Tools to probe the biophysical properties of cells

探测细胞生物物理特性的工具

• 批准号: 10375407
• 负责人: Liam J Holt
• 金额: $ 50.85万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10375407
• 关键词: Actins; Adipocytes; Adult; Affect; Aging; Animals; Archaea; Bicycling; Biochemical Reaction; Biological; Biology; Caenorhabditis elegans; Caliber; Cell Culture Techniques; Cell Nucleus; Cell physiology; Cells; Collaborations; Communities; Crowding; Cytoplasm; Data; Data Set; Development; Disease; Environment; Fluorescent Probes; Funding; Future; Generations; Genes; Genetic; Genetic Screening; Gold; Homeostasis; Investigation; Length; Light; Malignant Neoplasms; Mammalian Cell; Membrane; Methods; Microinjections; Mitochondria; Mitochondrial Matrix; Modeling; Molecular; Motion; Nature; Nerve Degeneration; Optics; Organelles; Organism; Organizational Efficiency; Particle Size; Partner in relationship; Pathway interactions; Pattern; Phase; Physical condensation; Physiological; Plants; Process; Property; Reaction; Regulation; Reporting; Reproducibility; Research; Role; Saccharomyces cerevisiae; Scientist; Signal Transduction; Speed; Stress; Structure; System; Techniques; Technology; Time; Tissues; Tracer; Yeasts; animal tissue; biological systems; biophysical properties; bone cell; computerized tools; design; experience; experimental study; improved; nanoparticle; nanoscale; physical property; physical state; polymerization; pressure; promoter; scaffold; success; tool; tumor progression

Abstract The physical properties of the cell interior are crucial for the organization and efficiency of biochemical reactions. The biophysical properties of cells can change during development, cancer progression and aging. Thus, it is crucial to understand the mechanisms that control the physical properties of the cell and the physiological consequences of perturbations to this unique environment. The current gold-standard method to study properties of the cell interior is the microinjection of inorganic nanoparticles. This technique dilutes the cytoplasm, damages the membrane and cortex, and is highly prone to experimental error. Microinjection is impossible in key genetic systems such as S. cerevisiae and has been mostly limited to cell culture models due to the difficulty of applying current techniques to animals. Studies of organelles have been almost impossible, limiting our current understanding to the cytoplasm. Finally, microinjection is labor intensive, making it impossible to undertake large- scale genetic screens to find genes and pathways that control the properties of the cell interior. We have created self-assembling, genetically-encoded fluorescent probes (GEMs) with 20- and 40-nm diameters that overcome all of the problems of the previous state-of-the-art technologies. After inserting the gene encoding GEMs, cells have nanoparticles permanently present, thus no microinjection is required. GEMs massively increase the speed, efficiency and reproducibility of microrheology experiments. Our recent discovery of pathways that control the physical properties of the cytoplasm required hundreds of experiments in an extensive genetic screen, which would not have been feasible without GEMs. We will use this focused technology research funding to extend the GEM technology and make it accessible to a broad community of scientists. In Aim 1, we will target GEMs to the nucleus and mitochondria, to characterize these organelles for the first time. In Aim 2, we will generate nanoparticles from 50 nm to 100 nm in size. The cellular environment varies substantially for objects of different sizes, just as car and a bicycle experience a traffic jam differently. Thus, we must investigate the environment for a wide range of particle sizes. Finally, in Aim 3, we will extend GEM technology to animals with well-defined developmental patterns to enable characterization of the physical properties of cells within tissues throughout development. Throughout, we will develop computational tools to identify and track GEMs, compare our technology to current gold-standard techniques and generate reference datasets that will aid the community in future studies. Overall, we will develop a suite of easy-to-use nanoparticles that will accelerate the discovery of mechanisms that control the physical properties of animals, cells and organelles. This will help elucidate the role of the intracellular environment to cell function, and the contributions of the loss of this physical homeostasis to disease.

抽象的 细胞内部的物理特性对于生化反应的组织和效率至关重要。 细胞的生物物理特性在发育，癌症进展和衰老过程中可能会发生变化。因此，是 了解控制细胞和生理的物理特性的机制至关重要 扰动对这个独特环境的后果。当前研究特性的金标准方法 细胞内部是无机纳米颗粒的显微注射。该技术稀释细胞质，损害 膜和皮层，并且高度容易出现实验误差。在关键遗传中不可能微分注射 酿酒酵母等系统，由于难以应用 当前对动物的技术。细胞器的研究几乎是不可能的，限制了我们的当前 了解细胞质。最后，显微注射是劳动密集型的，使得无法进行大规模的工作 缩放遗传筛选以找到控制细胞内部特性的基因和途径。 我们创建了自组装，遗传编码的荧光探针（GEM），具有20 nm和40 nm 克服了先前最新技术的所有问题的直径。插入基因后 编码宝石，细胞具有永久存在的纳米颗粒，因此不需要显微注射。宝石 大大提高微流变实验的速度，效率和可重复性。我们最近的发现 控制细胞质物理特性的途径需要数百个实验 没有宝石，广泛的遗传筛选是不可行的。我们将使用这种集中的技术 研究资金扩展了宝石技术，并使广泛的科学家社区可以使用。在 AIM 1，我们将将宝石靶向细胞核和线粒体，以首次表征这些细胞器。 在AIM 2中，我们将在50 nm到100 nm的大小中生成纳米颗粒。细胞环境有所不同 对于不同尺寸的物体而言，这基本上是汽车和自行车经历交通拥堵的方式。因此，我们 必须研究各种粒径的环境。最后，在AIM 3中，我们将扩展宝石 针对具有明确发育模式的动物的技术，以实现物理表征 整个发育过程中组织内细胞的特性。在整个过程中，我们将开发计算工具 识别和跟踪宝石，将我们的技术与当前的金标准技术进行比较并生成参考 将在未来的研究中帮助社区的数据集。总体而言，我们将开发一套易于使用的纳米颗粒 这将加速发现控制动物，细胞和 细胞器。这将有助于阐明细胞内环境对细胞功能的作用，并贡献 失去这种身体稳态的疾病。