Podosome volume and WASp retention within podosomes is predicted by WIP binding affinity, but podosome dynamics are unaffected. (A) Podosome volume was analyzed in transduced ^WASBMDCs plated on fibronectin-coated glass coverslips, fixed, and immunostained for EGFP, f-actin, and vinculin. Podosomes were defined using actin and vinculin signals on maximal intensity projections and cross-sectional areas were measured in ImageJ Version 1.45d software. Differences between constructs were analyzed by ANOVA with a Bonferroni correction. (B-F) ^WASBMDCs were cotransduced with EGFP-WASp (or EGFP alone) and LifeAct-mCherry using lentiviral infection and plated onto fibronectin-coated glass-bottomed Petri dishes for 4 hours before imaging. All imaging was performed using a Zeiss LSM710 microscope with a 63× lens. (B-C) Cells with EGFP-WASp localized to podosomes were identified and imaged with EGFP and LifeAct signals for 3 minutes (single Z stack, 12 frames/min). Podosome life was defined as the time an individual podosome's actin intensity remained greater than 75% of its maximum intensity. Kaplan-Meier fractional survival curves were plotted (B) and used to calculate median podosome half-life for each construct (C). Differences between survival curves were analyzed using the log-rank (Mantel-Cox) test using both WT and EGFP as standards and no significant differences were seen. Median podosome half-life was calculated for each individual cell and differences between constructs were analyzed by ANOVA. (D-F) Podosome patches were identified using the LifeAct-mCherry signal and FRAP was performed on the EGFP signal. The fluorescence recovery signal was standardized to natural bleaching to give a percent fluorescence recovery for each time point. Data presented are combined from 18-30 FRAP experiments in each of 2 independent experiments. (D-E) WASp^WT and WASp^T45M show significantly longer retention in podosomes than EGFP alone, WASp^dEVH1, and WASp^A134T. Average recovery curves were generated by plotting mean (and SD) of standardized percent fluorescence recovery for each time point. (F) The half-life of individual FRAP curves was determined by the time taken when the FRAP recovery curve reached half of the initial intensity. Median, interquartile range (box), and range (whiskers) for each construct is presented and differences were analyzed by ANOVA with a Bonferroni correction. ***P < .005, **P < .01, *P < .05, and ns indicates P > .05.