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
概要
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。
Abstract
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。
Introduction
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。
Protocol
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 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。 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…
Representative Results
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?…
Discussion
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…
開示
The authors have nothing to disclose.
Acknowledgements
我们感谢NINDS校内研究计划（ZIA NS003009-13和ZIA NS003105-08）支持这项工作。
Materials
| Adenosine 5'-triphosphate magnesium salt | Sigma | A9187-500MG | ATP for preparing internal solution |
| Atto 655 | ATTO-TEC GmbH | AD 655-21 | Atto dye to label bath solution |
| Basic Nucleofector for Primary Neurons | Lonza | VSPI-1003 | Electroporation transfection buffer along with kit |
| Boroscilicate capillary glass pipette | Warner Instruments | 64-0795 | Standard wall with filament OD=2.0 mm ID=1.16 mm Length=7.5 cm |
| Bovine serum albumin | Sigma | A2153-50G | Reagent for gland digestion |
| Calcium Chloride 2 M | Quality Biological | 351-130-721 | Reagent for preparing bath solution |
| Cell Strainers, 100 µm | Falcon | 352360 | Material for filtering chromaffin cell suspension |
| Cesium hydroxide solution | Sigma | 232041 | Reagent for preparing internal solution and Cs-glutamate/Cs-EGTA stock buffer |
| Collagenase P | Sigma | 1.1214E+10 | Enzyme for gland digestion |
| Coverslip | Neuvitro | GG-14-Laminin | GG-14-Laminin, 14 mm dia.#1 thick 60 pieces Laminin coated German coverslips |
| D-(+)-Glucose | Sigma | G8270-1KG | Reagent for preparing Locke’s solution and bath solution |
| DMEM | ThermoFisher Scientific | 11885092 | Reagent for preparing culture medium |
| EGTA | Sigma | 324626-25GM | Reagent for preparing Cs-EGTA stock buffer for bath solution |
| Electroporation and Nucleofector | Amaxa Biosystems | Nucleofector II | Transfect plasmids into cells |
| Fetal bovine serum | ThermoFisher Scientific | 10082147 | Reagent for preparing culture medium |
| FFN511 | Abcam | ab120331 | Fluorescent false neurotransmitter to label vesicles |
| Guanosine 5'-triphosphate sodium salt hydrate | Sigma | G8877-250MG | GTP for preparing internal solution |
| HEPES | Sigma | H3375-500G | Reagent for preparing Locke’s solution |
| Igor Pro | WaveMetrics | Igor pro | Software for patch-clamp analysis and imaging data presentation |
| Leica Application Suite X software | Leica | LAS X software | Confocal software for imaging data collection and analysis |
| Leica TCS SP5 Confocal Laser Scanning Microscope | Leica | Leica TCS SP5 | Confocal microscope for imaging data collection |
| L-Glutamic acid | Sigma | 49449-100G | Reagent for preparing Cs-glutamate stock buffer for bath solution |
| Lock-in amplifier | Heka | Lock-in | Software for capacitance recording |
| Magnesium Chloride 1 M | Quality Biological | 351-033-721EA | Reagent for preparing internal solution and bath solution |
| Metallized Hemacytometer Hausser Bright-Line | Hausser Scientific | 3120 | Counting chamber |
| Microforge | Narishige | MF-830 | Polish pipettes to enhance the formation and stability of giga-ohm seals |
| Millex-GP Syringe Filter Unit, 0.22 µm | Millipore | SLGPR33RB | Material for glands wash and digestion |
| mNG(mNeonGreen) | Allele Biotechnology | ABP-FP-MNEONSB | Template for PH-mNeonGreen construction |
| Nylon mesh filtering screen 100 micron | EIKO filtering co | 03-100/32 | Material for filtering medulla suspension |
| Patch clamp EPC-10 | Heka | EPC-10 | Amplifier for patch-clamp data collection |
| PH-EGFP | Addgene | Plasmid #51407 | Backbone for PH-mNeonGreen construction |
| Pipette puller | Sutter Instrument | P-97 | Make pipettes for patch-clamp recording |
| Potassium Chloride | Sigma | P5404-500G | Reagent for preparing Locke’s solution and bath solution |
| Pulse software | Heka | Pulse | Software for patch-clamp data collection |
| Recording chamber | Warner Instruments | 64-1943/QR-40LP | coverslip chamber, apply patch-clamp pipette on live cells |
| Sodium chloride | Sigma | S7653-1KG | Reagent for preparing Locke’s solution, bath solution and internal solution |
| Sodium hydroxide | Sigma | S5881-500G | Reagent for preparing Locke’s solution |
| Sodium phosphate dibasic | Sigma | S0876-500G | Reagent for preparing Locke’s solution |
| Sodium phosphate monobasic | Sigma | S8282-500G | Reagent for preparing Locke’s solution |
| Stirring hot plate | Barnsted/Thermolyne | type 10100 | Heater for pipette coating with wax |
| Syringe, 30 mL | Becton Dickinson | 302832 | Material for glands wash and digestion |
| Tetraethylammonium chloride | Sigma | T2265-100G | TEA for preparing bath solution |
| Trypsin inhibitor | Sigma | T9253-5G | Reagent for gland digestion |
| Type F Immersion liquid | Leica | 195371-10-9 | Leica confocal mounting oil |
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