Abstract
Brefeldin-A (BFA) is a specific and potent inhibitor of the intracellular transport of clathlin-uncoated transitional vesicles from the cisterns of rough-surfaced endoplasmic reticulum (RER) to the Golgi lamellae. This study was designed to clarify the effects of BFA on ultrastructure, subcellular localization of vacuolar-type H+-ATPase and a lysosomal cysteine proteinase, cathepsin K, in cultured osteoclasts and their resorptive function. H+-ATPase and cathepsin K are the most important enzymes for decalcification of apatite crystals and degradation of type-I collagen, respectively. In control cultures without BFA, osteoclasts were structurally characterized by the development of broad ruffled borders and clear zones, and formed many resorption lacunae in cocultured dentine slices. In BFA-treated cultures, osteoclasts lacked ruffled borders, and the cytoplasm was filled with regular-size and extremely large pale vacuoles over 2 microm in diameter, which were produced by fusion of adjacent vacuoles. BFA did not, however, inhibit clear zone formation and adhesion of osteoclasts to dentine slices. Resorption lacuna formation was markedly diminished by BFA treatment. Although H+-ATPase and cathepsin K were strongly expressed in osteoclast ruffled borders in control cultures, BFA treatment altered the subcellular localization and decreased the expression of these molecules. In BFA-treated cultures, H+-ATPase immunoreaction in osteoclasts was observed along the limiting membranes of some, but not all, regular-size pale vacuoles, but neither in extremely large vacuoles nor along the smooth plasma membranes facing the dentine slices. Similarly, cathepsin K was localized within lysosomes and some regular-size pale vacuoles, but its secretion toward the dentine slices through the ruffled borders was strongly inhibited by BFA treatment. These results suggest that 1.) formation of the osteoclast ruffled borders and their resorptive function are closely associated with the intracellular transport of these molecules from the RER cisterns and the Golgi lamellae to the ruffled borders, and 2.) both H+-ATPase and cathepsin K are selectively transported to the ruffled border membranes by pale vacuoles.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.