FIG. 1.

Dynamin and Pyk2 colocalize within the actin podosome belt and form a molecular complex in osteoclasts. (A) Confocal immunofluorescence microscopy of authentic mouse osteoclasts plated on glass and labeled for Pyk2 (blue), dynamin (green), and actin (rhodamine-phalloidin; red). Images from the three fluorescence channels were merged using Adobe Photoshop 6.0. The yellow arrow indicates overlapping expression of dynamin, actin, and Pyk2 within the podosome belt, while the arrowhead shows peripherally localized Pyk2. Also shown are high-magnification images showing the colocalization of Pyk2 with actin, dynamin with actin, and Pyk2 with dynamin. Scale bars, 5 μm. (B) Dynamin was immunoprecipitated from mouse OCLs and resolved by SDS-PAGE. Western blotting was performed with an anti-Pyk2 antibody. As a control for nonspecific binding, immunoprecipitations were also performed using an isotype-matched immunoglobulin G (IgG). TCLs were blotted for dynamin or Pyk2. Reciprocal experiments in which the anti-Pyk2 antibody was used for immunoprecipitation and the dynamin antibody was used for blotting were also performed, with similar results (data not shown). (C) OCLs were removed from the plates as described in Materials and Methods and kept in suspension in medium supplemented with 1% FBS for 1 h. An equal number of cells was replated onto culture plates previously coated with vitronectin and allowed to reattach for 0, 1, or 3 h. A total of 500 μg of cell lysates were subjected to immunoprecipitation as indicated. Immune complexes were resolved by SDS-PAGE and blotted as indicated. Portions (10 μg) of TCLs were blotted for dynamin or Pyk2. Molecular mass markers are indicated. Protein bands were quantified by densitometry. Numbers are in arbitrary units and reflect the normalized ratios of band intensity in blot 1 (top, IP:Dyn/B:Pyk2) to blot 2 (middle, TCLs blotted for dynamin). Total Pyk2 levels were not significantly changed by replating (blot 3, bottom). Similar results were obtained when immunoprecipitations were performed with an anti-Pyk2 antibody, followed by blotting for endogenous dynamin (data not shown). Labels: IP, immunoprecipitation; B, Western blot; Dyn, dynamin.