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First published online November 18, 2003
doi: 10.1242/10.1242/jcs.00818

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Podosome formation in cultured A7r5 vascular smooth muscle cells requires Arp2/3-dependent de-novo actin polymerization at discrete microdomains

Irina Kaverina1, Theresia E. B. Stradal2 and Mario Gimona1,*

1 Institute of Molecular Biology, Department of Cell Biology, Austrian Academy of Sciences; Billrothstrasse 11, A-5020 Salzburg, Austria
2 Gesellschaft für Biotechnologische Forschung (GBF), Department of Cell Biology, D-38124 Braunschweig, Germany



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  		Fig. 1. Confocal analysis of the 3D structure of smooth muscle podosomes reveals a ring-like arrangement. (A) Three-dimensional structure of podosomes in a GFP-transfected cell fixed in 4% formaldehyde after 1 hour in 1 µM PDBu. Overview in phase contrast. Boxed areas 1, 2 and 3 show Nipkow disk confocal images of single podosomes, with GFP as cytoplasmic marker. X-Y frames show bottom confocal section. X-Y top frames show the uppermost confocal sections for each podosome. X-Z and Y-Z frames show vertical sections of the central regions of each podosome. Note the GFP-free area in the center of the cones in boxed areas 1 and 2, and the lack of such a zone in all X-Y top frames. (B) A living cell co-transfected with GFP and DsRed-SM22. Phase dense podosomes in the left panel (phase contrast) correspond to the hollow regions in ring-shaped GFP staining (arrows in middle panel). SM22 accumulates at the GFP-free zones in the center of these patches (right panel). (C) Nipkow disk confocal images of X-Y plane overview and X-Z planes of individual podosomes (in boxed areas) of a DsRed-SM22-transfected cell fixed in 4% formaldehyde. SM22 is present along the entire length along the middle axis of the podosome (boxed areas 4 and 5). (D,E) X-Z planes of single podosomes in GFP-p20-transfected cell as seen in a Nipkow disk confocal microscope. Arp2/3 is distributed along the middle axis of the podosomes, with decreasing density towards the dorsal cell surface.

 


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  		Fig. 2. Ventral podosome domains contact the substrate. (A) Typical `late' podosomes are detected both by epifluorescence imaging (A) and TIRF microscopy (A') in GFP-{alpha}-actinin-expressing cells. The TIRF picture shows that {alpha}-actinin in the ventral podosome domain is distributed in ring-like structures, surrounding the podosome core. (B) Podosomes in A7r5 cells are adhesive structures. Arrows indicate the areas of adhesive contact detected by interference reflection microscopy (B'), corresponding to the podosomes visible in the phase contrast images (B).

 


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  		Fig. 3. Cortactin is enriched in specialized microdomains in unstimulated A7r5 cells. Even in the absence of PDBu, small clusters of cortactin can be observed at the microdomain bridging the interface between focal adhesions and actin stress fibers. The microdomains are characterized by the absence of Phalloidin decoration of actin filaments in this region. (A-C) Unstimulated cell (0 min). (D-F) Cell after 30 minutes in 1 µM PDBu. Note the dual localization of cortactin at the cell periphery in addition to the microdomains in C.

 


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  		Fig. 4. Arp2/3 engagement precedes translocation of SM22 to early sites of podosome formation. (A) DsRed SM22 and GFP-p20 co-transfected cells before (A) and after 40 minutes of 1 µM PDBu treatment (A'). Note the presence p20-rich dots in both panels. (B) Selected region of the PDBu-treated cell in A' showing p20 and SM22; merged image is shown in the left panel. SM22 is not concentrated in small p20 foci (large arrows), but co-localizes in larger, p20-positive patches proximal to stress fibers (small arrows). (C) Dynamics of GFP-p20 and DsRed-SM22 during a podosome life cycle. Time of PDBu treatment is indicated in minutes and seconds. Note that p20 is concentrated in the spot before, during and after SM22 accumulation.

 


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  		Fig. 5. Arp2/3-dependent actin polymerization is necessary for podosome formation but not for actin remodeling. (A) Formation of podosomes in PDBu-treated GFP-p20-expressing cells. Note the basal Arp2/3 clustering at time 0 and the increase in size of clusters in developing podosomes (arrows). (B) SCAR-WA and GFP-p20-co-expressing cell before and after PDBu treatment. The phase contrast image reveals the absence of lamellipodia despite the formation of extensive filopodia (black arrow). Note the lack of Arp2/3 clustering (arrowheads) and the absence of podosomes upon PDBu treatment. (C) Immunofluorescence images of cells treated with PDBu for 40 minutes. Ectopically expressed myc-tagged Scar domains are visualized by anti-myc antibody, and the actin cytoskeleton by phalloidin staining. The SCAR-WA transfected cell (1) displays no podosomes (arrowhead), while a non-transfected cell (2), and a cell transfected with the SCAR-W domain (3) develops numerous podosomes (arrows). (D) SCAR-WA and GFP-ß-actin co-transfected cells before (0') and after 45 minutes of PDBu treatment. Actin stress fibers undergo substantial remodeling and disassembly in response to PDBu (arrowhead) despite the lack of podosomes. (E) SCAR-WA and GFP-zyxin co-transfected cells before (0') and after 45 minutes of PDBu treatment. Focal adhesions are partially disassembled (arrowheads) and zyxin redistributes into the cytoplasmic pool. Fl, epifluorescence; Ph, phase contrast.

 


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  		Fig. 6. Podosome formation is initiated at discrete sites at the insertion point of actin fibers into focal adhesions. (A) The overview shows a DsRed SM22 and GFP-zyxin co-transfected cell before (0') and after PDBu treatment (40'). Note the formation of SM22-rich early podosomes in the transition zone between stress fibers and focal adhesions (arrows in enlargement, 2 minutes), and later reassembling podosomes proximal to stress fibers, which are separated from adhesions (enlargement, 38 minutes). (B) Similar data set as in A for a DsRed SM22 and GFP-{alpha}-actinin co-transfected cell. Podosomes formation (inset, 11 minutes) and re-assembly (enlargement, 25 minutes) is initiated within the {alpha}-actinin-enriched region (arrows). (C) Enlarged view of the rectangular boxed area in B. Note the functional uncoupling of stress fibers (SF) and focal adhesion (FA) and the parallel decrease of adhesion sites (arrows). (D) Selected examples of podosome formation and subsequent adhesion disassembly in DsRed zyxin and GFP-{alpha}-actinin co-transfected cells (ellipses). Note that {alpha}-actinin is incorporated at early stages of podosomes formation and stays associated with the structures until the complete disassembly of focal adhesion.

 


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  		Fig. 7. Focal adhesion components are recruited at late stages in the formation processes. (A) Video frames showing a cell co-transfected with DsRed SM22 and YFP-vinculin. Vinculin is absent from stress fiber-associated podosomes after 6 minutes (long arrows) but accumulates in late podosomes (32 minutes, short arrows). Right panels: Vinculin; left panels: Merged images. (B) Transient accumulation of vinculin in late podosomes in YFP-vinculin transfected cells. Images are a merger of YFP and phase contrast images. Time of PDBu treatment is indicated in minutes. (C) A GFP-zyxin transfected cell microinjected with TAMRA vinculin prior to PDBu treatment (50 minutes). The merged image (upper left panel) shows that vinculin and zyxin co-localize in both adhesions and late podosomes (arrow), which are identified by phase contrast microscopy as dense structures. (D) Late podosomes in DsRed zyxin and GFP-{alpha}-actinin co-transfected cells. At later stages of PDBu stimulation zyxin localizes to phase-dense podosomes together with {alpha}-actinin (arrow).

 

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© The Company of Biologists Ltd 2003