Functional nanoscopy of membrane deformations and fission by dynamin superfamily members

动力超家族成员膜变形和裂变的功能纳米观察

基本信息

批准号：
9982344
负责人：
Vadim A Frolov
金额：
$ 47.81万
依托单位：
UNIVERSITY OF CALIF-LAWRNC LVRMR NAT LAB
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-26 至 2022-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9982344
关键词：
Affect Atomic Force Microscopy Biological Cells Centronuclear myopathy Characteristics Chemicals Chimera organism Complex Coupled Coupling Crowding Dependence Diagnostic Dimensions Disease Dominant-Negative Mutation Dynamin Dynamin 2 Dynamin I Elements Environment Epilepsy Evolution Extravasation Genes Geometry Guanosine Triphosphate Guanosine Triphosphate Phosphohydrolases Human Human Pathology Hydrolysis Impairment In Vitro Individual Kinetics Knowledge Length Life Link Lipids Maintenance Measurement Measures Mechanics Mediating Membrane Modernization Molecular Mutation Nanoscopy Nanotechnology Neck Organelles Osmotic Pressure Outcome Pathologic Pathology Pathway interactions Phenotype Physiological Point Mutation Problem Solving Process Property Protein Analysis Protein Engineering Protein Isoforms Proteins Regulation Signal Transduction Speed Stochastic Processes Stress Surface System Tertiary Protein Structure Testing Therapeutic Time Tissues Tube Variant base biophysical techniques constriction dimer in vivo insight member membrane activity membrane model mutant nano nanomechanics nanoscale nervous system disorder next generation novel novel therapeutic intervention protein complex prototype public health relevance reconstitution self assembly single molecule submicron tool

项目摘要

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.

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。尽管我们目前对生命和疾病中裂变的机制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）再现受限和拥挤的环境颈部的。相反，体外重构主要是使用大（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物质在整个裂变过程中与脂质重排相结合。我们将 (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。