Type I myosins anchor actin assembly to the plasma membrane during clathrin-mediated endocytosis

Figures

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  Figure 1.. 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…

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  Figure 2.. 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…

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  Figure 3.. 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…

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  Figure 4.. Molecular determinants of myosin-mediated coupling of actin assembly to endocytic sites. (A) Schematic diagram of Myo5 domains and deletion mutants analyzed. Domain boundaries are marked with the corresponding amino acid number. All mutants were linked to a C-terminal 13Myc tag. (B) Quantification of the proportion of cells harboring the indicated MYO5 truncation mutant displaying cytoplasmic actin comets in epifluorescence videos. Each data point represents a separate experiment during which at least 70 cells were observed. Data points for MYO5 and myo5-TH1Δ are repeated from Fig. 1 C. Asterisks denote statistical significance compared with MYO5 or between bracketed variants after one-way ANOVA (F = 18.88) followed by Sidak’s multiple comparisons test. Error bars are SD. (C–E) Stills from epifluorescence videos of cells endogenously expressing Sla1-GFP (green) and Abp1-mRFP (magenta). Yellow arrowheads indicate cytoplasmic actin comets in mutants. See Videos 12, 13, and 14. (F) Growth of cells harboring truncation mutations in MYO5 on YPD plates. Photos are representative of results from three separate experiments. (G) Schematic diagram illustrating membrane anchoring of actin…

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  Figure 5.. Model for Myo5 function in anchoring actin assembly to the PM at endocytic sites. (A) Myo5 (yellow bananas) restricts activation of the Arp2/3 complex (gray avocados) by the WASP complex (blue widgets) to a discrete location, generating an actin array that grows predominantly in the same direction to generate force. (B) Absent this critical linkage, Arp2/3 activators splinter off of the PM, leading to Arp2/3 complex activation throughout the actin network. Delocalized Arp2/3 complex activation results in disordered actin arrays that fail to produce force. In the most catastrophic cases, the Arp2/3 complex and its activators pull away from the PM completely to form cytoplasmic actin comets (lower left of zoom).