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: 8910559
• 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/8910559
• 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; Health; 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; 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 是一种细胞间功能连接的新机制，可用于病毒传播、导致神经退行性疾病和癌症的错误折叠蛋白质聚集体。 TNT 已在多种细胞类型中发现，允许细胞质和膜结合分子、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。不幸的是，目前人们对TNT形成的基本机制、其结构成分、转运机制或信号通路知之甚少。 传统的蛋白质组学技术有望帮助阐明许多尚未解答的问题。但研究 TNT 的研究人员在使用传统技术时常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（通常约为 10,000 个细胞）。第二个是获得显着结果所需的样本高度同质性。事实上，使用 TNT 的困难在于这些 TNT 的短暂性。 结构。细胞并非在所有细胞中始终形成连接。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 的研究已被简化为“钓鱼”潜在的重要蛋白质，从而减缓了该领域的进展。幸运的是，最近的研究表明，TNT 可以通过蛋白质过度表达或在应激条件下诱导，并为克服 TNT 的瞬时性带来了希望。我们发现了一种名为 Myo10 的蛋白质，与对照细胞相比，它使 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。我们将使用微蛋白质组学技术（例如 MALDI-MS）分析这些富集的样品，该技术应该允许检测和比较实验条件之间的蛋白质表达差异。此外，与需要 10-50 µg 蛋白质的传统蛋白质组学技术不同，微蛋白质组学仅需要 1-2 µg 蛋白质即可进行分析。总的来说，虽然这个项目在技术上具有挑战性，并且需要耐心和故障排除来确定拟议分析的最佳条件，但我们相信有一个很好的机会结合这些新技术并开拓这个领域。此外，更好地表征 TNT 的形成和功能将提供重要的见解，并产生新的靶标，以改善针对朊病毒或其他蛋白质病以及 HIV-1 感染和癌症的神经保护。