Periodic Reporting for period 2 - DynChan (Dynamic regulation of paracellular channel gating)
Okres sprawozdawczy: 2023-03-16 do 2024-03-15
Tight junction proteins seal the space between adjacent epithelial and endothelial cells and form paracellular pores that regulate the paracellular transport: pore-forming claudins. Unfortunately, biophysics of these pore-forming claudins remains incompletely undefined. Mutations in pore-forming claudins cause human disease, including familial hypomagnesemia and hypercalcuria, neonatal sclerosing cholangitis associated with ichthyosis, and others. Underlying structure-function relationships will fundamentally advance our understanding of paracellular permeability and may, ultimately, lead to novel therapeutic approaches. The Dynchan project aims to develop a new technology that will enable high resolution analyses of claudin channel activity and regulation. The technology based on silicon chips with array of nanopillar electrodes is expected to provide the tool needed to develop foundational understanding of claudin biology. During the Dynchan project, we established conditions for cultivating functional, mature, properly assembled epithelial monolayers on high aspect ratio pillars localized to lateral intercellular spaces. We found that the pillar size strongly affected the assembling of tight junctions. For MDCK I cells, pillar structures of 1 µm in diameter and approximately up to 6 µm in height facilitated the formation of tight junctions in close proximity with the tips of the pillar structures. Additionally, through the application of micro and nanotechnologies, we have successfully fabricated miniature electrodes atop these pillars. This breakthrough allows us to record electrical currents crossing the tight junctions with an very low spatial resolution in the one micron range.
The project has accomplished so far the formation of epithelial monolayers on top of nanopillar structures with tips in close interaction with tight junctions. In the context of tight junctions, it was previously reported that transient application of a nanotopographic surface over the apical brush border membrane of epithelial monolayers triggers redistribution of ZO-1, claudins, and F-actin that increases paracellular macromolecular flux. So, nanopillar structures were first optimized to avoid the perturbation of the cellular structure or function. We have determined the nanopillar size that, without disturbing the tight junctions, gets the tips of the pillars in close contact with the tight junctions. In addition, we have identified culture conditions that allow epithelial cells form mature monolayers on top of nanopillar that are structurally similar from those grown on semipermeable supports, which is the gold-standard.
The unprecedented integration of high aspect ratio structures into the lateral intercellular space sets the stage for application of nanotechnologies for perturbation and analysis of epithelial tight junction biology. This nanoelectrode arrays will enable other basic and applied studies and ultimately support development of therapeutics designed to modulate claudin function. The recent breakthrough discovery that tight junction channels might be actively-gated (i.e. transitioning between open and closed states) has identified a new plausible avenue to treat barrier-related diseases. Development of the Dynchan technology will accelerate advances in understanding of paracellular channel gating, without which the biophysics of the pore-forming claudin functions will remain incompletely undefined.