Live Cell Interferometry for Quantifying Mass Transport in Cancer

用于量化癌症质量传递的活细胞干涉测量法

基本信息

批准号：
8242477
负责人：
Thomas Andrew Zangle
金额：
$ 11.8万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-18 至 2015-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8242477
关键词：
A549 Actins Acute Myelocytic Leukemia Address Affect Biochemical Biological Biological Models Cancer Biology Cancer Model Carrier Proteins Cell Nucleus Cell physiology Cells Cellular biology Chemicals Complement Cytoplasm Cytoskeleton Data Dimensions Discipline Doctor of Philosophy Drug Delivery Systems Effectiveness Electronics Engineering Fluorescence Fluorescence Microscopy Goals HOXA9 gene Harvest Hela Cells Hematopoietic Human Human Cell Line Immunodeficiency and Cancer Individual Interdisciplinary Study Interferometry Investigation Life Link Location MEIS1 gene Malignant Neoplasms Measurement Mechanics Membrane Mentors Microscopy Microtubules Modeling Molecular Molecular Biology Motion Movement Multiple Myeloma Mus Mutation N-WASP protein Optics Pathology Pharmacotherapy Phase Physics Population Positioning Attribute Pre-Clinical Model Protein Family Protein translocation Proteins RNA Regulation Research Research Personnel Research Project Grants Resolution Signal Pathway Signal Transduction Specificity Stimulus Structure System Techniques Therapeutic Therapeutic Agents Time Training Training Programs Whole Organism Wiskott-Aldrich Syndrome base cancer cell cancer therapy career cellular engineering design imaging modality improved inorganic phosphate insight leukemia link protein macromolecule molecular assembly/self assembly mouse model nanoparticle novel strategies novel therapeutics polymerization pre-clinical programs protein transport response retroviral transduction technology development therapeutic target trafficking translational medicine

项目摘要

DESCRIPTION (provided by applicant): Mauro Ferrari (1) describes cancer as a multi-scale transport problem inside cells and notes that, "the physics of transport within the cytoplasm, such as trafficking to different subcellular locations...requires much deeper understanding." Towards this end, new techniques are needed to study mass transport in single cells in order to 1- answer fundamental questions about the effects of cancer on transport within the cytoplasm, and 2- design novel therapeutic agents to attack altered mass transport mechanisms within cancer cells. In order to address the physics of transport within the cytoplasm, I propose to fully develop a novel approach that enables the analysis of mass transport through the cytoplasm linked to the trafficking of specifically tagged proteins. Our live cell interferometry system already tracks single cell mass changes in real time for hundreds of cells simultaneously with sub-cellular resolution. I will add simultaneous high-resolution fluorescence capabilities to link mass transport to the transport of specific cellular components. This will establish time as an essential 4th dimension for mass transport studies beyond traditional static snap-shot based approaches. I will apply this new approach to studies of mass transport and linked protein translocation in HeLa, HEK293T, and A549 human cell lines first as a model for system optimization and to establish utility. In the project second phase, I will use the live cell interferometer to study mass transport in a mouse primary model of human acute myeloid leukemia (AML) to provide translational relevance for this approach in cancer. This will include silencing hairpin RNA (shRNA) knockdown and eGFP-tagged studies of the Wiskott-Aldrich Syndrome proteins WASP and N-WASP, which are known components of the cellular transport machinery that traffic widely and are involved in regulation of the actin cytoskeleton network inside the cytoplasm and nucleus, respectively. My goal is to understand how cytoskeleton regulatory components affect, and are affected by, the transport of mass within cells under native and altered environmental conditions, far beyond standard studies that employ non-localized actin or microtubule dissociating agents. Most importantly, I will be immersed in a focused, comprehensive mentored training program with an emphasis on cancer cell biology, technology development, and applications. This program will include coursework and seminars in cancer and cell biology and formal mentoring, by individuals and a mentoring committee, from leading experts in cancer/cell biology and engineering disciplines in optics/microscopy, electronics and fluidics. These rigorous training components complement my extensive classroom background in molecular biology and engineering acquired during my Ph.D. studies at Stanford. Together with a focused, interdisciplinary research project, this structured training and mentored program will help propel me into an early career independent investigator position in cancer cell biology with powerful applications in targeted technology development for fundamental investigations in translational medicine.

描述（由申请人提供）：Mauro Ferrari (1) 将癌症描述为细胞内的多尺度运输问题，并指出，“细胞质内运输的物理原理，例如运输到不同的亚细胞位置......需要更深入的理解”。为此，需要新技术来研究单细胞中的质量运输，以便 1- 回答有关癌症对细胞质内运输影响的基本问题，2- 设计新型治疗剂来攻击癌细胞内改变的质量运输机制。为了解决细胞质内运输的物理问题，我建议充分开发一种新方法，能够分析与特定标记蛋白质运输相关的通过细胞质的质量运输。我们的活细胞干涉测量系统已经能够以亚细胞分辨率同时实时跟踪数百个细胞的单细胞质量变化。我将添加同步高分辨率荧光功能，将质量运输与特定细胞成分的运输联系起来。这将使时间成为超越传统静态快照方法的大众运输研究的重要第四维度。我将首先将这种新方法应用于 HeLa、HEK293T 和 A549 人类细胞系中的传质和连接蛋白易位研究，作为系统优化和建立实用性的模型。在项目第二阶段，我将使用活细胞干涉仪研究人类急性髓系白血病 (AML) 小鼠原代模型中的质量传递，为这种癌症方法提供转化相关性。这将包括对 Wiskott-Aldrich 综合征蛋白 WASP 和 N-WASP 进行沉默发夹 RNA (shRNA) 敲低和 eGFP 标记研究，这些蛋白是细胞运输机制的已知组成部分，广泛运输并参与肌动蛋白细胞骨架网络的调节分别位于细胞质和细胞核内。我的目标是了解细胞骨架调节成分如何影响细胞内质量在天然和改变的环境条件下的运输以及受其影响，这远远超出了使用非局部肌动蛋白或微管解离剂的标准研究。最重要的是，我将沉浸在一个重点突出、全面的指导培训项目中，重点是癌细胞生物学、技术开发和应用。该计划将包括癌症和细胞生物学方面的课程和研讨会，以及由个人和指导委员会提供的正式指导，指导委员会来自癌症/细胞生物学和光学/显微镜、电子和流体学领域的工程学科的领先专家。这些严格的培训内容补充了我在攻读博士学位期间获得的分子生物学和工程学方面广泛的课堂背景。在斯坦福大学学习。与一个重点突出的跨学科研究项目相结合，这个结构化的培训和指导计划将帮助我进入癌细胞生物学领域的早期职业独立研究员职位，并在转化医学基础研究的靶向技术开发中发挥强大的应用作用。