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Files in this Data Supplement:
Movie 1. Arp2/3 engagement precedes translocation of SM22 to early sites of podosome formation. Cell co-transfected with DsRed SM22 and GFP p20. Prior to the addition of PDBu p20 localises preferentially to the tips of lamellipodia and several dot-like, transiently appearing foci, which are present throughout the cytoplasm. These foci are extremely short-lived (about 30 seconds). SM22 is not enriched at these foci. Following the induction of podosome formation by PDBu a subset of p20-containing foci rapidly grow in size, and continue to serve as platforms for podosome initiation. Within a few minutes after the first appearance of Arp2/3-marked actin polymerisation sites, SM22 is recruited to the newly forming podosomes and is co-localised with all prominent p20-containing patches. Movie=40 minutes.
Movie 2. Partial disassembly and uncoupling of actin stress fibers and focal adhesions accompany podosome formation. Cell co-transfected with DsRed SM22 and GFP zyxin. Podosome formation is induced at discrete microdomains at the focal adhesion/stress fiber interface within 2 minutes post PDBu treatment. The movie sequence demonstrates the formation of SM22-rich early podosomes in the transition zone between stress fibre and focal adhesion, and later reassembling podosomes proximal to stress fibres. Focal adhesions, as well as the attached stress fibers become reduced in size, and disassemble either partially or completely. Movie=40 minutes.
Movie 3. Podosome formation is initiated at discrete sites at the insertion point of actin fibers into focal adhesions. Cell co-transfected with DsRed SM22 and GFP a-actinin. Podosome formation and re-assembly is initiated within the a-actinin-enriched region. Movie=40 minutes.
Movie 4. Cell co-transfected with DsRed Zyxin and GFP–a-actinin. Enlarged view of the inset shown in Fig. 6B. Note the functional uncoupling of stress fibres and focal adhesion, and the decrease of adhesion sites. Podosomes, which remain in close proximity to focal adhesions continue to be visible as prominent structures and gradually replace the focal adhesions. Movie=40 minutes.
Movie 5. Selected example of podosome formation and subsequent adhesion disassembly in DsRed SM22 and GFP–a-actinin co-transfected cells. a-actinin is incorporated at early stages of podosomes formation, and intense actin polymerisation during podosome formation is accompanied by a loss of the stable connection between the actin stress fibres and the focal adhesions in which they terminated. Movie=40 minutes.
Movie 6. Vinculin and zyxin are recruited to podosomes only at late stages. GFP zyxin transfected cell microinjected with TAMRA vinculin pre-treated for 60 minutes with PDBu. The movie sequence shows that GFP-Zyxin co-localises with microinjected 5¢-TAMRA vinculin in late podosomes and focal adhesions of A7r5 cells. Movie=25 minutes.
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