Cytoskeleton in Axon Growth

Abstract

Axons and dendrites are the neuronal processes that transmit nerve signals throughout the body. These processes are formed and maintained by the neuronal cytoskeleton, particularly microtubules and actin filaments. Microtubules are the supportive framework of neurites, axons and dendrites, as well as the rails for transporting organelles and cytoplasmic components. Actin filaments support the neuron's outer cortex, and the dynamic protrusion of actin‐filled filopodia and lamellipodia drives neuronal morphogenesis, including axon guidance, branching and regeneration. The initiation of axon formation requires advance of microtubules into filopodial protrusions adherent to the substratum. Polymerisation of microtubule plus ends and dynein‐driven transport of short microtubules provides ‘push’ for continued axonal growth. Actin‐filled protrusions at the axonal terminal, the growth cone, explore the environment for cues that bind receptors and trigger either protrusion and microtubule advance or cessation of protrusion and myosin II contractility that retracts protrusions and limits microtubule advance.

Key Concepts

  • The neuronal cytoskeleton generates and supports neuronal shape.
  • Axonal elongation involves accumulation of microtubules through polymerisation, stabilisation and transport.
  • The motile growth cone at the tip of an elongating axon is where the axon is steered to its synaptic target.
  • Dynamic actin filaments drive the growth cone's exploration of its environment.
  • Growth cone adhesive interactions allow traction that advances the growth cone.
  • Neuronal polarisation involves regulation of cytoskeletal dynamics.
  • Extrinsic guidance cues locally regulate actin dynamics and microtubule advance to steer the growth cone.
  • Axonal branching involves cytoskeletal dynamics to produce new streams of microtubule advance.
  • Success in axonal regeneration will require recapitulation of the cytoskeletal dynamics of developing neurons.

Keywords: neurite; axon; actin; microtubule; myosin II ; dynein; growth cone; guidance; branching; regeneration

Figure 1. Stages of axon formation. A hippocampal neuron in vitro passes through several stages of axon formation. (a) The neuron is initially a sphere on the substratum. Microtubules (red) circle the neuronal soma, and actin filaments (green) form a sub‐plasmalemmal cortex. (b) In stage one, dynamic actin filaments protrude filopodia from the neuronal perimeter, while microtubules remain parallel to the periphery. (c) To begin stage two, myosin II‐mediated tension exerted in an adherent filopodium directs a microtubule plus end into the base of a filopodium. (d) In stage two, further microtubule advance and actin‐based protrusion leads to formation of a neurite with growth cone. (e) In stage three one, neurite has become the axon, which contains microtubules that are stable, and with a growth cone rich in dynamic actin filaments and microtubules that probe into the actin‐filled P‐domain. Microtubule advance is limited in the growth cones of neurites.
Figure 2. Dynamics of actin filaments. Actin filaments grow by adding ATP‐actin (red) to the barbed end, which faces the leading edge of the growth cone. ADP‐actin (green) is disassembled from the filament at the opposite pointed end. Polymerisation at the distal membrane and depolymerisation proximally lead to treadmilling of F‐actin. ADP‐actin becomes recycled to ATP‐actin and can be used again for polymerisation. Actin‐binding proteins (ABPs) regulate these dynamics by either promoting polymerisation like Ena/Vasp proteins or by severing and depolymerising F‐actin like ADF/cofilin. Furthermore, ABPs can organise the actin filaments into higher order structures, for example by bundling through proteins like fascin.
Figure 3. Dynamics of microtubules. Microtubules are dynamic polymers of tubulin dimers. The polymers rapidly switch from a growing mode to a shrinking mode (catastrophe) or from shrinking to growing (rescue). Microtubule‐associated proteins (MAPs) regulate microtubule dynamics and organisation. For instance, CRMP‐2 and plus‐end tracking proteins (+TIPs) promote microtubule assembly. In contrast, SCG10 and stathmin destabilise microtubules. Furthermore, proteins such as katanin and spastin sever microtubules and are important in axon growth and branching.
Figure 4. The growth cone. (a) Growth cone of a hippocampal neuron. The actin cytoskeleton is labelled by fluorescent phalloidin (red). Microtubules are stained with antibodies against tubulin (green). The peripheral domain (P‐domain) of the growth cone contains dynamic actin filaments, while microtubules dominate the central domain (C‐domain) of the growth cone and the axon shaft. Scale bar, 10 µm. (b) Schematic diagram of a growth cone. The growth cone can be divided into two domains: The P‐domain is composed of finger‐like filopodia that are separated by veils and lamellipodia. Filopodia are formed by F‐actin bundles while lamellipodia are based on an F‐actin meshwork. The central domain (C‐domain) is filled with microtubules that enter the growth cone bundled from the axon shaft. Single, dynamic microtubules grow forward from the C‐domain to explore the P‐domain.
Figure 5. A schematic model of growth cone navigation. Actin polymerisation pushes the leading margin of the growth cone forward. Forces generated by myosin II pull actin filaments backward, where filaments are disassembled. When growth cone receptors make adhesive contacts, a molecular ‘clutch’ links the adhesive contact to actin filaments, the retrograde flow of actin filaments slows and traction forces pull the growth cone forward. This permits the advance of microtubules and organelles and promotes axonal elongation. Intracellular signals generated by attractive and repulsive axonal guidance cues interact with the mechanisms of actin polymerisation, myosin II force generation, adhesive contacts and microtubule advance to regulate the paths of growth cone migration. Reproduced with permission from Gomez and Letourneau (2013) © John Wiley and Sons.
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Letourneau, Paul C(Mar 2016) Cytoskeleton in Axon Growth. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0021855.pub2]