Functional nanoscopy of membrane deformations and fission by dynamin superfamily members

项目总结 膜裂变与连接两个细胞的微小纳米级膜颈断裂有关 在分裂后期分离/隔膜隔室。及时切断这条脖子 而无渗漏方式对内膜系统的正常运行至关重要，因此膜 分裂是由专门的和严格控制的蛋白质机器在颈部组装完成的。而当 我们目前对生命和疾病中的裂变的机械理解在很大程度上是基于体外实验。 重建方法，这种方法很少(如果有的话)再现受限和拥挤的环境 脖子上的。取而代之的是，体外重建大多使用大(亚微米到微米)进行 不同物理化学性质的膜模板，导致有争议的结果和 排除了对裂变的严格的机械分析。这个项目专注于创造下一个- 重建和量化生理长度/时间的膜分裂的体外生成方法 比例。我们将把纳米技术与现代生物物理方法和蛋白质工程相结合，以 解开了长期以来由Dynamin超家族蛋白介导的膜分裂之谜 它们与细胞内融合/分裂密切相关，并与各种人类病理直接相关。我们 我将从几个不同的角度来处理这个问题： -我们将在膜表面进行动力蛋白齐聚的单分子分析 精确(2 Nm)校准曲率(10-1至10-2 nm范围)以识别和表征 用动力剂组装的基本机械力化学单元。我们将决定(I) 动力蛋白在弯曲膜表面上的齐聚/自组装途径，(Ii) 能够协同GTP水解的最小低聚物的尺寸/几何排列和(Iii) 膜曲率对小分子动力蛋白低聚体自组装和GTP酶活性的影响。 -我们将评估单个动力蛋白寡聚体(二聚体和更高阶)的膜活性 多聚体)在纳米受限膜模板上以确定力场如何 在整个裂变过程中，动力素产生的蛋白质与脂类重排相连。我们会 (I)测量不同动力蛋白寡聚体产生的局部作用力，并量化相关 膜变形和不稳定性，以及(Ii)决定脂质重排途径(S)和 它们对动力蛋白复合体的大小/几何和几何/力学的依赖性 膜模板参数。 -我们将分析辅助蛋白质和动力素的关键突变的影响，比较自身 动力蛋白超家族不同成员的组装和分裂途径的区分 一般的和特定于蛋白质的参数(可能，甚至特定的通路) 膜分裂与功能进化背后的分子机制 以及对动力素裂变机械的监管。

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.