Collaborative Research: Regulation of Cellular Mechanics by Crosslinked Actin Networks - Role of Palladin and Alpha-actinin

智力优势身体中的细胞具有非凡的能力来感知环境中的“硬度”-也就是说，细胞可以区分坚硬的基质（如骨骼）和柔软的基质（如大脑）。这种能力对于细胞功能的许多方面至关重要，例如迁移，伤口愈合以及组织和器官的正确形成。为了感知环境的机械特性，细胞通过重组其内部细胞骨架来调整其内部刚度，以匹配外部表面的刚度，内部细胞骨架由微观细丝的动态网络组成。这些细丝的主要成分是一种叫做肌动蛋白的蛋白质。各种肌动蛋白结合蛋白交联肌动蛋白丝并将它们组织成多丝束。Palladin是一种新发现的肌动蛋白交联蛋白，在组织这些细丝网络中起重要作用。先前的实验表明，palladin在胚胎发育中起着至关重要的作用：当palladin基因在小鼠中沉默时，它会导致致命的发育缺陷，其特征是细胞无法正确地粘附到基质上并适当地迁移。这些缺陷可能是因为细胞调节其内部硬度的能力在缺乏palladin的情况下受到损害。该提议将测试以下假设：palladin通过其交联肌动蛋白的能力及其与另一种肌动蛋白交联剂α-辅肌动蛋白的相互作用，决定肌动蛋白网络的结构和机械性质，并使细胞能够感知其物理环境。将采用两种方法来解决这一问题。首先，肌动蛋白网络组装在载玻片上的结构和机械性能将被测量，以阐明肌动蛋白交联的palladin有助于肌动蛋白组织。此外，palladin水平将在活细胞中进行遗传操作，以研究肌动蛋白组织，细胞刚度和力的产生如何影响细胞的机械敏感性。更广泛的影响这一合作提案将加强对细胞运动和组织形成的基本生物过程的理解。从物理学和细胞生物学的跨学科方法的研究生和本科生的培训将是工作的一个组成部分。作为该项目的一部分，将开发的细胞系将在发表后免费提供给其他研究人员。细胞力学的研究生课程将根据这一建议的概念框架开发。PI和co-PI还将鼓励少数民族学生以及该地区的高中生参与研究，作为马里兰州大学和北卡罗来纳州大学研究学徒计划的路易斯·斯托克斯少数民族参与联盟计划的一部分。作为物理系暑期女生方案的一部分，PI将组织为期一周的生物物理实验室演示，以鼓励女生参与科学和技术领域。

Intellectual Merit Cells in the body have a remarkable ability to sense "stiffness" in their environment - that is, a cell can distinguish between a hard substrate such as bone, and a softer substrate such as brain. This ability is critical for many aspects of cell function such as migration, wound healing and the proper formation of tissues and organs. In order to sense the mechanical properties of their environment, cells adjust their internal stiffness to match that of the external surface by reorganizing their internal cytoskeleton, which is made of a dynamic network of microscopic filaments. The main component of these filaments is a protein called actin. Various actin-binding proteins crosslink the actin filaments and organize them into multifilament bundles. Palladin is a newly identified actin cross-linking protein that is important in organizing these filament networks. Previous experiments have shown that palladin plays an essential role in embryonic development: when the palladin gene is silenced in mice, it results in lethal development defects that are characterized by a failure of cells to adhere properly to a substrate and to migrate appropriately. These deficits may arise because the cell's ability to adjust its internal stiffness is compromised in the absence of palladin. This proposal will test the hypothesis that palladin, by its ability to cross-link actin and its interaction with another actin cross-linker, alpha-actinin, determines the structure and mechanical properties of actin networks and enables the cell to sense its physical environment. Two types of approaches will be used to address this question. First, the structural and mechanical properties of actin networks assembled on a glass slide will be measured to elucidate how actin cross-linking by palladin contributes to actin organization. In addition, palladin levels will be genetically manipulated in living cells to study how altered actin organization, cellular stiffness and force generation impacts cellular mechano-sensitivity. Broader Impact This collaborative proposal will enhance the understanding of essential biological processes that underlie cell movement and tissue formation. The training of graduate and undergraduate students in interdisciplinary approaches from Physics and Cell Biology will be an integral part of the work. The cell lines that will be developed as part of this project will be made freely available to other investigators following their publication. A graduate course in Cell Mechanics will be developed based on the conceptual framework of this proposal. The PI and co-PI will also encourage minority students as well as high school students from the area to participate in research as part of the Louis Stokes Alliance for Minority Participation program at the University of Maryland and the University of North Carolina Research Apprenticeship Program. The PI will organize a one week biophysics laboratory demonstration as part of the Summer Girls Program in the Department of Physics to encourage participation of female students in science and technology fields.