Name: imaging fitc-dextran as a reporter for regulated exocytosis
Uploaded: 2018-06-20T15:00:00.000+00:00
Duration: 4 min 50 s
Description: Watch this Scientific Journal Video about Imaging FITC-dextran as a Reporter for Regulated Exocytosis at JoVE.com

我们感谢 u. 阿瑟瑞博士对 cDNA 的慷慨馈赠。我们感谢 Drs. g. 质量, l. Mittleman, m Shaharbani, 和 Zilberstein 从 Sackler 细胞 & 分子成像中心为他们的宝贵协助与显微术。这项工作得到了美国-以色列两国科学基金会 (赠款2013263至 r. Sagi-艾森伯格、i. Hammel 和 s.j. 加利) 的支持, 并由以色列科学院创立的以色列科学基金会授予 933/15 (以 r Sagi-艾森伯格) 和 NIH 赠款 U19 AI 104209 和 R01 AR067145 (S.J. 加利)。

1. 筹备工作
<ol><li>RBL 培养基的制备<ol>
<li>混合500毫升低葡萄糖 Dulbecco 的改良鹰的培养基 (DMEM) 与56毫升的胎牛血清 (血清), 5.5 毫升青霉素-链霉素-制霉菌素溶液, 5.5 毫升 l-谷氨酰胺200毫米溶液。这导致低葡萄糖 DMEM 补充10% 血清, 100 µg/毫升链霉素, 100 单位/毫升青霉素, 12 单位/毫升制霉菌素, 2 毫米 l-谷氨酰胺。</li>
		<li>使用0.22 µm 孔径大小的顶部真空过滤器过滤介质, 并存储在4摄氏度。</li>
	</ol>
</li>
	<li>对 RBL 细胞的维护。<ol>
<li>在10厘米的盘子中, 将 RBL 细胞生长到最大融合90%。如果细胞培养是健康的, 细胞应该有一个纺锤形与偶尔突起。</li>
		<li>对于细胞分裂, 从盘子中分离细胞, 通过吸媒体和取代2毫升的 trypsine/EDTA 溶液 b. 在 5% CO2的湿润气氛中孵育5-10 分钟, 37 摄氏度。</li>
		<li>一旦细胞分离, 就可以通过添加2毫升培养基, 使用吸管来中和胰蛋白酶, 并将细胞分成 1:2-1:10 的比例。</li>
	</ol>
</li>
	<li>20x Tyrode 缓冲器的研制<ol>
<li>在双蒸馏水 (DDW) 中, 准备20x 溶液, 54 毫米氯化钾, 20 毫米氯化镁2, 2.74 米氯化钠, 8 毫米 NaH2PO4 。混合好, 并存储在4摄氏度。</li>
	</ol>
</li>
</ol>2. 活细胞显微术中的 RBL 细胞培养
<ol><li>FITC 葡聚糖溶液的制备。<ol>
<li>混合1毫克的 FITC-葡聚糖粉 (150 K) 每1毫升培养基 (见步骤 1.1.1)。对于全腔 coverglass, 准备3毫升。</li>
		<li>使用醋酸纤维素注射器过滤器单元, 0.22 µm 孔径, 过滤溶解 FITC-葡聚糖。</li>
		<li>添加小鼠 IgE 到浓度为1µg/毫升。</li>
	</ol>
</li>
	<li>为成像而播种 RBL 细胞<ol>
<li>在成像前的前一天, 从培养基中吸取培养基, 用2毫升的 trypsine/EDTA 溶液代替, 在 5% CO2的湿润气氛中孵育 5-10 分钟, 37 摄氏度。细胞分离后, 加入2毫升培养基, 中和胰蛋白酶。</li>
		<li>使用 hemocytometer 计数 RBL 单元格, 并相应地调整音量以培养培养基, 以获得 7.5 x 105/毫升的细胞浓度。</li>
		<li>添加10µL 细胞悬浮液室 coverglass 预填充新鲜的 FITC 葡聚糖补充培养基 (这导致在室内播种 7.5 x 103细胞)。</li>
		<li>在 5% CO2在37摄氏度的 humified 大气中, 一夜之间生长出 RBL 细胞。细胞应保持在一个亚汇合的水平, 以确保细胞分离, 并容易识别每个细胞在显微镜下单独。</li>
	</ol>
</li>
	<li>对 RBL 细胞的转染-可选。<ol>
<li>对于成像 exocytic 事件结合其他荧光标记的蛋白质, 请参阅转染协议的 RBL 细胞在阿祖兹·恩尼法尔等。67
</li>
	</ol>
</li>
</ol>3. 胞吐作用活细胞显微术
<ol><li>解决方案的准备:<ol>
<li>准备一个最终 Tyrode 的缓冲解决方案, 稀释 DDW 中的库存溶液在1:20 稀释和补充与20毫米 Hepes pH 7, 1.8 毫米 CaCl2, 1 毫克/毫升 BSA, 5.6 毫米葡萄糖。</li>
		<li>新鲜地准备一个 20x secretagogue 试剂在 Tyrode 的缓冲 [1 µg/毫升 dinitrophenyl 共轭人血清白蛋白 () 在我们的情况下, 为 50 ng/毫升1x 浓度]。</li>
		<li>在 Tyrode 的缓冲液中溶解粉末, 制备400毫米氯化铵溶液。</li>
	</ol>
</li>
	<li>细胞的制备。<ol>
<li>清洗腔 coverglass 3 次, 通过从腔室中吸出介质, 并用300µL Tyrode 的缓冲器填充, prewarmed 到37摄氏度。最后, 补充300µL Tyrode 的缓冲室, prewarmed 到37摄氏度。</li>
		<li>将腔内 coverglass 放在显微镜的孵化室中。确保房间稳定。</li>
	</ol>
</li>
	<li>设置显微镜<ol>
<li>要选择一个感兴趣的区域跟踪, 打开荧光光源 (传统的水银灯), 并选择适当的荧光过滤器 (选择绿色显影的过滤器, 以查看 FITC 葡聚糖)。一旦感兴趣的区域集中并处于视野的中间, 关闭光源以避免光漂白和毒性。 注: 部分 FITC 葡聚糖在细胞中可能保留荧光。这是因为 FITC-葡聚糖也可能被归类为非溶酶室, 如内涵体。</li>
		<li>打开适当的照明 FITC 励磁。如果使用激光显微镜, 打开488纳米激光。排放量应收集约 500-550 nm (FITC 发射峰在 510-520 nm)。</li>
		<li>使用共焦显微镜时, 将针孔打开至最大值。这将允许使用较低的激光功率, 以避免漂白和毒性, 并将确保捕获的胞吐作用事件从所有平面的细胞。</li>
		<li>校准图像购置之间的时间间隔。<ol>
<li>不同的细胞在其调节胞吐作用68的动力学中可能有所不同。对于复合胞吐作用的特定成像, 我们建议至少5秒的时间间隔。但是, 作为一个经验法则, 要允许快速成像, 请确保单个帧的采集时间是快速的。</li>
			<li>当使用激光扫描共焦显微镜时, 将扫描方向设置为双向, 不允许平均值, 并设置分辨率为 512 x 512 (推荐; 后两项规定也将有助于尽量减少漂白和毒性)。</li>
		</ol>
</li>
	</ol>
</li>
	<li>胞吐作用的影像学。<ol>
<li>所需工期的图像单元格, 具体取决于单元格或 secretagogue 的类型。在 IgE/Ag 引发的 RBL 细胞中, 大多数 exocytic 事件发生在 15-20 分钟的激活范围内。</li>
		<li>为激活细胞, 添加16µL 从 20x secretagogue 解决方案的房间。</li>
	</ol>
</li>
	<li>确认 FITC-葡聚糖在细胞中的存在。<ol>
<li>为了确认 FITC 葡聚糖在 SGs 中的存在和定位, 添加16µL 氯化铵溶液 (小心不要移动房间, 以免失去焦点) 轻轻地 (这将使最终浓度为20毫米)。这将引起 FITC 荧光的 SGs 和 dequenching 的碱性, 这将在几秒钟内发生。</li>
	</ol>
</li>
</ol>

作者声明没有竞争的财政利益

在这里, 我们描述如何追踪 FITC-葡聚糖的荧光加载到 SGs 可以用来专门捕捉化合物胞吐作用事件。这是通过设置显微镜, 每15秒获得一个图像, 从而确保只有长期的事件将记录一段时间, 因此不包括短事件, 将对应于完全胞吐作用或接吻和运行胞吐作用。为了建立这种方法, 我们表明, 在 RBL 细胞中表达的 Rab5 亚型的击倒, 是复合胞吐作用的关键, 消除了捕捉 FITC-葡聚糖释放事件的能力, 而细胞的整体分?...

调节胞吐作用是一个主要的机制, 使货物从分泌细胞释放, 以响应特定的触发。这些货物是预先形成的, 并被封存成分泌性的囊泡, 在其中储存, 直到触发器中继的信号, 以释放 SGs 的内容。不同类型的信号可能导致不同的调节胞吐作用模式, 或不同的调节胞吐作用模式可能同时发生。三调节胞吐作用的主要模式为: 全胞吐作用, 包括与血浆膜完全融合单一分泌颗粒;胞吐作用, 它涉及分泌颗粒与血浆膜的瞬态融合, 其次是其循环再造;和复合胞吐作用, 其特点是着融合的几个 SGs 之前 (即, 多粒胞吐作用) 或顺序 (即, 顺序胞吐作用), 以融合与等离子膜1。复合胞吐作用被认为是最广泛的货物释放模式2, 因为它允许快速分泌的货物, 包括从 SGs 在远离等离子膜。复方胞吐作用已记录在外分泌和内分泌细胞3,4,5,6,7,8,9, 以及在免疫细胞。在免疫细胞中, 如嗜酸性粒10、11、12和中性白细胞13, 复方胞吐作用允许快速和强健地释放需要杀死入侵病原体的介质, 如细菌或寄生虫。肥大细胞 (MCs) 在先天免疫反应、过敏反应和其他过敏性应答14、15、16中, 部署复合胞吐作用, 有效释放预贮存的炎症介质;,17. 由于胞吐作用的不同模式可能同时发生18、19, 因此在实时区分它们或确定其各自的融合机制方面已成为一项挑战, 因此阐明其基本机制。
在这里, 我们提出了一个方法, 基于细胞加载 FITC 葡聚糖的活细胞成像, 使实时跟踪 exocytic 事件, 并区分其不同的模式。特别是, 我们的方法允许独家监测复合胞吐作用。
FITC 葡聚糖是 pH 敏感荧光 FITC 与葡聚糖多糖的共轭。荧光标记的 dextrans 已显示为进入细胞由 micropinocytosis20,21和 macropinocytosis22,23。随着吞室成熟为溶酶体, 已证明 FITC-葡聚糖在溶酶中积累, 没有明显的降解。然而, 由于 FITC 是一个高度 pH 敏感的荧光24, 溶酶腔是酸性的, FITC-葡聚糖荧光止渴到达溶酶体24。因此, 建立 dextrans 作为溶酶体靶向的货物, 连同 FITC 的 pH 敏感性, 为使用 FITC-葡聚糖在溶酶研究胞吐作用25,26,27奠定了基础,28,29。
在几种细胞类型中, 包括 MCs、中性粒细胞、嗜酸性细胞、细胞毒性 T 淋巴细胞、黑色素等, SGs 显示溶酶体特征, 被归类为溶酶体相关细胞器 (LROs) 或分泌溶体酶30,31.由于 LROs 具有酸性的腔内 pH 值, FITC-葡聚糖可以用来可视化他们的胞吐作用, 因为更高的 pH 值与 LROs exteriorization 相关。事实上, FITC-葡聚糖已被用于监测 MCs18,32,33的胞吐作用。在这种方法中, FITC 葡聚糖被添加到细胞培养, 由细胞吞饮, 并归类到 SGs。在溶酶体中, FITC 荧光在 SGs 细胞内被淬火。然而, 当 sg 与等离子膜融合并随之暴露于外部环境时, FITC-葡聚糖在 sg pH 升高时就恢复了荧光, 允许通过活细胞显微镜对 exocytic 事件进行简单的跟踪。在这里, 我们调整了这种方法, 以实现独特的跟踪复合胞吐作用。
以前有两种方法用于跟踪复合胞吐作用。电镜是描述 exocytic 结构的第一种方法, 它表明了胞吐作用不同模式的发生。特别是, 胰腺腺泡细胞34和 MCs35,36,37的 "分泌通道" 的观察导致了复方胞吐作用的假说。然而, 虽然电子显微镜的高分辨率具有揭示熔泡的能力, 但它无法跟踪其融合的动力学, 因此不能定义它们是否对应于复胞吐作用或重新捕获颗粒融合过程中的 SG 融合。跟随他们的吞。这种障碍是克服其他方法, 可以测量胞吐作用在活细胞, 如膜片钳测量的等离子膜电容11,13,38,39或探头40的媒体。然而, 补丁夹紧需要一个特殊的设置, 可能不适合所有的细胞类型。只有当货物在极接近电极的情况下释放时, 探头测量才能跟踪胞吐作用。因此, 使用活细胞成像比这些方法更有优势, 因为它不仅允许实时跟踪胞吐作用, 而且还允许快速简单地从整个单元中获取数据。
用活细胞显微镜对 FITC 葡聚糖的跟踪也为其他活细胞成像方法提供了一些优势。例如, 一个广泛使用的方法是全内反射荧光显微镜 (TIRFM) 的细胞加载与荧光 SG 探针或表达荧光蛋白标记的 SG 货物或膜蛋白26,41,42,43. 这种方法的优点在于它能够监测与等离子膜 (此处称为脚印) 相近的事件, 因此 exocytic 事件。然而, 这也是这一方法的缺点, 因为只有与 coverglass 相邻的细胞分数和接近显微镜透镜可以成像44。这些脚印是否确实代表了整个细胞膜表面仍然有争议的45,46,47。在这方面, 使用 pH 敏感的染料, 如 FITC 葡聚糖和标准荧光显微镜或共聚焦显微镜与开放针孔, 使整个细胞的成像, 从而捕获的总 exocytic 事件发生在该细胞。
其他 ph 敏感的记者, 用于研究受控胞吐作用的全细胞成像或 TIRFM 包括 sg 货物或 sg 膜蛋白融合到 phlourin, 一个 pH 敏感的 GFP 变种。例子包括 NPY-phlourin-, β-hexoseaminidase-phlourin 和 synapto-phluorin48,49,50,51,52。虽然这些探针的表达可能更紧密地代表 SGs 的内源成分, 但它需要转染细胞, 因此可能不太适合难以染的细胞。因此, 在研究难以染的细胞, 或在需要多重基因组操作的实验条件下, 使用一种可以简单补充到细胞培养基中的化合物, 如 FITC 葡聚糖, 是有利的。.FITC-葡聚糖也提供了优于吖啶橙 (AO), 另一种 pH 敏感染料, 已用于跟踪胞吐作用的活细胞显微镜53,54,55,56,57,58. AO 已被证明可以诱导对水泡的光解, 导致假闪光, 这不对应于实际分泌过程27。相反, FITC-葡聚糖反映了更好的分泌事件, 可能是由于其低光诱导生产活性氧27。
值得注意的是, 研究胞吐作用的另一种方法是通过在这个过程中打开的融合孔跟踪染料的流入, 从外部介质进入 SG。在这种情况下, 染料被添加到外部培养基旁边的分泌触发器。然后, 当融合孔打开, 染料扩散到 SG59,60。这种方法的一个明显的优点是, 它也提供的能力, 以估计的融合孔径大小, 使用的染料的可变大小。例如, 不同分子量 (兆瓦) 的 dextrans, 共轭到不同的显影, 可以作为胞外染料, 即能穿透 SG 的葡聚糖的最大尺寸对应于融合孔59的大小,61,62,63,64. 此外, 这种方法不需要使用 pH 敏感探针。然而, 一个很大的缺点是, 信噪比是非常低的, 因为在图像采集过程中存在大量的染料, 导致较高的背景。
总的来说, 使用 FITC 葡聚糖作为胞吐作用的标志, 克服了以往报告方法的一些缺点, 如信噪比, 毒性, 动态跟踪和复杂性。
在这里, 我们描述了使用 FITC 葡聚糖监测复合胞吐作用在 RBL-2H3 肥大细胞系 (这里称为 RBL, 主要建立埃克莱斯顿等。65和进一步克隆 Barsumian等。66), 对免疫球蛋白 E (IgE)/抗原 (Ag) 活化反应。

<table><tbody><tr><td>DMEM low glucose</td><td>Biological Inductries</td><td>01-050-1A</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td>12657</td><td></td></tr><tr><td>Pen-strep-nystatin solution</td><td>Biological Inductries</td><td>03-032-1B</td><td></td></tr><tr><td>L-Glutamine 200 mM solution&amp;nbsp;</td><td>Biological Inductries</td><td>03-020-1A</td><td></td></tr><tr><td>FITC dextran 150K</td><td>Sigma-Aldrich</td><td>46946-500MG-F</td><td></td></tr><tr><td>Trypsin/EDTA Solution B</td><td>Biological Inductries</td><td>03-052-1A</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Top-vacuum filter of 0.22 &amp;micro;m pore size&amp;nbsp;</td><td>Sigma-Aldrich</td><td>CLS430769-1EA</td><td></td></tr><tr><td>Cellulose acetate syringe filter unit, 0.22 &amp;micro;m pore size</td><td>Sartorius</td><td>16534K</td><td></td></tr><tr><td>Chambered coverglass&amp;nbsp;</td><td>Thermo scientific</td><td>155411</td><td></td></tr><tr><td>Corning tissue-culture treated culture dishes</td><td>Sigma-Aldrich</td><td>CLS430167</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A4503</td><td></td></tr><tr><td>Anti-DNP monoclonal IgE</td><td>Sigma-Aldrich</td><td>D8406</td><td></td></tr><tr><td>DNP-HSA (Ag)</td><td>Sigma-Aldrich</td><td>A6661&amp;nbsp;</td><td>Avoid direct light exposure</td></tr><tr><td>Hepes buffer 1M, pH 7.4</td><td>Biological Inductries</td><td>03-025-1B</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>MERK</td><td>102382</td><td></td></tr><tr><td>Glucose</td><td>BDH Laboratories</td><td>284515V</td><td></td></tr><tr><td>Ammonium chloride</td><td>MERK</td><td>1145</td><td></td></tr><tr><td>PIPES dipotassium salt</td><td>Sigma-Aldrich</td><td>108321-27-3</td><td></td></tr><tr><td>Calcium acetate hydrate</td><td>Sigma-Aldrich</td><td>114460-21-8</td><td></td></tr><tr><td>Magnesium acetate tetrahydrate</td><td>Sigma-Aldrich</td><td>M5661</td><td></td></tr><tr><td>Potassium glutamate (L-Glutamic acid potassium salt monohydrate)</td><td>Sigma-Aldrich</td><td>G1501</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>MERK</td><td>6346</td><td></td></tr><tr><td>NaCl</td><td>MERK</td><td>106404</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>MERK</td><td>105833</td><td></td></tr><tr><td>KCl</td><td>MERK</td><td>104936</td><td></td></tr><tr><td>Electroporator&amp;nbsp;</td><td>BTX</td><td>ECM830</td><td></td></tr><tr><td>Confocal microscope, Zeiss</td><td>Zeiss</td><td>LSM 5 Pascal, Axiovert 200M</td><td>Used in Figure 3. Equipped with an electronic temperature-controlled&lt;br/&gt; airstream incubator</td></tr><tr><td>Confocal microscope, Zeiss</td><td>Zeiss</td><td>LSM 800, Axio Observer.Z1 /7</td><td>Used in Figure 2. Equipped with a GaAsP detector an electronic temperature-controlled&lt;br/&gt; airstream incubator</td></tr><tr><td>Confocal microscope, Leica</td><td>Leica</td><td>SP5</td><td>Used in Figure 1. Equipped with a leica HyD detector and an top-stage incubator (okolab)</td></tr><tr><td>RBL-2H3 cells</td><td></td><td></td><td>RBL-2H3 cells were cloned in the lab of Reuben P. Siraganian. See reference 67</td></tr></tbody></table>

imaging fitc-dextran as a reporter for regulated exocytosis

被调控的胞吐作用是一个过程, 其中储存在分泌颗粒 (SGs) 的货物是针对分泌扳机释放的。调节胞吐作用是细胞间沟通的基础, 是神经递质、激素、炎症介质和其他化合物分泌的关键机制, 由多种细胞引起。至少有三种不同的机制是已知的调节胞吐作用: 全胞吐作用, 其中一个单一的 sg 完全融合与等离子膜, 亲吻和运行胞吐作用, 其中一个单一的 sg 瞬态保险丝与等离子膜, 和复合胞吐作用, 其中在 SG 与等离子膜融合之前或之后的几个 SGs 保险丝。细胞所进行的调节胞吐作用的类型通常由分泌触发器的类型决定。然而, 在许多细胞中, 单个分泌触发器可以同时激活多种模式的调节胞吐作用。尽管它们在细胞类型和种类上的丰富和重要, 但确定不同分泌方式的机制基本上没有解决。对胞吐作用的不同模式进行调查的主要挑战之一, 是区分两者之间的困难, 并分别进行探讨。在这里, 我们描述使用荧光素异硫氰酸酯 (FITC)-葡聚糖作为胞吐作用的记者, 和活细胞成像, 以区分不同的途径, 调节胞吐作用, 侧重于复合胞吐作用, 基于鲁棒性和exocytic 事件的持续时间。

图 1a代表了 FITC-葡聚糖如何作为胞吐作用的记者, 并概括了 exocytic 事件的不同模式。首先, 细胞与 FITC 葡聚糖一起孵化, 这是由吞饮内化, 并归类到 SGs。由于 MCs 的 SGs 是 LROs, 他们的低 pH 值抑制 FITC 的荧光, 这里显示为黑色颗粒 (A-C, I)。当细胞由 secretagogue 和与等离子膜的 SGs 保险丝触发时, 形成一个融合孔, 允许将质子和 sgs 的碱性流出。因此, FITC 恢?...

Watch this Scientific Journal Video about Imaging FITC-dextran as a Reporter for Regulated Exocytosis at JoVE.com

Imaging FITC-dextran as a Reporter for Regulated Exocytosis

成像 FITC-葡聚糖作为胞吐作用调节的记者

在这里, 我们详细介绍了一种带电细胞成像的调节胞吐作用方法。该方法利用 FITC-葡聚糖, 在溶酶体相关细胞器中积累, 作为记者。这一简单的方法也允许区分不同模式的调节胞吐作用在细胞难以操纵基因。

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated ...

imaging-fitc-dextran-as-a-reporter-for-regulated-exocytosis

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Unit 1

Endocytosis and Exocytosis

SCIENTISTS IN ACTION

12.5K 次观看.  Tel Aviv University. 在这里, 我们详细介绍了一种带电细胞成像的调节胞吐作用方法。该方法利用 FITC-葡聚糖, 在溶酶体相关细胞器中积累, 作为记者。这一简单的方法也允许区分不同模式的调节胞吐作用在细胞难以操纵基因。

视频: Imaging FITC-dextran as a Reporter for Regulated Exocytosis

Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective imaging of fluorescent molecules that are closest to a high refractive index substance such as glass1. In this article, we apply this technique to image exocytosis of synaptic vesicles in retinal bipolar cells isolated from the goldfish retina. These neurons are very suitable for this kind of study due to their large axon terminals. By simultaneously patch clamping the bipolar cells, it is possible to investigate the relationship between pre-synaptic voltage and synaptic release2,3. Synaptic vesicles inside the bipolar cell terminals are loaded with a fluorescent dye (FM 1-43&reg;) by co-puffing the dye and a ringer solution containing a high K+ concentration onto the synaptic terminals. This depolarizes the cells and stimulates endocytosis and consequent dye uptake into the glutamatergic vesicles. After washing the excess dye away for around 30 minutes, cells are ready for being patch clamped and imaged simultaneously with a 488 nm laser. The patch pipette solution contains a rhodamine-based peptide that binds selectively to the synaptic ribbon protein RIBEYE4, thereby labeling ribbons specifically when terminals are imaged with a 561 nm laser. This allows the precise localization of active zones and the separation of synaptic from extra-synaptic events.

In this video, we demonstrate how to label and visualize single synaptic vesicle exocytosis and trafficking in goldfish retinal bipolar cells using total internal reflectance fluorescence (TIRF) microscopy.

TIRF显微镜视网膜双极细胞的影像胞外分泌

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective ...

科研

生物学

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.

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 ...

神经科学

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

内质网子域的培养细胞中的可视化

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly understood. Here assays are described to monitor the ability and kinetics of healing of cultured cells following localized injury. The first protocol describes an end point based approach to simultaneously assess cell membrane repair ability of hundreds of cells. The second protocol describes a real time imaging approach to monitor the kinetics of cell membrane repair in individual cells following localized injury with a pulsed laser. As healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of injury, the third protocol describes the use of above end point based approach to assess one such trafficking event (lysosomal exocytosis) in hundreds of cells injured simultaneously and the last protocol describes the use of pulsed laser injury together with TIRF microscopy to monitor the dynamics of individual subcellular compartments in injured cells at high spatial and temporal resolution. While the protocols here describe the use of these approaches to study the link between cell membrane repair and lysosomal exocytosis in cultured muscle cells, they can be applied as such for any other adherent cultured cell and subcellular compartment of choice.

The process of healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of cell membrane injury. This protocol describes assays to monitor these processes.

成像生物膜损伤和参与亚细胞修复过程

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly ...

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.

成像质膜变形随着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 ...

Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological assays, measuring global changes in endocytosis, have identified over 30 known components participating in CME, and biochemical studies have generated an interaction map of many of these components. It is becoming increasingly clear, however, that CME is a highly dynamic process whose regulation is complex and delicate. In this manuscript, we describe the use of Total Internal Reflection Fluorescence (TIRF) microscopy to directly visualize the dynamics of components of the clathrin-mediated endocytic machinery, in real time in living cells, at the level of individual events that mediate this process. This approach is essential to elucidate the subtle changes that can alter endocytosis without globally blocking it, as is seen with physiological regulation. We will focus on using this technique to analyze an area of emerging interest, the role of cargo composition in modulating the dynamics of distinct clathrin-coated pits (CCPs). This protocol is compatible with a variety of widely available fluorescence probes, and may be applied to visualizing the dynamics of many cargo molecules that are internalized from the cell surface.

Clathrin-mediated endocytosis, a rapid and highly dynamic process internalizes many proteins, including signaling receptors. The protocol described here directly visualizes the kinetics of individual endocytic events. This is essential for understanding how core members of the endocytic machinery coordinate with each other, and how protein cargo influence this process.

通过全内反射荧光显微镜可视化G蛋白偶联受体的单事件分辨率网格蛋白介导的内吞作用

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological ...

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins.

Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.

利用pHluorin标记的受体监视亚细胞定位和贩运

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular ...

Automated Imaging and Analysis for the Quantification of Fluorescently Labeled Macropinosomes

Macropinocytosis is a non-specific fluid-phase uptake pathway that allows cells to internalize large extracellular cargo, such as proteins, pathogens, and cell debris, through bulk endocytosis. This pathway plays an essential role in a variety of cellular processes, including the regulation of immune responses and cancer cell metabolism. Given this importance in biological function, examining cell culture conditions can provide valuable information by identifying regulators of this pathway and optimizing conditions to be employed in the discovery of novel therapeutic approaches. The study describes an automated imaging and analysis technique using standard laboratory equipment and a cell imaging multi-mode plate reader for the rapid quantification of the macropinocytic index in adherent cells. The automated method is based on the uptake of high molecular weight fluorescent dextran and can be applied to 96-well microplates to facilitate assessments of multiple conditions in one experiment or fixed samples mounted onto glass coverslips. This approach is aimed at maximizing reproducibility and reducing experimental variation while being both time-saving and cost-effective.

Automated assays using multi-well microplates are advantageous approaches for identifying pathway regulators by allowing the assessment of a multitude of conditions in a single experiment. Here, we have adapted the well-established macropinosome imaging and quantification protocol to a 96-well microplate format and provide a comprehensive outline for automation using a multi-mode plate reader.

用于荧光标记大促卵体定量的自动成像和分析

Macropinocytosis is a non-specific fluid-phase uptake pathway that allows cells to internalize large extracellular cargo, such as proteins, pathogens, and ...

Live Fluorescence, Inverse Imaging of Cell Ruffling, and Macropinocytosis

Macropinocytosis is a highly conserved but still incompletely understood process that is essential for the uptake and ingestion of fluid, fluid-phase nutrients and other material in cells. The dramatic extension of cell surface ruffles, their closure to form macropinosomes, and the maturation of internalized macropinosomes are key events in this pathway that can be difficult to capture using conventional confocal imaging based on tracking a bolus of fluorescent cargo. Fluorescent dextrans are commonly used experimentally as fluid phase markers for macropinosomes and for other endocytic pathways. A method the lab has adopted to optimize the imaging of dextran uptake involves using live imaging of cells bathed in high concentrations of fluorescent dextran in the medium, with the unlabeled cells appearing in relief (as black). The cell ruffles are highlighted to visualize ruffle closure, and internalized macropinosomes appear as fluorescent vacuoles in the cell interior. This method is optimal for visualizing macropinosome features and allows for easy segmentation and quantification. This paper describes dual-labeling of pathways with different sized dextrans and the co-expression of lipid probes and fluorescent membrane proteins to demark macropinosomes and other endosomes. The detection of internalized dextran at an ultrastructural level using correlative light and electron microscopy (CLEM) is also demonstrated. These cell processes can be imaged using multiple live imaging modalities, including in 3D. Taken together, these approaches optimize macropinosome imaging for many different settings and experimental systems.

This protocol demonstrates dextran imaging in live cells using continuous uptake and inverse images to optimize visualization of ruffling, macropinosome maturation, and analysis of dextran and other cell labelings.

Macropinocytosis is a highly conserved but still incompletely understood process that is essential for the uptake and ingestion of fluid, fluid-phase ...

An Introduction to Endocytosis and Exocytosis

Cells can take in substances from the extracellular environment by endocytosis and actively release molecules into it by exocytosis. Such processes involve lipid membrane-bound sacs called vesicles. Knowledge of the molecular architecture and mechanisms of both is key to understanding normal cell physiology, as well as the disease states that arise when they become defective. This video will first briefly review a few pivotal discoveries in the history of endo- and exocytosis research. Next, some key questions will be examined, followed by a discussion of the prominent methods used to investigate these problems, including cell labeling, fusion assays, and fluorescence imaging. Finally, it will explore current research being conducted by scientists in the field today.

Endocytosis and Exocytosis; Clathrin Coated Pits and SNAREs

内吞作用和胞吐过程简介

Cells can take in substances from the extracellular environment by endocytosis and actively release molecules into it by exocytosis. Such processes ...