Atp6v1c1 is an essential component of the osteoclast proton pump and in F-actin ring formation in osteoclasts.

Vacuolar-type proton-translocating ATPases (V-ATPases) are composed of an ATP-hydrolytic domain (V1) and a proton-translocation domain (V0), as well as the accessory subunits ac45 and M8-9 [1]. The V1 domain is located in the cytoplasm and contains eight different subunits (A–H): the catalytic nucleotide-binding subunit A, the non-catalytic nucleotide-binding subunit B (stoichiometry of A3:B3), and the stalk subunits C-H (proposed stoichiometry of C1:D1:E1:F1:G2:H1) [2-4]. The V0 domain, an integral membrane bound domain, connects to the V1 domain by the central and peripheral stalks. In mammals, the V0 domain consists of a, c, c′, c″,e and d subunits. V-ATPases are involved in a wide variety of physiological processes, including endocytosis, intracellular membrane trafficking, macromolecular processing and degradation, and ligand-coupled transport, but they are primarily responsible for the acidification of intracellular compartments in all eukaryotic cells [3;5]. For their diverse functions, V-ATPases utilize specific subunit isoform(s) in specific cells and cell organelles. For instance, a4, B1, C2b, d2, and G3 are highly expressed in the kidney and epididymis [6-10]; a1 and G2 are highly expressed in the brain [11]; and a3, B2, and d2 are highly expressed in osteoclasts [9;12;13]. Osteoclasts are the principal cells responsible for bone resorption. Their ruffled membrane V-ATPases tightly coupling to a passive chloride channel are required for acidification of the resorption lacunae and bone matrix demineralization [14]. Therefore, we reason that osteoclast V-ATPases present in the ruffled border are comprised of unique subunits such as a3, B2, and other unknown subunits, which are different from other V-ATPases [12;15]. In this study, we defined another subunit of the osteoclast proton pump. As we know, in mouse V-ATPase there are three isoforms of subunit C: Atp6v1c1 (C1), Atp6v1c2a (C2a), and Atp6v1c2b (C2b). C1 is expressed ubiquitously, but C2a is a lung-specific isoform containing a 46-amino acid insertion and C2b is a kidney-specific isoform without the insert [10;16]. Despite these insights, the possible role of C1 as an essential component of the osteoclast proton pump; C1's function in an osteoclast proton pump; its localization in activated osteoclasts; and its function in the differentiation, maturation, acidification, and bone resorption of mature osteoclasts, remain unclear. In multinucleated osteoclasts, bone resorption is active in the ruffled border surrounded by a tight sealing zone, which contains highly dynamic actin-containing adhesion structures known as podosomes. These podosomes are composed of a small filamentous actin (F-actin) column surrounded by proteins such as vinculin and paxillin. Protons and enzyme secretions are restricted to the resorption lacuna, limited by the sealing zone. However, in mature osteoclasts seeded on glass or plastic, podosomes form a belt on the cell periphery [17-19]. Previous reports showed that the formation of actin sealing rings was severely impaired in osteoclasts deficient in Atp6v0d2 [20], while formation was uncertain in Atp6v0a3 deficient osteoclasts [21;22]. These results prompted us to wonder whether V-ATPase subunits are also involved in F-actin ring formation during osteoclast activation. Recently, Vitavska et al. reported that in vitro subunit C can directly bind and stabilize F-actin, increase the initial rate of actin polymerization in a concentration-dependent manner, and cross-link actin filaments to bundles of varying thickness [23;24]. According to these findings we speculate that subunit C1 may be involved in F-actin ring formation during osteoclast activation. Here we show that subunit C1 is highly expressed in mouse osteoclasts and that its expression can be induced by receptor activator of NF-κB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF) during osteoclast differentiation. The acidification activity and the bone resorption function of C1-depleted cells were severely impaired, but osteoclast differentiation and maturation were normal. Furthermore, immunohistochemistry showed that C1 was expressed mainly in the ruffled membrane. The co-IP assay showed that C1 interacts with subunit a3 and that C1 co-localized with microtubules in the plasma membrane and its vicinity in mature osteoclasts. We also found that F-actin ring formation was severely defected in C1-depleted osteoclasts and that C1 co-localized with F-actin both in the cell periphery and in the cytoplasm of mature osteoclasts.