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

基本信息

批准号：
8683197
负责人：
NATALIE G. AHN
金额：
$ 39.87万
依托单位：
UNIVERSITY OF COLORADO
依托单位国家：
美国
项目类别：
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/8683197
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

项目摘要

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）：企业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物，对介导细胞器相互作用的大分子复合物的了解很少。首先，许多相关蛋白是整合膜蛋白，它们很难纯化和维持弱但生理上重要的结合相互作用。在技术上，通过常规生化和遗传方法捕获这种相互作用很困难。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遵循活细胞实时动态的能力。但是，许多用于实时成像的荧光蛋白（FPS）因许多细胞器的氧化环境（包括ER，高尔基体和分泌囊泡）的氧化环境而损害。该建议的目的是使用质谱法和基于荧光蛋白的新技术的组合来定义定义和调节新型细胞器亚群的蛋白质复合物，以实时细胞成像。我们的具体目的是：（1）鉴定新的细胞器和生产组织蛋白质复合物的候选蛋白标志物。我们将制定一种蛋白质组学策略，以在动态和瞬态的细胞器亚群中以及桥接细胞器的大分子复合物中概述蛋白质。（2）通过时间分析成像和高分辨率显微镜，开发出新的生物传感器来跟踪活细胞中这些蛋白质标志物。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道器中的关键问题来最大化用于多色活细胞成像研究中的荧光蛋白光谱的可用颜色。（3）将这些方法应用于细胞生物学的最前沿问题，解决（i）ER应力和Ca2+介导的细胞器重塑，（ii）Zn2+稳态和（III）细胞极性。我们将结合在目标1和2中开发的技术，以创建一个新的实验工作流程，该工作流程整合质谱/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中的相关问题。我们的建议建立了一个独特的建议。，一个由四个研究人员组成的团队之间的多学科合作，他们是蛋白质组学/质谱技术，蛋白质工程和生物传感器设计的领先专家，以及用于高分辨率单元格成像的尖端方法。这些研究人员的联合专业知识为我们提供了一个独特的机会，可以发现与企业间接触涉及的新型细胞器和大分子复合物，并定义其细胞生物学。