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Investigating the function of Zpg gap junction protein in Drosophila spermatogenesis by structure function analysis Kaur, Priya


Gap junction channels are intercellular transmembrane proteins that play an essential role in the development of tissues. They are a complex of proteins that can form homomeric or heteromeric channels between coupled cells to allow the passage of ions and small molecules. In Drosophila melanogaster, gap junctions are composed of innexins (Inx), which are structural homologues of vertebrate connexins. In Drosophila testes, Inx proteins contribute to spermatogenesis via bi-directional soma-germline communication. Inx2 on the somatic cells, which support the developing germline, and Zpg (Inx4) on the germline cells form a coupled heterotypic transmembrane channel in the fly testis. While the significance of these proteins is established, little is known about the nature of this intercellular communication. Our lab has previously demonstrated that flies lacking innexin expression have rudimentary gonads and impaired germline and somatic cell differentiation. The recently determined 3D protein structure of an innexin protein predicts that the N-terminus may be involved in regulating channel permeability. We are undertaking a detailed structure function analysis of innexin function in the context of the Drosophila testes using site-directed mutagenesis to generate mutations within the N-terminal and C-terminal domains that are predicted to disrupt functionally important residues. The effects on spermatogenesis are analysed using immunostainings and various functional assays. A 4 amino-acid truncation of the N-terminal results in a loss-of-function phenotype, and a deletion of the C-terminal disrupts the subcellular localization of the Zpg protein. This provides a mechanistic insight into the function of structurally important domains in innexins and further our understanding of germline stem cell regulation and maintenance during spermatogenesis.

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