Effects of CK666 treatment on actin patches in WT and cap2∆ cells, and sequence alignment of capping protein β-tentacle across species. (A) CK666 inhibits Arp2/3 in wild-type and cap2Δ cells. Spinning-disk confocal average intensity projections of formaldehyde-fixed wild-type and cap2Δ cells expressing the Arp2/3 marker, Arc15-RFP^mScarlet. Cells were incubated with 100 μM CK666 or the DMSO carrier control for 30 min prior to fixation and imaging. Scale bar = 2 μm. (B) The coefficient of variation (CoV; SD/mean) of the Arc15-RFP^mScarlet signal in each cell is shown. Decreased CoV indicates the signal is more homogenous due to loss of focused Arc15-RFP^mScarlet signal at patches (n = 292 wild-type cells, 252 CK666 treated wild-type cells, 173 cap2Δ cells, and 111 CK666 treated cap2Δ cells). Statistical significance calculated by two-sided Student's t test (****, P ≤ 0.0001). (C) Additional examples of maximum intensity projections of Airyscan images of F-actin (cyan) in fixed and phalloidin stained wild-type and cap2Δ cells expressing the Arp2/3 marker, Arc15-RFP^mScarlet (magenta) treated with CK666 (Arp2/3 inhibitor), or DMSO (carrier control) accompanying Fig. 7. (D) Alignment of the capping protein β-tentacle from budding yeast (S. cerevisiae), fission yeast (S. pombe), human (H. sapiens), and chicken (G. gallus) reveals divergence in hydrophobic residues used to interact with the actin filament barbed end. Top, Cap2 (S. cerevisiae) NCBI ID NP_012230.3, second ACP2 (S. pombe) NCBI ID NP_593619.3, third CAPB (H. sapiens) NCBI ID NP_001269091.1, bottom CAPB (G. gallus) NCBI ID NP_990768.1. Sequences were aligned using QIAGEN CLC Main Workbench. Key residues used to bind actin are highlighted (Kim et al. 2010).