Membrane-bound Myo5 restricts the activity of the Arp2/3 complex and its regulators to endocytic sites. (A) Stills from epifluorescence videos of cells endogenously expressing Arc15-GFP (green) to label the Arp2/3 complex and Abp1-mRFP (magenta). Yellow arrowheads mark cytoplasmic actin comets in mutants. Percentages are proportions of Abp1-mRFP comets with associated Arc15-GFP signal ± SD; n = 192 (myo5Δ) and 207 (myo5-TH1Δ) total comets observed. See Video 3. (B) Montages from epifluorescence videos during which 250 mM CK-666 or 0.5% DMSO was added to myo5-TH1Δ cells endogenously expressing Abp1-mRFP. Yellow arrowheads indicate cytoplasmic actin comets that dissolve upon CK-666 addition and persist upon DMSO addition. Times are minutes:seconds. Montages are representative of seven experiments. (C) Quantification of the proportion of cells displaying cytoplasmic actin comets in epifluorescence videos in the presence of 0.5% DMSO or 250 µM CK-666. Each data point represents a separate experiment during which at least 50 cells were observed. Asterisks denote statistical significance according to Student’s t tests corrected for multiple comparisons using the two-stage setup method of Benjamani, Kreiger, and Yekutieli with a false discovery rate of 5%. Error bars are SD. (D) Stills from epifluorescence videos of MYO5 and myo5-TH1Δ cells endogenously expressing Abp1-mRFP (magenta) and GFP-tagged Arp2/3 complex regulators (green). Yellow boxes indicate Abp1-mRFP comets with the corresponding GFP-tagged protein at the tip or, in the case of Pan1, absent from the comet. These selections are enlarged twofold and shown in grayscale below. Percentages are proportions of Abp1-mRFP comets with associated GFP signal ± SD; n = 143, 343, 328, 188, and 179 total comets observed for Las17, Vrp1, Bbc1, Bzz1, and Pan1, respectively. See Videos 4, 5, 6, 7, and 8. All cells are myo3Δ.