ABSTRACT Epithelia form selective barriers that compartmentalize organs and protect them from external environments which allows for specialized physiologic function. Epithelial barrier permeability has two regulated components, the transcellular path mediated by transcytosis, and the paracellular path between cell-cell contact sites regulated by tight junctions (TJs). We previously determined that stimulation of epithelial cells by contact between the apical plasma membrane with nanostructured films (NSFs) increases transepithelial permeability to large macromolecules, such as Alexa-594 labeled Fab, through both the transcellular and paracellular pathways. Several lines of evidence identified a potential role for apically localized 1 integrin in the ability of NSFs to increase substrate permeability. However, a drawback to using NSFs is that they act by engaging large, heterogeneous patches of the apical plasma membrane making it difficult to precisely define molecular mechanisms linking specific surface proteins to changes in epithelial barrier function. Thus, to specifically investigate whether 1 integrin is directly involved in increased barrier permeability, we developed an anti- integrin nanowire system consisting of anti-1 integrin antibodies conjugated to functionalized polycaprolactone nanowires. These anti-integrin nanowires served as a platform to specifically cluster apically localized 1 integrin to determine whether integrin stimulation has the capacity to regulate epithelial barrier function. Treatment of epithelial monolayers with anti-integrin nanowires significantly decreased the transepithelial resistance of the monolayer and increased the rate of transepithelial flux of Alexa-594 Fab across polarized monolayers. These functional effects were associated with nanowire-induced changes in the localization of the TJ scaffolding protein zonula occludens-1 (ZO-1) and the integrin-associated actin binding protein talin, as well as rearrangement of the actin cytoskeleton from stress fibers into a more cortical pattern of organization. In order to define the mechanisms of action for integrin-mediated regulation of epithelial permeability we will test the hypothesis that integrin clustering by anti-integrin nanowires increases permeability through changes in integrin- associated actin binding proteins leading to cytoskeletal remodeling through the following Aims. In Aim 1, we will measure the transcellular and paracellular contributions to solute permeability following integrin stimulation by measuring how anti-integrin nanowire treatment causes structural and functional changes in TJs and transcellular permeability. In Aim 2, we will determine if integrin mediated changes in permeability are driven by changes in the recruitment of integrin-associated actin binding proteins and measure their impact on actin organization. We will also assess if changes in integrin-associated actin binding proteins and actin cytoskeleton organization are requirements for integrin mediated changes in permeability. The goal of this proposal is to identify novel mechanisms whereby apically localized integrins regulate epithelial barrier function.

PROJECT NARRATIVE Epithelia form selectively permeable barriers that compartmentalize organs and allow for specialized physiologic function. There are many known components that regulate transepithelial permeability, but the role for apically localized integrins in the regulation of barrier function is not presently known. The goal of this project is to use anti-integrin-1 antibodies conjugated with nanowires to identify novel mechanisms whereby apically localized integrins regulate epithelial barrier function.