WASp^EMM induces dysregulation of actin polymerization in vivo, which results in an increased susceptibility to form multiple lamellipodia and subsequent migration defect. (A) Transduced ^WASBMDCs were matured with lipopolysaccharide and harvested on day 9 of culture. F-actin within cells was quantified by flow cytometry after fixation and staining with Alexa Fluor 633 phalloidin. The sympercentage difference in median f-actin signal between transduced and simultaneously stained untransduced ^WASBMDCs is shown, with error bars representing the SEM. Differences between wild-type transduced and other constructs were analyzed by ANOVA. Data represent 8 independent experiments. (B) WASp^EMM is less tyrosine phosphorylated than WASp^WT in vivo. U937 cells with standardized EGFP-WASp expression were harvested and lysates underwent anti-EGFP immunoprecipitation. Immunoprecipitant were resolved and immunoblotted for EGFP, phosphotyrosine (4G10; pTyr), and WASp pSER^433/434 (pSer). Representative immunoblots shown. (C-D) EGFP, pTyr, and pSer bands from 8 experiments were quantified by densitometry. The percent tyrosine phosphorylation was calculated by comparison with fully tyrosine phosphorylated standards, and the means and 95% confidence interval are graphically represented. Differences between wild-type and other constructs were analyzed by ANOVA (C). WASp serine phosphorylation was compared with WASp^WT and mean differences (from 4 experiments) are expressed as sympercentage differences from WASp^WT (with 95% confidence intervals). The 95% confidence interval for WASp^WT is shown by dotted vertical lines. Differences from WASp^WT were analyzed by ANOVA (D). (E) Transduction of ^WASBMDCs with WASp^EMM results in a greater proportion of cells forming > 2 lamellipodia compared with WASp^WT-transduced cells. Cell shape of fixed transduced ^WASBMDCs plated onto fibronectin-coated coverslips in 3 independent experiments was analyzed. EGFP-positive, spread cells with lamellipodia were scored as having > or ≤ 2 lamellipodia. (F) Relative odds (and 95% confidence interval) of an individual spread cell forming > 2 lamellipodia compared with wild-type transduced cells is shown. Odds ratios and significance of differences WT and other constructs were calculated using logistical regression. (G-I) ^WASBMDCs transduced with WASp^EMM show similar velocity but reduced displacement and persistence during migration compared with WASp^WT-transduced cells. Migration of transduced DCs plated onto fibronectin-coated coverslips toward a CCL3 gradient was assayed in Dunn chambers. Cells were imaged in phase and green fluorescence for 6 hours using an Zeiss Axiovert 135 (10 × /0.25 NA lens), and cell tracks were analyzed using Volocity Version 5.3.2 software. EGFP-negative cells on the same coverslip as transduced cells were analyzed as WAS controls. Average velocity (G), total cell displacement (H), and persistence of migration (meandering index; I) collated from 5 independent experiments are presented with 95% confidence intervals. Differences between wild-type and other construct transduced cells were analyzed by ANOVA with correction for variation between experiments. ***P < .005, **P < .01, *P < .05, and ns indicates P > .05.