Tools to probe the biophysical properties of cells

摘要 细胞内部的物理性质对生化反应的组织和效率至关重要。 细胞的生物物理特性会在发育、癌症进展和衰老过程中发生变化。因此，它是 对于理解控制细胞的物理属性和生理的机制至关重要 对这一独特环境的扰动的后果。当前研究性能的金标准方法 细胞内部的是无机纳米颗粒的微量注射。这项技术稀释了细胞质，损害了 膜和皮质，极易发生实验误差。显微注射在关键基因中是不可能的 系统，如酿酒酵母，由于应用的困难，大多局限于细胞培养模型 目前的技术对动物来说。细胞器的研究几乎是不可能的，限制了我们目前的 对细胞质的理解。最后，微量注射是劳动密集型的，因此不可能进行大剂量的注射。 扩大基因筛查的范围，以找到控制细胞内部属性的基因和途径。 我们已经创造了自组装的、遗传编码的荧光探针(GEM)，具有20和40 nm 克服了以前最先进技术的所有问题的直径。在插入基因后 编码宝石，细胞有纳米颗粒永久存在，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、效率和重复性。我们的最新发现 控制细胞质物理特性的途径需要数百次实验 广泛的基因筛查，如果没有宝石，这是不可能的。我们将使用这种聚焦技术 提供研究资金，以推广宝石技术，并使广大科学家社区能够接触到它。在……里面 目的1，我们将宝石定位于细胞核和线粒体，首次对这些细胞器进行特征描述。 在目标2中，我们将产生尺寸从50纳米到100纳米的纳米颗粒。蜂窝环境各不相同 基本上对于不同大小的物体，就像汽车和自行车经历不同的交通拥堵一样。因此，我们 必须调查各种颗粒大小的环境。最后，在目标3中，我们将扩展GEM 向具有明确发育模式的动物提供技术，使其能够表征身体 组织内细胞在整个发育过程中的性质。在整个过程中，我们将开发计算工具来 识别和跟踪宝石，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 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及其贡献 身体动态平衡的丧失是疾病造成的。

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.