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

智力优势人体内的细胞有一种非凡的能力来感知环境中的“僵硬”--也就是说，细胞可以区分坚硬的底物，如骨骼，以及软质的底物，如大脑。这种能力对细胞功能的许多方面都是至关重要的，如迁移、伤口愈合和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硬度，使其与外表面的硬度相匹配。内部细胞骨架由微观细丝组成的动态网络组成。这些细丝的主要成分是一种名为肌动蛋白的蛋白质。不同的肌动蛋白结合蛋白使肌动蛋白细丝交联化，并将它们组织成复丝束。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着重要的作用。此前的实验表明，Palladin在胚胎发育中起着至关重要的作用：当Palladin基因在小鼠体内沉默时，它会导致致命的发育缺陷，其特征是细胞无法正确地附着在底物上，并无法适当地迁移。这些缺陷的出现可能是因为细胞在缺乏巴拉丁的情况下调节其内部硬度的能力受到损害。这项提议将检验这样一种假设，即Palladin通过其交联肌动蛋白的能力以及它与另一种肌动蛋白交联剂α-肌动蛋白的相互作用，决定了肌动蛋白网络的结构和机械特性，并使细胞能够感知其物理环境。将使用两种类型的方法来解决这个问题。首先，将测量组装在玻片上的肌动蛋白网络的结构和力学性质，以阐明由Palladin交联的肌动蛋白如何有助于肌动蛋白的组织。此外，帕拉丁水平将在活细胞中进行基因操作，以研究改变的肌动蛋白组织、细胞硬度和力产生如何影响细胞的机械敏感性。广泛影响这一合作提案将加强对细胞运动和组织形成基础的基本生物过程的理解。对研究生和本科生进行物理学和细胞生物学跨学科方法的培训将是这项工作不可或缺的一部分。将作为该项目的一部分开发的细胞系将在发表后免费提供给其他研究人员。将根据这一提议的概念框架开发一门细胞力学研究生课程。PI和共同PI还将鼓励该地区的少数族裔学生以及高中生参与研究，这是马里兰大学和北卡罗来纳大学研究学徒计划的路易斯·斯托克斯少数族裔参与联盟计划的一部分。该协会将组织为期一周的生物物理实验室演示，作为物理系暑期女孩计划的一部分，以鼓励女学生参与科学和技术领域。

Intellectual MeritCells 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 ImpactThis 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.