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. 2022 Feb 8;298(3):101700. doi: 10.1016/j.jbc.2022.101700

Figure 3.

Figure 3

The effect of mutations on cellular actin reorganizing activity induced by BAR-PH. Overexpression of FLAG ASAP1 BAR-PH WT, [K283A, K284A], and [K283E, K284E], but not [K75A, K76A, K79A], [K75E, K76E, K79E], [K159A, K163A, K164A], and [K159E, K163E, K164E], causes formation of actin protrusions. Human osteosarcoma cells U2OS were transiently transfected with empty vector, or FLAG-tagged 1 to 431 (BAR-PH) WT and indicated mutants and costained with anti-FLAG antibody and fluorescent phalloidin for F-actin. Images are representative of two independent experiments with at least 20 cells per group per experiment, except ∗[K159A, K163A, K164A] mutant, which showed low expression (5–6 cells). Insets highlight the presence or absence of actin protrusions. The scale bars (main and inset) represent 10 μm. B, graphs summarizing the quantification of percentage of BAR-PH expressing cells that contain long actin protrusions, quantification of shape changes (length-to-width ratio) induced by BAR-PH expression, number of actin protrusions per cell, and average length of the protrusions per cell, presented as mean ± SD. Random empty vector cells were counted as a reference. Statistics are based on the quantification of cells from all experiments using One-way ANOVA with Dunnet’s multiple comparison test (compared to the WT BAR-PH), n.s., not significant, ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. BAR, Bin/Amphiphysin/Rvs domain; F-actin, filamentous actin; PH, pleckstrin homology.