Machine-Learning-Based Analysis in Genome-Edited Cells Reveals the Efficiency of Clathrin-Mediated Endocytosis.
Machine-Learning-Based Analysis in Genome-Edited Cells Reveals the Efficiency of Clathrin-Mediated Endocytosis.
复制标题
DOI：
10.1016/j.celrep.2015.08.048
复制
发表时间：
2015-09-29
期刊：
影响因子：
8.8
通讯作者：
Drubin DG
中科院分区：
文献类型：
--
作者：
Hong SH;Cortesio CL;Drubin DG
细胞通过网格蛋白介导的内吞作用（CME）内化各种分子。先前的活细胞成像研究表明，CME是低效的，大约一半的事件终止。这些CME效率估计可能被荧光标记蛋白的过度表达和无法过滤掉虚假CME位点所混淆。在这里，我们使用基因组编辑和机器学习来识别和分析真正的CME站点。我们在表达两种定义CME蛋白AP2和网格蛋白的荧光融合的细胞中检测了CME动力学。建立支持向量机分类器来识别和分析真实的CME站点。从开始到消失，真正的CME位点同时含有AP2和网格蛋白，具有相同程度的有限迁移，在生命周期内持续积累AP2和网格蛋白，并且几乎总是形成囊泡（通过dynamin2募集来评估）。仅含有网格蛋白或AP2的位点表现出明显的动态，表明它们不是CME途径的一部分。
Cells internalize various molecules through clathrin-mediated endocytosis (CME). Previous live-cell imaging studies suggested that CME is inefficient, with about half of the events terminated. These CME efficiency estimates may have been confounded by overexpression of fluorescently tagged proteins and inability to filter out false CME sites. Here, we employed genome editing and machine learning to identify and analyze authentic CME sites. We examined CME dynamics in cells that express fluorescent fusions of two defining CME proteins, AP2 and clathrin. Support vector machine classifiers were built to identify and analyze authentic CME sites. From inception until disappearance, authentic CME sites contain both AP2 and clathrin, have the same degree of limited mobility, continue to accumulate AP2 and clathrin over lifetimes > ~20s, and almost always form vesicles as assessed by dynamin2 recruitment. Sites that contain only clathrin or AP2 show distinct dynamics, suggesting they are not part of the CME pathway.