Myo5 couples the assembling actin network to the PM during CME. (A) Montages from the same epifluorescence videos shown in Fig. 2 A. Yellow arrowheads indicate actin comets pulling away from the PM. Similar behavior was observed in at least 50 cells across many different experiments. See Video 3. (B) Spinning disc confocal images of cells subjected to a 5-min pulse of FM4-64 staining followed by a 5-min chase with imaging media. (C) Quantification of percentage of FM4-64 signal internalized during a 5-min chase. n = 20 (MYO5), 19 (myo5Δ), and 22 (myo5-TH1Δ) cells. Asterisks denote statistical significance compared with MYO5 by one-way ANOVA (F = 767.1) followed by Dunnett’s test. Error bars are SD. ****, P < 0.0001. (D) Kymographs of individual endocytic events following washout of CK-666 or no washout in wild-type MYO5 and myo5Δ cells endogenously expressing Sla1-GFP (green) and Abp1-mRFP (magenta). See Fig. S2 for montages. Kymographs are representative of 13 (MYO5) and 14 (myo5Δ) separate experiments. See Video 10. (E) Kymographs as in D from videos of cells endogenously expressing Vrp1-GFP (green) and Abp1-mRFP (magenta). Kymographs are representative of 8 (MYO5) and 16 (myo5Δ) separate experiments. See Video 11. All cells are myo3Δ.