Probing structural and biophysical mechanisms of mitochondrial membrane ultrastructure

项目摘要/摘要 细胞器形态专门研究在复杂组织中发现的高度适应的功能。线粒体 超微结构微妙地调整到新陈代谢和生理状态。形态异常是一种 神经疾病、心脏疾病和癌症。有了深冷-EM‘解决方案革命’，我们有 对膜蛋白结构和调控有了戏剧性的新理解，但我们对 细胞器结构滞后。这是由于细胞器的多形性，以及 将特定的形态特征赋予蛋白质状态。我的实验室的首要目标是了解， 在机制水平上，蛋白质的构象变化如何受亚细胞环境的影响，如何影响膜 超微结构受蛋白质因素的调节，这些元素在生理和生理过程中的功能相互作用。 疾病。在接下来的五年里，我的团队将开发一个结合电子冷冻的技术平台- 显微镜(冷冻-EM)和生物物理方法研究线粒体超微结构及其调控。我们会 应用我们最新开发的体外重组系统可视化重组膜蛋白 通过单颗粒冷冻-EM制备脂质体和双分子膜。我们将使我们新建立的电子冷冻机成熟- 用于膜性质计算分析的层析(冷冻-ET)管道以了解其 依赖于蛋白质之间的相互作用。我们将开发新的结构和生物物理方法来 表征细胞器脂质的异质性。最后，我们将探索天然的组装或蛋白质复合体。 上下文。总之，这些方法将促进对蛋白质构象状态的机械性理解 帮助我们确定细胞器形状的基本决定因素。我们广泛地对问题感兴趣 膜间距、组成和曲率。我们将开发专门针对这些问题的工具 以线粒体为试验床，探索调控候选因子的结构和功能 线粒体膜形态，在神经退行性疾病中起因果作用。OPA1是 在显性视神经萎缩中，内膜FusoGen和巩膜重建器发生突变。SLC25A46是一种外部- 溶质转运蛋白家族的膜成员，在协调脂质平衡方面起着重要作用 在利综合症中。MICOS是脊连接的稳定器和调节器(到 线粒体内膜折叠)，其丢失导致早发性致死性线粒体脑病 肝病。该项目的直接影响包括分享了解线粒体形状的新模型 与细胞生物学家合作，为药剂学家提供新的、高度特异的治疗构象靶点 发展，并为生理学家提供了解组织特化的基本规则。这个 长期目标是构建一种可推广到其他器官的可扩展方法，并为 从第一原理出发对细胞器形态的合理控制。

PROJECT SUMMARY/ABSTRACT Organelle morphology is specialized for the highly adapted functions found in complex tissues. Mitochondrial ultrastructure is exquisitely tuned to metabolic and physiological state. Abnormal morphology is a hallmark of neurological disorders, cardiac conditions and cancer. With the cryo-EM `resolution revolution', we have developed dramatic new understanding of membrane protein structure and regulation, but our knowledge of organelle structure lags behind. This is due to the pleomorphic nature of organelles, and the challenge of assigning specific morphological features to protein states. The overarching goals of my lab are to understand, at a mechanistic level, how protein conformational change is influenced by subcellular context, how membrane ultrastructure is regulated by protein factors, and the functional interplay of these elements in physiology and disease. Over the next five years, my group will develop a technical platform that combines electron cryo- microscopy (cryo-EM) and biophysical methods to study mitochondrial ultrastructure and its regulation. We will apply our recent developed in vitro reconstitution systems to visualize reconstituted membrane proteins in liposomes and bilayers by single-particle cryo-EM. We will mature our newly established electron cryo- tomography (cryo-ET) pipeline for computational analyses of membrane properties to understand their dependence on protein-protein interactions. We shall develop new structural and biophysical methods to characterize organelle lipid heterogeneity. And finally, we will explore assembly or protein complexes in native contexts. Together these approaches will advance mechanistic understanding of protein conformational state and help us identify the fundamental determinants of organelle shape. We are broadly interested in questions of membrane spacing, composition and curvature. We will develop tools precisely tailored for these questions using mitochondria as a test bed, exploring the structure and function of candidate factors that regulate mitochondrial membrane morphology, which play causal roles in neurodegenerative conditions. Opa1 is the inner-membrane fusogen and cristae remodeler mutated in Dominant Optic Atrophy. SLC25A46 is an outer- membrane member of the solute transporter family that plays important roles in coordinating lipid homeostasis in Leigh Syndrome. MICOS is the stabilizer and regulator of cristae junctions (the `choke-point' to the mitochondrial inner-membrane folds) whose loss results in early-onset fatal mitochondrial encephalopathy with liver disease. This project's immediate impacts include sharing new models to understand mitochondrial shape with cell biologists, equipping pharmacologists with new, highly specific conformational targets for therapeutic development, and providing physiologists with fundamental rules for understanding tissue specialization. The long-term goal is to build an extensible approach generalizable to other organelles, and a foundation for rational control of organelle morphology from first principles.