Functional nanoscopy of membrane deformations and fission by dynamin superfamily members

项目摘要 膜分裂与连接两个细胞的微小纳米尺度膜颈的断裂有关。 在分裂的后期阶段分离/划分膜隔室。及时切断他的脖子 和无泄漏的方式是至关重要的正常功能的内膜系统，因此膜 分裂是由专门的和严格调控的蛋白质机器组装在颈部。而 我们目前对生命和疾病中裂变的机械理解，在很大程度上是基于体外的 重建方法，这种方法很少（如果有的话）重现狭窄和拥挤的环境 脖子上。相反，体外重构主要使用大的（亚微米至微米）微球进行。 规模）的各种物理化学性质的膜模板，导致有争议的结果， 排除了对裂变的严格的机械分析。这个项目的重点是创造下一个- 在生理长度/时间重建和量化膜分裂的体外方法 鳞片我们将联合收割机纳米技术与现代生物物理方法和蛋白质工程相结合， 解决了长期以来存在的发动蛋白超家族蛋白介导的膜分裂的难题， 与细胞内融合/分裂密切相关，并与各种人类病理学直接相关。我们 将从几个不同的角度来探讨这个问题： - 我们将进行单分子分析的发动蛋白寡聚化的膜表面， 精确（2 nm）校准曲率（10-1至10-2 nm范围），以识别和表征 由发动蛋白组装的基本机械化学单元。我们将确定（i） 在弯曲的膜表面上的发动蛋白寡聚化/自组装的途径，（ii） 能够协同GTP水解的最小寡聚物的尺寸/几何排列，和（iii） 膜曲率对小发动蛋白寡聚体的自组装和GTdR活性的影响。 - 我们将评估单个发动蛋白寡聚体（二聚体和更高阶）的膜活性 多聚体）在纳米限制膜模板，以确定如何力场 在整个裂变过程中与脂质重排偶联。我们将 (i)测量由不同发动蛋白寡聚体产生的局部力并量化相关的 膜变形和不稳定性，以及（ii）确定脂质重排的途径， 它们依赖于发动蛋白复合物的大小/几何形状和几何/机械性质， 膜模板参数。 - 我们将分析辅助蛋白和发动蛋白关键突变的影响，比较自我， 组装和裂变途径的不同成员的动力超家族，以区分 一般和蛋白质特异性参数（也许，甚至是特定的途径） 膜分裂和解开功能进化背后的分子机制 和调节发动蛋白裂变机制。

PROJECT SUMMARY Membrane fission is associated with the breakage of a tiny nanometer-scale membrane neck connecting two separating/dividing membrane compartments at the late stages of division. Severing this neck in a timely and leakage-free manner is critical for normal functioning of endomembrane systems, hence membrane fission is performed by specialized and tightly-regulated protein machinery assembling on the neck. While our current mechanistic understanding of fission, in life and disease, is heavily based upon in vitro reconstitution approaches, such approaches rarely (if at all) reproduce confined and crowded environment of the neck. Instead, in vitro reconstitution has been mostly performed using large (sub-micron to micron scale) membrane templates of various physico-chemical properties, resulting in controversial outcomes and precluding rigorous mechanistic analysis of fission. This project is focused on creation of the next- generation in vitro approaches that reconstruct and quantify membrane fission at physiological length/time scales. We will combine nanotechnology with modern biophysical approaches and protein engineering to solve the long-standing puzzle of membrane fission mediated by the proteins of dynamin superfamily, which are intimately involved in intracellular fusion/fission and directly linked to various human pathologies. We will approach this problem from several different angles: - We will perform single-molecule analysis of dynamin oligomerization on membrane surfaces with precisely (2 nm) calibrated curvature (10-1 to 10-2 nm range) to identify and characterize elementary mechano-chemical units assembled by dynamin. We will determine (i) the pathways of dynamin oligomerization/self-assembly on a curved membrane surface, (ii) the size/geometrical arrangement of minimal oligomers capable of cooperative GTP hydrolysis and (iii) the effects of membrane curvature on self-assembly and GTPase activity of small dynamin oligomers. - We will assess membrane activity of individual dynamin oligomers (dimers and higher order multimers) at nano-confined membrane templates to determine how the force fields produced by dynamin are coupled to lipid rearrangements throughout fission. We will (i) measure the local forces produced by different dynamin oligomers and quantify associated membrane deformations and instabilities, and (ii) determine pathway(s) of lipid rearrangements and their dependence on the size/geometry of dynamin complexes and geometrical/mechanical parameters of membrane templates. - We will analyze effects of auxiliary proteins and critical mutations of dynamins, compare the self- assembly and fission pathways for different members of dynamin superfamily to distinguish general and protein-specific parameters (perhaps, even specific pathways) of membrane fission and unravel molecular mechanisms behind functional evolution and regulation of dynamin fission machinery.