Substrate Rigidity and Gene Expression: Role of Nuclear Tension

描述（由申请人提供）：使用固体支架为细胞提供正确的机械和化学线索，是引导组织修复、促进组织-支架整合和实现充分新血管形成的有前景的方法。越来越清楚的是，调整支架刚度是控制细胞功能的一种有效方法，但如何做到这一点呢？ 支架刚性调节基因表达还不是很清楚。该提案的重点是基因表达由基质的机械性质控制的分子机制。我们建议测试的假设，基板刚性控制基因表达的调节核张力。这一假设的强有力的支持来自我们的初步结果：我们已经发现，基板刚度显着改变核形状，通过调节细胞骨架的力量。我们还建立了细胞骨架力转移到核表面介导的核膜嵌入LINC（连接器的核骨架细胞骨架）复合物蛋白。我们的方法是1）确定哪些基因以底物刚性依赖性方式打开或关闭，2）检查LINC复合物蛋白对于基因的刚性控制所需的程度，以及3）表征核形状的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。提出了两个具体的目标：目标1：为了测试的假设，底物刚性控制基因表达的LINC复杂依赖的方式。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。总的来说，这项工作对工程和科学学科都有浓厚的兴趣。该项目整合了三位来自不同背景（生物工程、分子生物学、细胞生物学）的合作者（Lele、Nickerson和Roux）的专业知识。具有不同背景的研究人员之间的互动有望在拟议的问题领域产生新的和非常重要的发现。每位研究人员将在分子生物学、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特性如何指导血管细胞的理解。因此，我们期望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.