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. Author manuscript; available in PMC: 2024 Jan 9.
Published in final edited form as: Dev Cell. 2023 Jan 9;58(1):18–33.e6. doi: 10.1016/j.devcel.2022.12.001

Figure 3: Barbed end-directed force is required for filopodial induction by Myo10MD.

Figure 3:

(A) Montage of a filopodial growth event from a HeLa cell expressing Myo10MD (green) and CDHR2TM (magenta) imaged in 0.5% methylcellulose. Arrows denote the tip of a single, growing filopodium. Scale bar = 5 μm. (B) Normalized length (magenta) and tip intensity (green) vs. time curves for individual filopodia on cells expressing Myo10MD and CDHR2TM; n = 10 filopodia. (C) Filopodial length and myosin distal tip-intensity from individual protrusions in cells expressing Myo10FL (gray) or Myo10MD (blue); each circle represents an individual filopodium. Orange and black circled data points represent filopodia shown in D; n = 304 filopodia for Myo10FL and 273 for Myo10MD. (D) Confocal maximum intensity projections of length and tip intensity-matched filopodia in cells expressing Myo10FL or Myo10MD (green) and stained for F-actin with phalloidin (magenta); single inverted channel images are shown to the right. Scale bar = 5 μm. (E) Cartoons depicting the myosin-10 motor domain dead G437A mutant (Myo10MDx) and pointed end-directed myosin-6 motor domain (Myo6MD) constructs; numbers below represent amino acids. (F) Confocal maximum intensity projection of HeLa cells expressing Myo10MDx (green) and CDHR2TM (magenta) before (i) and 25 min after (ii) rapalog treatment. (G) Confocal maximum intensity projection of HeLa cells expressing Myo6MD (green) and CDHR2TM (magenta) before (i) and 25 min after (ii) rapalog treatment. For F and G, scale bar = 10 μm. (H) Fractional change in filopodia number in HeLa cells expressing CDHR2TM and Myo10MDx (red) or Myo6MD (blue) 0 min and 25 min after rapalog treatment; n = 6–7 individual cells for each condition. (I) Filopodial length measured at 0 min and 25 min post rapalog treatment in HeLa cells expressing Myo10MDx and CDHR2TM; n = 6 individual cells, n = > 420 individual protrusions. (J) Filopodial length measured at 0 min and 25 min post rapalog treatment in HeLa cells expressing Myo6MD and CDHR2TM; n = 7 individual cells, n = > 365 individual protrusions. Data in (I) and (J) are represented as a SuperPlot, where transparent circles represent the length of individual filopodia, opaque circles represent the average length of filopodia from individual cells, violin plots show the distribution of the data, and all color-matched circles represent measurements from the same cell. All graph error bars represent the mean ± SD; ns, p-value > 0.05.