JCS00839 Supplemental Data

Files in this Data Supplement:

• Figure 1 -

  Fig. S1. PDBu stimulation of a cell transiently expressing GFP-tagged regulatory myosin light chain (A) shows a similar displacement of myosin by cortactin clustering (B) as seen with an antibody against non-muscle myosin II (compare with Fig. 3). Note the complete overlap of cortactin clusters with myosin-free regions at the cell periphery (arrows in A and B).

• Figure 2 -

  Fig. S2. RhoKinase inhibition by Y-27632 induces stress fibre de-bundling and partial disassembly, but is insufficient to drive podosome formation in the absence of PDBu. F-actin was visualised using Alexa 488 phalloidin.

• Figure 3 -

  Fig. S3. AFAP-110 is localised along actin stress fibres (A-C) and enriched at the ends of actin bundles prior to their insertion in focal adhesions (J-L; enlarged view). Induction of cytoskeletal rearrangement by PDBu causes the rapid accumulation of AFAP-110 in early forming podosomal structures (E-F). Note that AFAP-110 accumulations at the periphery of a stress fibre are readily remodelled, while AFAP-concentration in the more central region of the same bundle remains stable (arrows in E and F). Upon further progression of cytoskeletal modulation AFAP is almost quantitatively incorporated in podosomes (G-I).

• Movie 1 -

  Movie 1. Double immunofluorescence live imaging of a cell expressing GFP-myosin RLC and DsRed SM22 (to mark sites of podosome formation). The sequence demonstrates that the dynamic translocation of podosome formation sites coincides with the appearance of myosin-depleted regions, supporting the contention that myosin dispersion is a direct consequence of the cytoplasmic dynamics of cortactin-containing podosomal complexes.