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

我们感谢NINDS校内研究计划（ZIA NS003009-13和ZIA NS003105-08）支持这项工作。

注意：动物使用程序遵循NIH指南，并已获得NIH动物护理和使用委员会的批准。
1. 牛嗜铬细胞培养
<ol><li>在嗜铬细胞培养前1天准备Locke的溶液（表1）和高压灭菌工具。</li>
	<li>在培养日从当地的屠宰场获取牛肾上腺，并在解剖前将它们浸没在冰冷的Locke溶液中。 注意：肾上腺来自21-27个月大，健康，黑人安格斯，男女皆宜（主要是男性），体重约为...

<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>

描述了一种共聚焦显微成像方法，用于检测牛肾上腺嗜铬细胞中融合孔隙和递质释放的动力学，以及三种融合模式，即紧密融合，停留融合和收缩融合4，24。描述了一种电生理学方法，用于使细胞去极化，从而唤起外吞和内吞作用。系统共聚焦图像处理提供有关聚变和裂变事件的不同孔隙行为模式的信息。
同时监测具有全细胞结构...

膜融合介导许多生物学功能，包括突触传递，血糖稳态，免疫反应和病毒进入1，2，3。胞吐作用涉及质膜处的囊泡融合，释放神经递质和激素以实现许多重要功能，例如神经元网络活动。融合打开一个孔以释放囊泡内容物，之后孔隙可能关闭以取回融合的囊泡，这被称为亲吻和运行1，4。不可逆和可逆的融合孔隙开口都可以通过细胞附着的电容记录与单个囊泡融合的融合孔隙电导记录来测量。
这通常被解释为反映全塌陷融合，涉及融合的扩张，直到融合囊泡变平，以及吻和运行，涉及融合孔隙的开放和闭合，分别为5，6，7，8，9，10，11，12，13.最近在染色质细胞中的共聚焦和受激辐射耗尽（STED）成像研究直接观察到融合孔隙开放和闭合（吻和运行，也称为紧密融合），融合孔隙开放，长时间保持具有开放孔隙的Ω形，称为停留融合，以及融合囊泡的收缩，直到它与质膜完全合并，这取代了完全塌陷融合，用于将融合囊泡与质膜合并4，8，14，15，16，17.
在神经元中，已经通过成像检测到融合孔的开放和闭合，显示预装在大于融合孔的囊泡中的量子点的释放，并且在神经末梢释放面5，18，19处具有融合孔导率测量。肾上腺嗜铬细胞被广泛用作研究外吞和内吞作用的模型20，21。虽然嗜铬细胞含有大的致密核心囊泡，而突触含有小的突触囊泡，但嗜铬细胞和突触中的胞吐作用和内吞作用蛋白非常相似10，11，12，20，21，22，23。
在这里，描述了一种使用共聚焦成像方法结合牛肾上腺嗜铬细胞电生理学来测量这三种融合模式的方法（图1）。该方法涉及将荧光假神经递质（FFN511）加载到囊泡中以检测胞吐作用;在浴液中加入Atto 655（A655）以填充融合产生的Ω形轮廓，并用磷脂酶C δ（PH）的PH结构域标记质膜，该结构域在质膜8，15，24处与PtdIns（4，5）P2结合。聚变孔隙动力学可以通过不同荧光强度的变化来检测。虽然描述了嗜铬细胞，但这里描述的这种方法的原理可以广泛应用于许多分泌细胞，远远超出嗜铬细胞。

<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

动态融合孔隙打开和闭合介导胞吐和内吞作用，并确定其动力学。在这里，详细展示了共聚焦显微镜如何与膜片钳记录结合使用，以检测原代培养牛肾上腺嗜铬细胞中的三种融合模式。三种融合模式包括1）紧密融合（也称为亲吻和运行），涉及融合孔隙开放和闭合，2）停留融合，涉及融合孔隙开放并保持开放孔，以及3）收缩融合，涉及融合产生的Ω形轮廓的收缩，直到它在质膜上完全合并。
为了检测这些融合模式，通过过表达mNeonGreen标记质膜，该蛋白与磷脂酶C δ（PH-mNG）的PH结构域连接，其与磷脂酰肌醇-4，5-二磷酸（PtdIns（4，5）P2）在质膜的细胞质基质层面小叶处结合;囊泡加载荧光假神经递质FFN511以检测囊泡内容物的释放;并将Atto 655包含在浴液中以检测熔融孔隙闭合。在活嗜铬细胞中以每帧约20-90 ms的速度同时对这三种荧光探针进行成像，以检测融合孔隙开放，含量释放，融合孔闭合和融合囊泡尺寸变化。描述了分析方法以将三种融合模式与这些荧光测量区分开来。原则上，这里描述的方法可以应用于嗜铬细胞以外的许多分泌细胞。

按照图1和图2所示的实验程序，用PH-mNG转染来自牛肾上腺的嗜铬细胞以标记质膜;将A655加入到熔池溶液中以检测熔融孔隙闭合;并将荧光假神经递质FFN511加载到囊泡中以检测释放。接下来，在细胞底部（Z焦平面~细胞膜上方约100-200nm）每20-90 ms对FFN511，PH-mNG和A655进行XY平面共聚焦延时成像。进行全细胞膜片钳记录和应用从-80至+ 10 mV的1s去极化以唤起外吞和?...

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

该协议描述了一种共聚焦成像技术，用于检测牛肾上腺嗜铬细胞中的三种融合模式。这些融合模式包括1）闭合融合（也称为亲吻和运行），涉及融合孔隙开放和闭合，2）停留融合，涉及融合孔隙开放和维持开放孔，以及3）收缩融合，涉及融合囊泡收缩。

共聚焦显微镜测量肾上腺嗜铬细胞融合孔隙动力学的三种模式

Dynamic fusion pore opening and closure mediate exocytosis and endocytosis and determine their kinetics. Here, it is demonstrated in detail how confocal ...

膜融合介导许多生物学功能。该协议描述了一种检测融合孔隙开度和发射机释放动力学并区分三种融合模式的方法：紧密融合，停留融合和收缩融合。共聚焦显微镜和膜片钳记录的结合有助于融合孔隙开放和闭合的可视化以及动力学的测定。虽然描述了嗜铬细胞，但这里描述的方法的原理可以广泛应用于许多分泌细胞，远远超出嗜铬细胞。该方法的成功实施取决于几个一般步骤，例如产生健康的原代嗜铬细胞培养物，足够的质粒转染效率和高电生理记录成功率。选择三个完整的腺体，表面没有割伤或出血，并用剪刀去除脂肪组织。用洛克的溶液灌注来清洗腺体，直到没有血液流出。为了消化，使用30毫米注射器通过肾上腺静脉注射酶溶液，注射器与0.22微米过滤器连接，直到腺体开始膨胀。然后将腺体在37摄氏度下放置10分钟。再次注射，将腺体保持在37摄氏度下10分钟。消化后，用剪刀纵向切开从肾上腺静脉到另一端的腺体以展开腺体。通过将白色延髓分成几块放入含有洛克溶液的10厘米培养皿中来隔离白色延髓。用剪刀切碎延髓。用80至100微米尼龙网过滤延髓悬浮液到烧杯中。将滤液转移到50毫升锥形管中，并在48×g室温下离心三分钟，减速3分钟。离心后，除去上清液，并通过移液将细胞沉淀与Locke溶液重悬。用80至100微米过滤器过滤细胞悬浮液，并在48×g室温下离心三分钟，减速三分钟。除去上清液并用30毫升培养基重悬细胞沉淀。使用血细胞计数器计数室确定细胞数。将2， 800， 000个细胞转移到15毫升管中。通过以48×g离心两分钟，以三个减速沉淀细胞。向细胞沉淀中加入制造商提供的100微升横断缓冲液。然后加入两微克PH-mNG质粒。通过将溶液上下管道轻轻混合悬浮液，并将混合物立即转移到电穿孔比色皿中。立即将比色皿转移到电敏器，在屏幕列表中选择005程序，然后按Enter键进行电穿孔。电穿孔后，立即向比色皿中加入1.8毫升培养基，并与配备无菌尖端的微量移液管轻轻混合。在每个培养皿中将300微升电穿孔电池的悬浮液加入盖玻片上，总共镀五至六个培养皿，以进行一次电穿孔反应。小心地将培养皿转移到37摄氏度的加湿培养箱中，用9%二氧化碳加热30分钟，并在30分钟后轻轻地向每个培养皿中加入两毫升预热培养基。从硼硅酸盐玻璃毛细管制备贴片移液器，方法是用移液器拉动它们，用液体蜡涂覆它们的尖端，并用微锻造抛光它们。打开膜片钳记录放大器并启动软件。设置软件适当的钙电流和电容记录参数。设置总共60秒持续时间的记录协议，其中刺激从10秒开始。将电压钳位记录的保持电位设置为 80 毫伏。设置一秒钟的去极化，从零下80毫伏到10毫伏，作为刺激诱导钙流入和电容跳跃。打开共聚焦显微镜系统并在软件中设置适当的参数。打开激光器，包括458纳米，514纳米和633纳米，并根据每个荧光探针设置每个激光器的发射收集范围。选择细胞状态良好且表达适当的培养皿，并在培养皿中的培养基中加入两微升荧光假神经递质FFN511。将培养皿放回培养箱中20分钟。加载FFN511后，制备记录室，并将两微升荧光染料A655加入50微升浴液中。用镊子将盖玻片从培养皿转移到记录室中，并立即加入含有浴液的A655。在100X油浸物镜上滴一滴油。将腔室安装在显微镜中，并使用调节旋钮使油刚好接触到盖子的底部滑动。将细胞聚焦，并使用明场和共聚焦成像找到具有mNG表达的合适细胞。放大所选单元格并将其居中显示在视野中，从而最大限度地减少空白区域。在 FFN511、PH-mNG 和 A665 的固定 Z 平面上设置 XY 平面共聚焦成像的参数，并最小化时间间隔。使用微调旋钮将焦点调整到单元格的底部。在软件中调整激发激光功率，找到一个设置，以获得最佳的信噪比，并避免明显的荧光漂白。将9微升内部溶液加入贴片移液器中，并将移液器连接到膜片钳放大器级的支架上。用注射器施加少量正压，并移动移液器尖端以用显微操纵器触摸浴液溶液。通过电压脉冲测试，确保放大器显示移液器电阻大约在两到四兆欧之间。按 LJ/自动取消液体液络部。用显微操作器将移液器向所选细胞移动。要形成细胞附着模式，请移动移液器吸头以触摸细胞。将保持电位从零更改为零80毫伏，同时使用注射器施加轻柔的负压。一旦阻力超过 10 亿兆欧，请等待大约 30 秒，以使配置稳定下来。按 C-快速/自动以补偿快速电容。要形成批发模式，请用注射器施加短而强大的负压脉冲，直到膜破裂。按 C 慢/自动以补偿慢速电容。稍微调整成像焦点以聚焦在细胞底部。通过单击两个不同的软件应用程序的“开始”按钮，同时启动共聚焦延时成像和膜片钳记录。记录后，确保数据已保存。将保持电位改回零毫伏。将移液器移出浴液并丢弃。使用任何制造商或提供的软件打开原始映像文件。转到“处理”，单击“处理工具”，然后使用工具为每个延时图像生成滚动平均文件。保存这些文件。在量化下，转到“工具”，然后单击“堆栈配置文件”按钮，以检查刺激前后的时间点，并识别每个通道中的荧光变化。单击绘制椭圆按钮以圈出融合事件的感兴趣区域。右键单击图像，然后单击保存要保存的 ROI。单击“打开项目”以找到原始文件。右键单击图像，然后单击加载ROI以加载原始成像文件中的ROI文件以测量荧光强度。转到“工具”，然后单击软件中的“排序投资回报率”，然后绘制每个ROI的所有三个通道的跟踪。单击“报告”将 ROI 数据（包括数字数据和每个 ROI 的成像跟踪）保存到一个文件夹中。进行整个细胞膜片钳记录和应用从80到10毫伏的一秒钟去极化，以唤起外源和内吞作用。施加的去极化诱导向内钙电流，电容跳跃，表明胞吐，以及跳跃后电容衰减，表明内吞作用。随着细胞底部的延时成像，由1秒去极化方案诱导的融合事件被指示为FFN减少，反映了FFN511的释放，伴随着FPH和A655斑点荧光的增加，反映了PH-mNG和A655从质膜和浴溶液扩散到熔融囊泡中。该图显示了被识别为F655调光的紧密聚变，而FPH持续或衰减延迟。该图表示通过存在PH-mNG和A655斑点检测到的停留融合。该图表示检测到的平行FPH和F655衰变的收缩融合，伴随着PH-mNG斑点和A655斑点的平行尺寸减小 成功记录需要通过膜片钳和成像在每个通道的细胞底部生成具有适当荧光强度的整个细胞配置。这里描述的电生理学和超分辨率显微镜的结合是测量融合孔隙动力学和神经回路的宝贵工具。

Research

JoVE Journal

Neuroscience

A Simple Way to Measure Ethanol Sensitivity in Flies

一个简单的方法来衡量在果蝇的乙醇灵敏度

Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain ...

科研

神经科学

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

在完整的感觉器官前体细胞的活细胞成像果蝇蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Three-dimensional Confocal Analysis of Microglia/macrophage Markers of Polarization in Experimental Brain Injury

偏振的实验性脑损伤小胶质细胞/巨噬细胞标志物的三维共聚焦分析

After brain stroke microglia/macrophages (M/M) undergo  rapid activation with dramatic morphological and phenotypic changes that  include expression of ...

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

方法在小鼠肾上腺嗜铬细胞细胞贴附电容测量

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

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

神经肌肉接头处：测量突触大小，碎片和改变突触蛋白密度利用共聚焦荧光显微镜

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

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

TIRFM和pH敏感的GFP-探针来评估神经递质囊泡动力学SH-SY5Y神经母细胞瘤：细胞成像和数据分析

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

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

协议星形胶质细胞的三维共聚焦形态分析

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify ...

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

单脂蛋白的SNARE介导融合在一个微流控流细胞拴支持双分子层通过偏光显微镜TIRF监控

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. ...

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

测定在运动神经元样NSC-34细胞中实时测量核细胞质运输

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a ...

Three-dimensional Navigation-guided, Prone, Single-position, Lateral Lumbar Interbody Fusion Technique

三维导航引导，俯卧，单位，侧腰椎椎体间融合技术

Lateral interbody fusion provides a significant biomechanical advantage over the traditional transforaminal lumbar interbody fusion due to the large ...