Fig. with this mechanism. Intro The nerve growth cone is the migrating tip of growing neurites and takes on a central part in axon outgrowth and guidance (Ramon y Cajal, 1890; Bray and Hollenbeck, 1988). Actin polymerizes near the leading edge of growth cones, and actin filaments display remarkable retrograde movement in filopodia and lamellipodia (Forscher and Smith, 1988; Katoh et al., 1999; Mallavarapu and Ethynylcytidine Mitchison, 1999). Myosin 1c and myosin II have been implicated in actin filament retrograde circulation in growth cones (Diefenbach et al., 2002; Medeiros et al., 2006). Linkage between the actin filament retrograde circulation Ethynylcytidine and cell adhesion molecules (CAMs) is thought to transmit the push of actin filament movement to extracellular substrates via CAMs (Mitchison and Kirschner, 1988; Jay, 2000; Suter and Forscher, 2000), thereby providing mechanical pressure (Bray, 1979; Lamoureux et al., 1989) and protrusion of the leading edge (Lin and Forscher, 1995) for axon outgrowth and steering. Earlier studies shown that CAMs such as apCAM (Suter et al., 1998), Nr-CAM (Faivre-Sarrailh et al., 1999), integrin (Grabham et al., 2000), and L1-CAM (Kamiguchi and Yoshihara, 2001) are coupled with actin filament retrograde circulation in growth cones. Consistently, coupling between apCAM and actin filament retrograde circulation produced mechanical pressure and induced protrusion of growth cones (Suter et al., 1998). L1 is definitely a single-pass transmembrane protein expressed mainly in developing neurons and involved in axon outgrowth and guidance (Lemmon et al., 1989; Dahme et al., 1997; Kamiguchi et al., 1998). A recent study showed that ankyrinB promotes neurite initiation by coupling F-actin circulation to L1 (Nishimura et al., 2003). However, ankyrinB was involved neither in their coupling in growth cones nor in L1-mediated neurite elongation, and the molecular basis for the actin flowCCAM linkage in growth cones remains elusive (Suter et al., 1998; Gil et al., 2003; Ethynylcytidine Nishimura et al., 2003). It is also unanswered whether coupling of this linkage is involved in the regulation of axon outgrowth. Recently we explained a novel brain-specific intracellular protein, shootin1, which is usually involved in axon formation and polarization of cultured hippocampal neurons (Toriyama et al., 2006). Shootin1 accumulates in axonal growth cones, and its accumulation in growth cones dynamically enhances neurite elongation. Shootin1 was shown to take action upstream of phosphoinositide-3-kinase (PI 3-kinase). However, shootin1-induced axon formation was not fully suppressed by a PI 3-kinase inhibitor (Toriyama et al., 2006), which suggests the presence of an additional mechanism for axon outgrowth. Here, we show that shootin1 interacts with both actin filament retrograde circulation and L1-CAM in growth cones. Our data suggest that shootin1 mediates the linkage between actin retrograde circulation and L1-CAM to promote axon outgrowth. Results Shootin1 interacts with both actin filament retrograde circulation and L1-CAM in growth cones Fig. 1 A shows shootin1 immunoreactivity in a cultured rat hippocampal neuron. Shootin1 accumulated to a high level in axonal growth cones (Fig. 1 A, arrows), where it localized in filopodia and lamellipodia in close apposition with Rabbit Polyclonal to NCAML1 actin filaments (Fig. 1 B, arrowheads). To elucidate the association of shootin1 with cytoskeletal dynamics in growth cones, we performed fluorescent speckle microscopy (Waterman-Storer and Salmon, 1997; Watanabe and Mitchison, 2002). We performed live imaging of EGFP-shootin1 expressed at a low level in cultured hippocampal neurons. EGFP-shootin1 appeared as discrete speckle signals that serve as fiduciary marks, allowing measurement of molecular movement of shootin1. Shootin1 speckles displayed retrograde movement in filopodia and lamellipodia of axonal growth cones (Fig. 1 C, arrowheads; and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200712138/DC1). The mean velocity of shootin1 speckles was 4.5 0.4 m/min (mean SD, = 60), which is similar to that of actin filament retrograde circulation in Ethynylcytidine axonal growth cones (Katoh et al., 1999; Mallavarapu and Mitchison, 1999) and fibroblast lamellipodia (Watanabe and Mitchison, 2002). Open in a separate window Physique 1. Shootin1 and actin filaments in axonal growth cones Ethynylcytidine and XTC fibroblasts. (A) Immunofluorescent localization of shootin1 in a cultured hippocampal neuron. Arrows and arrowheads denote an axonal growth cone and minor process growth cones, respectively. (B) Deconvolved images of an axonal growth.