Membrane-bound Myo5 restricts actin assembly to endocytic sites. (A) Stills from epifluorescence videos of cells endogenously expressing Sla1-GFP (green) to label endocytic coats and Abp1-mRFP (magenta) to label endocytic actin networks. Yellow arrowheads mark cytoplasmic actin comets in mutants. See Video 1. (B) Kymographs of individual attempted endocytic events. Extracellular space is up and the cytoplasm down. (C) Quantification of Sla1-GFP patch internalization during a 2-min video. Each data point represents the proportion of patches internalized in one cell. n = 61 (MYO5), 64 (myo5Δ), and 65 (myo5-TH1Δ) cells observed. Asterisks denote statistical significance compared with MYO5 by one-way ANOVA (F = 5168) followed by Dunnett’s test. Error bars are SD. (D) Quantification of the proportion of cells manifesting cytoplasmic actin comets in epifluorescence videos. Each data point represents a separate experiment during which at least 110 cells were observed. Asterisks denote statistical significance compared with MYO5 as in C (F = 82.29). Error bars are SD. (E) Montages from TIRFM videos of cells endogenously expressing Sla1-GFP (green) and Abp1-mRFP (magenta). Yellow lines indicate selections used for intensity profiles in F. Montages are representative of observations from 80 (MYO5), 156 (myo5Δ), and 71 (myo5-TH1Δ) cells. See Video 2. (F) Abp1-mRFP fluorescence intensity profiles from the indicated time points along the line indicated in E. ****, P < 0.0001. All cells are myo3Δ.