Axon Branching Requires Interactions between Dynamic Microtubules and Actin Filaments

Abstract

Cortical neurons innervate many of their targets by collateral axon branching, which requires local reorganization of the cytoskeleton. We coinjected cortical neurons with fluorescently labeled tubulin and phalloidin and used fluorescence timelapse imaging to analyze interactions between microtubules and actin filaments (F-actin) in cortical growth cones and axons undergoing branching. In growth cones and at axon branch points, splaying of looped or bundled microtubules is accompanied by focal accumulation of F-actin. Dynamic microtubules colocalize with F-actin in transition regions of growth cones and at axon branch points. In contrast, F-actin is excluded from the central region of the growth cone and the axon shaft, which contains stable microtubules. Interactions between dynamic microtubules and dynamic actin filaments involve their coordinated polymerization and depolymerization. Application of drugs that attenuate either microtubule or F-actin dynamics also inhibits polymerization of the other cytoskeletal element. Importantly, inhibition of microtubule or F-actin dynamics prevents axon branching but not axon elongation. However, these treatments do cause undirected axon outgrowth. These results suggest that interactions between dynamic microtubules and actin filaments are required for axon branching and directed axon outgrowth. Key words: microtubule; actin filament; growth cone; collateral axon branching; cortical development; fluorescence timelapse imagingAxons are guided in new directions by reorientation of their growth cones as well as extension of collateral branches (O'Leary et al., 1990). We have shown previously (Szebenyi et al., 1998) that cortical axon branching occurs in vitro through changes in growth cone morphologies and behaviors. Growth cones at the tips of rapidly extending cortical axons are typically small and highly motile. However, in preparation for branching, growth cones pause for many hours, greatly enlarge, and maintain motility without forward advance. Subsequently, a new growth cone develops from the tip of the large paused growth cone and forms the new leading axon. Remnants of the large paused growth cone remain behind on the axon shaft as filopodial and lamellar expansions that subsequently give rise to interstitial axon collaterals. In living cortical slices (Halloran and Kalil, 1994), similar growth cone pausing behaviors were observed in the corpus callosum in regions where collateral axon branches develop and extend toward cortical targets, suggesting that growth cone pausing is closely related to branching mechanisms in vivo.Changes in the direction of axon outgrowth depend on reorganization of the microtubule and actin cytoskeleton (Lin and

A Role for Epsin N-terminal Homology/AP180 N-terminal Homology (ENTH/ANTH) Domains in Tubulin Binding

“…Although not exclusively associated with microtubule filaments, punctate staining of endogenous epsins 1 and 2 in neuronal processes was coincident with microtubules as revealed with ␣-and ␤III-tubulin antibodies, and co-localization was particularly evident within growth cones (Fig. 8) where microtubules are splayed and distinct filaments are easily discernable (43,44). Staining of the actin cytoskeleton in the same cell preparations revealed the full extent of the growth cone (Fig.…”

Integrin-Mediated Dendrite Branch Maintenance Requires Abelson (Abl) Family Kinases

“…New axon branches emerge as F-actin-rich filopodia (Bastmeyer and O'Leary, 1996), which evolve into growth cones after microtubule invasion (Dent and Kalil, 2001) (for review, see Luo, 2002).…”

“…Genetic studies in Drosophila indicate that Abl regulates axon guidance through interactions with F-actin and microtubules (Wills et al, 1999a). Splaying of microtubule bundles at axon branch sites is followed by localized actin polymerization and the emergence of an F-actin-rich protrusion that evolves into a new growth cone (Bastmeyer and O'Leary, 1996;Dent et al, 1999;Dent and Kalil, 2001). In fibroblasts, Arg requires F-actin-and microtubule-binding domains located in its C-terminal half to promote adhesion-dependent protrusions at the periphery Miller et al, 2004).…”

Phosphorylation of Microtubule-associated Protein STOP by Calmodulin Kinase II

“…Hippocampal neurons from E18.5 embryos were cultured for 12 days in vitro, a stage of differentiation at which N-STOP is expressed. At this stage of differentiation neurons are forming a complex network comprising neurite extensions and branching points (18) where individual microtubules are distinct. Total N-STOP labeling showed co-localization of STOP and tubulin staining in neurites (Fig.…”