Hundreds of thousands of Myo10 monomer molecules found in U2OS cells overexpressed with Myo10.

(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) Left SDS-PAGE lanes: 1, 5, and 10 ng of Halo standard protein were loaded (left to right). Right SDS-PAGE lanes: 40,000 cells from three separate U2OS transient transfections (bioreplicate 1, 2 and 3) were loaded. Stain-free shows total protein signal, and TMR illumination shows only HaloTag-Myo10 signal. Red asterisk (*) indicates aggregate avoided in signal integration. (C) Standard curve for TMR fluorescence signal of Halo Standard Protein (black dots) compared to signal from HaloTag-Myo10 U2OS cells (red dots). y = 76.12x, where the y-intercept is set to 0. R2 = 0.99. Standard error of slope = 8.25. Grey shading indicates 95% confidence interval. (D) Distribution of the number of Myo10 molecules per cell as determined by quantification of 150 cell images. Min = 30,000 (95% CI: 21,000-57,000), median = 480,000 (95% CI: 330,000-900,00), max =10,000,000 (95% CI: 7,000,000-19,000,000).

Only a small portion of intracellular Myo10 is activated and enters filopodia, and Myo10 is not evenly distributed along the cell edge.

(A) Distribution of the number of Myo10-positive filopodia per cell. Min = 12, median = 55, max = 116. Total of 8,733 Myo10-positive filopodia were analyzed. (B) Distribution of the percent of Myo10 localized in the filopodia per cell as determined by quantification of 150 cell images. Min = 0.9%, median = 5.4%, max = 19.9%. (C) 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. (D) Spatial correlation of Myo10-poor regions of the cell edge. As in (C), 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 C, D 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°).

Tens of Myo10 are sufficient to nucleate filopodia.

(A) 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 0.30. (B) Correlation between number of Myo10-positive filopodia in a cell and the number of filopodial Myo10 molecules in the cell. The slope of power law function is 0.39. (C) Dim Myo10 puncta in filopodia, identified as those containing <10 Myo10 molecules, were examined. 135 total dim puncta across 26 cells were identified. Of the 135 puncta, 7 puncta were hard to visually interpret, 7 puncta were image noise, and 56 puncta were over-segmented (e.g. not actual distinct puncta but rather part of continuous Myo10 signal in filopodia). The distribution of the remaining 66 true dim puncta is displayed. (D) Filopodial localization of dim Myo10 puncta containing <10 molecules. The 66 true dim puncta from (C) were analyzed.

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

(A) Distribution of the number of Myo10 molecules per filopodium as determined by quantification of 150 cell images. Min = 2 (95% CI: 1-4), median = 360 (95% CI: 240-670), max = 63,000 (95% CI: 43,000-120,000). 59 data points with values >10,000 molecules were excluded from plotting. 8,733 total filopodia. (B) Cumulative distribution function plot of Myo10 molecules per filopodium. 59 data points with values >10,000 molecules were excluded from plotting. 8,733 total filopodia. (C) The length of 90 randomly chosen filopodia tip-localized Myo10 puncta were measured in ImageJ. The volume occupied by the Myo10 punctum was estimated using the volume of a cylinder, where length = height and width = 2*radius. Radius for all Myo10 puncta was treated as 0.1 μm (published average) since resolution limit ∼250 nm. The signal intensity of the Myo10 punctum was converted to molecules to obtain local concentration (in μM). Min = 3.2 (95% CI: 2.1-6), median = 35 (95% CI: 24-65), max = 220 (95% CI: 150-420). 9 different cell images were analyzed in total. Blue dashed vertical line indicates the concentration of F-actin accessible for Myo10 binding in a filopodium (∼96 uM). (D) Model of a Myo10 traffic jam at the filopodium tip. Not enough available actin monomers results in a population of free and unbound Myo10. (E) 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.