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Depending on the substrate, upon which osteoclasts are spread, they adhere either via podosomes or via sealing zones. Cells cultured on ECM coated glass form podosomes, which are special integrin-mediated adhesion structures of cells from the monocytic lineage. Podosomes are highly dynamic, actin rich structures with a half- life of 2-12 min [240]. They are also found in transformed fibroblasts, carcinoma cell lines and smooth muscle cells [39; 241]. Beside their function in adhesion, podosomes play a role in migration. They form at the leading edge and anchor the cell during translocation [242]. Furthermore, they are important for matrix remodelling and tissue invasion and accordingly, matrix metalloproteases such as MT1-MMP and MMP-9 localize to podosomes [39]. They have many molecular components in common with focal adhesions but differ from them in function and structure. They arrange already one hour after cell-substrate contact and are not dependent on de novo protein synthesis in contrast to focal adhesions [39].

Podosomes have a diameter of approximately 0.5-1 µm. They are composed of a central actin core surrounded by a ring of plaque proteins (Figure 17). Podosomes contain cytoskeletal components and regulators, protein kinases and RhoGTPases. Podosome assembly is controlled by several RhoGTPases, among them Cdc42, controls. The podosome core consists primarily of F-actin and actin associated molecules, such as WASp, WASp interacting protein (WIP) and Arp2/3 complex. These proteins regulate actin assembly and reorganization within the core and are activated downstream of Cdc42. The cell surface receptor CD44 plays a major role in nucleating podosome cores by binding to WASp. CD44 is a widely expressed receptor for hyaluronic acid but it can also bind to osteopontin, collagen and laminin. Ligand bound CD44 triggers signaling pathways, which are sufficient to initiate podosome core formation [243]. Lack of functional CD44, WASp, WIP or Arp2/3 lead to impaired podosome formation [39; 244; 245]. Cytochalasin or latrunculin treatment leads to disassembly of podosomes, suggesting that the F-actin core is essential for their stability [240; 246]. The actin core is surrounded by a cloud of G-actin and actin oligomers, which is a source for actin turnover. While the actin core depends on CD44, the actin cloud is organized by integrins but both actin subdomains are interconnected by actin branching and crosslinking proteins such as cortactin and fimbrin [240; 243].

Integrins, predominantly integrin V3, and integrin associated proteins are mainly

located in the ring structure. Integrin/ligand interactions lead to autophosphorylation of Pyk2 (proline-rich tyrosine kinase 2), necessary for complex formation with Src [247], and activation of Src and PI3K inducing the phosphorylation of p130Cas and Pyk2 at distinct sites by Src [248-250]. Also paxillin is phosphorylated upon integrin engagement. It binds to cytoplasmic domains of integrin subunits and can provide a

link to actin by interaction with vinculin, which in turn binds to talin and -actinin [39].

Dephosphorylation events are involved in podosome turnover.

Podosomes are closely associated with microtubules and are influenced by them but the exact mechanism is still unclear [246]. Microtubules are not required for de novo assembly of podosomes, while reorganization, fission and fusion of podosomes are based on microtubule dynamics [251]. Podosomes also crosstalk with intermediate filaments and vimentin localizes to them.

Figure 17: Podosome model, cross section perpendicular to the substrate. The membrane is delineated by a black line. Membrane invaginations can provide space for matrix metallopro- teinase secretion. The upper left panel shows the podosomal ring structure. Integrins mediate the connection to the ECM. A complex consisting of paxillin, Src, Pyk2, gelsolin, PI3K and p130Cas is recruited to the adhesion site, vinculin, talin and -actinin link the complex to actin. The upper right panel represents the podosomal core structure. WASp is activated by Cdc42 at the plasma membrane leading to the activation of the Arp2/3 complex. Cortactin and fimbrin crosslink the actin filaments [39].

Podosomes arrange into higher ordered structures. The actin cores of neighbouring podosomes are connected by radial actin filaments. In osteoclast precursors and immature osteoclasts, podosomes are usually organized in clusters that are embedded in an actin cloud composed of monomeric and polymerized actin [240]. The clusters can rearrange into short-lived rings. In fully differentiated osteoclasts these rings expand to a stable belt at the cell periphery by a treadmilling-like mechanism (Figure 18). Podosomes are newly formed at the periphery and disassembled in the centre. The centrifugal movement is reminiscent of Src activity waves [252]. Furthermore, this transition is dependent on microtubules, which are acetylated during the rearrangement process. Nocodazole treatment results in disorganisation of podosome belts and reappearance of clusters and rings [253].

When mature osteoclasts adhere to mineralized matrix, calcium apatite crystals induce the formation of sealing zones in a microtubule dependent process (Figure 18) [265]. Sealing zones consist of an actin ring with a width and height of approximately 4 µm and an inner and outer ring of vinculin and other plaque proteins. Sealing zones are composed of structural units that are closely related to individual podosomes but they are packed at very high density and are more heavily interconnected by a network of actin filaments than clustered podosomes [254].

Figure 18: Scheme of podosome organizations along osteoclast differentiation. Preosteoclasts arrange their podosomes in clusters that evolve into dynamic rings. These rings expand to a belt at the cell periphery in mature osteoclast stabilized by acetylated microtubules. Osteoclasts adherent on mineralized matrix form sealing zones [253].