A Drosophila Model of Epidermolysis Bullosa Simplex

Abstract

The blistering skin disorder Epidermolysis bullosa simplex (EBS) results from dominant mutations in K5 or K14 genes, encoding the intermediate filament network of basal epidermal keratinocytes. The mechanisms governing keratin network formation and collapse due to EBS mutations remain incompletely understood. Drosophila lacks cytoplasmic intermediate filaments, providing a ‚null’ environment to examine the formation of keratin networks and determine mechanisms by which mutant keratins cause pathology. Here, we report that ubiquitous co-expression of transgenes encoding wild-type human K14 and K5 resulted in the formation of extensive keratin networks in Drosophila epithelial and non-epithelial tissues, causing no overt phenotype. Similar to mammalian cells, treatment of transgenic fly tissues with phosphatase inhibitors caused keratin network collapse, validating Drosophila as a genetic model system to investigate keratin dynamics. Co-expression of K5 and a K14R125C mutant that causes the most severe form of EBS resulted in widespread formation of EBS-like cytoplasmic keratin aggregates in epithelial and non-epithelial fly tissues. Expression of K14R125C/K5 caused semi-lethality; adult survivors developed wing blisters and were flightless due to lack of intercellular adhesion during wing heart development. This Drosophila model of EBS is valuable for the identification of pathways altered by mutant keratins and for development of EBS therapies.

Insulin and TOR signal in parallel through FOXO and S6K to promote epithelial wound healing

“…Because many signalling pathways are conserved between Drosophila and mammals and many labs use Drosophila larvae to accelerate drug discovery for treatment in cancer or diabetes and other chronic diseases, this model may be useful for such purposes in the future 44 45 46 . Similarly, Drosophila larvae may be used to test the effects of dominant mutations of human genes to understand their mechanism and function, as recently described, for example, for the human keratin networks and their role in epidermolysis bullosa simplex 47 .…”

Desmosomes and Intermediate Filaments: Their Consequences for Tissue Mechanics

“…EBS is characterized by cytoplasmic keratin aggregates, cytolysis of basal keratinocytes, and bullous lesions following mild trauma to the skin. Although it is recognized that the pathomechanisms contributing to EBS and additional keratinopathies are more complex than originally considered Roth et al 2012;Bohnekamp et al 2015;Hobbs et al 2016;Kumar et al 2016), it is evident that loss of an intact keratin cytoskeleton renders keratinocytes fragile on mild physical stress, shown by KRT5 and KRT14 KO mice (Lloyd et al 1995;Peters et al 2001). Of note, even mutations causing severe disease do not prevent formation of long keratin intermediate filaments (KIFs) in vitro (Herrmann et al 2002), suggesting that mutations and physical stress act at the level of keratin bundling, network organization, dynamics, or by affecting association with other proteins.…”

MEK inhibitor cobimetinib rescues a dRaf mutant lethal phenotype in Drosophila melanogaster

“…Because of the very early lethality with ubiquitous GAL4 drivers, we used a temperature‐sensitive Act5C‐GAL4 driver to ubiquitously express the FLAG‐tagged dRaf A572E for 16 hours (verification of dRaf A572E expression) or 16‐64 hours (ERK phosphorylation) and used total proteins of third‐instar larvae for Western blot analysis . For a more detailed description, see Appendix ,and Experimental Design.…”