Probing structural and biophysical mechanisms of mitochondrial membrane ultrastructure

项目概要/摘要 细胞器形态学专门用于复杂组织中发现的高度适应功能。线粒体 超微结构被精细地调整到代谢和生理状态。异常形态是 神经系统疾病、心脏病和癌症。随着cryo-EM的"分辨率革命"， 对膜蛋白的结构和调节有了新的认识，但我们对膜蛋白的认识， 细胞器结构落后。这是由于细胞器的多形性，以及 将特定的形态学特征赋予蛋白质状态。我实验室的首要目标是了解， 在机械水平上，蛋白质构象变化如何受到亚细胞环境的影响，膜如何 超微结构受蛋白质因子调节，这些因子在生理学和免疫学中的功能相互作用， 疾病在接下来的五年里，我的团队将开发一个技术平台，结合电子冷冻， 显微镜（cryo-EM）和生物物理方法来研究线粒体超微结构及其调节。我们将 应用我们最近开发的体外重建系统， 脂质体和双层通过单颗粒cryo-EM。我们将使我们新建立的电子冷冻- 断层扫描（cryo-ET）管道，用于膜特性的计算分析，以了解其 依赖于蛋白质间的相互作用。我们将开发新的结构和生物物理方法， 表征细胞器脂质异质性。最后，我们将探索天然蛋白质复合物的组装， contexts.这些方法将共同推进对蛋白质构象状态的机理理解 帮助我们确定细胞器形状的基本决定因素。我们广泛关注的问题 膜的间距、组成和曲率。我们将开发针对这些问题的工具 使用线粒体作为试验床，探索调节细胞凋亡的候选因子的结构和功能。 线粒体膜形态，其在神经变性病症中起因果作用。Opa1是 显性视神经萎缩中内膜融合因子和嵴重塑因子突变。SLC25A46是一种外- 溶质转运蛋白家族的膜成员，在协调脂质体内平衡中起重要作用 利氏综合症MICOS是嵴连接的稳定剂和调节剂（ 线粒体内膜褶皱），其丧失导致早发性致命性线粒体脑病， 肝脏疾病该项目的直接影响包括共享新模型以了解线粒体形状 与细胞生物学家合作，为药理学家提供新的，高度特异性的构象靶点，用于治疗 发育，并为生理学家提供了解组织特化的基本规则。的 长期目标是建立一个可扩展的方法推广到其他细胞器，并为 从第一性原理合理控制细胞器形态。

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.