Mechanisms of Cytoskeletal Regulation of Tight Junction Homeostasis and Repair

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 DESCRIPTION (provided by applicant): Tight junctions (TJs) undergo constant homeostatic maintenance. While it is clear that TJ protein internalization and vesicular recycling are essential for this process, the underlying mechanisms are not defined. Studies using non-physiological stimuli have defined the actin cytoskeleton as a critical contributor to TJ assembly and maintenance. Actomyosin contraction, which has been linked to physiologic and pathophysiologic TJ barrier loss, such as that triggered by tumor necrosis factor (TNFa), drives downstream caveolar endocytosis of the TJ protein occludin. Similar processes have been implicated in barrier loss caused by enteropathogenic E. coli (EPEC) infection. While both forms of barrier loss are reversible, it is not clear if actomyosin relaxation or TJ protein recycling are involved in this process. Further, despite an established role for microtubules in apical protein recycling, few studies have examined the contributions of microtubules to TJ homeostasis and repair. Recently, a specific subset of microtubules, planar apical networks of microtubules, was found to interact with the TJ protein cingulin. This interaction is regulated by AMP-activated protein kinase (AMPK)-mediated phosphorylation of cingulin and is essential to epithelial morphogenesis. Our data show that microtubule disruption impedes TJ recovery in calcium switch assays. In addition, the EPEC virulence factor EspG, which disrupts microtubules, attenuates TJ barrier restoration. These findings indicate that microtubules play an unexpected and critical role in TJ homeostasis and repair. However, the mechanisms by which microtubules contribute to these processes have not been explored. The long-term goal of this proposal is to define the molecular and cellular processes involved in the restoration of TJ structure and function and use this knowledge to design therapies to promote epithelial repair. The objective of the proposed studies is to define the role of the actin cytoskeleton and microtubules in TJ recovery from pathophysiologic insults. The central hypothesis is that microfilaments and microtubules actively participate in reestablishing intestinal epithelial TJs after disruption by pathological insults such as inflammation and infection. This research is highly relevant to the VA population as diarrhea among military troops is quite prevalent rendering a substantial portion unfit for duty at any given time and is associated with chronic health sequelae including irritable bowel disease and inflammatory bowel disease. Definition of the events involved in TJ maintenance and restoration will guide the development of strategies to preserve intact TJs and aid in TJ recovery. The central hypothesis will be tested by 3 aims: 1. Define the role of the actin cytoskeleton in TJ recovery following molecularly targeted, inflammatory (TNF) and infectious (EPEC), stimuli; 2. Determine the contribution of microtubules to TJ restoration from inflammatory insults (TNF) and enteric infection (EPEC) in vitro; and 3. Resolve the role of microfilaments and microtubules in TJ recovery from pathophysiologic conditions (TNF and EPEC infection) in vivo.