Technologies to Define and Map Novel Interorganelle Macromolecular Interactions

定义和绘制新型细胞器间大分子相互作用的技术

• 批准号: 8488980
• 负责人: NATALIE G. AHN
• 金额: $ 41.18万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8488980
• 关键词: Address; Apoptosis; Binding; Biochemical; Biochemical Genetics; Biochemical Process; Biochemistry; Biosensor; Calcium; Cell Polarity; Cell membrane; Cell physiology; Cells; Cellular biology; Charcot-Marie-Tooth Disease; Collaborations; Color; Communication; Complex; Endoplasmic Reticulum; Environment; Eukaryotic Cell; FarGo; Fluorescence Microscopy; Fluorescent Probes; Fractionation; Goals; Golgi Apparatus; Health; Homeostasis; Human; Image; Integral Membrane Protein; Interdisciplinary Study; Label; Life; Link; Macromolecular Complexes; Maps; Mass Spectrum Analysis; Mediating; Membrane; Membrane Proteins; Metabolism; Methods; Microscopy; Mitochondria; Morphology; Movement; Multiprotein Complexes; Mutation; Oranges; Organelles; Pathway interactions; Phospholipids; Population; Process; Production; Protein Engineering; Proteins; Proteomics; Reporter; Reporting; Research; Research Personnel; Resolution; Resources; Role; Secretory Vesicles; Site; Students; Supraoptic Vertical Ophthalmoplegia; System; Technology; Time; base; cell growth; cell type; cellular imaging; design; endoplasmic reticulum stress; human disease; imaging modality; innovation; late endosome; new technology; novel; novel marker; programs; protein complex; protein crosslink; protein profiling; public health relevance; red fluorescent protein; response; screening; single cell analysis; uptake

DESCRIPTION (provided by applicant): Interorganelle interactions are key processes controlling eukaryotic cell function, and dysregulation of these interactions has been implicated in many human diseases. However, relatively little is known about macromolecular complexes that mediate organelle interactions, due to obstacles that have been difficult to overcome. First, many relevant proteins are integral membrane proteins, which are hard to purify and maintain weak but physiologically important binding interactions. Capturing such interactions by conventional biochemical and genetic approaches is technically difficult. Second, simultaneously tracking transient organelle populations and interactions requires the ability to follow real time dynamics in living cells using multi-color fluorescent probes. However, many fluorescent proteins (FPs) used for live imaging are compromised by the oxidizing environment of many organelles, including ER, Golgi, and secretory vesicles. The goal of this proposal is to define protein complexes that define and modulate novel organelle subpopulations, using a combination of new technologies in mass spectrometry and fluorescent protein based probes for live cell imaging. Our Specific Aims are: (1) Identify candidate protein markers of novel organelles and interorganellar protein complexes. We will develop a proteomics strategy to profile proteins within organelle subpopulations that are dynamic and transient, as well as macromolecular complexes that bridge organelles. (2) Develop novel biosensors to track these protein markers in living cells, by time resolved imaging and high resolution microscopy. We will maximize the available colors of the fluorescent protein spectrum for use in multi-color live cell imaging studies, by solving key problems in fluorescent protein reporters caused by organellar environments that restrict their folding and function. (3) Apply these methods to cutting edge problems in cell biology, addressing mechanisms underlying (i) ER stress and Ca2+-mediated organelle remodeling, (ii) Zn2+ homeostasis, and (iii) cell polarity. We will combine technologies developed in Aims 1 and 2 to create a new experimental workflow which integrates mass spectrometry/proteomics, biosensor design, and high resolution fluorescence microscopy, and apply this to relevant problems in collaborator labs in Aim 3. Our proposal establishes a unique, multidisciplinary collaboration between a team of four investigators, who are leading experts in technologies of proteomics/mass spectrometry, protein engineering and biosensor design, and cutting edge methods for high resolution cell imaging. The combined expertise from these investigators gives us a unique opportunity to discover novel organelles and macromolecular complexes involved in interorganelle contacts, and define their cell biology.

描述（由申请人提供）：细胞器间相互作用是控制真核细胞功能的关键过程，这些相互作用的失调与许多人类疾病有关。然而，由于难以克服的障碍，对介导细胞器相互作用的大分子复合物的了解相对较少。首先，许多相关蛋白是完整的膜蛋白，难以纯化并维持微弱但生理上重要的结合相互作用。通过传统的生物化学和遗传方法捕捉这种相互作用在技术上是困难的。其次，同时跟踪瞬态细胞器种群和相互作用需要使用多色荧光探针跟踪活细胞中的实时动态。然而，许多用于实时成像的荧光蛋白（FPs）受到许多细胞器的氧化环境的损害，包括内质网、高尔基体和分泌囊泡。本提案的目标是定义定义和调节新的细胞器亚群的蛋白质复合物，使用新的技术在质谱和荧光蛋白为基础的探针活细胞成像的组合。我们的具体目标是：(1)鉴定新的细胞器和细胞器间蛋白复合物的候选蛋白质标记物。我们将开发一种蛋白质组学策略来分析动态和瞬时的细胞器亚群中的蛋白质，以及连接细胞器的大分子复合物。(2)开发新型生物传感器，通过时间分辨成像和高分辨率显微镜技术来跟踪活细胞中的这些蛋白质标记物。我们将通过解决由限制其折叠和功能的细胞器环境引起的荧光蛋白报告的关键问题，最大限度地提高荧光蛋白光谱的可用颜色，用于多色活细胞成像研究。(3)将这些方法应用于细胞生物学的前沿问题，解决以下机制：(i)内质网应激和Ca2+介导的细胞器重塑，（ii） Zn2+稳态，以及（iii）细胞极性。我们将结合Aims 1和2中开发的技术，创建一个新的实验工作流，该工作流集成了质谱/蛋白质组学，生物传感器设计和高分辨率荧光显微镜，并将其应用于Aim 3合作实验室的相关问题。我们的提案建立了一个独特的、多学科的合作，在一个由四名研究人员组成的团队之间，他们是蛋白质组学/质谱、蛋白质工程和生物传感器设计技术的领先专家，以及高分辨率细胞成像的前沿方法。这些研究人员的综合专业知识为我们提供了一个独特的机会来发现涉及细胞器间接触的新型细胞器和大分子复合物，并定义它们的细胞生物学。