Structure-based integration of claudin functions in establishing epithelial barriers and selective permeability

• Applicant: Dr. Laura-Sophie Beier
• Subject Area: Cell Biology ; Biochemistry
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 531212047

Multicellular organisms require clear discrimination from the external environment to support an internal milieu. In more advanced organisms, internal compartments must also be separated from one another. To this end, external surfaces and body cavities are lined by tissue barriers, i.e., epithelia and, in blood vessels, endothelia. Epithelia are not impermeable, but enable directional exchange between compartments. Essential for this are the tight junctions, which seal the gaps between the epithelial cells, i.e., the paracellular space, against uncontrolled exchange, while facilitating charge- and size-selective permeability. This selective permeability is mediated by proteins of the claudin family. Within one plasma membrane, claudins polymerize and form strands and meshworks surrounding the epithelial cells in the apicolateral region. Claudins in apposing plasma membranes interact via their extracellular domains to seal the paracellular space. Homo- and heterotypic interactions between the ensemble of claudins in an epithelium define its barrier- and permeability properties, but the principles and consequences of these interactions are still incompletely understood. Here, I propose to conduct a study focusing on homo- and heterotypic interactions of claudin-2, which, among others, is involved in gastrointestinal and kidney diseases, thus posing a relevant therapeutic target. I will systematically analyze the determinants of claudin-2 homo-polymerization as well as the molecular interactions underlying claudin-4 mediated de-polymerization of claudin-2 strands (interclaudin interference). I will analyze claudin-2 meshworks using super-resolution STED microscopy and quantitative post-acquisition evaluation, and change them by mutating claudin-2. The morphologic data will feed into computational interaction models, which will define homo- and heterotypic claudin-2 interactions ranging from molecular interactions to claudin strand morphology on the micrometer scale. Subsequently, I will analyze the functional relevance of the identified interfaces on tight junction, and ultimately, epithelial function in in vitro models. I will employ electrophysiologic methods to measure channel- and barrier-forming claudin-2 properties. By correlating these data with claudin strand morphology in epithelial cells, which I will assess by STED microscopy, I will characterize the tight junction structure-function relationship. Finally, to test the validity and functional relevance of the models derived from in vitro cell models, I will translate them to the context of the intestinal epithelium using stem cell-derived enteroids. The proposed studies will be the first to range from in silico modeling to claudin strand imaging and analysis of tight junction function in stem-cell derived enteroid cultures. Ultimately, the data will guide our understanding of claudinopathies and development of rational, mechanism-based therapies to improve human health.

• DFG Programme: WBP Fellowship
• International Connection: USA
• Host: Professor Jerrold R. Turner, Ph.D.