Microproteomic analysis of laser capture microdissected cells forming TNTs

激光捕获显微切割细胞形成 TNT 的微蛋白质组学分析

• 批准号: 8741090
• 负责人: KARINE GOUSSET
• 金额: $ 14万
• 依托单位: CALIFORNIA STATE UNIVERSITY FRESNO
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-15 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8741090
• 关键词: Alzheimer' s Disease; Binding; Cell Count; Cells; Co-Immunoprecipitations; Data; Detection; Development; Down-Regulation; Fishes; Fluorescence Microscopy; Funding Opportunities; Future; Grant; HIV; HIV-1; Hydrogen Peroxide; Immunoprecipitation; In Vitro; Individual; Industry; Infection; Lasers; Lead; Life; Malignant Neoplasms; Mass Spectrum Analysis; Membrane; Mentors; Methods; Microdissection; Molecular Machines; Nanotubes; Nature; Neurodegenerative Disorders; Organelles; Population; Prions; Protein Analysis; Proteins; Proteomics; Protocols documentation; Relative (related person); Research; Research Personnel; Research Proposals; Resolution; Role; Sample Size; Sampling; Signal Pathway; Signal Transduction; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stress; Structure; Techniques; Technology; Time; Tissues; Training; Tubular formation; United States National Institutes of Health; Virus; Western Blotting; Work; base; cell type; improved; in vivo; insight; laser capture microdissection; mutant; neuroprotection; novel; pathogen; protein aggregate; protein expression; public health relevance; sample fixation; tool; vector control

DESCRIPTION (provided by applicant): With this project we propose to analyze small membrane protrusions know as tunneling nanotubes (TNTs) by exploring the limits of laser capture microdissection (LCM) and microproteomic techniques. Our research is focused on the characterization of TNTs, a novel mechanism for functional connectivity between cells, in the spreading of viruses, misfolded protein aggregates that lead to neurodegenerative diseases, and cancer. TNTs have been found in numerous cell types, allowing the transport of cytosolic and membrane-bound molecules, organelles and the spreading of pathogens. In vitro, these structures are heterogeneous and numerous disparities have emerged both in their structures and functions. Similar structures also exist in vivo and in tissue explants. Unfortunately, little s currently known about the basic mechanism of TNT formation, its structural components, transport mechanism or signaling pathways. Traditional proteomic techniques tantalizingly offer the potential to help elucidate many of these unanswered questions. But researchers studying TNTs have often faced two significant obstacles in using traditional techniques. The first is the substantial amounts of cells that are required for downstream MS analysis (usually on the order of 10,000 cells). The second is the high degree of sample homogeneity necessary to obtain significant results. In fact, the difficultly of working with TNTs is the transient nature of these structures. Cells do not form connections at all times, in all cells. Since researchers in the past have found no help in overcoming these obstacles, research on TNTs has been reduced to "fishing" for potentially significant proteins, slowing progress in the field. Fortunately, recent studies have demonstrated that TNTs can be induced by protein over-expression or under stress conditions, and have offered hope in overcoming the transient nature of TNTs. We identified a protein, Myo10, that increases the relative percentage of cells connected by TNTs by over 50% compared to control cells. This reproducible manner of TNT induction finally offers us a tool to elucidate the proteins necessary for TNT function and formation. Here, we plan to use this protein to induce TNT formation and then further enrich sample populations using LCM. We will analyze these enriched samples using microproteomic techniques such as MALDI-MS which should permit the detection and comparison of protein expression differences between experimental conditions. Also, unlike traditional proteomic technologies, that require 10-50 ?g of protein, microproteomics requires only 1-2 ?g of protein for analysis. Overall, while this project will be technically challenging and will require patience and troubleshooting to identify the best conditions for the proposed analyses, we believe that there is a great opportunity to combine these new techniques and open up the field. Furthermore, a better characterization of both formation and function of TNTs will provide important insights and lead to novel targets to improve neuroprotection against prion or other proteopathies as well as against HIV-1 infection and cancer.

描述（由申请人提供）：在这个项目中，我们建议通过探索激光捕获显微切割（LCM）和微量蛋白质组学技术的局限性来分析被称为隧道纳米管（TNT）的小膜突起。 我们的研究重点是TNT的表征，TNT是细胞之间功能连接的一种新机制，在病毒传播，导致神经退行性疾病和癌症的错误折叠蛋白质聚集体中。TNTs存在于许多细胞类型中，允许胞质和膜结合分子、细胞器的运输和病原体的传播。在体外，这些结构是异质的，并且在它们的结构和功能上都出现了许多差异。类似的结构也存在于体内和组织外植体中。然而，目前对TNT形成的基本机制、结构组成、转运机制和信号通路等方面的研究还很少。 传统的蛋白质组学技术提供了诱人的潜力，以帮助阐明许多这些悬而未决的问题。但是研究TNT的研究人员在使用传统技术时经常面临两个重大障碍。第一个是下游MS分析所需的大量细胞（通常在10，000个细胞的数量级上）。第二是获得显著结果所需的高度样品均匀性。事实上，与TNT合作的困难在于这些TNT的短暂性。 结构.细胞并不总是在所有的细胞中形成连接。由于过去的研究人员 由于没有发现克服这些障碍的帮助，对TNT的研究已经减少到“捕捞”潜在的重要蛋白质，减缓了该领域的进展。幸运的是，最近的研究表明，TNTs可以通过蛋白质过度表达或在应激条件下诱导，并为克服TNTs的瞬时性提供了希望。我们发现了一种名为Myo 10的蛋白质，与对照细胞相比，该蛋白质使通过TNT连接的细胞的相对百分比增加了50%以上。这种可重复的TNT诱导方式最终为我们提供了一种工具来阐明TNT功能和形成所必需的蛋白质。 在这里，我们计划使用这种蛋白质诱导TNT形成，然后使用LCM进一步富集样品群体。我们将使用微量蛋白质组学技术（如MALDI-MS）分析这些富集的样品，该技术应允许检测和比较实验条件之间的蛋白质表达差异。此外，与传统的蛋白质组学技术不同，这需要10-50？g蛋白质，微蛋白质组学只需要1-2？g蛋白质用于分析。总的来说，虽然该项目在技术上具有挑战性，需要耐心和故障排除，以确定拟议分析的最佳条件，但我们相信，将这些新技术结合起来并开拓该领域是一个很好的机会。此外，更好地表征TNT的形成和功能将提供重要的见解，并导致新的靶点，以改善对朊病毒或其他蛋白质病以及对HIV-1感染和癌症的神经保护。