Podosomes in osteoclast-like cells: structural analysis and cooperative roles of paxillin, proline-rich tyrosine kinase 2 (Pyk2) and integrin alphaVbeta3.

Macrophages and osteoclasts develop unique contact sites with the extracellular matrix called podosomes. Podosomes have been associated with migratory and invasive cell characteristics, but a basic mechanism outlining their function is lacking. We have used chicken and human monocytes differentiating in vitro into osteoclast-like cells in the presence of RANKL-ODF ...
to study these cytoskeletal structures. During the differentiation process, podosomes are redistributed from the cell body in early macrophages to the cell periphery in increasingly spread and multinucleated cells expressing high levels of integrin alphaVbeta3. 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. Paxillin and the FAK-related kinase Pyk2 colocalized with integrin alphaVbeta3 in the juxtamembrane region surrounding individual podosomes. In lysates of macrophages and differentiated osteoclasts both paxillin and Pyk2 associated with synthetic and recombinant polypeptides containing the C-terminal region 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-dependent tyrosinephosphorylation of Pyk2 and paxillin however did not detectably alter their interaction with beta3 tail peptides in cell lysates. Our results provide novel insight into the molecular architecture and the phosphorylation dynamics in podosomes. Moreover, they outline a novel potential mechanism for the recruitment of paxillin and Pyk2 to beta3 integrin-dependent cell contacts.
Mesh Terms:
Actins, Amino Acid Sequence, Animals, Carrier Proteins, Cell Adhesion, Cells, Cultured, Chickens, Cytoskeletal Proteins, Cytoskeleton, Fluorescent Antibody Technique, Focal Adhesion Kinase 2, Macrophages, Membrane Glycoproteins, Molecular Sequence Data, Monocytes, Osteoclasts, Paxillin, Phosphoproteins, Phosphorylation, Protein-Tyrosine Kinases, RANK Ligand, Receptor Activator of Nuclear Factor-kappa B, Receptors, Vitronectin, Tyrosine
J. Cell. Sci.
Date: Aug. 01, 2001
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