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

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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.