Cortical excitability as a mechanism for epithelial barrier maintenance: A joint experiment-theory systems approach

Multicellular organisms evolved multiple adaptations to separate and protect themselves from the surrounding environment, which is frequently hostile to the interior of the organism. Epithelial tissues in our bodies play the guardian role enveloping the entire body (skin) as well as wrapping each of our organs. This protective role relies on the formation and continuous maintenance of tight junctions between individual epithelial cells. These junctions have to be sufficiently tight to prohibit entry of pathogens, foreign proteins, chemicals and gases as well as to keep inside the bodily fluids. Multiple debilitating human diseases are associated with loss of these protection barriers in the gut, lungs, skin, mucosa, etc. However, epithelial tissues are live and individual cells within epithelium undergo cell divisions, death and rearrangements, all process that require that the junctions between cells must be plastic and dynamic. How the barrier function is maintained within living and actively developing epithelia remains still poorly understood. Studying frog embryonic epithelium, we recently discovered that cell-cell junctions in this dynamic tissue undergo random failures (ruptures) due to the increased mechanical tension but are rapidly repaired by a sentinel mechanism that is somehow triggered by the rupture. Our data suggest that the molecular mechanism of this repair process may involve a fundamental systemic property known as excitability, which is observed in complex nonlinear systems across all areas of science and technology. We recently described a likely similar excitable behavior in frog and starfish oocytes and this discovery, published in November 2015 by Nature Cell Biology, was featured in the BBSRC News and Business Magazine. In this proposal we will explore the hypothesis of the excitable nature of the epithelium repair mechanism in depth and breadth using both modern experimental methods and computational modelling. We will identify the molecular details of this mechanism and will seek points of intervention within this mechanism to be able to eventually translate our results into novel medical therapies.

In this project we will investigate the mechanism of cell-cell junctions repair in the developmentally active vertebrate epithelia using frog X. laevis embryonic epithelium as the experimental system. Epithelia must strike a balance between maintaining barrier function and dynamically reorganizing in response to cell divisions, cell death and vigorous morphogenetic cell movements, such as invaginations and cell intercalations. How the barrier function is maintained throughout these vital processes is an important yet poorly understood question. Our published and unpublished preliminary data indicate that, in frog embryonic epithelium, cell-cell junctions frequently fail (possibly due to the tensile rupture of the underlying actomyosin belt) and are rapidly repaired. The repair is associated with short-term localized rises in cortical activity of small GTPase Rho ("Rho flairs") and the following rapid F-actin polymerization and ZO-1 recruitment. We will explore the hypothesis that Rho flares are another manifestation of cortical excitability, the phenomenon recently discovered by us in the form of Rho-actin waves traversing cortices of frog and starfish oocytes and early embryos. We will combine a variety of experimental approaches, including expression of fluorescently labeled proteins and reporters, drug perturbations, laser ablation, optogenetic manipulations and extensive live-cell fluorescence imaging, together with mathematical modelling and computer simulations to test our hypothesis and characterize the molecular mechanism or Rho flares. We will also extend our modelling to the scale of the entire epithelial sheet and utilize its predictions in conjunction with extensive experimental perturbations and long-term imaging to further improve our understanding of the epithelial barrier function on the scale of the whole epithelium.

The primary biological function of all epithelia is to provide a protective barrier that defends the underlying tissues and that keeps essential nutrients, bodily fluids and water inside and pathogens, noxious substances and natural elements outside. This function is crucial for the viability of all multicellular organisms. The proposed project aims to identify molecular mechanisms that enable epithelial cells to sense the presence of cell-cell junction disruptions and effectively repair this damage. Therefore, the primary cohort of stakeholders includes a wider international cell and developmental biology communities. These stakeholders will benefit from the developed knowledge, experimental methods and protocols and computational models developed by us. To reach this broad community we will publish our results in the open access journals and present them at the international meetings. We expect that the mechanisms by which Rho GTPase regulates junction dynamics and remodeling in frog are conserved in human. Therefore, the proposed project has a clear value for medical applications. Indeed, a great variety of human diseases is directly associated with compromised barrier function of epithelial tissues. To mention a few, these include but are not limited to irritable bowel syndrome, food allergies, Crohn's disease and other gut diseases; multiple eye, lung and kidney diseases. The project has an opportunity to influence these diverse groups of stakeholders that include applied biomedical researchers and medical doctors. Throughout the project we will be seeking druggable targets among the positive and negative regulators of junction integrity. Should we identify potential candidates, we will enter in collaboration with local U. of Edinburgh researchers who perform small molecule candidate screens and also consider reaching out to the broader pharmaceutical community. We will work with the Edinburgh Research and Innovations (ERI) to identify potential links with the industry and other stakeholders in search for commercialization opportunities for our results. We will communicate the results of our work to the press, in which the project PI already has a successful track record, as well as to the broader public, including school pupils. This activity will be lead through participation in a variety of public outreach programs and campaigns conducted by the University in the framework of science festivals, fairs, etc. Finally, the project provides an opportunity to train postdoctoral researchers as well as gives them the opportunity to develop not only professionally but also in the areas of soft skills, communication, relations with media and commercialization.