Functional nanoscopy of membrane deformations and fission by dynamin superfamily members

项目概要 膜裂变与连接两个膜的微小纳米级膜颈的破裂有关 在分裂后期分离/划分膜室。及时割断这条脖子 无泄漏方式对于内膜系统的正常功能至关重要，因此膜 裂变是由组装在颈部的专门且严格调节的蛋白质机器进行的。同时 我们目前对生命和疾病中裂变的机械理解很大程度上基于体外 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 颈部。相反，体外重建主要是使用大（亚微米到微米）进行的。 规模）膜模板的各种物理化学性质，导致有争议的结果和 排除了裂变的严格机械分析。该项目的重点是创建下一代 产生在生理长度/时间重建和量化膜裂变的体外方法 秤。我们将纳米技术与现代生物物理方法和蛋白质工程相结合 解决了由动力超家族蛋白介导的膜裂变的长期难题， 密切参与细胞内融合/裂变，并与各种人类病理直接相关。我们 将从几个不同的角度来解决这个问题： - 我们将对膜表面的动力寡聚进行单分子分析 精确 (2 nm) 校准曲率（10-1 至 10-2 nm 范围）来识别和表征 由动力组装的基本机械化学单元。我们将确定 (i) 弯曲膜表面上的动力寡聚/自组装途径，(ii) 能够协同 GTP 水解的最小低聚物的尺寸/几何排列，以及 (iii) 膜曲率对小动力寡聚体自组装和 GTP 酶活性的影响。 - 我们将评估单个动力寡聚体（二聚体和更高阶）的膜活性 多聚体）在纳米限域膜模板上确定力场如何 动力产生的能量在整个裂变过程中与脂质重排相结合。我们会 (i) 测量不同动力低聚物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和 它们对动力复合物的尺寸/几何形状和几何/机械的依赖 膜模板参数。 - 我们将分析辅助蛋白和动力蛋白关键突变的影响，比较自我 动力蛋白超家族不同成员的组装和裂变途径以区分 一般和蛋白质特异性参数（也许，甚至是特定途径） 膜裂变并揭示功能进化背后的分子机制 和动力裂变机械的调节。

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.