Endocytic trafficking in actively resorbing osteoclasts

Abstract

Endocytosis and the subsequent intracellular trafficking of the endocytosed material are important determinants of cellular function. Osteoclasts, cells of the monocyte/macrophage family, are specialized for the internalization and processing of bone matrix. Transcytosis of endocytosed material has been observed in osteoclasts but the precise mechanism controlling this process is unclear. Here, we investigate the regulation of these trafficking events. To establish the directionality and kinetics of trafficking events in resorbing osteoclasts, we devised a system using fluorescent low-molecular-weight markers as probes to follow the route taken by the digested bone matrix. We demonstrate that this route is largely distinct from the pathway followed by proteins taken up by receptor-mediated endocytosis at the basolateral plasma membrane. Endocytosis and transcytosis from the ruffled border are fast processes, with a half-life of the endocytosed material inside the cells of 22 minutes. We demonstrate the crucial role of the microtubule network in transport from the ruffled-border area and provide evidence for a role of the cytoskeleton in the overall efficacy of trafficking. Moreover, we analyse the effect of the V-ATPase inhibitor bafilomycin A1 on endocytic uptake, which gives insight into the pH-dependent regulation of membrane trafficking and resorption in osteoclasts.

Microtubule Dynamic Instability Controls Podosome Patterning in Osteoclasts through EB1, Cortactin, and Src

“…MTs play a central role in processes essential for OC activity and bone resorption: polarization of the cells (66), vesicular traffic (67)(68)(69)(70)(71)(72), motility (68), and podosome belt formation (21,23,24,70,(73)(74)(75). Formation of the podosome belt has been the focus of this study because if other cell types use podosomes/invadosomes to migrate, adhere, and degrade the extracellular matrix (7), OCs are the only cells where podosomes follow a precise patterning such that clusters form rings that fuse and expand to form a characteristic peripheral belt (6,7,12), establishing the sealing zone when OCs are plated on bone or dentin (11).…”

The dynamic skeleton

“…All these components play unique and important roles in osteoclast action. In fact, loss of their function results in diseases which are characterized by excess bone mass such as osteopetrosis and pycnodysostosis [17].…”

The Cdc42 Guanine Nucleotide Exchange Factor FGD6 Coordinates Cell Polarity and Endosomal Membrane Recycling in Osteoclasts

“…Bone digestion also requires the secretion of lysosomal hydrolases into these resorption lacunae and the targeting of typical lysosomal membrane proteins to the ruffled membrane, in particular to acidify the resorption lacunae. After digestion, the digested bone material is endocytosed into transcytotic vesicles that fuse with the secretory domain of osteoclasts to release their content into the extracellular space (3). This transcytotic process using large surface areas of the ruffled border suggests the possibility that internalized membrane components are recycled back to the ruffled membrane.…”