A Molecular Toolkit for Controlling and Probing Cell Junction-Actin Interactions

用于控制和探测细胞连接-肌动蛋白相互作用的分子工具包

• 批准号: 10276194
• 负责人: Brian Belardi
• 金额: $ 38.06万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10276194
• 关键词: Actins; Adhesions; Award; Binding; Biological; Biology; Cadherins; Carcinoma; Cell Shape; Cells; Characteristics; Communities; Cytoskeleton; Development; Disease; Disease Progression; Elements; Endocytosis; Engineering; Epithelial; Exhibits; Extracellular Matrix; Filament; Focal Adhesions; Glues; Human; Individual; Integrins; Intercellular Junctions; Link; Maintenance; Malignant Neoplasms; Mechanics; Membrane; Membrane Biology; Membrane Proteins; Methods; Microfilaments; Mitosis; Molecular; Morphogenesis; Natural Products; Organism; Permeability; Play; Polymers; Process; Property; Proteins; Research; Research Personnel; Role; Side; Signal Transduction; Specificity; Structure; System; Tight Junctions; Tissues; Translational Research; Work; base; cell motility; inhibitor/antagonist; innovation; interest; mechanical properties; monolayer; programs; reconstitution; repaired; side effect; small molecule; therapeutic target; tool; vector

PROJECT SUMMARY: A MOLECULAR TOOLKIT FOR CONTROLLING AND PROBING CELL JUNCTION- ACTIN INTERACTIONS Higher metazoans exhibit robust, yet dynamic connections between neighboring cells, leading to the exquisite morphogenesis, vectorial transport, and resilient mechanical properties that define tissue. Spatially separated junctions line individual epithelial membranes and are tasked with linking cells to one another and to the underlying extracellular matrix. These junctions are composed of well-characterized membrane proteins, each with unique functions: claudins create paracellular barriers; cadherins bind cells together; and integrins attach cells to matrix. Despite unique classes of membrane proteins, different junctions all possess a common element, the cytoskeleton, which resides on the cytosolic side of the contact. One cytoskeletal polymer in particular – actin – appears indispensable for junction activity. While decades of elegant work have transformed our understanding of the structure and binding characteristics of junctional membrane proteins, the question of how actin is involved in cell junction formation, junction maintenance and repair, and junctional signaling remains unresolved. Actin filaments are ubiquitous throughout the cell as they contribute to cell shape, endocytosis, mitosis, motility, and other critical phenomena. However, this wide distribution presents a fundamental problem when studying actin – namely how to pinpoint the exact role actin filaments play in the process-of-interest. While actin- targeted natural products and small molecules are widely used to disrupt filaments globally, they lack the specificity needed to uncover the role of actin filaments locally at cell junctions. My research group is developing a suite of molecular tools to both control and dissect actin interactions at cell junctions. In this way, we provide researchers with new methods to turn-on and -off actin association and to probe actin’s role in adhesion and cell-cell mechanics. These tools come in various molecular forms: i) protein-based switches, ii) small-molecule molecular glues and inhibitors, and iii) synthetic cells, which can be applied to wide-ranging systems, such as reconstituted membranes, cells, monolayers, tissues, and organisms, to illuminate and manipulate actin- dependent processes. In my lab, we will harness these molecular tools to focus on three specific research directions in epithelial biology, although we anticipate that the toolkit will benefit the greater biological community, including biochemists, cell biologists and developmental biologists. First, we will focus on applying our tools to dissect actin’s role during tight junction maturation and, ultimately, to modulate barrier function. Second, we will investigate, in mechanistic detail, how actin potentiates integrin activation during focal adhesion formation. Finally, we will assemble cells using actin switches to generate “synthetic tissues” with programmable and toggleable properties, such as dynamic tissue permeability and adhesion. Broadly, this research program relies on our diverse expertise in molecular engineering, basic membrane biology, and translational science to create a virtuous cycle of innovation and discovery over the course of the MIRA award.

项目概要：用于控制和探测细胞结的分子工具箱- 肌动蛋白相互作用 高等后生动物在相邻细胞之间表现出强大而动态的连接，导致了精致的 形态发生、矢量运输和定义组织的弹性机械特性。空间上分离 连接排列在单个上皮细胞膜上，负责将细胞彼此连接，并将细胞连接到 细胞外基质。这些连接由特征明确的膜蛋白组成， 具有独特的功能：claudin创建细胞旁屏障;钙粘蛋白将细胞结合在一起;整合素附着在 细胞到矩阵。尽管膜蛋白有独特的种类，但不同的连接都具有共同的元件， 细胞骨架，位于接触的胞质侧。特别是一种细胞骨架聚合物-肌动蛋白 - 似乎是连接活动不可或缺的。几十年的优雅工作已经改变了我们对 连接膜蛋白的结构和结合特性，肌动蛋白如何参与的问题， 在细胞连接形成、连接维持和修复以及连接信号传导方面仍然没有解决。 肌动蛋白丝在整个细胞中普遍存在，因为它们有助于细胞形状、内吞作用、有丝分裂， 运动性和其他关键现象。然而，这种广泛的分布提出了一个基本问题， 研究肌动蛋白-即如何确定肌动蛋白丝在感兴趣的过程中发挥的确切作用。而肌动蛋白- 有针对性的天然产物和小分子被广泛用于破坏全球的细丝，但它们缺乏 特异性需要揭示肌动蛋白丝的作用，局部在细胞连接。我的研究小组正在研究 一套分子工具，既控制和解剖肌动蛋白在细胞连接处的相互作用。通过这种方式，我们提供 研究人员用新的方法来打开和关闭肌动蛋白协会，并探测肌动蛋白在粘附中的作用， 细胞-细胞力学这些工具以各种分子形式出现：i）基于蛋白质的开关，ii）小分子 分子胶和抑制剂，以及iii）合成细胞，其可应用于广泛的系统，例如 重组膜、细胞、单层、组织和生物体，以照亮和操纵肌动蛋白- 依赖过程。 在我的实验室，我们将利用这些分子工具，专注于上皮细胞的三个特定研究方向， 生物学，尽管我们预计该工具包将有利于更大的生物界，包括 生物化学家、细胞生物学家和发育生物学家。首先，我们将专注于应用我们的工具来剖析 肌动蛋白在紧密连接成熟过程中的作用，并最终调节屏障功能。二是 研究，在机制细节，肌动蛋白如何增强整合素活化过程中粘着斑形成。 最后，我们将使用肌动蛋白开关组装细胞，以产生具有可编程和可移植的“合成组织”。 可切换特性，例如动态组织渗透性和粘附性。总的来说，这项研究计划依赖于 凭借我们在分子工程、基础膜生物学和转化科学方面的丰富专业知识， 在MIRA奖的过程中，创新和发现的良性循环。