Name: confocal microscopy to measure three modes of fusion pore dynamics in adrenal chromaffin cells
Uploaded: 2022-03-16T14:30:00
Description: Watch this Scientific Journal Video about Confocal Microscopy to Measure Three Modes of Fusion Pore Dynamics in Adrenal Chromaffin Cells at JoVE.com

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

NOTE: The animal use procedure followed NIH guidelines and was approved by the NIH Animal Care and Use Committee.
1. Bovine chromaffin cell culture
<ol>
	<li>Prepare Locke&#39;s solution (Table 1) and autoclave tools 1 day before chromaffin cell culture.</li>
	<li>Obtain bovine adrenal glands from a local abattoir on the culture day, and keep them submerged in ice-cold Locke&#39;s solution before dissection. 
	NOTE: Adrenal glands are from 21-27-month-o...

<ol>
	<li>Wu, L. G., Hamid, E., Shin, W., Chiang, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exocytosis+and+endocytosis:+modes,+functions,+and+coupling+mechanisms.">Exocytosis and endocytosis: modes, functions, and coupling mechanisms.</a> Annual Review of Physiology. 76, 301-331 (2014).</li><li>Chang, C. W., Chiang, C. W., Jackson, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fusion+pores+and+their+control+of+neurotransmitter+and+hormone+release.">Fusion pores and their control of neurotransmitter and hormone release.</a> Journal of General Physiology. 149 (3), 301-322 (2017).</li><li>Harrison, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+membrane+fusion.">Viral membrane fusion.</a> Nature Structural &amp; Molecular Biology. 15 (7), 690-698 (2008).</li><li>Zhao, W. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemi-fused+structure+mediates+and+controls+fusion+and+fission+in+live+cells.">Hemi-fused structure mediates and controls fusion and fission in live cells.</a> Nature. 534 (7608), 548-552 (2016).</li><li>Klyachko, V. A., Jackson, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capacitance+steps+and+fusion+pores+of+small+and+large-dense-core+vesicles+in+nerve+terminals.">Capacitance steps and fusion pores of small and large-dense-core vesicles in nerve terminals.</a> Nature. 418 (6893), 89-92 (2002).</li><li>Albillos, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+exocytotic+event+in+chromaffin+cells+revealed+by+patch+amperometry.">The exocytotic event in chromaffin cells revealed by patch amperometry.</a> Nature. 389 (6650), 509-512 (1997).</li><li>He, L., Wu, X. S., Mohan, R., Wu, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+modes+of+fusion+pore+opening+revealed+by+cell-attached+recordings+at+a+synapse.">Two modes of fusion pore opening revealed by cell-attached recordings at a synapse.</a> Nature. 444 (7115), 102-105 (2006).</li><li>Chiang, H. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-fusion+structural+changes+and+their+roles+in+exocytosis+and+endocytosis+of+dense-core+vesicles.">Post-fusion structural changes and their roles in exocytosis and endocytosis of dense-core vesicles.</a> Nature Communications. 5, 3356 (2014).</li><li>Sharma, S., Lindau, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fusion+pore,+60+years+after+the+first+cartoon.">The fusion pore, 60 years after the first cartoon.</a> Federation of European Biochemical Societies Letters. 592 (21), 3542-3562 (2018).</li><li>Jorgacevski, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Munc18-1+tuning+of+vesicle+merger+and+fusion+pore+properties.">Munc18-1 tuning of vesicle merger and fusion pore properties.</a> Journal of Neuroscience. 31 (24), 9055-9066 (2011).</li><li>Rituper, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicle+cholesterol+controls+exocytotic+fusion+pore.">Vesicle cholesterol controls exocytotic fusion pore.</a> Cell Calcium. 101, 102503 (2021).</li><li>Gucek, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dominant+negative+SNARE+peptides+stabilize+the+fusion+pore+in+a+narrow,+release-unproductive+state.">Dominant negative SNARE peptides stabilize the fusion pore in a narrow, release-unproductive state.</a> Cellular and Molecular Life Sciences. 73 (19), 3719-3731 (2016).</li><li>Grabner, C. P., Moser, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Individual+synaptic+vesicles+mediate+stimulated+exocytosis+from+cochlear+inner+hair+cells.">Individual synaptic vesicles mediate stimulated exocytosis from cochlear inner hair cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (50), 12811-12816 (2018).</li><li>Wen, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+dynamics+provides+membrane+tension+to+merge+fusing+vesicles+into+the+plasma+membrane.">Actin dynamics provides membrane tension to merge fusing vesicles into the plasma membrane.</a> Nature Communications. 7, 12604 (2016).</li><li>Shin, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+Membrane+Pore+in+Live+Cells+Reveals+a+Dynamic-Pore+Theory+Governing+Fusion+and+Endocytosis.">Visualization of Membrane Pore in Live Cells Reveals a Dynamic-Pore Theory Governing Fusion and Endocytosis.</a> Cell. 173 (4), 934-945 (2018).</li><li>Shin, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicle+Shrinking+and+Enlargement+Play+Opposing+Roles+in+the+Release+of+Exocytotic+Contents.">Vesicle Shrinking and Enlargement Play Opposing Roles in the Release of Exocytotic Contents.</a> Cell Reports. 30 (2), 421-431 (2020).</li><li>Ge, L., Shin, W., Wu, L. -. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+sequential+compound+fusion+and+kiss-and-run+in+live+excitable+cells.">Visualizing sequential compound fusion and kiss-and-run in live excitable cells.</a> bioRxiv. , (2021).</li><li>Zhang, Q., Li, Y., Tsien, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamic+control+of+kiss-and-run+and+vesicular+reuse+probed+with+single+nanoparticles.">The dynamic control of kiss-and-run and vesicular reuse probed with single nanoparticles.</a> Science. 323 (5920), 1448-1453 (2009).</li><li>He, L., Wu, X. S., Mohan, R., Wu, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+modes+of+fusion+pore+opening+revealed+by+cell-attached+recordings+at+a+synapse.">Two modes of fusion pore opening revealed by cell-attached recordings at a synapse.</a> Nature. 444 (7115), 102-105 (2006).</li><li>Androutsellis-Theotokis, A., Rubin de Celis, M. F., Ehrhart-Bornstein, M., Bornstein, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Common+features+between+chromaffin+and+neural+progenitor+cells.">Common features between chromaffin and neural progenitor cells.</a> Molecular Psychiatry. 17 (4), 351 (2012).</li><li>Bornstein, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromaffin+cells:+the+peripheral+brain.">Chromaffin cells: the peripheral brain.</a> Molecular Psychiatry. 17 (4), 354-358 (2012).</li><li>Park, Y., Kim, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+plasticity+of+small+synaptic+vesicle+(SSV)+and+large+dense-core+vesicle+(LDCV)+exocytosis.">Short-term plasticity of small synaptic vesicle (SSV) and large dense-core vesicle (LDCV) exocytosis.</a> Cellular Signalling. 21 (10), 1465-1470 (2009).</li><li>Thahouly, T., Niedergang, F., Vitale, N., Gasman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bovine+Chromaffin+Cells:+Culture+and+Fluorescence+Assay+for+Secretion.">Bovine Chromaffin Cells: Culture and Fluorescence Assay for Secretion.</a> Exocytosis and Endocytosis. Methods in Molecular Biology. 2233, (2021).</li><li>Shin, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preformed+Omega-profile+closure+and+kiss-and-run+mediate+endocytosis+and+diverse+endocytic+modes+in+neuroendocrine+chromaffin+cells.">Preformed Omega-profile closure and kiss-and-run mediate endocytosis and diverse endocytic modes in neuroendocrine chromaffin cells.</a> Neuron. 109 (19), 3119-3134 (2021).</li><li>O'Connor, D. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+culture+of+bovine+chromaffin+cells.">Primary culture of bovine chromaffin cells.</a> Nature Protocols. 2 (5), 1248-1253 (2007).</li><li>Wu, X. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+Tension+Inhibits+Rapid+and+Slow+Endocytosis+in+Secretory+Cells.">Membrane Tension Inhibits Rapid and Slow Endocytosis in Secretory Cells.</a> Biophysical Journal. 113 (11), 2406-2414 (2017).</li><li>Revelo, N. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+probe+for+super-resolution+imaging+of+membranes+elucidates+trafficking+pathways.">A new probe for super-resolution imaging of membranes elucidates trafficking pathways.</a> Journal of Cell Biology. 205 (4), 591-606 (2014).</li><li>Gao, J., Liao, J., Yang, G. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CAAX-box+protein,+prenylation+process+and+carcinogenesis.">CAAX-box protein, prenylation process and carcinogenesis.</a> American journal of translational research. 1 (3), 312-325 (2009).</li><li>Schermelleh, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+microscopy+demystified.">Super-resolution microscopy demystified.</a> Nature Cell Biology. 21 (1), 72-84 (2019).</li><li>Ceccarelli, B., Hurlbut, W. P., Mauro, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depletion+of+vesicles+from+frog+neuromuscular+junctions+by+prolonged+tetanic+stimulation.">Depletion of vesicles from frog neuromuscular junctions by prolonged tetanic stimulation.</a> Journal of Cell Biology. 54 (1), 30-38 (1972).</li><li>Rust, M. J., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sub-diffraction-limit+imaging+by+stochastic+optical+reconstruction+microscopy+(STORM).">Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM).</a> Nature Methods. 3 (10), 793-795 (2006).</li><li>Betzig, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+intracellular+fluorescent+proteins+at+nanometer+resolution.">Imaging intracellular fluorescent proteins at nanometer resolution.</a> Science. 313 (5793), 1642-1645 (2006).</li><li>Fish, K. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+internal+reflection+fluorescence+(TIRF)+microscopy.">Total internal reflection fluorescence (TIRF) microscopy.</a> Current Protocols in Cytometry. , 18 (2009).</li><li>Schmidt, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MINFLUX+nanometer-scale+3D+imaging+and+microsecond-range+tracking+on+a+common+fluorescence+microscope.">MINFLUX nanometer-scale 3D imaging and microsecond-range tracking on a common fluorescence microscope.</a> Nature Communications. 12 (1), 1478 (2021).</li></ol>

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.</...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adenosine 5&#39;-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 &#181;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>1.1214E+10</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&#8217;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&#39;-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&#8217;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 &#181;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&#8217;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&#8217;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&#8217;s solution</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>Sigma</td>
			<td>S0876-500G</td>
			<td>Reagent for preparing Locke&#8217;s solution</td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic</td>
			<td>Sigma</td>
			<td>S8282-500G</td>
			<td>Reagent for preparing Locke&#8217;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 plan...

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 ...

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