Measurements and Models of Cell Nuclear Mechanics

细胞承受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，因此适应和消除机械应力的机制对于细胞生存是必要的，特别是在细胞核本身。重要的是，细胞核机械性能的缺陷可能会影响正常细胞过程中的细胞存活，比如细胞迁移，有时会导致疾病。此外，细胞的机械环境由其所在的组织定义，是细胞和组织发育的关键决定因素。因此，理解细胞的外部机械环境是如何与细胞核沟通的是一个重要的挑战，细胞核在很大程度上决定了细胞的命运。此外，细胞核的机械性能必须根据其组织环境进行调整，这一过程人们知之甚少。这个项目将通过结合遗传模型生物体、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，受资助的研究生将被培养成下一代研究人员和教育工作者，他们既擅长定量方法，又拥有解决尖端生物学问题的生物学尖端技术。被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计划(IGPPEB)，在该计划中，PI担任执行委员会成员，指导学生，并教授计划课程。IGPPEB提供沟通技能、外展活动和教学方面的培训。还将为高中生提供暑期研究机会。该项目将测试并进一步开发细胞核力学的物理模型，方法是将新的活细胞成像和力谱工具相结合，利用遗传模型系统分裂酵母(Schizosaccharmyces Pombe)，能够在生物相关的时间、空间和力尺度上探测细胞核的力学性质。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力学。其次，它将实施生物传感器，直接测量野生型细胞以及核力学和/或染色质状态受扰的细胞中活的酵母细胞染色质-核膜蛋白界面上的张力。第三，它将开发一种新的光镊法，能够对活的酵母细胞中的细胞核施加校准力，从而能够测量体内的核粘弹性和染色质流动。在这些实验中，收集到的新信息将被反馈到发展中的核力学数学模型中，最终导致定义原子核力学的机制的全面图景。最后，该项目将测试开发的解释核泡化的模型的能力，核泡化可能导致转化的哺乳动物细胞失去核完整性。

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.