Materials Science Division, Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore, CA 94550.
Biofisika Institute Consejo Superior de Investigaciones Científicas, Universidad del País Vasco, Euskal Herriko Unibersitatea (CSIC, UPV/EHU), University of the Basque Country, Leioa, 48940, Spain.
Proc Natl Acad Sci U S A. 2024 Dec 3;121(49):e2321514121. doi: 10.1073/pnas.2321514121. Epub 2024 Nov 27.
Dynamin 1 (Dyn1) GTPase, a principal driver of membrane fission during synaptic endocytosis, self-assembles into short mechanoactive helices cleaving the necks of endocytic vesicles. While structural information about Dyn1 helix is abundant, little is known about the nanoscale dynamics of the helical scaffolding at the moment of fission, complicating mechanistic understanding of Dyn1 action. To address the role of the helix dynamics in fission, we used High-Speed Atomic Force Microscopy (HS-AFM) and fluorescence microscopy to track and compare the spatiotemporal characteristics of the helices formed by wild-type Dyn1 and its K44A mutant impaired in GTP hydrolysis on minimal lipid membrane templates. In the absence of nucleotide, membrane-bound Dyn1 and Dyn1 self-assembled into tubular protein scaffolding of similar diameter encaging the lipid bilayer. In both cases, the GTP addition caused scaffold constriction coupled with formation of 20 to 30 nm nanogaps in the protein coverage. While both proteins reached scaffold diameters characteristic for membrane superconstriction causing fission, the fission was detected only with Dyn1. We associated the fission activity with the dynamic evolution of the nanogaps: Dyn1 gaps were static, while Dyn1 gaps actively evolved via repetitive nonaxisymmetric constriction-bending deformations caused by localized GTP hydrolysis. Modeling of the deformations implicated filament twist as an additional deformation mode which combines with superconstriction to facilitate membrane fission. Our results thus show that the dynamics of the Dyn1 helical scaffold goes beyond radial constriction and involves nonaxisymmetric deformations, where filament twist emerges as a critical driver of membrane fission.
动力蛋白 1（Dyn1）GTP 酶是突触内吞作用中膜裂变的主要驱动蛋白，它自我组装成短的机械活性螺旋，切断内吞小泡的颈部。虽然关于 Dyn1 螺旋的结构信息很丰富，但在裂变时刻，关于螺旋支架的纳米级动力学知之甚少，这使得 Dyn1 作用的机制理解变得复杂。为了解决螺旋动力学在裂变中的作用，我们使用高速原子力显微镜（HS-AFM）和荧光显微镜来跟踪和比较野生型 Dyn1 及其在 GTP 水解中受损的 K44A 突变体在最小脂质膜模板上形成的螺旋的时空特征。在没有核苷酸的情况下，膜结合的 Dyn1 和 Dyn1 自组装成管状蛋白支架，其直径与脂质双层相似。在这两种情况下，GTP 的加入都会导致支架收缩，并在蛋白覆盖物中形成 20 到 30nm 的纳米间隙。虽然这两种蛋白质都达到了导致裂变的膜超缩的支架直径，但只有 Dyn1 检测到了裂变。我们将裂变活性与纳米间隙的动态演化联系起来：Dyn1 间隙是静态的，而 Dyn1 间隙通过局部 GTP 水解引起的重复非轴对称收缩-弯曲变形而主动演化。变形的建模表明，纤维扭曲是一种额外的变形模式，它与超缩合结合，促进了膜裂变。因此，我们的结果表明，Dyn1 螺旋支架的动力学不仅限于径向收缩，还涉及非轴对称变形，其中纤维扭曲成为膜裂变的关键驱动因素。
