Search

Calmodulin (CAM) is recognized as a major intermediary in intracellular calcium signaling, but as yet little is known of its role in developmental and behavioral processes. We have generated and studied mutations to the endogenous Cam gene of Drosophila melanogaster that change single amino acids within the protein coding region. One of these mutations produces a striking pupal lethal phenotype involving failure of head eversion. Various mutant combinations produce specific patterns of ectopic wing vein formation or melanotic scabs on the cuticle. Anaphase chromosome bridging is also seen as a maternal effect during the early embryonic nuclear divisions. In addition, specific behavioral defects such as poor climbing and flightlessness are detected among these mutants. Comparisons with other Drosophila mutant phenotypes suggests potential CAM targets that may mediate these developmental and behavioral effects, and analysis of the CAM crystal structure suggests the structural consequences of the individual mutations.

The myotonic dystrophies are prototypic toxic RNA gain-of-function diseases. Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are caused by different unstable, noncoding microsatellite repeat expansions - (CTG) DM1 in DMPK and (CCTG) DM2 in CNBP Although transcription of mutant repeats into (CUG) DM1 or (CCUG) DM2 appears to be necessary and sufficient to cause disease, their pathomechanisms remain incompletely understood. To study the mechanisms of (CCUG) DM2 toxicity and develop a convenient model for drug screening, we generated a transgenic DM2 model in the fruit fly Drosophila melanogaster with (CCUG) n repeats of variable length ( n =16 and 106). Expression of noncoding (CCUG) 106 , but not (CCUG) 16 , in muscle and retinal cells led to the formation of ribonuclear foci and mis-splicing of genes implicated in DM pathology. Mis-splicing could be rescued by co-expression of human MBNL1, but not by CUGBP1 (CELF1) complementation. Flies with (CCUG) 106 displayed strong disruption of external eye morphology and of the underlying retina. Furthermore, expression of (CCUG) 106 in developing retinae caused a strong apoptotic response. Inhibition of apoptosis rescued the retinal disruption in (CCUG) 106 flies. Finally, we tested two chemical compounds that have shown therapeutic potential in DM1 models. Whereas treatment of (CCUG) 106 flies with pentamidine had no effect, treatment with a PKR inhibitor blocked both the formation of RNA foci and apoptosis in retinae of (CCUG) 106 flies. Our data indicate that expression of expanded (CCUG) DM2 repeats is toxic, causing inappropriate cell death in affected fly eyes. Our Drosophila DM2 model might provide a convenient tool for in vivo drug screening., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Appropriate cell-cell signaling is crucial for proper tissue homeostasis. Protein sorting of cell surface receptors at the early endosome is important for both the delivery of the signal and the inactivation of the receptor, and its alteration can cause malignancies including cancer. In a genetic screen for suppressors of the pro-apoptotic gene hid in Drosophila, we identified two alleles of vps25, a component of the ESCRT machinery required for protein sorting at the early endosome. Paradoxically, although vps25 mosaics were identified as suppressors of hid-induced apoptosis, vps25 mutant cells die. However, we provide evidence that a non-autonomous increase of Diap1 protein levels, an inhibitor of apoptosis, accounts for the suppression of hid. Furthermore, before they die, vps25 mutant clones trigger non-autonomous proliferation through a failure to downregulate Notch signaling, which activates the mitogenic JAK/STAT pathway. Hid and JNK contribute to apoptosis of vps25 mutant cells. Inhibition of cell death in vps25 clones causes dramatic overgrowth phenotypes. In addition, Hippo signaling is increased in vps25 clones, and hippo mutants block apoptosis in vps25 clones. In summary, the phenotypic analysis of vps25 mutants highlights the importance of receptor downregulation by endosomal protein sorting for appropriate tissue homeostasis, and may serve as a model for human cancer.