Break, Flare, Repair: Rho Flares Locally Reinforce the Tight Junction Barrier
Stephenson, Rachel
2018
Abstract
Epithelial tissues serve essential functions, such as preventing infection and water loss, regulating absorption and secretion, and creating specialized compartments within complex multicellular organisms. Epithelia are sheets of cells connected by specialized apical cell-cell junctions. Adherens junctions adhere cells to one another and mechanically integrate cells in the tissue, while tight junctions regulate both how much and what kind of materials can cross through the paracellular space (the space between cells). Both tight junctions and adherens junctions are dynamically regulated by filamentous actin and myosin II (actomyosin), which forms a contractile array near the apical cell-cell junctions. The forces generated by the apical actomyosin array can be transmitted to neighboring cells through adherens junctions to drive cell- and tissue-scale changes. Cellular events, such as epithelial cytokinesis, cell extrusion, and wound healing, alter tension on adherens junctions and the dynamics of the apical actomyosin array. However, very little is known about how these changes in cell shape and actomyosin dynamics influence tight junctions and epithelial barrier function. To better understand how dynamic cell shape change influences epithelial barrier function, we developed a tight junction barrier assay compatible with live imaging. This approach, the Zinc-based Ultrasensitive Microscopic Barrier Assay, or ZnUMBA, allows for the detection of localized, transient leaks. Using this approach in the epithelium of Xenopus laevis gastrula-staged embryos, we discovered that leaks that result from cell shape change are rapidly repaired by transient accumulation of the active conformation of the small GTPase RhoA, or Rho flares. Then, using fluorescently-tagged tight junction proteins, I found that occludin and ZO-1 show localized decline prior to Rho flares and are reinforced afterwards. Using molecular inhibitors of the targets of Rho activity, I concluded that both actin polymerization and Rho Kinase-mediated junction contraction reinforce ZO-1 and occludin, promoting efficient restoration of epithelial barrier function. We hypothesize that Rho flares serve as a rapid repair mechanism to quickly restore barrier function and that this allows epithelial cells to dynamically change shape without prolonged breaches in barrier function. Rho flares are accompanied by an apical protrusion of the plasma membrane. However, both the cause of the membrane protrusion and its purpose are unclear. I examine three potential causes of membrane protrusion and assess each one by reviewing the temporal and spatial accumulation of F-actin and myosin II, as well as several candidate actin nucleators. Based on the data presented, I propose that a bleb-like protrusion mechanism is likely, and I hypothesize that the protrusion acts to temporarily seal the paracellular space while the tight junction is reinforced. The work presented in this dissertation advances tight junction biology in several ways. ZnUMBA is a widely adaptable technique that will allow other researchers to examine changes in barrier function with greater temporal and spatial precision. We hope that this will usher in a better understanding about what causes tight junction leaks and how they are repaired. Finally, I describe a previously unknown mechanism for rapid repair of local breaches in epithelial barrier function by active Rho, adding nuance to our understanding of the many roles this small GTPase plays in epithelial tissues.Subjects
tight junction Rho GTPase epithelium Xenopus
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