Department of Biomolecular Chemistry, University of Wisconsin-Madison School of Medicine and Public Health, Madison, WI 53706, USA.
Department of Biomolecular Chemistry, University of Wisconsin-Madison School of Medicine and Public Health, Madison, WI 53706, USA.
Methods. 2014 Aug 1;68(3):518-28. doi: 10.1016/j.ymeth.2014.03.028. Epub 2014 Apr 2.
Over the past decade, the early Caenorhabditis elegans embryo has proven to be a useful animal model to study a variety of membrane trafficking events, at least in part due to its large size, optical transparency, and ease of manipulation. Importantly, the stereotypic nature of membrane remodeling that occurs during early embryogenesis has enabled quantitative measurement of endocytic flux. In the absence of exogenous stimulation, resumption of the cell cycle triggered by fertilization is coupled to a dramatic redistribution of plasma membrane content. Numerous proteins are rapidly internalized via clathrin-mediated endocytosis, and the fate of these cargoes can be followed precisely using live imaging in utero. Key to these studies is the maintenance of animal health and their immobilization, which can become technically challenging during extended imaging sessions. Here we highlight recent advances in live imaging techniques that have facilitated the interrogation of endocytic transport in live animals. We focus on the use of transgenic C. elegans strains that stably express fluorescently-tagged proteins, including components of the endosomal system and cargo molecules that traverse this network of membranes. Our findings demonstrate the utility of the C. elegans embryo in defining regulatory mechanisms that control the numerous steps of endocytic trafficking.
在过去的十年中,早期秀丽隐杆线虫胚胎已被证明是研究各种膜运输事件的有用动物模型,这至少部分归因于其较大的尺寸、光学透明度和易于操作。重要的是,在早期胚胎发生过程中发生的膜重塑的定型性质使得内吞通量的定量测量成为可能。在没有外源刺激的情况下,受精引发的细胞周期恢复与质膜含量的剧烈再分配相关联。许多蛋白质通过网格蛋白介导的内吞作用被迅速内化,并且可以使用体内实时成像来精确跟踪这些货物的命运。这些研究的关键是维持动物的健康及其固定,这在延长的成像过程中可能会在技术上具有挑战性。在这里,我们强调了最近在活体成像技术方面的进展,这些进展促进了对活体动物中内吞运输的研究。我们专注于使用稳定表达荧光标记蛋白的转基因秀丽隐杆线虫品系,包括内体系统的组成部分和穿过该膜网络的货物分子。我们的研究结果证明了秀丽隐杆线虫胚胎在定义控制内吞运输的众多步骤的调控机制方面的效用。