Abstract
Podosomes are dynamic actin-based structures found constitutively in cells of monocytic origin such as macrophages, dendritic cells and osteoclasts. They have been involved in osteoclast cell adhesion, motility and matrix degradation, and all these functions rely on the ability of podosomes to form supra-molecular structures called podosome belts or sealing zones on mineralized substrates. Podosomes contain two distinct domains, an actin-rich core enriched in actin polymerization regulators, surrounded by a ring of signaling and plaque molecules. The organization of podosome arrays into belts is linked to actin dynamics. Cofilin is an actin-severing protein that is known to regulate cytoskeleton architecture and cell migration. Cofilin is present in lamellipodia and invadopodia where it regulates actin polymerization. In this report, we show that cofilin is a novel component of the podosome belt, the mature osteoclast adhesion structure. Time-course analysis demonstrated that cofilin is activated during primary osteoclast differentiation, at the time of podosome belt assembly. Immunofluorescence studies reveal a localization of active cofilin in the podosome core structure, whereas phosphorylated, inactive cofilin is concentrated in the podosome cloud. Pharmacological studies unraveled the role of a specific cofilin phosphatase to achieve cofilin activation during osteoclast differentiation. We ruled out the implication of PP1/PP2A and PTEN in this process, and rather provided evidence for the involvement of SSH1. In summary, our data involve cofilin as a regulator of podosome organization that is activated during osteoclast differentiation by a RANKL-mediated signaling pathway targeting the SSH1 phosphatase.
Highlights
Osteoclasts (OCs) are multinucleated cells of hematopoietic origin that degrade bone matrix
Primary OCs were generated by stimulating mouse bone marrow-derived macrophages (BMMs) with M-CSF and RANKL, and the organization of the actin network was recorded in parallel with cofilin activation
Podosome belts accumulated at day 3 (Figure 1B), and this maturation of podosome arrays into belts was associated with a strong reduction in the phosphocofilin level which dropped to 30% (Figure 1C), revealing an activation of cofilin at this stage of osteoclastogenesis
Summary
Osteoclasts (OCs) are multinucleated cells of hematopoietic origin that degrade bone matrix. To perform this function, OCs must adhere firmly to bone using specialized adhesion structures called podosomes [1]. Podosomes are highly dynamic structures, containing an actin-rich core extending perpendicularly to the substrate, surrounded by a ring of associated proteins [2,3]. The core of podosomes is enriched in several actin-associated proteins (Arp2/3, cortactin, WASp, WIP, dynamin, gelsolin) [2], which regulate actin polymerization [4]. The actin cores undergo continuous polymerization and severing processes, and this dynamic was recently shown to play a critical role in regulating podosome stability in OCs [5]
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