Hundreds of thousands of Myo10 monomer molecules found in Myo10-transfected U2OS cells.

(A) Epifluorescence image of exogenously expressed HaloTag-Myo10 in U2OS cells. Actin is labeled with phalloidin-AF633 (magenta). Myo10 is labeled with HaloTag ligand-TMR (green). Scale bar = 10 µm. (B) The top SDS-PAGE lanes show the indicated quantity (in ng) of HaloTag standard protein. The bottom SDS-PAGE lanes from the same gel show 50,000 cells from 6 separate U2OS transient transfections (except bioreplicate 5, indicated by a red asterisk (*), has 10,000 cells). Bioreplicates 1, 2 and 3 are from live-cell analysis, while bioreplicates 4, 5 and 6 are from fixed-cell analysis. Stain-free shows total protein signal, while TMR shows only TMR-HaloTag-Myo10 signal. Signal was integrated for full-length Myo10 (at ∼250 kDa) and any Myo10 aggregated in the wells at the top of the panels. (C) Standard curve for TMR fluorescence signal of HaloTag standard protein (black dots) compared to signal from HaloTag-Myo10 U2OS cells (red dots = fixed cell experiments, blue dots = live cell experiments). The linear fit is y = 24.32x, where the y-intercept is set to 0. R2 = 0.98. Standard error of slope = 1.42. Grey shading indicates the 95% confidence interval. (D) Distribution of the number of Myo10 molecules per fixed cell (N = 150 cell images; min = 39,000, 95% CI: 33,000-46,000; median = 1,000,000, 95% CI: 870,000-1,200,000; max = 21,000,000, 95% CI: 18,000,000-25,000,000; bins = 30). (E) Distribution of the number of Myo10 molecules per live cell as determined by quantification of the first frame of N = 168 cell movies (min = 79,000, 95% CI: 67,000-92,000; median = 450,000, 95% CI: 370,000-520,000; max = 3,300,000, 95% CI: 2,800,000-3,800,000; bins = 30).

Only a small portion of intracellular Myo10 is activated and enters filopodia, and Myo10 is unevenly distributed around the cell.

The following values are from fixed cell images. (A) Distribution of the number of Myo10-positive filopodia per cell (N = 8,733 Myo10-positive filopodia, 150 cells, min = 12, median = 55, max = 116). (B) Distribution of the percent of Myo10 localized in the filopodia per cell (N = 150 cells, min = 0.86%, median = 5.35%, max = 19.87%). (C) Correlation between number of Myo10-positive filopodia in a cell and the filopodial number of Myo10 molecules in the cell. The slope of power law function is 0.36. (D) Correlation between number of Myo10-positive filopodia in a cell and the total number of Myo10 molecules in the cell. The slope of power law function is 0.29. (E) Spatial correlation of Myo10-rich regions of the cell edge. Each cell was divided into 20 angular sections, and the section with the most molecules was aligned to 0°. Section quantities were then averaged across cells. Molecules, puncta, and molecules per punctum are shown. (F) Spatial correlation of Myo10-poor regions of the cell edge. As in E, but the section with the fewest molecules was aligned to 0° for each cell’s rose plot. If >1 section contained no Myo10, a randomly selected empty Myo10 section was aligned to 0°. Error bars in E, F are the standard error of the mean for 500 bootstrapped samples of the 150 cells. Note the correlation of both molecules and puncta at opposite ends of cells (0°, 180°), and the anticorrelation with the two sides (90°, 270°).

Hundreds of Myo10 molecules are found in a filopodium, potentially in excess over available actin at the filopodial tip.

The following values are from fixed cell images. (A) Distribution of the number of Myo10 molecules per filopodium (N = 150 cells, 8,733 filopodia; min = 6, 95% CI: 5-7; median = 730, 95% CI: 610-850; max = 80,000, 95% CI: 67,000-93,000; 62 values >20,000 not shown; bins = 100). (B) Cumulative distribution function plot of data in part A. (C) Distribution of the number of Myo10 molecules at the filopodial tip (90 randomly chosen filopodia tip-localized Myo10 puncta from 9 different cell images; min = 66, 95% CI: 55-76; median = 788, 95% CI: 660-915; max =11,000, 95% CI: 9,600-13,000; bins = 30). (D) The local concentration of Myo10 at a filopodial tip. To estimate the volume, we measured the length of the filopodia tip-localized Myo10 puncta from part C in ImageJ. We then modeled filopodium as a cylinder of radius = 0.1 µm (published average). Min = 6.2 µM, 95% CI: 5.2-7.2; median = 84 µM, 95% CI: 70-97; max = 560 µM, 95% CI: 470-650, bins = 30. Blue dashed vertical line indicates the concentration of F-actin accessible for Myo10 binding in a filopodium (∼96 µM). (E) Scatterplots of molecules vs. length for the puncta from part C. The phase boundary shows the 96 µM threshold from part D. (F) As in part E, but the line represents an estimate of allowable Myo10 on the filopodial tip membrane area. See supplementary Fig. 5 for membrane occupancy estimates. (G) Model of a Myo10 traffic jam at the filopodium tip. Not enough available actin monomers results in a population of free Myo10 (in blue). The free Myo10 is detached from actin but potentially still membrane-associated. (H) Model of frayed actin filaments at the filopodium tip. If actin filaments are not neatly packed into parallel bundles at the filopodium tip, disorganized and frayed actin filaments yield more accessible binding sites to Myo10.

Myo10 dynamics from live cell movies.

(A-C) Dense Myo10 puncta appear at the start of each phase of the filopodial lifecycle. Distributions of the number of Myo10 molecules in puncta upon: (A) Filopodial initiation (min = 52, 95% CI: 44-61; median = 160, 95% CI: 140-190; max = 1200, 95% CI: 970-1,300). (B) Second-phase elongation (min = 65, 95% CI: 54-75; median = 290, 95% CI: 240-340; max = 1,400, 95% CI: 1,200-1,600). (C) Filopodial retraction (min = 71, 95% CI: 60-82; median = 240, 95% CI: 200-280; max = 1,200, 95% CI: 1,000-1,400). Values are the means of the first two frames after spot detection and identification of filopodial lifecycle stage. Histograms A-C have 30 bins each. (D-F) Accumulation of Myo10 in puncta after filopodial initiation or second-phase elongation, but not after retraction. Evolution of number of molecules for each filopodial phase over time for: (D) Filopodial initiation. (E) Second-phase elongation. (F) Filopodial retraction. Starting values from (A-C) were subtracted from all traces to obtain delta over time. The generalized additive model (GAM) trend lines (blue) exclude long times (> 100 s) with few surviving trajectories. The outlier trajectory indicated by the magenta asterisk is from Movie 3, and the cyan asterisk is from Movie 4. (G-I) Myo10 punctum speeds are inversely correlated with the number of Myo10 molecules. Plots of instantaneous speeds vs. molecules for: (G) Filopodial initiation (min = 0.7, median = 160, max = 2,200 nm/s, Spearman’s ρ = -0.51, p < 2.2*10-16). (H) Second-phase elongation (min speed = 0.4, median = 110, max = 3,000 nm/s, Spearman’s ρ = -0.55, p < 2.2*10-16). (I) Filopodial retraction (min speed = 1.2, median = 140, max = 1,900 nm/s, Spearman’s ρ = -0.45, p < 2.2*10-16). Color signifies Myo10 puncta belonging to the same trajectory within each event type. Colors are independent in each panel. Blue lines are GAM trend lines. Panels A, D, G: 237 trajectories from 31 cells; B, E, H: 51 trajectories from 19 cells; C, F, I: 58 trajectories from 26 cells.