Time-resolved Cryo-EM of Dynamin Family Members

动力蛋白家族的蛋白质在整个细胞中被发现，并且可能都参与膜重塑、分裂或融合。我们已经研究了两个家庭成员，发动蛋白和DNM 1的构象变化。 以前，我们已经表明，发动蛋白和Dnm 1自组装成螺旋结构的存在或不存在的脂质。 当GTP加入到动力蛋白-脂质组装体中时，动力蛋白管的直径从50 nm收缩到40 nm，Dnm 1管的直径从100 nm收缩到50 nm。为了进一步了解这一动态过程的机制，我们在加入GTP后的不同时间点通过时间分辨冷冻电子显微镜检查了样品。该方法允许样品在玻璃状冰的薄层中快速检测，而无需添加染色剂或碳载体。 我们发现，在GTP加入后，发动蛋白立即以协调一致的方式收缩下面的脂质双层，多余的脂质在收缩的管的焦点沿着凸出。 收缩后，动力蛋白福尔斯从脂质双层上脱落，表明动力蛋白-动力蛋白相互作用在收缩状态下是不稳定的。 我们的研究结果表明，发动蛋白可以迅速收缩的颈部涂层坑和拆卸一旦其在膜分裂的作用是完整的。 初步结果表明，类似的行为Dnm 1，然而，GTP诱导的构象变化显着更大。

The dynamin family of proteins are found throughout the cell and potentially all are involved in membrane remodeling, fission or fusion. We have examined the conformational change of two family members, dynamin and DNM1. Previously, we have shown that dynamin and Dnm1 self-assemble into helical structure in the presence or absent of lipid. When GTP is added to the dynamin-lipid assemblies, the dynamin tubes constrict in diameter from 50 nm to 40 nm and Dnm1 tubes constrict from 100 nm to 50 nm. To further understand the mechanism of this dynamic process we examined the sample by time-resolved cryo-electron microscopy at different time points following GTP addition. The method allows the sample to be examined quickly in a thin layer of vitreous ice without the addition of stain or carbon support. We showed that immediately upon GTP addition dynamin constricts the underlying lipid bilayer in a concerted action and excess lipid bulges out at focal points along the constricted tubes. Following constriction, dynamin falls off the lipid bilayer, suggesting that dynamin-dynamin interactions are unstable in the constricted state. Our results demonstrate that dynamin can rapidly constrict the necks of coated pits and disassemble once its role in membrane fission is complete. Preliminary results suggest a similar behavior for Dnm1, however, the GTP-induced conformational change is significantly greater.