4) 5) flow maps are sparse and flow tracking contains errors. In other words, the vertex is most likely not on a successfully tracked speckle, and even if it was, that track might be wrong. The ROI is displaced and deformed along the averaged vectors determined at each vertex. This mimics a closed system, assuming that all assembled actin moves with retrograde flow; therefore, changes in intensity must be due to the assembly or disassembly of actin filaments. Steps 3 and 4 are repeated for each frame pair in the series in order to generate plots of area and intensity over time. Changes in area reflect network convergence or expansion in two dimensions; changes in the total intensity within the area reflect the shifting balance of polymerization and depolymerization over time. To evaluate changes in network compression and net kinetics, three regions were tracked per growth cone. Regions were defined during the control period and tracked for three minutes (37 frames at five seconds per frame). The positions of the regions were reset and tracked for another three minutes, starting 90 seconds after the addition of jasplakinolide. Media exchange induces focus shifts in the time-lapse movie. The delay eliminated any intensity shifts from the analysis. Regression Analysis of Retrograde Flow Decay The curve fitting toolbox in Matlab (MathWorks) was used to model the flow rate changes resulting from drug treatments as an exponential process with an offset according to Equation 1: F (t)~(1{c)e{t=tzc Where F(t) is the flow at time t in minutes, c is the flow offset, and t is a time constant, defined as the amount of time required for flow to decrease to 1/e of its initial value. The flow rates for individual growth cones were normalized to rates assessed just before jas- plakinolide addition. Regression analysis was performed on popu- lation averages and individual growth cone results. Image Processing All fluorescent images were subjected to background subtraction prior to quantitative analysis. The lone exception is automated speckle tracking analysis, which was performed upon raw image data. For display only, images were convolved with a Gaussian kernel and scaled according to a linear look up table. For ratio imaging, Gaussian convolution was utilized prior to background subtraction in order to reduce noise levels. A binary mask was used to eliminate noise amplification outside the cell. Ratio images were normalized by sliding and stretching the image histograms and displayed using a linear lookup table. Fluorescent image processing was performed in Metamorph (Universal Imaging), Matlab (MathWorks) or Image J (open source, NIH: http://imagej.nih. gov/ij/index.html) software. Electron micrographs were scaled and sharpened in Photoshop (Adobe) with an 8 pixel radius and between 50 and 100% sharpening. Line Scan Analysis Line scans were used to analyze the spatial intensity distribution of fluorescent probes. Line scans were performed in Metamorph (Universal Imaging) or Image J (NIH) and the data exported to Excel (Microsoft). The growth axis was defined by drawing a line along the presumed axis of growth. Average pixel intensity was Retrograde Flow Requires Actin Depolymerization sampled using 15–20 pixel width lines were plotted as a function of distance from the leading edge. Supporting Information Figure S1 Myosin II localization in unaffected by jaspla- kinolide treatment. TRITC-Phalloidin stabilized F-actin (A, C) and Myosin II localization (B, D) in growth cones treated for 3 minutes with vehicle (0.05% DMSO, A–B) or 500 nM Jasplaki- nolide (C–D) and then live-cell extracted. All phalloidin and myosin II images were acquired using the same imaging parameters to enable direct comparison across conditions. (E, F) Line-scan analysis of F-actin (E) and myosin II (F) localization in growth cone. Line scans (20 pixels wide) were sampled in regions indicated by blue and orange lines in A and C respectively and intensities plotted verses distance from the leading edge. Blue diamonds and yellow squares represent individual growth cones sampled under control conditions and in jasplakinolide respectively. Black and red lines represent 20 point rolling averages for control and jasplakinolide treated conditions (n = 27 and 34 growth cones). (TIF) Figure S2 Schematic representation of region tracking algorithm. Flow displacement and deformation of regions generates a closed system where changes in intensity can be interpreted as reflecting kinetic activity. The principle steps in the algorithm are illustrated here. Panel 1) The user is prompted to select a background region (dashed white box outside the cell). The average fluorescence in this box is determined for each frame in the image series and subtracted from each pixel in the frame. Panel 2) The user is prompted to select a region of interest to be tracked (Red outline in panel 2). Panel 3) The average flow is calculated at each vertex. A user selectable grid size (dashed white box) is centered upon each vertex and all flow vectors detected within the box are averaged. Panel 4) The ROI is displaced and deformed along the averaged flow vectors. The dark grey of the ROI interior reflects the idea that this is a closed system. (TIF) Figure S3 Intensity profiles of lysine-substituted dex- trans are unchanged during post-fixation membrane extraction. A bag cell neuron, injected with 10 kD, lysine- fixable, Texas Red dextran (Molecular Probes), was imaged throughout processing for immunocytochemistry. (A–B) Epifluor- escence images after 4% formalin fixation only (A) and after 1% Triton X-100 extraction (B). (C) Line scans reporting the average intensity in a 20 pixel wide band along the red and green lines in A and B. The position of the leading edge is indicated by the dashed line. The average background intensity was subtracted from the images prior to the line scan analysis. (D) Ratio of the Fixed line scan divided by the Extracted line scan. Inside the cell, the ratio is flat; however, as the fluorescent signal decreases, the noise increases. The ratio is greater than 1, reflecting a ,17% loss of signal during 1% Triton X-100 extraction. (TIF) Figure S4 G-actin concentration profiles in control growth cones. A–D) Four examples of G-actin profiles in growth cones of varying size and shape. Note consistently higher levels in P as compared to C domains despite differences in P domain concentration profile shape. Plots are 4 point rolling averages of intensities sampled from the leading edge into the C domain. Bar = 10 mm. (TIF) Video S1 Actin dynamics during jasplakinolide treat- ment. A. californica BCN injected with Alexa-568 actin (Molecular PLoS ONE | www.plosone.org 13 February 2012 | Volume 7 | Issue 2 | e30959