Intracellular Fate of Endocytosed Collagen in Rat Liver Endothelial Cells

Abstract

The fate of endocytosed collagen (COL) in sinusoidal liver endothelial cells was studied using COL labeled with FITC (F-COL) or iodine (125I-COL) or both (125I-FCOL). In pulse–chase experimentsin vitro,F-COL localized after 10 min along the limiting membrane of vesicles, taking the appearance of rings. After 20 min chase the probe appeared more concentrated in fewer but larger ring structures, and after 60 min the probe was observed mainly in the interior of smaller vesicles. Gel filtration of solubilized cultures after pulse-chase experiments using125I-FOL or125I-COL revealed that degradation was initiated and largely completed in the small, filled vesicles, judged as a pre- or early lysosomal compartment. In the presence of monensin, or by incubation at 20°C, the probe was arrested at the level of the larger ring structures, and degradation could not be observed. Lysosomal preloading by iv injection of TRITC-COL (T-COL) 24 h prior to pulse–chase experiments with cultured cells using F-COL disclosed colocalization of the two dyes only after 8 h in perinuclear vesicles of a size larger than the early lysosomal vesicles observed after 60 min, suggesting a slow, unidirectional transport of F-COL to the T-COL-labeled lysosomal compartment. Esterase reaction product was observed mainly in vesicles resembling the double-stained lysosomes. We conclude that (1) the ring structures observed after 10 and 20 min represent early endosomes, (2) the vesicles appearing after 60 min are prelysosomes mediating degradation, and (3) the lysosomes accumulating in the probe after 8 h are responsible for final degradation and storage of residual stain.