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

项目摘要：用于控制和探测细胞连接的分子工具包- 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联系，导致精致的 定义组织的形态发生、载体运输和弹性机械特性。空间上分离的 连接排列在单个上皮膜上，负责将细胞彼此连接起来，并连接到 潜在的细胞外基质。这些连接是由特性良好的膜蛋白组成的，每个 具有独特的功能：Claudins创建细胞旁屏障；钙粘素将细胞结合在一起；整合素附着 细胞到基质。尽管膜蛋白具有独特的类别，但不同的连接都具有共同的元件， 细胞骨架，驻留在接触的细胞质一侧。一种特殊的细胞骨架聚合物--肌动蛋白 -对于连接活动来说似乎是不可或缺的。虽然几十年的优雅作品改变了我们对 关于连接膜蛋白的结构和结合特性，肌动蛋白是如何参与的问题。 在细胞连接形成中，连接的维持和修复以及连接信号仍未解决。 肌动蛋白细丝在整个细胞中普遍存在，因为它们对细胞形状、内吞作用、有丝分裂、 运动性，以及其他关键现象。然而，这种广泛的分布在以下情况下会带来一个根本问题 研究肌动蛋白--即如何准确定位肌动蛋白细丝在感兴趣的过程中扮演的确切角色。当肌动蛋白- 靶向天然产物和小分子被广泛用于在全球范围内破坏细丝，它们缺乏 发现肌动蛋白细丝在细胞连接局部的作用所需的特异性。我的研究小组正在开发 一套控制和剖析细胞连接处肌动蛋白相互作用的分子工具。通过这种方式，我们提供 研究人员用新方法打开和关闭肌动蛋白关联，并探索肌动蛋白在粘连和 细胞-细胞力学。这些工具有各种分子形式：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，可应用于广泛的系统，如 重组膜、细胞、单层、组织和生物体，以照亮和操纵肌动蛋白- 从属进程。 在我的实验室里，我们将利用这些分子工具，专注于上皮细胞的三个具体研究方向 生物学，尽管我们预计该工具包将使更大的生物界受益，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解剖 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。第二，我们将 详细研究肌动蛋白如何在局部粘连形成过程中增强整合素的激活。 最后，我们将使用肌动蛋白开关组装细胞，通过可编程和 可调节的特性，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 基于我们在分子工程、基础膜生物学和翻译科学方面的不同专业知识，创建 在Mira奖的过程中，创新和发现的良性循环。

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.