Fig. 1

Identification of dystroglycan-associated proteins. a Schematic of Drosophila dystrophin glycoprotein complex (DGC). The transmembrane protein dystroglycan (Dg) is a major component of the DGC. Its C-terminal end binds the cytoplasmic protein dystrophin (Dys), while the heavily glycosylated N-terminus is associated with extracellular matrix (ECM) proteins. In humans, perturbed DGC function results in various types of neuromuscular disorders such as congenital muscular dystrophy (CMD), limb-girdle muscular dystrophy (LGMD), and Duchenne/Becker muscular dystrophy (DMD/BMD). b The pipeline used to identify muscle-specific Dg-associated proteins. Firstly, we generated transgenic animals expressing in the muscle tissue the full-length Dg tagged by the GFP at the C-terminal end (Mhc-Gal4/+, UAS Dg::GFP/+, abbreviated Mhc>Dg). Secondly, tissue lysates were subjected to co-immunoprecipitation procedure with GFP-Trap beads. Thirdly, immunoprecipitated Dg protein complexes were analyzed by mass spectrometry in order to identify Dg-interacting proteins. Lastly, revealed Dg-associated components were bioinformatically analyzed. c Coomassie Blue-stained gel confirms increased protein levels in the samples with muscle-specific Dg overexpression. Experiments were performed in triplicate. As a control, the driver line crossed to a wild-type line was used (Mhc-Gal4/OregonR, abbreviated Mhc>/+). d Schematic of muscle cell with subcellular compartments (plasma membrane, cytoskeleton, mitochondria, cytoplasmic vesicles, nucleus, endoplasmic reticulum, and Golgi apparatus). The scheme was created with the SERVIER Medical Art. Subcellular localization of the identified Dg-associated proteins. e Protein association network for Dg-interacting proteins clustered in functional groups, among which are structural components of the basal lamina, proteins with functions in muscle cell cytoskeleton, cellular transport regulators, mitochondria-associated proteins, factors regulating protein biosynthesis and degradation, cellular metabolism, nucleosome components, and proteins from the Hippo signaling pathway. Nodes represent proteins, lines and dashed lines connect associated proteins, and line thickness shows the confidence of association. Markov clustering algorithm identifies several protein complexes (thick blue lines) within basal lamina, cytoskeleton, protein degradation, mitochondria, and nucleosome groups