Biogenesis of multilamellar bodies via autophagy

Transfection of Mv1Lu mink lung type II alveolar cells with beta 1-6-N-acet ylglucosaminyl transferase V is associated with the expression of large lys osomal vacuoles, which are immunofluorescently labeled for the lysosomal gl ycoprotein lysosomal-associated membrane protein-2 and the beta 1-6-branche d N-glycan-specific lectin phaseolis vulgaris leucoagglutinin. By electron microscopy, the vacuoles present the morphology of multilamellar bodies (ML Bs). Treatment of the cells with the lysosomal protease inhibitor leupeptin results in the progressive transformation of the MLBs into electron-dense autophagic vacuoles and eventual disappearance of MLBs after 4 d of treatme nt. Heterologous structures containing both membrane lamellae and periphera l electron-dense regions appear 15 h after leupeptin addition and are indic ative of ongoing lysosome-MLB fusion. Leupeptin washout is associated with the formation after 24 and 48 h of single or multiple foci of lamellae with in the autophagic vacuoles, which give rise to MLBs after 72 h. Treatment w ith 3-methyladenine, an inhibitor of autophagic sequestration, results in t he significantly reduced expression of multilamellar bodies and the accumul ation of inclusion bodies resembling nascent or immature autophagic vacuole s. Scrape-loaded cytoplasmic FITC-dextran is incorporated into lysosomal-as sociated membrane protein-2-positive MLBs, and this process is inhibited by 3-methyladenine, demonstrating that active autophagy is involved in MLB fo rmation. Our results indicate that selective resistance to lysosomal degrad ation within the autophagic vacuole results in the formation of a microenvi ronment propicious for the formation of membrane lamella.