Membrane fusion mediates many biological functions. This protocol describes a method to detect fusion pore opening and transmitter release dynamics and distinguish three fusion modes;close fusion, stay fusion, and shrink fusion. The combination of confocal microscope and the patch-clamp recording facilitates visualization of fusion pore opening and closure and determination of their kinetics.
Although described for chromaffin cells, the principle of the method described here can be applied widely to many secretory cells well beyond chromaffin cells. Successful implementation of this method depends on several general steps, such as generating healthy primary chromaffin cell culture, sufficient plasmid transfection efficiency, and high electrophysiological recording success rate. Choose three intact glands without cuts or bleeds on the surface and remove the fat tissue with scissors.
Wash the glands by perfusion with Locke's solution until no blood comes out. For digestion, inject the enzyme solution through the adrenal vein using a 30 millimeter syringe attached with a 0.22 micrometer filter until the gland starts to swell. Then leave the glands at 37 degrees Celsius for 10 minutes.
Inject again and leave the glands at 37 degrees Celsius for another 10 minutes. After digestion, cut the gland longitudinally from the adrenal vein to the other end with scissors to unfold the gland. Isolate the white medulla by tweezing it out in pieces into a 10 centimeter Petri dish containing Locke's solution.
Cut and mince the medulla with scissors. Filter the medulla suspension with an 80 to 100 micrometer nylon mesh into a beaker. Transfer the filtrate to a 50 milliliter conical tube and centrifugate at 48 x g room temperature for three minutes with a deceleration of three.
After centrifugation, remove the supernatant and resuspend the cell pellet with Locke's solution by pipetting. Filter the cell suspension with an 80 to 100 micrometer strainer and centrifugate at 48 x g room temperature for three minutes with deceleration three. Remove the supernatant and resuspend the cell pellet with 30 milliliters of culture medium.
Determine the cell number using a hemocytometer counting chamber. Transfer 2, 800, 000 cells into a 15 milliliter tube. Pellet the cells by centrifugation at 48 x g for two minutes with the deceleration of three.
Add 100 microliters of transection buffer provided by the manufacturer to the cell pellet. And then add two micrograms of PH-mNG plasmid. Gently mix the suspension by piping the solution up and down and transfer the mixture into an electroporation cuvette without delay.
Immediately transfer the cuvette to the electroporator, select the 005 program in the screen list and press Enter to perform electroporation. After electroporation, immediately add 1.8 milliliters of medium to the cuvette and mix gently with a micropipette equipped with a sterile tip. Add 300 microliters of the suspension of the electroporated cells onto the cover slip in each dish plating five to six dishes in total for one electroporation reaction.
Carefully transfer the dishes to a humidified incubator at 37 degrees Celsius with 9%carbon dioxide for 30 minutes and gently add two milliliters of prewarmed medium to each dish after 30 minutes. Prepare patch pipettes from borosilicate glass capillaries by pulling them with a pipette puller, coating their tips with liquid wax, and polishing them with a microforge. Turn on the patch clamp recording amplifier and start the software.
Set the software's appropriate calcium current and capacitance recording parameters. Set a recording protocol of 60 seconds duration in total where the stimulation starts at 10 seconds. Set the holding potential for voltage clamp recording to 80 millivolts.
Set a one second depolarization from minus 80 millivolts to 10 millivolts as the stimulus to induce calcium influx and capacitance jump. Turn on the confocal microscope system and set the appropriate parameters in the software. Turn on the lasers, including 458 nanometers, 514 nanometers, and 633 nanometers and set the emission collection range for each laser according to each fluorescence probe.
Choose a dish with good cell state and proper expression and add two microliters of fluorescence false neurotransmitter FFN511 into the medium in the dish. Put the dish back in the incubator for 20 minutes. After loading FFN511, prepare the recording chamber and add two microliters of fluorescence dye A655 into 50 microliters of bath solution.
Transfer the cover slip from the dish into the recording chamber with tweezers and immediately add the A655 containing bath solution. Place a drop of oil on the 100X oil immersion objective. Mount the chamber in the microscope and use the adjustment knob to make the oil just contact the bottom of the cover slip.
Bring the cells into focus and use bright field and confocal imaging to find a suitable cell with mNG expression. Zoom in on the selected cell and center it in the field of view, minimizing blank regions. Set parameters for XY plane confocal imaging at a fixed Z-plane of FFN511, PH-mNG, and A665 with a minimized time interval.
Adjust the focus to the bottom of the cell with a fine adjustment knob. Adjust the excitation laser power in the software to find a setting to get the best signal-to-noise ratio and avoid significant fluorescence bleaching. Add nine microliters of internal solution into a patch pipette and attach the pipette to the holder of the patch clamp amplifier stage.
Apply a small amount of positive pressure with a syringe and move the pipette tip to touch the bath solution with a micromanipulator. Ensure the amplifier shows that the pipette resistance is approximately between two to four megaohms with a voltage pulse test. Press LJ/Auto to cancel the liquid junction.
Move the pipette toward the selected cell with a micromanipulator. To form a cell attached mode, move the pipette tip to touch the cell. Change the holding potential from zero to minus 80 millivolts while applying gentle negative pressure with a syringe.
Once resistance passes one gigaohm, wait for approximately 30 seconds for the configuration to stabilize. Press C-fast/Auto to compensate for fast capacitance. To form a wholesale mode, apply short yet powerful pulses of negative pressure with the syringe until the membrane ruptures.
Press C-slow/Auto to compensate for slow capacitance. Adjust the imaging focus slightly to focus on the cell bottom. Start the confocal time lapse imaging and patch clamp recording simultaneously by clicking the Start buttons with two different software applications.
After recording, ensure the data are saved. Change the holding potential back to zero millivolts. Move the pipette out of the bath solution and discard it.
Open the raw imaging files with any manufacturer or supplied software. Go to Process, click on ProcessTools and use the tools to generate rolling average files for each time lapse image. Save these files.
Under quantify, go to Tools and click on Stack Profile buttons to check time points before and after stimulation and identify fluorescence changes in each channel. Click on the Draw ellipse button to circle regions of interest for fusion events. Right click on the image and click Save ROIs to save.
Click Open projects to locate the raw file. Right click on the image and click Load ROIs to load the ROI file in the raw imaging file to measure the fluorescence intensities. Go to Tools and click on Sort ROIs in the software and plot the traces for all three channels of each ROI.
Click Report to save the ROI data, including digital data and imaging traces for each ROI into a file folder. The whole cell patch-clamp recording and application of a one second depolarization from 80 to 10 millivolts was performed to evoke exo and endocytosis. The applied depolarization induced an inward calcium current, a capacitance jump, indicating exocytosis, and a capacitance decay after the jump, indicating endocytosis.
With time lapse imaging at the cell bottom, fusion events induced by the one second depolarization protocol were indicated as FFN decrease reflecting FFN511 release, accompanied by FPH and A655 spot fluorescence increase, reflecting PH-mNG and A655 diffusion from the plasma membrane and the bath solution into the fusing vesicle. The figure presents close fusion identified as F655 dimming, while FPH sustained or decayed with a delay. The figure represents stay fusion detected by the presence of both PH-mNG and A655 spots.
The figure represents shrink fusion detected as parallel FPH and F655 decay accompanied by a parallel size reduction of the PH-mNG spot and the A655 spot Successful recording requires generating the whole cell configuration with patch-clamp and imaging at the cell bottom with proper fluorescent intensity for each channel. The combination of electrophysiology and super resolution microscopy described here is a valuable tool for measuring fusion pore dynamics and neurocircuits.
