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. 2013 Jan 29;4:1412. doi: 10.1038/ncomms2402

Figure 2. Myosin IIA-mediated contractility is essential for podosome core oscillations.

Figure 2

(a) DCs were transfected with Lifeact-GFP to visualize actin. Shown are the intensities of the actin fluorescence and the inverse IRM signal (where values >1 mean tight contact, while values <1 mean loss of contact between cell and substrate) at a podosome and a non-podosome area. Data were normalized to the average intensity. (b) Cells were transfected with Lifeact-GFP or -RFP to visualize actin. Shown are the Lifeact-RFP fluctuations over time of three representative podosomes in untreated control cells or in cells treated for 30 min with 20 μM blebb. Also, Lifeact-GFP fluctuations of three representative podosomes are shown in cells treated for 30 min with 20 μM Y27632. Data are normalized to the average intensity. (c) DCs were transfected with actin-mCherry and subjected to FRAP analysis. Snapshots from the FRAP time-lapse movies at 5 s before (−5 s), directly after (0 s) and 70 s after photobleaching for untreated and blebb-treated cells are shown. The white circle indicates the FRAP region that corresponds to a single podosome. (d) FRAP analysis of actin-mCherry within podosome cores without (solid boxes) and with (open boxes) blebb. Shown are the average and standard deviation of the fluorescence recovery for at least 10 podosomes in five different cells. FRAP data were background corrected and double normalized to correct for total bleaching of the image (see Methods). (e) FRAP analysis of actin-GFP within podosome cores without (solid boxes) and with (open boxes) Y27632. Data are represented in the same way as in panel d. (f) Representative images of podosome cores in fixed DCs, visualized by Texas Red-conjugated phalloidin, in the control situation (left) and after 30 min of treatment with 20 μM blebb. Scale bar, 5 μm. (g) Median±interquartile range of the actin core fluorescence intensity (left plot) and core area (right plot) for at least 800 podosomes per condition. Statistical analyses were performed by two-tailed Student’s t-test. ***P<0.001.