Time-resolved Cryo-EM of Dynamin Family Members

动力蛋白家族的蛋白质在整个细胞中都有发现，并且可能都参与了膜重塑、裂变或融合。我们研究了两个家族成员，dynamin和DNM1的构象变化。之前，我们已经证明了动力蛋白和Dnm1在存在或不存在脂质的情况下自组装成螺旋结构。当GTP加入到动力蛋白-脂质组件中时，动力蛋白管的直径从50 nm收缩到40 nm， Dnm1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后的不同时间点用时间分辨低温电子显微镜检查了样品。该方法允许样品在玻璃冰薄层中快速检测，而无需添加染色剂或碳支撑。我们发现，在GTP加入后，动力蛋白立即收缩底层的脂质双分子层，并在收缩管的焦点处多余的脂质凸起。收缩后，动力蛋白从脂质双分子层脱落，表明动力蛋白与动力蛋白的相互作用在收缩状态下是不稳定的。我们的研究结果表明，动力蛋白在膜裂变中的作用一旦完成，就能迅速收缩被涂膜凹坑的颈部并分解。初步结果表明Dnm1具有类似的行为，但gtp诱导的构象变化明显更大。

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.