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Emergence of Large-Scale Cell Morphology and Movement from Local Actin Filament Growth Dynamics

Figure 3

Keratocytes with High VASP at the Leading Edge Are Canoe-Shaped with Smooth Leading Edges

(A, B) Using immunofluorescence, VASP intensity levels were measured along lines (∼5 μm wide) positioned across lamellipodia in the middle of cells roughly perpendicular to the leading edge of keratocytes (arrows). The relative levels of VASP at the leading edge of a population of keratocytes were compared using VASP peak-to-base ratios, which were calculated by dividing the highest mean fluorescence intensity at the leading edge (peak) by the lowest mean intensity found interior to the leading edge (base). For cells with low VASP at the leading edge, peak and base positions were assigned based on mean positions from cells (n = 30) with medium or high VASP peak-to-base ratios and thus clear peak and base positions. (A) An intensity linescan shows that VASP is enriched at the leading edge of a smooth cell with a VASP peak-to-base ratio of 2.56. (B) On the other hand, the rough leading edge of a keratocyte has a VASP peak-to-base ratio of 0.81 and thus VASP is practically absent from the leading edge. Scale bar=10 μm.

(C) The keratocyte population examined (n = 43) had a wide range of VASP peak-to-base ratio values ranging from 0.5 to 2.8.

(D) The shapes of keratocytes were compared using PCA, which identified the major modes of shape variation of polygonal cell contours extracted from intensity-thresholded fluorescent images. A shape mode value of zero corresponds to the mean shape and the negative or positive values correspond to standard deviations describing canoe (+SD) or round D shape (−SD) on the y-axis. Representative cell contours for the specified values are shown on the y-axis. For example, the cells in (A) and (B) have shape mode values of 1.32 and −0.77, showing that they can be quantitatively described as canoe or D shaped, respectively. This shape mode significantly correlates with VASP peak-to-base ratios (r2 = 0.28, p = 0.0002, n = 43, solid line).

(E) To compare leading-edge shapes, we measured and normalized their degree of local curvature. Local leading-edge curvature negatively correlates (r2 = 0.28, p = 0.0003, n = 43, solid line) with VASP peak-to-base ratios.

(F) A significant negative correlation is also observed between cell shape and local leading-edge curvature (r2 = 0.28, p = 0.0003, n = 43, solid line).

Figure 3