Substrate Rigidity and Gene Expression: Role of Nuclear Tension

描述(申请人提供)：使用固体支架，为细胞提供正确的机械和化学信号，是指导组织修复、促进组织-支架整合和实现足够的新生血管的一种有前途的方法。越来越清楚的是，调节支架的刚性是控制细胞功能的一种有效方法，但如何 支架刚性调控基因表达的机制还不是很清楚。这一建议的重点是底物的机械性能控制基因表达的分子机制。我们建议检验底物刚性通过调节核张力来控制基因表达的假设。这一假说得到了我们的初步结果的有力支持：我们发现，底物刚性通过细胞骨架作用力的调节显著改变了核的形状。我们还证实，细胞骨架向核表面的力传递是通过核膜嵌入LINC(核骨架到细胞骨架的连接物)复合蛋白来实现的。我们的方法是1)确定哪些基因是以底物刚性依赖的方式打开或关闭的，2)检查基因刚性控制所需的LINC复合蛋白的程度，以及3)表征核形状刚性调节控制核内染色质结构、基因的空间位置和共同调节基因表达的表观遗传修饰的机制。目标1：验证底物刚性以LINC复合体依赖的方式控制基因表达的假设。目的2：研究核张力调控基因表达的机制。这些目标的成功实现将在细胞-生物材料相互作用、核和细胞力学以及基因调控的分子和细胞生物学等领域产生广泛的影响。总体而言，这项工作对工程和科学学科都有很大的兴趣。该项目融合了三位合作者(Lele、Nickerson和Roux)的专业知识，他们来自非常不同的背景(生物工程、分子生物学、细胞生物学)。具有如此不同背景的调查人员之间的互动预计将在拟议的问题领域产生新的和非常重要的发现。每名研究人员都将在分子生物学、细胞和分子成像、生物材料以及细胞和核力学领域贡献创新的尖端技术。这些目标的完成将增强我们对支架性质如何引导血管细胞的理解。因此，我们期待它们将促进许多组织工程应用的改进支架的发展。

DESCRIPTION (provided by applicant): The use of solid scaffolds that provide the correct mechanical and chemical cues to cells is a promising approach for guiding tissue repair, promoting tissue-scaffold integration and achieving adequate neovascularization. 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. The focus of this proposal is on the molecular mechanisms by which gene expression is controlled by the mechanical properties of the substrate. We propose to test the hypothesis that substrate rigidity controls gene expression by tuning nuclear tension. Strong support for this hypothesis comes from our preliminary results: we have found that substrate rigidity significantly alters nuclear shape through the modulation of cytoskeletal forces. We have also established that cytoskeletal force transfer to the nuclear surface is mediated by nuclear membrane embedded LINC (for linker of nucleoskeleton to cytoskeleton) complex proteins. Our approach is to 1) determine which genes are turned on or off in a substrate rigidity dependent manner, 2) examine the extent to which LINC complex proteins are required for rigidity control of genes, and 3) characterize the mechanisms by which rigidity modulation of nuclear shape controls intra-nuclear chromatin structure, spatial location of genes and epigenetic modifications that collectively regulate gene expression. Two specific aims are proposed: Aim 1: To test the hypothesis that substrate rigidity controls gene expression in a LINC complex dependent manner. Aim 2: To characterize the mechanisms by which nuclear tension regulates the expression of genes. The successful completion of these aims will have broad-ranging impact, in fields as diverse as cell-biomaterial interactions, nuclear and cell mechanics and molecular and cell biology of gene regulation. Collectively, this work is of strong interest to both engineering and scientific disciplines. The project integrates the expertise of three collaborators (Lele, Nickerson and Roux) from very different backgrounds (bioengineering, molecular biology, cell biology). The interaction between investigators of such varied background is expected to result in new and highly significant discoveries in the proposed problem area. Each investigator will contribute innovative, cutting-edge techniques in the fields of molecular biology, cell and molecular imaging, biomaterials and cell and nuclear mechanics. The completion of these aims will enhance our understanding of how scaffold properties direct vascular cells. As a result, we expect that they will promote the development of improved scaffolds for many tissue engineering applications.