Figure 6. Podosome core growth drives the recruitment of vinculin and zyxin to the ring.

DCs were cotransfected with Lifeact-RFP and GFP-tagged adaptor proteins. Cells were imaged for 25 min and core and ring fluctuations were correlated for (a) vinculin, (b) talin, (c) zyxin and (d) paxillin. (e) Adaptor protein levels and actin levels were correlated for at least 65 podosomes for 25 min. Shown are the linear fit for zyxin and vinculin and the nonlinear fit for paxillin and talin. Data were normalized to the average intensity. (f) Cells were cotransfected with Lifeact-RFP and vinculin-GFP, seeded on coverslips and fixed after 16 h. Image stacks (sized 100 nm) of the podosome core by confocal microscopy and one widefield image of the vinculin ring were taken. Podosome core height was calculated using the intensity profile of the Lifeact-RFP stack image. Subsequently, podosome height was correlated with the vinculin ring intensity for at least 500 podosomes in five different cells. Shown are an overview of the analysis we performed (left) and a graph (right) plotting the individual data points as well as the median (middle line) and the interquartile range (lower and upper whiskers).