Formation of functional innexin hemichannels, as well as gap junctional channels, in an insect cell line, NIAs-AeAl-2, derived from Asian tiger mosquito Aedes albopictus (Diptera: Culicidae): A partial but significant contribution of innexin 2.

In vertebrates, gap junctions and hemichannels consisting of connexins are important cell surface structures for communication with neighboring cells and for the regulation of various cell functions. To date, various gap-junction-related proteins have been found, including innexins in invertebrates and pannexins in vertebrates. Significant contributions of gap junctions by innexins and (hemi-)channels by pannexins to numerous functions have been reported. Verification of the presence and functional significance of innexin hemichannels, however, remains a gap in our knowledge in innexin physiology. In this study, we revealed the localization of an innexin protein (innexin 2) on the cell surface in mosquito tissues and cultured cells. Furthermore, we demonstrated the presence of functional hemichannels, as well as gap junctions, in mosquito cells using dye transfer assays. The inward uptake of fluorescent dye was inhibited by anti-innexin 2 antibody. These results suggest that innexin hemichannels are formed to function in cultured mosquito cells, in at least a partially innexin 2-dependent manner. Although only a few studies on insect hemichannels have been published, innexin-based hemichannels, as well as innexin gap junctions, could also significantly contribute to insect intercellular signal transduction.

Targeting of Nphp3 to the primary cilia is controlled by an N-terminal myristoylation site and coiled-coil domains.

Primary cilia are organelles that extend from the cell surface. More than 600 proteins have been identified in cilia, but ciliary targeting mechanisms are poorly understood. Nephronophthisis (NPHP) is an autosomal recessive cystic kidney disease with 11 responsible genes (NPHP1-11) thus far being identified. The mouse Nphp3 gene product is localized in the cilia and contains coiled-coil (CC) domains and tetratricopeptide repeats, but the ciliary targeting sequences (CTSs) are unknown. In the present study, we generated a series of GFP-tagged deletion constructs of Nphp3 and tried to find the CTSs of Nphp3. We found that the N-terminal 201 amino acid fragment (Nphp3 [1-201]), which contains two CC domains, is necessary and sufficient for cilia localization. Further analysis revealed that an N-terminal glycine (G2), which is a conserved myristoylation site among vertebrates, is also essential for trafficking of Nphp3 to the ciliary shaft. Interestingly, the N-terminal fragments, Nphp3 (8-201), Nphp3 (52-201), and Nphp3 (96-201), that contain the CC domains, targeted the basal body, but could not enter into the ciliary shaft. Our results showed the importance of myristoylation in ciliary trafficking, and suggest that Nphp3 trafficking to the ciliary shaft occurs in a two-step process.