Retrograde Flow Requires Actin Depolymerization Figure 8. Actin turnover is required to maintain an enhanced concentration of actin monomers in the peripheral domain. A) Cytoplasmic volume detected using injected, lysine-fixable, 10 kD TxRed-dextran (left); actin monomers detected by OG488-DNAseI (middle), and actin monomer concentration estimated by the DNAseI/Dextran (i.e. G-actin/volume) ratio in cells treated for 6 minutes with vehicle (0.05% DMSO, panel A) or 500 nM jasplakinolide (panel B). Red outlines: P = peripheral domain, T = transition zone, and C = central domain areas used for quantification. Arrowheads in DNAseI images indicate mitochondria found in C domain. DNAse/Dextran ratios were normalized by histogram sliding and stretching and a linear color lookup table used for display. C) Spatial profiles of G-actin concentration in Control vs Jasplakinolide treated growth cones in panels A–B. Plots are 4 point rolling averages of line scan intensities sampled from the leading edge into the C domain. Transition zone position is indicated by arrows (see Methods for details). D) Average changes in actin monomer concentration in the transition zone or central domain relative to the P domain in the absence and presence of jasplakinolide. Error bars are standard deviations, N = 12 and 13 growth cones in control and drug treatment conditions respectively. * = P,0.001 (E) Analysis of DNAseI G-actin probe solubility. Neurons were treated for 6 minutes in vehicle or 500 nM jasplakinolide, then DNAseI labeling assessed in cells that were fixed first then permeabilized or in live cells permeabilized in a cytoskeletal stabilization buffer and then fixed [34] (Note that most of the DNAseI in the P domain is soluble; see Methods). Labeling was normalized by total DNAaseI levels assessed in cells fixed before permeabilization. Error bars are standard deviations; n = 8 (control, fixRextractRlabel), 12 (control, extractRfixRlabel), 11 (drug, fixRextractRlabel), and 12 (drug, extractRfixRlabel). Scale bar = 10 um. doi:10.1371/journal.pone.0030959.g008 Build-up of actin filaments in central regions may progressively physically obstruct network compression and contribute to the observed retrograde flow deceleration. A similar inverse relation- ship between actin filament movement and network density is seen in simplified in vitro actomyosin contractile systems [40,41]. Fluorescence and EM images show decreased actin filament density near the leading edge -also coincident with retrograde flow deceleration. Quantification of free barbed ends also revealed markedly lower actin assembly site densities near the leading edge after jasplakinolide treatment (Fig. 7). Since polymerization tends to generate pushing forces near the leading edge [42] that can drive retrograde flow in growth cones [3], less net actin assembly could contribute to the observed slower flow rates. Note that actin network retraction from the leading edge is expected whenever net actin assembly rates fall below retrograde flow rates, however, once the network separates from the leading membrane edge it can no longer contribute pushing force to drive retrograde flow [12]. In summary, we suggest that the superimposed effects of: 1) reduced leading edge PLoS ONE | www.plosone.org 10 February 2012 | Volume 7 | Issue 2 | e30959