Cytoskeletal dynamics in growth-cone steering

Abstract

Interactions between dynamic microtubules and actin filaments are essential to a wide range of cell biological processes including cell division, motility and morphogenesis. In neuronal growth cones, interactions between microtubules and actin filaments in filopodia are necessary for growth cones to make a turn. Growth-cone turning is a fundamental behaviour during axon guidance, as correct navigation of the growth cone through the embryo is required for it to locate an appropriate synaptic partner. Microtubule-actin filament interactions also occur in the transition zone and central domain of the growth cone, where actin arcs exert compressive forces to corral microtubules into the core of the growth cone and thereby facilitate microtubule bundling, a requirement for axon formation. We now have a fairly comprehensive understanding of the dynamic behaviour of the cytoskeleton in growth cones, and the stage is set for discovering the molecular machinery that enables microtubule-actin filament coupling in growth cones, as well as the intracellular signalling pathways that regulate these interactions. Furthermore, recent experiments suggest that microtubule-actin filament interactions might also be important for the formation of dendritic spines from filopodia in mature neurons. Therefore, the mechanisms coupling microtubules to actin filaments in growth-cone turning and dendritic-spine maturation might be conserved.

Fig. 1.

(A) Organisation of the growth-cone cytoskeleton. The growth cone is an expanded, motile structure at the tip of the axon and is divided into several morphological regions (as indicated by dashed red lines). In the axon shaft, microtubules (green lines) are organised into parallel bundles by MAPs (purple dumbbells), whereas in the C-domain they become de-fasciculated and extend individually through the T-zone and into the P-domain, where they become aligned alongside the F-actin bundle (black lines) in filopodia. In the P-domain, the F-actin in filopodia is organised into parallel bundles that extend rearwards across the P-domain to terminate in the T-zone, where they are severed into short filaments by a mechanism that is incompletely understood. F-actin in the lamellipodia of the P-domain is organised into a branched dendritic network. In the T-zone and at the periphery of the C-domain there are so-called actin arcs (blue lines) composed of anti-parallel bundles of F-actin and myosin II. Actin arcs produce compressive forces in the C-domain that coral the unbundled microtubules and thereby facilitate microtubule bundling in the growth-cone wrist. (B) Immunofluorescence confocal images of microtubule (tubulin) and actin filament (F-actin) distribution in a growth cone from an embryonic cortical neuron in culture. The growth cone was simultaneously fixed and detergent-extracted to remove soluble proteins and preserve the cytoskeleton. F-actin is labelled with phalloidin and microtubules are identified with an antibody specific for tubulin. Microtubules extend individually into the P-domain, where they align alongside the bundles of F-actin in filopodia (white arrowheads). Drebrin predominantly localises to the T-zone but also associates with the proximal region of the F-actin in those filopodia where microtubules are also commonly inserted (black arrowheads). In the merged image, microtubules are blue, F-actin is red and drebrin is green.

Fig. 2.

Models of the molecular mechanisms of microtubule–F-actin coupling in growth cone filopodia. The diagrams show microtubules (green lines) located to one side of the proximal end of a filopodial bundle of F-actin (black lines). (A) The simplest mechanism for crosslinking microtubules to F-actin involves a protein that has both F-actin-binding (red blobs) and microtubule-binding (purple box) domains. Examples of such proteins found in growth cones include members of the spectraplakin family such as shortstop. It is assumed that the F-actin-binding domains recognise adjacent F-actin in the bundle, and that this confers specificity of binding to bundled F-actin. (B) A development of the simplest mechanism for crosslinking microtubules to F-actin is to have two proteins that bind to each other – one that binds to F-actin and the other to microtubules. The F-actin-binding protein drebrin and the +TIP protein EB3 are present in growth cones and have these properties. (C) A final possibility for coupling microtubules to F-actin is to have information conveyed through intermediary messengers (brown oval). In this model there is no direct physical interaction between microtubules and F-actin. The direction of information flow is depicted from F-actin to microtubules but could also occur in the opposite direction.

Fig. 3.

EB3 surfs on the growing end of dynamic microtubules in growth cones. Consecutive video frames [GFP fluorescence (upper panels), and merged fluorescence and phase contrast (lower panels)] from an embryonic cortical neuron growth cone expressing EB3-GFP. EB3-GFP comets can be seen entering the proximal region of emerging filopodia at the periphery of the growth cone (arrowheads).

Fig. 4.

Intracellular signalling pathways in growth cones converging on the cytoskeleton. The balance between dynamic and stable microtubules is regulated via several growth-cone guidance pathways, including signalling pathways activated by the binding of Slit to the Robo receptor, by the neurotrophin nerve growth factor (NGF) binding to the TrkA receptor, and by Wnt ligands binding to the Frizzled receptor (Frz) and its co-receptor low density lipoprotein receptor-related protein (LRP). Through the activity of kinases such as Abl and GSK3, these pathways converge at microtubule-associated proteins (MAPs) that bind directly to microtubules and alter their dynamic instability. Localised changes in the stability of microtubules modify their interactions with F-actin and regulate axon growth and growth-cone pathfinding.