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. Author manuscript; available in PMC: 2012 Feb 5.
Published in final edited form as: Cell. 2011 Aug 5;146(3):421–434. doi: 10.1016/j.cell.2011.07.008

Figure 3. The actin-severing protein cofilin is associated with TrkA signaling endosomes and is required for their retrograde transport.

Figure 3

(A) Early endosomes (top panels) were purified from cultured rat sympathetic neurons deprived of NGF for 18h then treated for 20 min with NGF (100 ng/ml) or NT3 (1000 ng/ml) or left untreated. Levels of total cofilin, Ser3-phosphorylated cofilin, and Rac1 were assessed by immunoblot analysis. Relative levels of cofilin and inactive cofilin (Ser3 phosphorylated) associated with NGF and NT3 endosomes were quantified. Whole cell lysates (bottom panels) from sympathetic neurons treated with NGF and NT3 were analyzed for cofilin and phosphor-cofilin. Data are represented as mean +/− SEM, (n=3), ** p< 0.01, *** p<0.0001.

(B) Colocalization of internalized Flag-TrkA endosomes (red) and cofilin (green) was assessed by immunocytochemistry of distal axons of sympathetic neurons that were untreated, or treated with either NGF or NT3. Data are represented as mean +/− SEM, (n=5), ** p< 0.01. Scale bar represents 10μm.

(C) Lentivirus infected sympathetic neurons were grown in microfluidic chambers, and the retrograde transport of Flag-TrkA endosomes was assessed. Neurons were counterstained with FITC-phalloidin. Scale bar represents 10μm.

(D) The effect of cofilin knockdown on NGF-dependent retrograde survival of neurons was tested for sympathetic neurons grown in microfluidic chambers. Neurons were allowed to project axons into distal chambers and then were infected with either a control lentivirus or a virus expressing an shRNA against cofilin. Survival was monitored 36h to 48h later under conditions in which NGF was exposed to both cell body and distal axon compartments, or NGF was exposed exclusively to the distal axon compartment. Data are represented as mean +/− SEM, (n=4), ** p< 0.01.

(E) Inhibition of actin polymerization by LatA rescues the TrkA transport defect of cofilin knockdown neurons. Neurons were treated as in (D) with the addition of a 30 min pretreatment of vehicle or LatA in distal axons prior to performing the Flag-TrkA retrograde transport assay. Data are represented as mean +/− SEM, (n=3). Scale bar represents 10μm. Statistical analysis was done using Student's t test with the exception of (C), which was done using one-way ANOVA followed by Tukey's post-hoc test. * p< 0.05.