Tools to probe the biophysical properties of cells

摘要 细胞内部的物理性质对于生物化学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会发生变化。照经上所 这对于理解控制细胞物理特性和生理特性的机制至关重要。 这是对这一独特环境的干扰。当前研究性质的金标准方法 是无机纳米粒子的显微注射。这项技术稀释了细胞质， 膜和皮层，并且非常容易出现实验错误。显微注射在关键遗传学中是不可能的 系统，如S。酿酒酵母，并且由于应用困难，主要限于细胞培养模型 目前的动物技术。对细胞器的研究几乎是不可能的，限制了我们目前的研究。 对细胞质的理解。最后，显微注射是劳动密集型的，使得不可能进行大规模的- 大规模的基因筛选，以找到控制细胞内部特性的基因和途径。 我们已经创建了自组装，基因编码的荧光探针（GEM），20和40纳米 这些直径克服了现有技术的所有问题。在插入基因后 编码GEM的细胞具有永久存在的纳米颗粒，因此不需要显微注射。宝石 大大提高了微观流变学实验的速度、效率和再现性。我们最近的发现 控制细胞质物理性质的途径需要数百个实验， 广泛的遗传筛查，如果没有基因工程，这是不可能的。我们将使用这种聚焦技术 提供研究资金，以扩大GEM技术，使其为广大科学家所利用。在 目的1，我们将GEM定位于细胞核和线粒体，首次对这些细胞器进行表征。 在目标2中，我们将生成尺寸为50 nm至100 nm的纳米颗粒。细胞环境变化 基本上对于不同尺寸的物体，就像汽车和自行车经历不同的交通堵塞一样。因此我们 必须调查环境中各种粒径的颗粒。最后，在目标3中，我们将扩展GEM 技术的动物具有明确的发展模式，使表征的物理 在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GEM，将我们的技术与当前的黄金标准技术进行比较，并生成参考 这些数据集将有助于社区未来的研究。总的来说，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，细胞和动物的物理特性的机制。 细胞器这将有助于阐明细胞内环境对细胞功能的作用， 这种身体内稳态的丧失。

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.