SUBSTRATE RIGIDITY AND GENE EXPRESSION: Role of Nuclear Tension

项目总结/摘要 使用固体支架为细胞提供机械和化学线索是一种有前途的方法， 引导组织修复和促进组织-支架整合。越来越明显的是， 支架刚性是控制细胞功能的有力途径，但支架刚性如何调节基因表达， 不太了解。以前，我们测试了底物刚性控制基因表达的假设， 调整核张力我们利用了LINC（linker of nucleotiron-to-cytoskeleton） 复合物是细胞核与细胞骨架的已知分子连接体，并询问它如何调节敏感性 的基因组范围内的转录基板刚性。我们的结果首次表明LINC复合物 促进整个基因组的表达的机械调节。结合肌球蛋白抑制研究，我们 能够识别出依赖于核张力的机械敏感性基因。在这一延续中，我们 提出确定这些高度新颖的发现的分子机制。提出了两个具体目标： 1：确定刚性介导的基因表达控制所需的核分子接头。目标2： 确定核-细胞骨架连接调节基因机械敏感性的机制。 科学上，这项工作解决了重要的和长期存在的问题的机制， 细胞微环境控制基因表达。临床上，这项工作的长期影响是促进 合理开发具有组织工程和修复所需机械性能的新型生物材料。 另一个好处是开发了一种综合方法，使用工程中的两种技术 以及来自分子细胞生物学的技术，用于解决刚性传感中的基本问题。这项工作 是不同领域的基本兴趣，包括细胞生物材料相互作用，核和细胞力学， 基因调控的分子和细胞生物学。该项目建立了乐乐集团之间的合作 （佛罗里达大学）、尼克森（马萨诸塞州大学医学院）和鲁克斯（桑福德研究所）。 每个实验室都为这项研究带来了独特的资源，包括使用复杂的 光学和电子显微镜，湿台细胞和分子生物学实验的强大专业知识 特别是与细胞核相关的，以及细胞和核机械传感和生物材料方面的专业知识 发展和表征。该团队还将受益于知名分子和细胞的支持， 生物学家和生物工程师在LINC的广泛领域开创了实验技术 复杂生物学、核/染色质结构和功能以及组织生物力学。

PROJECT SUMMARY/ABSTRACT The use of solid scaffolds that provide cells with mechanical and chemical cues is a promising approach for guiding tissue repair and promoting tissue-scaffold integration. It is becoming increasingly clear that tuning scaffold rigidity is a powerful way to control cell function, but how scaffold rigidity regulates gene expression is not well-understood. Previously, we tested the hypothesis that substrate rigidity controls gene expression by tuning nuclear tension. We took advantage of the fact that the LINC (linker of nucleoskeleton-to-cytoskeleton) complex is a known molecular linker of the nucleus to the cytoskeleton, and asked how it regulates the sensitivity of genome-wide transcription to substrate rigidity. Our results were the first to show that the LINC complex facilitates mechano-regulation of expression across the genome. Combined with myosin inhibition studies, we were able to identify genes that depend on nuclear tension for their mechanosensitivity. In this continuation, we propose to identify molecular mechanisms for these highly novel findings. Two specific aims are proposed: Aim 1: To identify the nuclear molecular linkers necessary for rigidity-mediated control of gene expression. Aim 2: To determine the mechanisms by which nuclear-cytoskeletal linkage regulates gene mechanosensitivity. Scientifically, this work addresses important and longstanding questions about the mechanisms by which the cell microenvironment controls gene expression. Clinically, the long-term impact of this work is to promote the rational development of new biomaterials with mechanical properties tuned for tissue engineering and repair. An additional benefit is the development of an integrated approach using both technologies from engineering and techniques from molecular cell biology for addressing a fundamental question in rigidity sensing. This work is of fundamental interest to diverse fields including cell-biomaterial interactions, nuclear and cell mechanics and molecular and cell biology of gene regulation. The project builds collaboration between the groups of Lele (University of Florida), Nickerson (University of Massachusetts Medical School) and Roux (Sanford Research). Each laboratory brings unique resources to this research including access and expertise in using sophisticated optical and electron microscopes, strong expertise with wet-bench cell and molecular biology experiments specifically related to the nucleus, and expertise in cell and nuclear mechanosensing and biomaterial development and characterization. The team will also benefit from the support of well-known molecular and cell biologists and bioengineers who have pioneered experimental techniques in a broad number of areas in LINC complex biology, nuclear/chromatin structure and function and tissue biomechanics.