Research Report Attenuation of actinomyosinII contractile activity in growth cones accelerates filopodia-guided and microtubule-based neurite elongation Harald Rösner ⁎ , Wolfgang Möller, Torsten Wassermann, Julia Mihatsch, Martin Blum Institute of Zoology, Cell- and Developmental Neuro-Biology, University of Hohenheim, Garbenstr. 30, D-70593 Stuttgart, Germany ARTICLE INFO ABSTRACT Article history: Accepted 2 July 2007 Available online 22 August 2007 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. © 2007 Elsevier B.V. All rights reserved. Keywords: Neurite outgrowth Actin Myosin Microtubules Blebbistatin 1. 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 BRAIN RESEARCH 1176 (2007) 1 – 10 ⁎ Corresponding author. Fax: +49 711 459 3450. E-mail address: roesnerh@uni-hohenheim.de (H. Rösner). 0006-8993/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2007.07.081 available at www.sciencedirect.com www.elsevier.com/locate/brainres