Emergence of Large-Scale Cell Morphology and Movement from Local Actin Filament Growth Dynamics
(A) Time-lapse images show that overexpressed EGFP-VASP, which is enriched at the leading edge before addition of 1.0 μM cytochalasin D (time: 1:00, left panel), becomes weaker at the leading edge ∼2 min after cytochalasin treatment (time: 2:50, middle panel). Cytochalasin D was added at 70 s (∼1.2 min). Approximately 3 min after cytochalasin treatment (time: 4:20, right panel), EGFP-VASP can barely be seen at the leading edge of this keratocyte that begins to exhibit a D shape rather than the original canoe shape. Time = min:s. Scale bar = 10 μm. Fluorescence intensities measured across the leading edge (arrows) quantitatively confirm the enrichment of EGFP-VASP at the leading edge before cytochalasin treatment and its delocalization a few minutes after addition of the drug (bottom graphs).
(B) The levels of EGFP-VASP at the leading edge and the width of the keratocyte in (A) were compared as a function of time. Cell width (axis perpendicular to migration) was measured from one side of the cell to the other (dotted line in (A)). EGFP-VASP intensity levels were measured inside a circle (4 μm diameter) placed in the middle of the cell at the leading edge (dotted circles in (A)) in each frame of the time-lapse sequence. These two parameters temporally correlate with each other (r2 = 0.21, p = 0.0009). The levels of EGFP-VASP at the leading edge (thick gray line) dramatically drop ∼2 min after cytochalasin addition (time ∼3 min, dotted line), followed by a decrease in cell width (thin black line).
(C) The frequencies of VASP peak-to-base ratios obtained from immunofluorescence images of keratocytes treated with cytochalasin D confirm that VASP becomes displaced from the leading edge. The keratocyte population with high VASP peak-to-base ratios observed in wild-type cells (see Figure 3C) becomes absent after cytochalasin treatment, leaving only cells with low and medium values.
(D) Shape mode analysis reveals that cytochalasin treatment (open diamonds) eliminates the population of keratocytes with canoe shapes (>1 in y-axis) compared to control cells (closed diamonds) causing most cells to resemble the mean shape of the population or rounder D shapes. No significant correlation was found between this shape mode and enrichment of VASP at the leading edge (VASP peak-to-base ratio) in cytochalasin treated cells (r2 = 0.09, n = 27, dashed line) compared to control cells.
(E) Cytochalasin treatment (open diamonds) eliminates the correlation between local leading-edge curvature and VASP enrichment at the leading edge (r2 = 0.0004, n = 27, dashed line) previously observed in control cells (closed diamonds).
(F) The negative correlation between cell shape and local leading edge curvature observed in control cells (closed diamonds) is abolished in cells treated with cytochalasin (open diamonds, r2 = 0.0009, n = 27, dashed line).
(G) After cytochalasin treatment, a typical cell has VASP absent from the leading edge (VASP peak-to-base ratio = 0.83) and an F-actin peak ratio of 0.96 corresponding to a flat F-actin distribution along the leading edge. Cytochalasin treatment eliminated cells with peaked F-actin distributions corresponding to high F-actin peak values from our population. In addition, no significant correlation was found between F-actin peak ratios and enrichment of VASP at the leading edge (VASP peak-to-base ratio) in our population of cytochalasin treated cells (r2 = 0.006, n = 27, dashed line) compared to control cells. Scale bar = 10 μm.