Characteristics of Unusual Endocytic Trafficking in Osteoclasts

Objective: Bone resorption by osteoclasts depends on subdomains of the plasma membrane called ruffled membranes that are packed with vacuolar H+-ATPase (V-ATPase), an enzyme that is excluded from the plasma membrane of most cell types. Preliminary studies suggest that interactions between V-ATPase and the actin cytoskeleton are associated with vesicular sorting that is required for ruffled membrane formation. Here, live cell imaging has been utilized to further characterize V-ATPase-rich compartments and sorting in osteoclasts. Materials and Methods: Osteoclasts were differentiated from precursors derived from mouse marrow and from RAW 264.7 cells stimulated with RANKL. Acidic compartments were detected with Lysotracker. Endocytic compartments were identifed with cholera toxin beta chain (CTB). Actin polymerization was disrupted using cytochalasin B. Microtubules were disrupted with nocodozole. Wortmannin was used to block the activity of phosphatidylinositol 3-kinase (PI 3-kinase). Immunotaining was performed with anti-E subunit antibody to detect V-ATPase. Results: We found that in osteoclast precursors, CTB was endocytosed to non-acidic compartments, but after osteoclast formation, CTB was directed to acidic, compartments that were rich in V-ATPase and GM1. In inactive osteoclasts, these CTB-rich acidic compartments were stably-positioned within the osteoclast, and were not disturbed by blocking PI 3-kinase activity for 15 minutes. Disruption of microtubules led to rapid movement (collapse) of the acidic organelles toward the center of the cell. Blocking actin polymerization with cytochalasin B had little short term effect on the distribution of acidic organelles. In resorbing osteoclasts, GM1 and V-ATPase were enriched in ruffled membranes. Conclusions: Osteoclastogenesis is associated with changes in endocytic sorting leading to V-ATPase-rich acidic vesicles that give rise to ruffled membranes as osteoclasts activate to resorb bone. Maintenance of positioning of vesicles within osteoclasts is dependent on both microfilaments and microtubules. This research was supported by a UFCD Student Summer Research Fellowship and AR-47959 (LSH).