No abstract

Background: Underneath the membrane of most animal cells is a network (membrane skeleton) assembled using tetramers of the protein spectrin. Results: Although spectrins are essential proteins, tetramer formation is surprisingly unimportant for Drosophila development. Conclusion:The major roles of the membrane skeleton do not require a conventional network. Significance: The ubiquitous model of the spectrin-based membrane skeleton has limited applicability.

Disruption of epithelial barriers is a common disease manifestation in chronic degenerative diseases of the airways, lung and intestine. Extensive human genetic studies have identified risk loci in such diseases, including in chronic obstructive pulmonary disease (COPD) and inflammatory bowel diseases (IBD). The genes associated with these loci have not fully been determined, and functional characterization of such genes requires extensive studies in model organisms. Here, we report the results of a screen in Drosophila melanogaster that allowed for rapid identification, validation and prioritization of COPD risk genes that were selected based on risk loci identified in human genome-wide association studies (GWAS) studies. Using intestinal barrier dysfunction in flies as a readout, our results validate the impact of candidate gene perturbations on epithelial barrier function in 56% of the cases, resulting in a prioritized target gene list. We further report the functional characterization in flies of one family of these genes, encoding for nicotinic acetylcholine receptor subunits (nAchR). We find that nAchR signaling in enterocytes of the fly gut promotes epithelial barrier function and epithelial homeostasis by regulating the production of the peritrophic matrix. Our findings identify COPD associated genes critical for epithelial barrier maintenance, and provide insight into the role of epithelial nAchR signaling for homeostasis.

Nuclear-cytoplasmic communication is not limited to nuclear pores, with both proteins and RNA using alternative routes between these compartments. We previously characterized cytoplasmic capes (large invaginations of the nuclear envelope in Drosophila), which are enriched for the membrane-bound EGF receptor ligand mSpitz, endosome-related organelles and ubiquitylated proteins. Closely associated with capes are groups of perinuclear vesicles resembling those seen at sites of RNP export via a budding mechanism. Here, we demonstrate that mSpitz delivery to capes requires passage through the endosomal system. We also show that capes are closely associated with sites of non-canonical RNP export as well as the dFrizzled2 receptor C terminal fragment, a core component of this export pathway. Video microscopy of glands in intact larvae indicates that cytoplasmic capes are stable structures that persist for at least 90 minutes without conspicuous growth. We further show that capes appear with the growth of the salivary gland rather than its developmental stage. Finally, we show that the large F-actin binding protein βH-spectrin, which modulates endosomal trafficking, as well as its partner βH-spectrin are required for cape formation. Cytoplasmic capes therefore represent a subspecialization of the nuclear envelope where endosomal trafficking and RNP export are closely associated.