Heteromerization of Innexin Gap Junction Proteins Regulates Epithelial Tissue Organization inDrosophila

Abstract

Gap junctions consist of clusters of intercellular channels, which enable direct cell-to-cell communication and adhesion in animals. Whereas deuterostomes, including all vertebrates, use members of the connexin and pannexin multiprotein families to assemble gap junction channels, protostomes such as Drosophila and Caenorhabditis elegans use members of the innexin protein family. The molecular composition of innexin-containing gap junctions and the functional significance of innexin oligomerization for development are largely unknown. Here, we report that heteromerization of Drosophila innexins 2 and 3 is crucial for epithelial organization and polarity of the embryonic epidermis. Both innexins colocalize in epithelial cell membranes. Innexin3 is mislocalized to the cytoplasm in innexin2 mutants and is recruited into ectopic expression domains defined by innexin2 misexpression. Conversely, RNA interference (RNAi) knockdown of innexin3 causes mislocalization of innexin2 and of DE-cadherin, causing cell polarity defects in the epidermis. Biochemical interaction studies, surface plasmon resonance analysis, transgenesis, and biochemical fractionation experiments demonstrate that both innexins interact via their C-terminal cytoplasmic domains during the assembly of heteromeric channels. Our data provide the first molecular and functional demonstration that innexin heteromerization occurs in vivo and reveal insight into a molecular mechanism by which innexins may oligomerize into heteromeric gap junction channels.

Innexins Ogre and Inx2 are required in glial cells for normal postembryonic development of the Drosophila central nervous system

“…We favour the latter because even at high levels of RNA ogre did not induce homotypic intercellular channels although increasing the amount of RNA injected did induce non-junctional currents. There is also some precedent for such a mechanism as Inx2 and Inx3 have been shown to form hetero-oligomers (Lehmann et al, 2006).…”

Bi-directional gap junction-mediated Soma-Germline communication is essential for spermatogenesis

“…Previous work has suggested that innexins may affect cadherinmediated cell-cell adhesion Giuliani et al, 2013;Lehmann et al, 2006). To determine if disruption of gap junctions altered levels of E-Cad (Shg -FlyBase) in the testes, we stained zpg and inx2RNAi testes for E-Cad.…”

Pannexin 1 and pannexin 3 are glycoproteins that exhibit many distinct characteristics from the connexin family of gap junction proteins

“…Further insights into the functional role of pannexins might in fact be extracted from studies on their invertebrate orthologs, innexins. In Drosophila, the heteromerization of Innexin 2 and Innexin 3 has proven crucial for epithelial organization and polarity of the embryonic epidermis (Lehmann et al, 2006). The knockdown of innexin 3 causes mislocalization of Innexin 2 and of DE-cadherin, causing cell polarity defects in the epidermis (Lehmann et al, 2006).…”

“…In Drosophila, the heteromerization of Innexin 2 and Innexin 3 has proven crucial for epithelial organization and polarity of the embryonic epidermis (Lehmann et al, 2006). The knockdown of innexin 3 causes mislocalization of Innexin 2 and of DE-cadherin, causing cell polarity defects in the epidermis (Lehmann et al, 2006). Therefore, it is possible that two pannexin family members need to be co-expressed to form heteromeric arrangements before they can function as intercellular gap junction channels.…”