A model of muscle atrophy based on live microscopy of muscle remodelling inDrosophilametamorphosis

Abstract

Genes controlling muscle size and survival play important roles in muscle wasting diseases. In Drosophila melanogaster metamorphosis, larval abdominal muscles undergo two developmental fates. While a doomed population is eliminated by cell death, another persistent group is remodelled and survives into adulthood. To identify and characterize genes involved in the development of remodelled muscles, we devised a workflow consisting of in vivo imaging, targeted gene perturbation and quantitative image analysis. We show that inhibition of TOR signalling and activation of autophagy promote developmental muscle atrophy in early, while TOR and yorkie activation are required for muscle growth in late pupation. We discovered changes in the localization of myonuclei during remodelling that involve anti-polar migration leading to central clustering followed by polar migration resulting in localization along the midline. We demonstrate that the Cathepsin L orthologue Cp1 is required for myonuclear clustering in mid, while autophagy contributes to central positioning of nuclei in late metamorphosis. In conclusion, studying muscle remodelling in metamorphosis can provide new insights into the cell biology of muscle wasting.

Genetic screen in Drosophila muscle identifies autophagy-mediated T-tubule remodeling and a Rab2 role in autophagy

“…During metamorphosis, changes in IOM cell size and myofibril content have been noted (Kuleesha et al, 2014, 2016). We previously showed that wildtype IOMs undergo dramatic cortical and membrane remodeling with costamere integrin adhesion complex disassembly and reassembly at discrete pupal stages (Ribeiro et al, 2011).…”

Live Imaging of muscle histolysis inDrosophilametamorphosis

“…Compared to TOR TED , Rm62-RNAi induced histolysis occurred 15 hours later and did not delay histolysis of DEOMs. In the context of abdominal muscles, TOR TED did not exactly behave like a dominant negative version of the endogenous TOR protein since TOR-RNAi mainly affected cell size and had negligible effects on cell survival [30]. We can only speculate that the regulation of cell survival and cell growth are mediated by different protein complexes.…”

“…Since TOR TED is a potent inducer of autophagy in the fat body [41], activation of autophagy may trigger cell death of wildtype DEOMs and TOR TED overexpressing DIOMs. In a parallel study, we found that RNAi of 5 autophagy related genes (Atg5, Atg9, Atg12, Atg17, and Atg18) caused inhibition of atrophy during the remodeling of DIOMs [30]. We examined 25 time-lapse datasets of pupae expressing shRNAs against Atg9 (7) and Atg5, Atg12 and Atg18 (6 each).…”

“…The UAS-shRNA and UAS-effector transgenic lines affecting cell survival and death of larval abdominal muscles studied in this report are listed in Table 1. A complete list of the UAS-lines used for gene perturbations in out pilot screen was previously described [30] and is provided as a supplement (Additional file 1: Table S1). In addition, we knocked down TOR by RNAi using the stock B-35578 (TRiP# GL00156).…”

“…Using custom software, we created time-lapse maximum intensity projections (MIPs) of 3D image stacks and performed time-series image analysis of muscles development during metamorphosis. We had earlier performed a pilot screen with 119 publicly available UAS fly stocks, involving 98 unique genes, which were targeted by 100 RNAi and 19 protein-| 14 P a g e overexpression constructs [30] (Additional file 1: Table S1). Five of the gene perturbations specifically interfered with cell death or survival of larval muscles without affecting the development of newly formed adult muscles (Table 1).…”