Elsevier

Brain Research

Volume 1176, 24 October 2007, Pages 1-10
Brain Research

Research Report
Attenuation of actinomyosinII contractile activity in growth cones accelerates filopodia-guided and microtubule-based neurite elongation

https://doi.org/10.1016/j.brainres.2007.07.081Get rights and content

Abstract

The myosinII-specific inhibitor blebbistatin was used to attenuate actinomyosinII contractility in E7-chicken retina explant, medulla and spinal cord neuronal cell cultures. Addition of 20–100 μM blebbistatin, a concentration range that reversibly disrupts actin stress fibers, led to a reduction of growth cone lamellipodial areas and to an elongation of filopodia within 5 to 10 min. These morphological changes were completely reversed after removing the inhibitor. In the continued presence of blebbistatin for several hours, a dose-dependent acceleration (up to 6-fold) of neurite outgrowth was observed. The rapidly elongating neuritic processes displayed narrowed growth cones with one to three long filopodia at the leading edge. At the same time, thin neuritic branches emerged in a “push”-like fashion guided by filopodial extensions. Immunocytochemical characterization of these thin sprouts revealed that they contained actin filaments, myosinIIA, phosphorylated neurofilament/tau epitopes, MAP2, NCAM-PSA, and microtubules, demonstrating that these processes presented neurites and not filopodia. The crucial involvement of microtubules in blebbistatin-induced accelerated neurite extension was confirmed by its inhibition in the presence of nocodazole or taxol. The promotion by blebbistatin of neurite outgrowth occurred on polylysine, laminin, as well as on fibronectin as substrate. The presence of the Rho/ROCK-inhibitor Y-27632 also caused a dose-dependent promotion of neurite growth which was, however, 3-fold less pronounced as compared to blebbistatin. In contrast to blebbistatin, Y-27632 led to the enlargement of growth cone lamellipodial extensions. Our data demonstrate that neurite outgrowth and branching are inversely correlated with the degree of actinomyosinII contractility which determines the speed of retrograde flow and turnover of actin filaments and, by this, microtubule extension.

Introduction

Axon guidance as well as axon and dendrite elongation and branching depend on cytoskeleton dynamics of neuronal growth cones and distal neurite compartments. Thus, actin assembly at the leading edge of growth cones generates actin networks (lamellipodia) and bundles (filopodia) which have both been shown to undergo retrograde flow within the peripheral domain (Bray and White, 1988, Mitchison and Kirschner, 1988, Smith, 1988, Forscher and Smith, 1988, Lewis and Bridgman, 1992, Rösner and Fischer, 1996, Brandt, 1998, Dontchev and Letourneau, 2003). In addition, a slower backward movement of actin structures was observed within the transition zone and central domain (Schäfer et al., 2002). Retrograde flow of actin filaments, which works in concert with cell adhesion to generate traction force (Lin et al., 1996, Heidemann and Buxbaum, 1998, Suter and Forscher, 2000, Jay, 2000) and ends up in actin filament severing and disassembly, has been shown to critically depend on the activity of myosins, with myosinIIA and B (Brown and Bridgman, 2003a, Brown and Bridgman, 2003b, Brown and Bridgman, 2004) and possibly also myosin 1c (Diefenbach et al., 2002) representing the major players. Pharmacological inhibition of myosin-light-chain-kinase (MLCK) by ML-7 was found to result in a loss of actin bundles and partial growth cone collapse (Ruchhoeft and Harris, 1997, Oberstar et al., 1997, Zhou et al., 2002, Schmidt et al., 2002). However, a Rho/ROCK-dependent increase of MLC phosphorylation was also found to induce growth cone collapse, probably due to hyperactivation of actinomyosinII force generation (Hirose et al., 1998, Wahl et al., 2000). Detailed data concerning the mechanism of myosinII action in growth cones have been recently provided by a study on Aplysia (Medeiros et al., 2006) following application of blebbistatin (BBS), a low molecular weight specific inhibitor of myosinII ATP'ase (Straight et al., 2003, Kovacs et al., 2004, Limouze et al., 2004, Allingham et al., 2005). Here it was shown that an inhibition of myosinII by blebbistatin decreased actin bundle contraction and severing in the transition zone. At the same time, peripheral retrograde actin flow was decreased by 51% and filopodial actin roots elongated by approximately 83%, suggesting that both are in part controlled by actinomyosinII contractility in the transition zone.

The above data suggest that myosinII activity may be an important target of signaling regulating the mode and intensity of neurite outgrowth. In order to further evaluate the role of myosinII activity in regulating neurite outgrowth, we used blebbistatin to specifically attenuate actin–myosinII interaction in cultured embryonic chicken neurons. We show that, in vertebrate growth cones, a blebbistatin-mediated partial inhibition of actinomyosinII contractility leads within a few minutes to a 2- to 4-fold increase in filopodial length followed by a dose-dependent acceleration of microtubule-based neurite elongation and branching. Our data provide evidence that the velocity of neurite elongation as well as the intensity of sprouting of neurite branches depend inversely on the level of actinomyosinII contractile activity controlling filopodial actin bundle dynamics and filopodial actin bundle-associated microtubule extension.

Section snippets

Attenuation of actin–myosinII interaction by blebbistatin reversibly disrupts actin stress fibers

In order to determine the appropriate dose-range of BBS, the effects of 10–100 μM BBS on stress fibers were investigated in chicken fibroblast cultures. Stress fibers were stained for actin filaments by phalloidin-Alexa 488 (Figs. 1A–E) and immunostained for myosinIIA (Figs. 1B–F). Treatment of the fibroblasts for 30 min with 50 μM BBS, a dose reported to inhibit platelet myosinII S1 ATP'ase to more than 90% (Kovacs et al., 2004, Straight et al., 2003), led to complete disruption of stress

Discussion

In this study we have used blebbistatin (BBS) in order to inhibit f-actin–myosinII interaction in neuronal growth cones. BBS was recently discovered and acts as a specific low molecular weight inhibitor of myosinII by reversibly binding to the myosinII head in a product complex with low affinity in an actin detached state (Straight et al., 2003, Kovacs et al., 2004, Limouze et al., 2004, Allingham et al., 2005). In cell cultures of embryonic chicken medulla and spinal cord neurons and retina

Materials

Dulbecco's minimal essential medium (DMEM), DMEM/Ham's F12 with stabilized l-glutamine, HEPES, heat-inactivated fetal calf serum (FCS), penicillin, and streptomycin were obtained from Biochrom. Fibronectin, bovine serum albumin (BSA), anti-β-tubulin mouse IgG (Tub. 2.1), anti-myosinIIA rabbit IgG (M8064), nocodazole, taxol, and cytochalasin B were from Sigma. Anti-rabbit IgG-Alexa 568 and phalloidin-Alexa 488 were purchased from Molecular Probes. Anti-mouse-Cy3 was from Jackson Immuno Research,

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