Retrograde Flow Requires Actin Depolymerization Figure 2. Jasplakinolide results in depletion of actin networks from the leading edge. (A) A bag cell neuron was injected with Alexa-568 actin monomers and imaged using time-lapse, SDC microscopy before and during drug treatment with 500 nM jasplakinolide (1006, 1.4NA objective). Note actin speckle depletion from the leading edge after 6 min in drug (top panel). Bottom panel is a 3 minute duration time montage sampled from the box in (A). (B) Rotary shadowed electron micrographs showing representative leading edge structures under control conditions and after 3 and 6 minutes of jasplakinolide treatment. Note depletion of leading edge veil actin and buckling of filopodia at the 6 min time point. doi:10.1371/journal.pone.0030959.g002 Actin filament turnover and myosin II contractility independently contribute to retrograde actin network flow To parse out myosin II’s role in jasplakinolide effects on retrograde flow, cells were pretreated with blebbistatin (70 mM, 10 min) and then exposed to jasplakinolide (500 nM) in the continued presence of blebbistatin (Fig. 5). Retrograde flow rates were assessed by kymography every 1.5 minutes under control conditions, then in blebbistatin, and finally, in blebbistatin plus jasplakinolide (Fig. 5B–C). Pretreatment with blebbistatin for 10 min decreased retrograde flow rates to about 75% of control levels and prevented retraction of actin networks from the leading edge seen with jasplakinolide (cf. Fig. 1–2 vs. Fig. 5A–B, Video S3). Addition of Jasplakinolide in the continued presence of blebbistatin reduced retrograde flow rates further to 12.8+/25.2% of control levels within 9 min (Fig. 5B–C). Residual network flow in blebbistatin plus jasplakinolide (Figure 5C) was significantly slower than that reported in Figure 3 for jasplakinolide alone (p,0.01; time-matched two- tailed Student’s t-test). Aside from the negative offset introduced by blebbistatin pre- treatment, we noted that the kinetics of jasplakinolide induced actin flow decay in blebbistatin were quite similar to those observed after jasplakinolide treatment alone. These results suggested the drug effects could be due to two separate additive processes. In agree- ment, the decay of retrograde flow was well fit by a first order exponential process with an offset (Equation 1, Materials and Methods) when flow rates were normalized to levels measured immediately before jasplakinolide addition (Fig. 6A). The adjusted R2 value with or without blebbistatin pretreatment were 0.9982 and 0.9991 respectively. The decay time constant t derived from the PLoS ONE | www.plosone.org 4 February 2012 | Volume 7 | Issue 2 | e30959