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First published online December 15, 2003
doi: 10.1242/10.1242/jcs.00839

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Actin cytoskeleton remodelling via local inhibition of contractility at discrete microdomains

Institute of Molecular Biology, Department of Cell Biology, Austrian Academy of Sciences, Billrothstrasse 11, 5020 Salzburg, Austria

View larger version (101K): [in a new window]   Fig. 1. Altered integrity of actin filaments at specialised microdomains. (A) The microdomain, located at the stress fibre (SF)/focal adhesion (FA) interface features a region where actin filaments fail to become decorated by phalloidin (black bar). Scale bar: 2 µm. (B-D) High molecular mass tropomyosins persist in this gap, demonstrating the continuity of the actin filaments within this microdomain. (E-G) Tropomyosin also concentrates in the `gap' between stress fibre and focal adhesion-targeted -actinin. Scale bar in B-G: 5 µm.

View larger version (73K): [in a new window]   Fig. 2. Cortactin accumulation precedes cytoskeletal remodelling. Cortactin distribution in serum stimulated (A-C) and PDBu-stimulated A7r5 cells during early (D-F) and late (G-I) phase of podosome formation. Clusters of cortactin are found at the stress fibre/focal adhesion microdomain (arrowheads in A and C; A'-C'). These clusters correspond to the initiation sites of podosomes at which cortactin accumulation increases during their formation. Scale bar: (A-I) 20 µm; (A'-C') 1 µm.

View larger version (102K): [in a new window]   Fig. 3. Mutually exclusive distribution of myosin II and cortactin. (A-F) Myosin arrangement in serum stimulated (A-C) and PDBu-stimulated (D-F) cells. Clustering of cortactin interferes with myosin assembly, and progression of podosome formation causes the long-term displacement of myosin filaments (arrows in D'-F'). Scale bar: 20 µm. (G) Selected frames from a video sequence (corresponding to the supplemental movie) of a live A7r5 cell double transfected with DsRed SM22 (to highlight podosome formation sites) and GFP regulatory myosin light chain (rMLC). The clustering of cortactin causes the transient dispersion of myosin (arrows indicate a selected event) which persist for several minutes. Upon disintegration of the cortactin cluster myosin re-assembles within a few minutes. Scale bar: 10 µm.

View larger version (99K): [in a new window]   Fig. 4. Tropomyosin is absent from early podosomes. (A-C) Similar to the situation with myosin II, TM is rearranged and diffused at the sites of cortactin clustering during the early phase of podosome formation. Higher magnification of a selected area is shown in A'-C'. Arrowheads indicate regions of TM dispersion and podosome formation. (D-F) Only at later stages are tropomyosins found at, and overlap with, the periphery of larger podosomes. Scale bar: 20 µm.

View larger version (100K): [in a new window]   Fig. 5. Clustering of cortactin coincides with the displacement of h1 calponin from actin stress fibres. Enlarged section from an A7r5 cell transiently transfected with GFP h1CaP (B, green), fixed and stained for cortactin (C, red) after 10 minutes of PDBu stimulation. Arrows indicate regions of cortactin clustering and depletion of calponin. Scale bar: 1 µm.

View larger version (88K): [in a new window]   Fig. 6. Accumulation of p190RhoGAP. PDBu treatment induces the rapid translocation of p190RhoGAP from actin stress fibres (A-C) to the sites of podosome formation (D-F). Note the almost complete displacement of p190RhoGAP following PDBu stimulation (E), despite the presence of prominent actin stress fibres in the centre of the cell (D,F). The localisation of p190RhoGAP with membrane ruffles remains unchanged (arrows). Scale bar: 20 µm.

View larger version (90K): [in a new window]   Fig. 7. Activated Dia interferes with cortactin clustering. Ectopic expression of active GFP-mDiaN3 suppresses podosome formation induced by PDBu (A,B), and causes the dispersion of cortactin with the concomitant failure to form cytoplasmic clusters (C-E). Scale bar: 20 µm.

View larger version (30K): [in a new window]   Fig. 8. Schematic representations of cytoskeletal remodelling events during the induction and formation of podosomes. Stress fibres align with the perpendicular arrangement of myosin and are anchored in vinculin-rich focal adhesions (1). The cortactin-rich microdomain residing at the stress-fibre/focal adhesion interface initiates the formation of early podosomes (e.p.; hatched orange), enriched in Arp2/3 and p190 RhoGAP (2). The reduction of contractile forces by the suppression of RhoA activity and the displacement of tropomyosin, calponin and myosin II causes the actin filaments to shrink and gradually reduce attachment to the focal adhesion sites. Further progression, growth and maturation of late podosomes (l.p.) along the existing actin trails causes further displacement of myosin filaments and the total separation of actin filaments from peripheral adhesions. The total loss of stress fibre/focal adhesion attachment leads to complete elimination of contractility and the subsequent disassembly of the focal adhesions, while regions of stable actomyosin interaction remain contractile.