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Removal of ubiquitinated targets by autophagosomes can be mediated by receptor molecules, like SQSTM1, in a mechanism referred to as selective autophagy. While cytoplasmic protein aggregates, mitochondria, and bacteria are the best-known targets of selective autophagy, their role in the turnover of membrane receptors is scarce. We here showed that fasting-induced wasting of skeletal muscle involves remodeling of the neuromuscular junction (NMJ) by increasing the turnover of muscle-type CHRN (cholinergic receptor, nicotinic/nicotinic acetylcholine receptor) in a TRIM63-dependent manner. Notably, this process implied enhanced production of endo/lysosomal carriers of CHRN, which also contained the membrane remodeler SH3GLB1, the E3 ubiquitin ligase, TRIM63, and the selective autophagy receptor SQSTM1. Furthermore, these vesicles were surrounded by the autophagic marker MAP1LC3A in an ATG7-dependent fashion, and some of them were also positive for the lysosomal marker, LAMP1. While the amount of vesicles containing endocytosed CHRN strongly augmented in the absence of ATG7 as well as upon denervation as a model for long-term atrophy, denervation-induced increase in autophagic CHRN vesicles was completely blunted in the absence of TRIM63. On a similar note, in trim63−/− mice denervation-induced upregulation of SQSTM1 and LC3-II was abolished and endogenous SQSTM1 did not colocalize with CHRN vesicles as it did in the wild type. SQSTM1 and LC3-II coprecipitated with surface-labeled/endocytosed CHRN and SQSTM1 overexpression significantly induced CHRN vesicle formation. Taken together, our data suggested that selective autophagy regulates the basal and atrophy-induced turnover of the pentameric transmembrane protein, CHRN, and that TRIM63, together with SH3GLB1 and SQSTM1 regulate this process.

Natural cartilage shows column orientation of cells and anisotropic direction of collagen fibers. However, matrices presently used in matrix-assisted autologous chondrocyte implantation do not show any fiber orientation. Our aim was to develop anisotropic scaffolds with parallel fiber orientation that were capable to support a cellular cartilaginous phenotype in vitro. Scaffolds were created by flock technology and consisted of a membrane of mineralized collagen type I as substrate, gelatine as adhesive, and parallel-oriented polyamide flock fibers vertically to the substrate. Confocal laser scan microscopy demonstrated that mesenchymal stem cells (MSCs) adhered and proliferated well in the scaffolds and cell vitality remained high over time. Articular chondrocytes seeded in a collagen type I gel into flock scaffolds deposited increasing amounts of proteoglycans and collagen type II over time. MSC-seeded flock scaffold constructs under chondrogenic conditions deposited significantly more proteoglycans and collagen type II than MSC collagen type I gel constructs only. Biomechanical testing revealed higher initial hardness of flock scaffolds than that of a clinically applied collagen type I/III scaffold combined with superior relaxation and an increasing hardness in MSC-loaded flock biocomposites during chondrogenesis. In conclusion, flock technology allows fabrication of scaffolds with anisotropic fiber orientation that mediates superior biomechanical and biochemical composition of tissue engineering constructs for cartilage repair.