Actin turnover is required to prevent axon retraction driven by endogenous actomyosin contractility - PubMed (original) (raw)

Actin turnover is required to prevent axon retraction driven by endogenous actomyosin contractility

Gianluca Gallo et al. J Cell Biol. 2002.

Abstract

Growth cone motility and guidance depend on the dynamic reorganization of filamentous actin (F-actin). In the growth cone, F-actin undergoes turnover, which is the exchange of actin subunits from existing filaments. However, the function of F-actin turnover is not clear. We used jasplakinolide (jasp), a cell-permeable macrocyclic peptide that inhibits F-actin turnover, to study the role of F-actin turnover in axon extension. Treatment with jasp caused axon retraction, demonstrating that axon extension requires F-actin turnover. The retraction of axons in response to the inhibition of F-actin turnover was dependent on myosin activity and regulated by RhoA and myosin light chain kinase. Significantly, the endogenous myosin-based contractility was sufficient to cause axon retraction, because jasp did not alter myosin activity. Based on these observations, we asked whether guidance cues that cause axon retraction (ephrin-A2) inhibit F-actin turnover. Axon retraction in response to ephrin-A2 correlated with decreased F-actin turnover and required RhoA activity. These observations demonstrate that axon extension depends on an interaction between endogenous myosin-driven contractility and F-actin turnover, and that guidance cues that cause axon retraction inhibit F-actin turnover.

PubMed Disclaimer

Figures

Figure 1.

jasp induces axon retraction. Numbers in individual panels indicate minutes relative to the addition of jasp (40 nM at 0 min). The bars in the leftmost panels denote 10 μm. (A) Within 2–3 min after treatment with jasp, growth cones became quiescent and underwent contraction followed by axonal retraction. (B) Axons began retracting 4–6 min after treatment with jasp. Axon retraction often resulted in the formation of axonal bends (indicated by arrowheads at 8 min). Note that at 6 min after treatment, an additional retracting growth cone becomes visible (arrow). (C) jasp caused axon retraction during a 20-min observation period in a concentration-dependent manner (n ≥ 37 axons at each concentration). (D) Overnight treatment of DRG explant cultures with jasp caused a dose-dependent decrease in the length of axon outgrowth (n > 8 explants per concentration). (E) Localized application of jasp to growth cones causes contraction and axon retraction. Bends in axons form distally after growth cone contraction (arrow). (F) Application of jasp to axons does not cause the formation of axon bends at the site of application, but after prolonged application and diffusion of jasp, axons retract with bends first forming at the distal end of axons. The pipette was removed before acquisition of the images from which the montage is created. The pipette position is denoted by the + symbol. Bars, 10 μm.

Figure 2.

jasp is rapidly internalized, binds to F-actin, and inhibits its turnover. (A) Treatment of DRG cultures with 40 nM jasp caused axon retraction (μm/20 min). Treatment with lat-A (LatA) did not cause axon retraction, but blocked the effects of jasp (n ≥ 32 axons per group). (B) Phalloidin staining of DRG axons treated with DMSO or 40 nM jasp (C) for 10 min. Bar (B) 10 μM. Note the largely diminished staining in jasp-treated axons. D and E are pseudocolored images of the same growth cone double stained with phalloidin and the antiactin antibody, respectively. Note the almost identical staining pattern demonstrating that using the combined fixation-extraction protocol, the antibody staining is reflective of F-actin in growth cones. (F–I) Actin antibody staining of growth cones treated for 4 min with either DMSO (F and G) or 40 nM jasp (H and I) and then for an additional 2 min with either DMSO (F and H) or 2 μM lat-A (LatA; G and I). Bars (D– I) 5 μM. Note that although lat-A causes the depolymerization of the majority of F-actin in DMSO-treated growth cones, it has only a partial effect on growth cones treated first with jasp. jasp causes the centripetal accumulation of growth cone F-actin as revealed by actin antibody staining. J and K are examples of growth cones treated for 10 min with DMSO or 40 nM jasp, respectively. Note the accumulation of F-actin as reflected by the presence of warmer colors (red and yellow) in the contracted growth cone treated with jasp. Bar (J), 10 μM. L is an example of the deformations (red arrows) of the axonal microtubule array that develop in axons undergoing jasp induced axon retraction (40 nM for 10 min).

Figure 3.

Myosin activity is required for jasp-induced axon retraction. A and B show phalloidin-stained chicken embryonic fibroblasts. (A) In control-loaded (BSA) fibroblasts, stress fibers form at the circumference by 2 h after plating the cells (arrows). However, in fibroblasts loaded with skMyosin II (SkMyo), stress fibers fail to form. (C) Trituration loading of C3 toxin or SkMyo largely decreased the percentage of fibroblasts with stress fibers 2 h after plating (n ≥ 76 fibroblasts per group). (D) Trituration loading of DRG neurons with SkMyo, or treatment with the myosin ATPase inhibitor BDM (2 mM for 40 min), inhibits the distance axons retract after treatment with 40 nM jasp for 20 min (n ≥ 43 axons per group). Data is presented normalized to the distance control axons retracted. (E) Example of BDM-treated growth cones before and 20 min after treatment with 40 nM jasp. Note that the growth cones do not undergo contraction. CNT, control. Bars, 10 μM.

Figure 4.

RhoA and MLCK are required for jasp-induced axon retraction. (A) Immunocytochemical localization of RhoA and MLCK in DRG growth cones. (B) Trituration-loaded C3 and the ROCK inhibitor Y-27632 (10 μM for 30 min) attenuate jasp-induced (40 nM for 20 min) axon retraction in DRG cultures. Conversely, trituration loading of constitutively active RhoA (CaRhoA) potentiates axon retraction (n ≥ 52 axons per group). Data are presented normalized to the distance control axons retracted. (C) Example of a Y-27632–treated growth cone before and after treatment with jasp. Note that the growth cone does not undergo contraction. (D) Trituration loading of two separate MLCK-inhibitory peptides (MLCKp1 and p2) into DRG neurons inhibits jasp-induced axon retraction. The pharmacological MLCK inhibitor ML-7 (300 nM for 30 min) also inhibits the effects of jasp. However, growth cones often underwent contraction (inset; n ≥ 43 axons per group). Data are presented normalized to the distance control axons retracted. CNT, control. Bars, 10 μM.

Figure 5.

RhoA and MLCK activity are required for jasp-mediated inhibition of axon extension. A 40-min treatment with 3 nM jasp reduced axon elongation rate. The effects of 3 nM jasp on elongation rate were attenuated by inhibition of ROCK with 10 μM Y-27632 or an MLCK inhibitory peptide (MLCKp1) delivered using Chariot™ (n ≥ 36 axons per group).

Figure 6.

jasp does not alter levels of myosin light chain phosphorylation. Shown is a Western blot representative of myosin light chains separated using urea-glycerol PAGE. This technique separates the light chains by size and charge, thereby revealing the extent of non, mono-, and di-phosphorylated chains. Note that treatment with 40 nM jasp for 10 min did not change the profile of light chain phosphorylation. The experiment was repeated three times and produced consistent results. The slight di-phosphorylated band was associated with both control and jasp-treated samples and did not correlate with treatment across all experiments.

Figure 7.

Ephrin-A2 induces axon retraction that requires RhoA activity and correlates with the presence of stable F-actin. (A) Example of temporal retinal axon retraction in response to 1 μg/ml ephrin-A2 (arrow shows distance the axon retracted during a 20-min period; ∼75 μm). Bar (A) 10 μM. (B) Example of axons treated first with C3, and then with ephrin-A2. Note that none of the axons undergo retraction. (C) Phalloidin staining of temporal retinal axons treated for 10 min with 1 μg/ml BSA or ephrin-A2, and then for 2 min with DMSO or 2 μM lat-A (LatA). Note that lat-A caused significant depolymerization of F-actin in BSA-treated axons, but only partial depolymerization in ephrin-A2–treated axons. Bar (C), 5 μM. (D) Quantification of the F-actin content in the distal axons of cultures treated with BSA or ephrin-A2 for 4 or 10 min before treatment with DMSO or lat-A for 2 min (n > 41 in each group). (E) Inhibition of RhoA (C3) or ROCK (Y-27632 and HA-1077) blocks ephrin-A2–induced axon retraction (n > 35 in each group). CNT, control.

Figure 8.

Working model for the regulation of axon extension by F-actin turnover and myosin-driven contractility. (A) Regulation of axon extension by the combination of F-actin turnover and actomyosin contractility. (B) Diagram of relationship between F-actin (blue) and myosin II (red) in the peripheral (P) and central (C) domains of growth cones. Under “normal” conditions, F-actin is depolymerized at the interface between the P- and C-domains and the monomers are turned over (arrow). In the presence of jasp, F-actin is not depolymerized and filament turnover is blocked. F-actin undergoes retrograde transport and accumulates in the myosin II–enriched C-domain resulting in increased actomyosin contractility.

References

1. Ahmad, F.J., J. Hughey, T. Wittmann, A. Hyman, M. Greaser, and P.W. Baas. 2000. Motor proteins regulate force interactions between microtubules and microfilaments in the axon. Nat. Cell Biol. 2:276–280. - PubMed
1. Aizawa, H., S. Wakatsuki, A. Ishii, K. Moriyama, Y. Sasaki, K. Ohashi, Y. Sekine-Aizawa, A. Sehara-Fujisawa, K. Mizuno, Y. Goshima, and I. Yahara. 2001. Phosphorylation of cofilin by LIM-kinase is necessary for semaphorin 3A-induced growth cone collapse. Nat. Neurosci. 4:367–373. - PubMed
1. Akasu, T., M. Ito, T. Nakano, C.R. Schneider, M.A. Simmons, T. Tanaka, T. Tokimasa, and M. Yoshida. 1993. Myosin light chain kinase occurs in bullfrog sympathetic neurons and may modulate voltage-dependent potassium currents. Neuron. 11:1133–1145. - PubMed
1. Ayscough, K.R. 2000. Endocytosis and the development of cell polarity in yeast require a dynamic F-actin cytoskeleton. Curr. Biol. 10:1587–1590. - PubMed
1. Billuart, P., C.G. Winter, A. Maresh, X. Zhao, and L. Luo. 2001. Regulating axon branch stability: the role of p190 RhoGAP in repressing a retraction signaling pathway. Cell. 107:195-207. - PubMed

Actin turnover is required to prevent axon retraction driven by endogenous actomyosin contractility - PubMed (original) (raw)