Molecular Mechanisms of Dynamin-related Protein 1-Mediated Mitochondrial Fission

项目摘要 线粒体是一种动态的细胞器，经历不断的分裂和融合。线粒体动力学是 对于细胞存活以及线粒体质量控制、运输、分布和遗传至关重要。 线粒体动力学的缺陷与各种神经系统疾病有关，包括阿尔茨海默氏症， 帕金森氏症和亨廷顿氏症，以及心血管疾病和癌症。分子 完成线粒体膜分裂和融合的机制知之甚少， 参与这些过程的分子。这项建议的长期目标是解决这些问题。的 动力蛋白相关蛋白1（Drp1）是线粒体分裂的主要调节因子。 胞浆Drp1通过与衔接蛋白Mff、MiD49/51或Fis1相互作用启动线粒体分裂 位于线粒体表面。随后的Drp1聚合作为"螺旋支架"围绕预定的 线粒体分裂位点和GTP水解驱动的支架收缩催化线粒体分裂。 令人兴奋的新研究也需要直接Drp1-磷脂相互作用的合作作用， 特别是与线粒体脂质，心磷脂（CL），在线粒体分裂。然而， 关于Drp1-adaptor和Drp1-CL相互作用的协同性，无论是在空间上还是在时间上，在这个过程中。 以下是几个对于理解Drp1介导的线粒体分裂至关重要的未知基本问题： 在这个应用程序中解决。这些包括1）Drp1 CL识别的潜在机制，以及 2）Drp1可变结构域的作用机制 (VD)在CL重组和非双层相变中，3）Drp1在 膜表面和构象重排后，随之而来的特定适配器和CL相互作用， CL和衔接子相互作用在影响线粒体分裂中的协同作用。拟议 实验将测试总体假设，合作Drp1与蛋白质衔接子和CL相互作用 促进在CL丰富的微环境和驱动器中本地化的生产性"裂变复合体"的形成 通过Drp1 GTP水解依赖性CL双层到非双层相的膜重塑和分裂 过渡机制我们将使用量身定制的创新荧光光谱和显微阵列 方法，再加上解决方案和固态NMR，以解决这些问题。其中包括使用一本小说 FRET方法的变化，以确定结构域特异性Drp1-膜距离，碰撞淬灭 以确定和测量Drp1 VD膜插入，并在模型上进行荧光成像 GUV可视化适配器和CL调节，Drp1介导的膜重塑和裂变。成功 这项研究的结果将提供（i）从根本上提高对合作分子的理解， 线粒体分裂的潜在机制，以及（ii）药物设计的分子基础， 在各种疾病状态下可以有益地调节线粒体动力学的治疗剂。

PROJECT SUMMARY Mitochondria are dynamic organelles that undergo continuous fission and fusion. Mitochondrial dynamics are essential for cell survival, as well as for mitochondrial quality control, transport, distribution and inheritance. Defects in mitochondrial dynamics are implicated in various neurological disorders including Alzheimer’s, Parkinson’s and Huntington’s diseases, as well as in cardiovascular disease and cancer. The molecular mechanisms that accomplish mitochondrial membrane fission and fusion are poorly understood, as are the roles of the molecules involved in these processes. The long-term goal of this proposal is to address such issues. The mechanoenzymatic GTPase, dynamin-related protein 1 (Drp1) is the master regulator of mitochondrial fission. Cytosolic Drp1 initiates mitochondrial fission via interactions with adaptor proteins, Mff, MiD49/51, or Fis1 localized at the mitochondrial surface. Subsequent Drp1 polymerization as ‘helical scaffolds’ around pre-destined mitochondrial division sites and GTP hydrolysis-driven scaffold constriction catalyzes mitochondrial fission. Exciting new studies have also necessitated a cooperative role for direct Drp1-phospholipid interactions, specifically with the mitochondrial lipid, cardiolipin (CL), in mitochondrial fission. However, very little is known about the cooperativity of Drp1-adaptor and Drp1-CL interactions, either in space or in time, during this process. Several unknown fundamental issues essential for understanding Drp1-mediated mitochondrial fission will be addressed in this application. These include 1) the mechanisms underlying Drp1 CL recognition, and the identity of Drp1 residues involved in specific phospholipid interactions, 2) the mechanism of the Drp1 variable domain (VD) in CL reorganization and nonbilayer phase transition, 3) the domain-specific topography of Drp1 on the membrane surface and conformational rearrangements that ensue upon specific adaptor and CL interactions, and 4) the cooperativity of CL and adaptor interactions in effecting mitochondrial fission. The proposed experiments will test the overarching hypothesis that cooperative Drp1 interactions with protein adaptors and CL promote the formation of a productive “fission complex” that is localized in CL-rich micro-environments and drives membrane remodeling and fission through a Drp1 GTP hydrolysis-dependent CL bilayer-to-nonbilayer phase transition mechanism. We will use a tailor-made array of innovative fluorescence spectroscopic and microscopic approaches, coupled to solution and solid state NMR, to address these issues. These include the use of a novel variation of the FRET approach to determine domain-specific Drp1-membrane distances, collisional quenching of fluorescence to determine and measure Drp1 VD membrane insertion, and fluorescence imaging on model GUVs to visualize adaptor- and CL-regulated, Drp1-mediated membrane remodeling and fission. Successful outcomes of this research will provide (i) a fundamentally improved understanding of the cooperative molecular mechanisms underlying mitochondrial fission, and (ii) a molecular foundation for the design of drugs and therapeutics that can beneficially modulate mitochondrial dynamics under various disease states.