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First published online March 30, 2007
doi: 10.1242/10.1242/jcs.03379

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Analysis of actin filament network organization in lamellipodia by comparing experimental and simulated images

Laboratory of Cell Biophysics, Ecole Polytechnique Fédérale, Lausanne, Switzerland

View larger version (14K): [in this window] [in a new window]   Fig. 1. Formation of simulated fluorescence image. (a) Diagram of the actin network illustrating filament elongation, branching and capping reactions. (b,c) Simulation, converted into an actin-polymer density map (b) and resolution was reduced to obtain an image similar to the fluorescence image (c).

View larger version (44K): [in this window] [in a new window]   Fig. 2. Actin concentration analysis. (a) Distribution of F-actin in keratocyte visualized with Rhodamine-phalloidin. Actin-concentration profile from front to back of a lamellipodium (boxed gray region) is plotted at the right panel. Black dashed box represents a typical (7x7 µm2) field of interest. Bar, 10 µm. (b) Contrast of the simulated images (circles) is plotted versus the inverse square root of actin-polymer concentration. Error bars give ± s.d. Gray area shows the range of contrast measured in experimental images and the corresponding range of actin concentration. (c,d) Histogram of the intensity ratio between front and back of a lamellipodium using measurements based on phalloidin-staining intensity (c), showing the same decay as the concentration profile measured by experimental contrast (d). Error bars give ± s.d. (e) Comparison of relative concentration profiles measuredon the basis of intensity (solid lines), contrast (dashed lines), in experimental (squares) and simulated images with zero-order and first-order depolymerization kinetics (circles and diamonds, respectively).

View larger version (132K): [in this window] [in a new window]   Fig. 3. Comparison of patterns of experimental and simulated fluorescence images of lamellipodia. (a-d) Dependence of the pattern on the mean filament length and orientation-disorder parameters in simulated fluorescence images. Low-disorder parameters (R=500 µm, =1°) and a mean filament length of (a) 4 µm, (b) 1 µm and (c) 0.3 µm and, (d) intermediate-disorder parameters (R=20 µm, =20°) and filament length of 4 µm. Simulated image with optimal parameters (L=1.3 µm, R=10 µm, =20°) built from stochastic processes (e) compared with the deterministic process of Arp2/3 activation (f). (g,h) Experimental image of equivalent sizes; (g) untreated cell and (h) cell treated with 0.1 µM cytochalasin D. Bar, 10 µm.

View larger version (59K): [in this window] [in a new window]   Fig. 4. Correlation-length analysis. (a-c) Square regions of the images (a) were filtered through Fourier mask (b) to highlight features along one orientation (c). Correlation curves measured along this direction (d, blue) were fitted (d, red) with theoretical correlation function to obtain the correlation length. (e,f) Mean correlation length measured in simulated images (solid lines) varies, depending on the mean filament length, (e) branching angle fluctuation and (f) curvature radius. Curves were compared with experimental correlation length measured in untreated cells (dashed black lines) and in cells treated with cytochalasin D at 0.05 µM (dark gray line) and 0.1 µM (light gray line). (g) Comparison of normalized correlation-length distributions from simulated images with different sets of parameters (colored symbols) to experimental images (black curve).

View larger version (46K): [in this window] [in a new window]   Fig. 5. Analysis of filament orientation and fit of simulated optical and EM images to the experimental images. (a) Angular distribution of correlation length in experimental (solid curves) and simulated (dashed curves) images. (b) Comparison of correlation-length distribution in cells treated with 0.1 µM of cytochalasin D with simulated distributions with short (or long) filaments and high (or low) orientational disorder parameters. The angle was measured with respect to the normal to the leading edge. (c-e) Simulated platinum replica images (c-e) created on the basis of the same simulations as in (a) and compared with a real platinum replica image (f) (provided T. M. Svitkina and G. G. Borisy). (g) A simulated negative staining EM image with the same parameters. Diagonal black line is parallel to the leading edge. Bar, 1 µm.