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Vol. 11, Issue 1, 255-268, January 2000
and
*Department of Pathology and Cell Biology, University of Montreal,
Montreal, Quebec, Canada H3C 3J7; and Transfection of Mv1Lu mink lung type II alveolar cells with
Samuel Lunenfeld
Research Institute, Mount Sinai Hospital, University of Toronto,
Toronto, Ontario, Canada M5G 1X5
1-6-N-acetylglucosaminyl transferase V is associated
with the expression of large lysosomal vacuoles, which are
immunofluorescently labeled for the lysosomal glycoprotein
lysosomal-associated membrane protein-2 and the 1-6-branched
N-glycan-specific lectin phaseolis vulgaris
leucoagglutinin. By electron microscopy, the vacuoles present the
morphology of multilamellar bodies (MLBs). 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 treatment.
Heterologous structures containing both membrane lamellae and
peripheral electron-dense regions appear 15 h after leupeptin
addition and are indicative of ongoing lysosome-MLB fusion. Leupeptin
washout is associated with the formation after 24 and 48 h of
single or multiple foci of lamellae within the autophagic vacuoles,
which give rise to MLBs after 72 h. Treatment with
3-methyladenine, an inhibitor of autophagic sequestration, results in
the significantly reduced expression of multilamellar bodies and the
accumulation of inclusion bodies resembling nascent or immature
autophagic vacuoles. Scrape-loaded cytoplasmic FITC-dextran is
incorporated into lysosomal-associated membrane protein-2-positive MLBs, and this process is inhibited by 3-methyladenine, demonstrating that active autophagy is involved in MLB formation. Our results indicate that selective resistance to lysosomal degradation within the
autophagic vacuole results in the formation of a microenvironment propicious for the formation of membrane lamella.
These authors contributed equally to this work.
§
Corresponding author. E-mail address:
ivan.robert.nabi{at}umontreal.ca.
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