Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos.
Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos.
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DOI：
10.3791/63291
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发表时间：
2021-12-13
期刊：
影响因子：
--
通讯作者：
Welte MA
中科院分区：
文献类型：
--
作者：
Kilwein MD;Welte MA
早期果蝇胚胎是包含大量常规细胞器和胚胎特有细胞器的大细胞。在胚胎发育的前三个小时，这些细胞器经历了戏剧性的运动，由基于肌动蛋白的细胞质流动和运动驱动的沿微管的运输提供动力。大量小的细胞器特异性荧光探针(FP)的开发使人们有可能在任何基因中可视化各种不同的含脂结构，从而无需遗传编码的荧光团即可进行实时成像。该方案展示了如何将重要的染料和分子探针注射到果蝇胚胎中，通过活体成像监测特定细胞器的运输。这种方法是通过标记脂滴(LDS)并用粒子图像测速仪(PIV)跟踪它们的整体运动来演示的。该方案为其他细胞器的研究提供了一种策略，包括溶酶体、线粒体、卵黄泡和内质网，以及跟踪单个LDS沿微管的运动。使用商业上可用的染料带来了分离到光谱的紫色/蓝色和远红区域的好处。通过显微注射对细胞器和/或细胞骨架元素进行多重共标记，果蝇的所有遗传资源都可以用于交易研究，而不需要引入荧光标记的蛋白质。与量子产率低且容易漂白的基因编码荧光团不同，许多可用的染料允许快速同时捕获多个具有高光子产率的通道。在果蝇早期胚胎中，许多细胞器是活动的。原则上，它们可以通过特定的荧光探针进行实时成像，但蛋壳阻止了对胚胎的直接应用。该方案描述了如何通过显微注射引入这种探针，然后通过粒子图像测速技术分析细胞器的整体运动。
Early Drosophila embryos are large cells containing a vast array of conventional and embryo-specific organelles. During the first three hours of embryogenesis, these organelles undergo dramatic movements powered by actin-based cytoplasmic streaming and motor-driven trafficking along microtubules. The development of a multitude of small, organelle-specific fluorescent probes (FPs) makes it possible to visualize a wide range of different lipid-containing structures in any genotype, allowing live imaging without requiring a genetically encoded fluorophore. This protocol shows how to inject vital dyes and molecular probes into Drosophila embryos to monitor the trafficking of specific organelles by live imaging. This approach is demonstrated by labeling lipid droplets (LDs) and following their bulk movement by particle image velocimetry (PIV). This protocol provides a strategy amenable to the study of other organelles, including lysosomes, mitochondria, yolk vesicles, and the ER, and for tracking the motion of individual LDs along microtubules. Using commercially available dyes brings the benefits of separation into the violet/blue and far-red regions of the spectrum. By multiplex co-labeling of organelles and/or cytoskeletal elements via microinjection, all the genetic resources in Drosophila are available for trafficking studies without the need to introduce fluorescently tagged proteins. Unlike genetically encoded fluorophores, which have low quantum yields and bleach easily, many of the available dyes allow for rapid and simultaneous capture of several channels with high photon yields. In the early Drosophila embryo, many organelles are motile. In principle, they can be imaged live via specific fluorescent probes, but the eggshell prevents direct application to the embryo. This protocol describes how to introduce such probes via microinjection, and then analyze bulk organelle motion via particle image velocimetry.
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