Epithelial tissues maintain selective barriers through the utilization of macromolecular complexes known as tight junctions. Tight junctions are essential for regulating cellular barriers, mediating drug delivery, and controlling paracellular transport. Tight junctions in vertebrates have a multiplex architecture consisting of the associated proteins claudins, occludins, zonula occludens (ZO-1), and junctional adhesion molecules (JAMs). The claudin integral membrane protein family is the fundamental component of the tight junction and is responsible for selectivity. Due to the small size of claudins (~20 kDa) and occludin (~55 kDa), and the disordered nature of ZO-1, tight junctions are unamenable to conventional structural methods. To overcome this limitation, this study proposes a two-tiered approach to image the analogous apical junction structure in Caenorhabditis elegans (C. elegans). The C. elegans CLC-2 and VAB-9 are claudin-like proteins that maintain conserved residues in key positions. This research plans to fluorescently label these homologs, with the intention of performing structural studies using cryogenic electron microscopy (Cryo-EM). The first approach is a single particle analysis of CLC-2 and VAB-9 purified proteins for Cryo-EM imaging. The second, is a cryogenic electron tomography (Cryo-ET) approach to obtain tomography data in situ to understand how the apical junctions are organized.
