Immunocytochemistry studies have established that multivesicular bodies (MVBs) in alveolar type II epithelial cells are important for processing pulmonary surfactant protein precursors (proSP; Voorhout et al., 1992 and 1993). Localization of pulmonary surfactant protein A (SP-A) gold and cationic ferritin to the MVBs demonstrated that MVBs also serve as endosomal/lysosomal compartments for recycling SPs (Williams et al., 1984, Ryan et al., 1989). However, these studies did not elucidate whether the biosynthesis of pulmonary SPs and endocytosis occur in the same MVBs. In this study, we used primary murine alveolar type II epithelial cell cultures as a model to determine if the biosynthetic and endocytic compartments colocalized in MVBs. The endocytic compartments of MVBs were identified using BSA gold conjugates, while the biosynthetic pathway was detected with antibodies directed against proSPs.

Alveolar type II epithelial cells isolated from 6 weeks old C57B/6 mice were grown on Transwell cell inserts in the presence of 6 nm BSA gold conjugates (BSAG6) adjusted to final concentration of OD520 5.0 in DMEM, 25 mM HEPES, and 10% fetal bovine serum, pH 7.3, at 37 °C for 20 hours.