成像 FITC-葡聚糖作为胞吐作用调节的记者
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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)。
Materials
| DMEM low glucose | Biological Inductries | 01-050-1A | |
| Fetal bovine serum | Gibco | 12657 | |
| Pen-strep-nystatin solution | Biological Inductries | 03-032-1B | |
| L-Glutamine 200 mM solution | Biological Inductries | 03-020-1A | |
| FITC dextran 150K | Sigma-Aldrich | 46946-500MG-F | |
| Trypsin/EDTA Solution B | Biological Inductries | 03-052-1A | Warm in 37 °C water bath before use |
| Top-vacuum filter of 0.22 µm pore size | Sigma-Aldrich | CLS430769-1EA | |
| Cellulose acetate syringe filter unit, 0.22 µm pore size | Sartorius | 16534K | |
| Chambered coverglass | Thermo scientific | 155411 | |
| Corning tissue-culture treated culture dishes | Sigma-Aldrich | CLS430167 | |
| Bovine serum albumin | Sigma-Aldrich | A4503 | |
| Anti-DNP monoclonal IgE | Sigma-Aldrich | D8406 | |
| DNP-HSA (Ag) | Sigma-Aldrich | A6661 | Avoid direct light exposure |
| Hepes buffer 1M, pH 7.4 | Biological Inductries | 03-025-1B | |
| CaCl2 | MERK | 102382 | |
| Glucose | BDH Laboratories | 284515V | |
| Ammonium chloride | MERK | 1145 | |
| PIPES dipotassium salt | Sigma-Aldrich | 108321-27-3 | |
| Calcium acetate hydrate | Sigma-Aldrich | 114460-21-8 | |
| Magnesium acetate tetrahydrate | Sigma-Aldrich | M5661 | |
| Potassium glutamate (L-Glutamic acid potassium salt monohydrate) | Sigma-Aldrich | G1501 | |
| NaH2PO4 | MERK | 6346 | |
| NaCl | MERK | 106404 | |
| MgCl2 | MERK | 105833 | |
| KCl | MERK | 104936 | |
| Electroporator | BTX | ECM830 | |
| Confocal microscope, Zeiss | Zeiss | LSM 5 Pascal, Axiovert 200M | Used in Figure 3. Equipped with an electronic temperature-controlled airstream incubator |
| Confocal microscope, Zeiss | Zeiss | LSM 800, Axio Observer.Z1 /7 | Used in Figure 2. Equipped with a GaAsP detector an electronic temperature-controlled airstream incubator |
| Confocal microscope, Leica | Leica | SP5 | Used in Figure 1. Equipped with a leica HyD detector and an top-stage incubator (okolab) |
| RBL-2H3 cells | RBL-2H3 cells were cloned in the lab of Reuben P. Siraganian. See reference 67 |
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