Podosomes in osteoclast-like cells: structural analysis and cooperative roles of paxillin, proline-rich tyrosine kinase 2 (Pyk2) and integrin alpha Vbeta 3

Macrophages and osteoclasts develop unique contact sites with the extracell ular matrix called podosomes. Podosomes have been associated with migratory and invasive cell characteristics, but a basic mechanism outlining their f unction is lacking. We have used chicken and human monocytes differentiatin g in vitro into osteoclast-like cells in the presence of RANKL-ODF to study these cytoskeletal structures. During the differentiation process, podosom es are redistributed from the cell body in early macrophages to the cell pe riphery in increasingly spread and multinucleated cells expressing high lev els of integrin alphaV beta3. Immunofluorescence with anti-phosphotyrosine antibodies revealed increased tyrosine-phosphorylation at the basal tips of these podosomes. RANKL-ODF treatment reinforced the peripheral location of podosomes and initiated their partial fusion to larger F-actin-containing structures that displayed reduced levels of tyrosine phosphorylation. Paxil lin and the FAK-related kinase Pyk2 colocalized with integrin alphaV beta3 in the juxtamembrane region surrounding individual podosomes. In lysates of macrophages and differentiated osteoclasts; both paxillin and Pvk2 associa ted with synthetic and recombinant polypeptides containing the C-terminal r egion of the integrin beta3 cytoplasmic domain. These in vitro interactions were direct and they were abolished by substitutions in the beta3 integrin peptides known to disrupt integrin function in vivo. The marked adhesion-d ependent tyrosine-phosphorylation of Pyk2 and paxillin however did not dete ctably alter their interaction with beta3 tail peptides in cell lysates. Ou r results provide novel insight into the molecular architecture and the pho sphorylation dynamics in podosomes. Moreover, they outline a novel potentia l mechanism for the recruitment of paxillin and Pyk2 to beta3 integrin-depe ndent cell contacts.