Dynamin GTPases (Dyn1 and Dyn2) are indispensable proteins of the core clathrin-mediated endocytosis (CME) machinery. Best known for their role in fission at the late stages of CME, many studies have suggested that dynamin also plays a regulatory role during the early stages of CME; however, detailed studies regarding isoform-specific early regulatory functions of the dynamins are lacking. With a recent understanding of the regulation of Dyn1 in nonneuronal cells and improved algorithms for highly sensitive and quantitative analysis of clathrin-coated pit (CCP) dynamics, we have evaluated the differential functions of dynamin isoforms in CME using domain swap chimeras. We report that Dyn1 and Dyn2 play nonredundant, early regulatory roles during CME in nonneuronal cells. The proline/arginine-rich domain of Dyn2 is important for its targeting to nascent and growing CCPs, whereas the membrane-binding and curvature-generating pleckstrin homology domain of Dyn1 plays an important role in stabilizing nascent CCPs. We confirm the enhanced ability of dephosphorylated Dyn1 to support CME, even at substoichiometric levels compared with Dyn2. Domain swap chimeras also revealed previously unknown functional differences in the GTPase and stalk domains. Our study significantly extends the current understanding of the regulatory roles played by dynamin isoforms during early stages of CME.

Dynamin GTPases（Dyn1和Dyn2）是核心网格蛋白介导的内吞（CME）机制不可缺少的蛋白质。最著名的是它们在CME的晚期分裂中的作用，许多研究表明，dynamin在CME的早期也起调节作用。然而，关于动力蛋白的异构体特异性早期调节功能的详细研究尚缺乏。随着对非神经元细胞中Dyn1调控的最新了解以及对网格蛋白包被的坑（CCP）动力学进行高度灵敏和定量分析的改进算法，我们已经使用域交换嵌合体评估了CME中的dynamin同工型的差异功能。我们报告Dyn1和Dyn2在非神经元细胞CME期间发挥非冗余的早期调节作用。Dyn2富含脯氨酸/精氨酸的结构域对于靶向新生和成长中的CCP非常重要，而Dyn1的膜结合和产生曲率的pleckstrin同源结构域在稳定新生CCP中起着重要作用。我们证实，即使在亚化学计量水平上，与Dyn2相比，脱磷酸化的Dyn1支持CME的能力也得到增强。域交换嵌合体还揭示了GTPase和茎域中以前未知的功能差异。我们的研究显着扩展了目前对CME早期动力型亚型发挥调节作用的理解。甚至与Dyn2相比处于亚化学计量水平。域交换嵌合体还揭示了GTPase和茎域中以前未知的功能差异。我们的研究显着扩展了目前对CME早期动力型亚型发挥调节作用的理解。甚至与Dyn2相比处于亚化学计量水平。域交换嵌合体还揭示了GTPase和茎域中以前未知的功能差异。我们的研究显着扩展了目前对CME早期动态型亚型发挥调节作用的理解。