Measurements and Models of Cell Nuclear Mechanics

细胞受到多种类型的机械应力，从施加在皮肤上的力到血管内的流体剪切。由于这些力可以传递到容纳基因组的细胞核，因此适应和消散机械应力的机制对于细胞存活是必要的，特别是在细胞核本身内。重要的是，细胞核机械特性的缺陷会在正常细胞过程中损害细胞存活，如细胞迁移，有时会导致疾病。此外，细胞的机械环境由其所在的组织限定，并且是细胞和组织发育的关键决定因素。因此，了解细胞的外部机械环境如何与细胞核沟通是一个重要的挑战，细胞命运在很大程度上决定于细胞核。此外，细胞核的机械特性必须与其组织环境相适应，这一过程目前还知之甚少。该项目将通过结合遗传模型生物，活细胞成像和生物物理工具的跨学科方法来解决这些具有挑战性的问题。此外，受资助的研究生将接受培训，成为下一代研究人员和教育工作者，他们既擅长定量方法，又具备解决尖端生物问题的生物学复杂性。 受资助的研究生将参加耶鲁大学物理与工程生物学综合研究生课程（IGPPEB），PI将担任执行委员会成员，指导学生并教授课程。IGPPEB提供沟通技巧、外联活动和教学方面的培训。还将为高中生提供暑期研究机会。 该项目将测试和进一步开发细胞核力学的物理模型，结合新的活细胞成像和力谱工具，能够在生物相关的时间，空间和力尺度上探测细胞核的机械特性，利用遗传模型系统裂变酵母（裂殖酵母）。首先，它将阐明改变异染色质-常染色质平衡如何改变细胞核的机制。其次，它将实现生物传感器，直接测量野生型细胞中活酵母细胞以及具有扰动核力学和/或染色质状态的细胞中染色质-内核膜蛋白质界面上的张力。第三，它将利用一种新的光镊试验，能够在活酵母细胞中对细胞核施加校准的力，从而能够在体内测量细胞核的粘弹性和染色质流动。通过这些实验，收集到的新信息将被反馈到一个正在发展的核力学数学模型中，最终导致对定义原子核力学的机制的全面了解。最后，该项目将测试开发的模型解释核起泡的能力，核起泡可以导致转化的哺乳动物细胞核完整性的丧失。

Cells are subject to numerous types of mechanical stress, from forces exerted on the skin to fluid shear within blood vessels. Because these forces can be transmitted to the cell nucleus, which houses the genome, mechanisms to adapt to and dissipate mechanical stress are necessary for cell survival, particularly within the nucleus itself. Importantly, defects in the mechanical properties of the nucleus can compromise cell survival during normal cellular processes, like cell migration, sometimes leading to disease. In addition, a cell's mechanical environment is defined by the tissue in which it resides, and is a key determinant of cell and tissue development. Therefore, it is an essential challenge to understand how a cell's external mechanical environment is communicated to the nucleus, where cell fate is largely determined. Moreover, the mechanical properties of the nucleus must be tuned to its tissue environment, a process that is poorly understood. This project will address these challenging questions through an interdisciplinary approach combining a genetic model organism, live-cell imaging, and biophysical tools. In addition, the supported graduate students will be trained to become the next generation of researchers and educators, who both excel at quantitative approaches and possess the biological sophistication to tackle cutting-edge biological problems. Supported graduate students will participate in Yale's Integrated Graduate Program in Physical and Engineering Biology (IGPPEB), for which the PIs serve on the executive committee, mentor students, and teach program courses. IGPPEB provides training in communication skills, outreach activities, and teaching. Summer research opportunities will also be provided to high school students. This project will test and further develop a physical model for cell nuclear mechanics by combining novel live-cell imaging and force-spectroscopy tools capable of probing the mechanical properties of nuclei at biologically-relevant temporal, spatial and force scales, taking advantage of the genetic model system fission yeast (Schizosaccharomyces pombe). First, it will elucidate how changing the heterochromatin-euchromatin balance can alter the mechanics of the nucleus. Second, it will implement biosensors that directly measure the tension on the chromatin-inner nuclear membrane protein interface in living yeast cells in wild-type cells as well as cells with perturbed nuclear mechanics and/or chromatin states. Third, it will exploit a novel optical tweezers assay capable of applying calibrated force to nuclei in living yeast cells, thereby enabling measurements of nuclear viscoelasticity and chromatin flow in vivo. Throughout these experiments, the new information gleaned will be fed back in to a developing mathematical model of nuclear mechanics, ultimately leading to a comprehensive picture of the mechanisms that define the mechanics of nuclei. Finally, this project will test the ability of the models developed to explain nuclear blebbing, which can drive loss of nuclear integrity in transformed mammalian cells.