Department of Cell Biology, University of Texas Southwestern Medical Center, TX 75390.
Lyda Hill Department of Bioinformatics, University of Texas Southwestern Medical Center, TX 75390.
Mol Biol Cell. 2020 Aug 15;31(18):2035-2047. doi: 10.1091/mbc.E20-06-0363. Epub 2020 Jun 24.
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.
动力蛋白 GTP 酶（Dyn1 和 Dyn2）是核心网格蛋白介导的胞吞作用（CME）机制中不可或缺的蛋白质。它们因在 CME 晚期的分裂中起作用而广为人知，但许多研究表明，动力蛋白在 CME 的早期阶段也发挥着调节作用；然而，关于动力蛋白异构体的具体早期调节功能的详细研究还很缺乏。最近对非神经元细胞中 Dyn1 调节的了解，以及对网格蛋白包被小窝（CCP）动力学进行高灵敏度和定量分析的算法的改进，使我们能够使用结构域交换嵌合体来评估动力蛋白异构体在 CME 中的差异功能。我们报告说，Dyn1 和 Dyn2 在非神经元细胞的 CME 中发挥非冗余的早期调节作用。Dyn2 的脯氨酸/精氨酸丰富结构域对于其靶向新生和生长的 CCP 很重要，而 Dyn1 的膜结合和产生曲率的pleckstrin 同源结构域在稳定新生的 CCP 方面起着重要作用。我们证实了去磷酸化的 Dyn1 增强了支持 CME 的能力，即使与 Dyn2 相比处于亚化学计量水平也是如此。结构域交换嵌合体还揭示了 GTP 酶和茎结构域中以前未知的功能差异。我们的研究大大扩展了对动力蛋白异构体在 CME 早期阶段发挥的调节作用的现有认识。
