Biochemical and functional analysis of innexin2-containing gap junction channels during organogenesis in <i>Drosophila</i>

Abstract

Gap junctions channels serve as mediators for direct cell-to cell communication between cells of different tissues and organs by the exchange of small metabolites and signalling molecules. They are formed by analogous protein families in vertebrates and invertebrates. The docking of two hemichannels leads to gap junction channel formation at the plasma membrane of two adjacent cells, resulting in so called “plaque like” structures that consist of accumulations of gap junction channels at contact sites.<br /> Whereas the biogenesis and functions of vertebrate gap junction channels consisting of connexins have been analysed in some detail, biochemical studies on innexin gap junction assembly and trafficking are lacking for any innexin known to date.<br /> The formation and trafficking of innexin2 containing gap junction hemichannels to the plasma membrane was analysed in <i>Drosophila melanogaster </i>and cultured cells in this thesis. Analysis of innexin2 oligomerisation by biochemical approaches like velocity sedimentation of different oligomers in density gradients combined to cell-free transcription/translation assays revealed the existence of hexameric innexin2 containing hemichannels that were termed innexons. It could be shown that the identified innexons formed by innexin2 can be either homomeric, meaning that they are composed of innexin2 subunits only, or heteromeric meaning that other innexin isoforms are part of one hemichannel in addition.Immunohistochemical analysis of innexin2 expression in<i> Drosophila ovaries</i>, invertebrate and mammalian cell culture as well as biochemical fractionation experiments indicate that innexin2-containing oligomers are transported along the secretory pathway to the plasma membrane. Moreover, by the combination co-immunoprecipitation and drug treatment experiments it could be demonstrated that innexin2-containing hemichannels are transported along the microtubule cytoskeleton to the plasma membrane with an involvement of the exocyst complex taking part in the anterograde transport of gap junction hemichannels. These findings provide the first biochemical evidence that innexins form hexameric innexon hemichannels and suggest that the oligomerisation and trafficking of connexins and innexins may rely on a common mechanism despite lacking of sequence similarity between both protein families.<br /> Investigation of the innexin2 function during the development of the proventriculus, an accessory organ of the <i>Drosophila</i> gastrointestinal tract that is formed during <i>Drosophila</i> embryogenesis, pointed in addition to an innexin2 dependent gap junctional cross regulation of major signalling molecules like hedgehog, Delta and wingless which are crucial for the formation of shaped organs.