ARNO but not cytohesin 1 is localized to podosomes and podosome-like structures in different cell types. (A–C) Localization of F-actin marker, mCherry-UtrCH, and GFP-ARNO in TGFβ1-stimulated THP1 cell (A), active Src-transformed fibroblast (B), and fibroblast on a RGD-functionalized fluid lipid bilayer (C). Left, F-actin cores of podosomes (A), podosome rosettes (B), and podosome-like structures formed on fluid bilayer (C). (middle) GFP-ARNO localized to periphery of F-actin cores (A–C). (right) Merged images. The boxed areas (A and C: 2.5 × 2.5 µm2, bar, 1 µm; B: 14 × 14 µm2, bar, 5 µm) of merged images are enlarged and line scanned as shown in inset. The graphs on the right demonstrate intensity profiles of F-actin and ARNO in individual podosome (A), podosome rosette (B), and podosome-like structure on bilayer (C). (D) Time course of ARNO localization to the podosome periphery. Dynamics of F-actin (labeled by mCherry-UtrCH) and GFP-ARNO fluorescence intensities in the podosome shown in the boxed area (3 × 3 µm2) in the left panel are presented in the sequences in the right panels. Time interval between frames is 30 s. See also Video 6. (E) Cytohesin-1 is not localized to podosomes. (left) F-actin cores of podosomes in TGFβ1-stimulated THP1 cell. Middle, GFP–cytohesin 1 localization in the same cell. (right) Merged image. Line scanning through the individual podosome in the boxed area (4 × 1.5 µm2) of the merged image shown in inset is quantified in the graph on the right. No enrichment of GFP–cytohesin 1 at podosome core or periphery was detected. Bars, 5 µm.
