitochondria that depolarize no longer have a sufficient membrane potential and tend not to fuse as efficiently if at all. Therefore, active fusing mitochondria can be tracked with these low levels of TMRE9,15. Accumulation of depolarized mitochondria that lack a TMRE signal may be a sign of phototoxicity or cell death. Higher concentrations of TMRE render mitochondria very sensitive to laser light, and therefore great care must be taken to avoid overlabeling with TMRE. If the effect of depolarization of mitochondria is the topic of interest, a technique using slightly higher levels of TMRE and more intense laser light can be used to depolarize mitochondria in a controlled fashion (Mitra and Lippincott-Schwartz, 2010). To ensure that toxicity due to TMRE is not an issue, we suggest exposing loaded cells (3-15 nM TMRE) to the imaging parameters that will be used in the assay (perhaps 7 stacks of 6 optical sections in a row), and assessing cell health after 2 hours. If the mitochondria appear too fragmented and cells are dying, other mitochondrial markers, such as dsRED or Mitotracker red could be used instead of TMRE.
The mtPAGFP method has revealed details about mitochondrial network behavior that could not be visualized using other methods. For example, we now know that mitochondrial fusion can be full or transient, where matrix content can mix without changing the overall network morphology. Additionally, we know that the probability of fusion is independent of contact duration and organelle dimension, is influenced by organelle motility, membrane potential and history of previous fusion activity8,15,16,17.
In this manuscript, we describe a methodology for scaling up the previously published protocol using mtPAGFP and 15nM TMRE8 in order to examine multiple cells at a time and improve the time efficiency of data collection without sacrificing the subcellular resolution. This has been made possible by the use of an automated microscope stage, and programmable image acquisition software. Zen software from Zeiss allows the user to mark and track several designated cells expressing mtPAGFP. Each of these cells can be photoactivated in a particular region of interest, and stacks of confocal slices can be monitored for mtPAGFP signal as well as TMRE at specified intervals. Other confocal systems could be used to perform this protocol provided there is an automated stage that is programmable, an incubator with CO2, and a means by which to photoactivate the PAGFP; either a multiphoton laser, or a 405 nm diode laser.

Mitochondrial fusion was measured by tracking the equilibration of photoconverted matrix-targeted GFP across the mitochondrial network over time. Thus far, only one cell could be subjected to an hour long kinetic analysis at a time. We present a method that simultaneously measures multiple cells, thereby speeding up the data collection process.

Mitochondrial fusion plays an essential role in mitochondrial calcium homeostasis, bioenergetics, autophagy and quality control. Fusion is quantified in ...

Biology

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of individual cells. Since an increase of intracellular calcium (Ca2+) in the cytosol often occurs because of the electrical activity, or in response to a myriad of other stimuli, this process can be monitored by the imaging of cells loaded with fluorescent calcium-sensitive dyes.&#160; However, it is difficult to image this response in an individual cell type within whole tissue because these dyes are taken up by all cell types within the tissue. In contrast, genetically encoded calcium indicators (GECIs) can be expressed by an individual cell type and fluoresce in response to an increase of intracellular Ca2+, thus permitting the imaging of Ca2+ signaling in entire populations of individual cell types. Here, we apply the use of the GECIs GCaMP3/6 to the mouse neuromuscular junction, a tripartite synapse between motor neurons, skeletal muscle, and terminal/perisynaptic Schwann cells. We demonstrate the utility of this technique in classic ex vivo tissue preparations. Using an optical splitter, we perform dual-wavelength imaging of dynamic Ca2+ signals and a static label of the neuromuscular junction (NMJ) in an approach that could be easily adapted to monitor two cell-specific GECI or genetically encoded voltage indicators (GEVI) simultaneously. Finally, we discuss the routines used to capture spatial maps of fluorescence intensity. Together, these optical, transgenic, and analytic techniques can be employed to study the biological activity of distinct cell subpopulations at the NMJ in a wide variety of contexts.

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

Here we present a protocol to image calcium signaling in populations of individual cell types at the murine neuromuscular junction.

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of ...

Imaging the Human Immunological Synapse

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) and an effector helper T lymphocyte (Th) cell, and to record the images corresponding to the first stages of the IS formation and the subsequent trafficking events (occurring both in the APC and in the Th cell). These events will eventually lead to polarized secretion at the IS. In this protocol, Jurkat cells challenged with Staphylococcus enterotoxin E (SEE)-pulsed Raji cells as a cell synapse model was used, because of the closeness of this experimental system to the biological reality (Th cell-APC synaptic conjugates). The approach presented here involves cell-to-cell conjugation, time-lapse acquisition, wide-field fluorescence microscopy (WFFM) followed by image processing (post-acquisition deconvolution). This improves the signal-to-noise ratio (SNR) of the images, enhances the temporal resolution, allows the synchronized acquisition of several fluorochromes in emerging synaptic conjugates and decreases fluorescence bleaching. In addition, the protocol is well matched with the end point cell fixation protocols (paraformaldehyde, acetone or methanol), which would allow further immunofluorescence staining and analyses. This protocol is also compatible with laser scanning confocal microscopy (LSCM) and other state-of-the-art microscopy techniques. As a main caveat, only those T cell-APC boundaries (called IS interfaces) that were at the right 90&#176; angle to the focus plane along the Z-axis could be properly imaged and analyzed. Other experimental models exist that simplify imaging in the Z dimension and the following image analyses, but these approaches do not emulate the complex, irregular surface of an APC, and may promote non-physiological interactions in the IS. Thus, the experimental approach used here is suitable to reproduce and to confront some biological complexities occurring at the IS.

This protocol images both the immunological synapse formation and the subsequent polarized secretory traffic towards the immunological synapse. Cellular conjugates were formed between a superantigen-pulsed Raji cell (acting as an antigen-presenting cell) and a Jurkat clone (acting as an effector helper T lymphocyte).

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) ...

Preparation of Pancreatic Acinar Cells for the Purpose of Calcium Imaging, Cell Injury Measurements, and Adenoviral Infection

The pancreatic acinar cell is the main parenchymal cell of the exocrine pancreas and plays a primary role in the secretion of pancreatic enzymes into the pancreatic duct. It is also the site for the initiation of pancreatitis. Here we describe how acinar cells are isolated from whole pancreas tissue and intracellular calcium signals are measured. In addition, we describe the techniques of transfecting these cells with adenoviral constructs, and subsequently measuring the leakage of lactate dehydrogenase, a marker of cell injury, during conditions that induce acinar cell injury in vitro. These techniques provide a powerful tool to characterize acinar cell physiology and pathology.

We describe a reproducible method of preparing mouse pancreatic acinar cells from a mouse for the purpose of examining acinar cell calcium signals and cellular injury with physiologically and pathologically relevant stimuli. A method for adenoviral infection of these cells is also provided.

The pancreatic acinar  cell is the main parenchymal cell of the exocrine pancreas and plays a primary  role in the secretion of pancreatic enzymes into ...

Imaging Plasma Membrane Deformations With pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during the fusion of vesicle and plasma membranes. These rapid and localized changes are achieved by dynamic interactions between lipids and specialized proteins that control membrane curvature. The absence of such interactions would not only have devastating consequences for vesicle fusion, but a host of other cellular functions that involve control of membrane shape. In recent years, the identity of a number of proteins with membrane-shaping properties has been determined. What remains missing is a roadmap of when, where, and how they act as fusion and content release progress.
Our understanding of the molecular events that enable membrane remodeling has historically been limited by a lack of analytical methods that are sensitive to membrane curvature or have the temporal resolution to track rapid changes. PTIRFM satisfies both of these criteria. We discuss how pTIRFM is implemented to visualize and interpret rapid, submicron changes in the orientation of chromaffin cell membranes during dense core vesicle (DCV) fusion. The chromaffin cells we use are isolated from bovine adrenal glands. The membrane is stained with a lipophilic carbocyanine dye,1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate, or&#160;diD. DiD intercalates in the membrane plane with a &#34;fixed&#34; orientation and is therefore sensitive to the polarization of the evanescent field. The diD-stained cell membrane is sequentially excited with orthogonal polarizations of a 561&#160;nm laser (p-pol, s-pol). A 488&#160;nm laser is used to visualize vesicle constituents and time the moment of fusion. Exocytosis is triggered by locally perfusing cells with a depolarizing KCl solution. Analysis is performed offline using custom-written software to understand how diD emission intensity changes relate to fusion pore dilation.

Polarization-based Total Internal Reflection Fluorescence Microscopy (pTIRFM) enables real-time detection of cell membrane dynamics. This article describes the implementation of pTIRFM for the study of membrane remodeling during regulated exocytosis. The technique is generalizable to other processes in cell biology that directly or indirectly involve changes in membrane shape.

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during ...

Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy

Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in transfected human embryonic kidney-293 (HEK) cells following stimulation with angiotensin II (Ang II). HEK cells are transiently transfected with plasmid DNA containing AT1aR tagged with enhanced green fluorescent protein (EGFP). Lysosomes are identified with a red fluorescent dye. Live-cell images are captured on a laser scanning confocal microscope after Ang II stimulation and analyzed by software in three dimensions (3D, voxels) over time. Live-cell imaging enables investigations into receptor trafficking and avoids confounds associated with fixation, and in particular, the loss or artefactual displacement of EGFP-tagged membrane receptors. Thus, as individual cells are tracked through time, the subcellular localization of receptors can be imaged and measured. Images must be acquired sufficiently rapidly to capture rapid vesicle movement. Yet, at faster imaging speeds, the number of photons collected is reduced. Compromises must also be made in the selection of imaging parameters like voxel size in order to gain imaging speed. Significant applications of live-cell imaging are to study protein trafficking, migration, proliferation, cell cycle, apoptosis, autophagy and protein-protein interaction and dynamics, to name but a few.

Here we present a protocol to image cells expressing green fluorescent protein-tagged angiotensin type 1a receptors during endocytosis initiated by angiotensin II treatment. This technique includes labeling lysosomes with a second fluorescent marker, and then utilizing software to analyze the co-localization of receptor and lysosome in three dimensions over time.

Confocal Microscopy to Measure Three Modes of Fusion Pore Dynamics in Adrenal Chromaffin Cells

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Imaging the Human Immunological Synapse

Preparation of Pancreatic Acinar Cells for the Purpose of Calcium Imaging, Cell Injury Measurements, and Adenoviral Infection

Imaging Plasma Membrane Deformations With pTIRFM

Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy