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Review
, 10 (5), 332-43

The Trip of the Tip: Understanding the Growth Cone Machinery

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Review

The Trip of the Tip: Understanding the Growth Cone Machinery

Laura Anne Lowery et al. Nat Rev Mol Cell Biol.

Abstract

The central component in the road trip of axon guidance is the growth cone, a dynamic structure that is located at the tip of the growing axon. During its journey, the growth cone comprises both 'vehicle' and 'navigator'. Whereas the 'vehicle' maintains growth cone movement and contains the cytoskeletal structural elements of its framework, a motor to move forward and a mechanism to provide traction on the 'road', the 'navigator' aspect guides this system with spatial bias to translate environmental signals into directional movement. The understanding of the functions and regulation of the vehicle and navigator provides new insights into the cell biology of growth cone guidance.

Figures

Figure 1
Figure 1. Directions for the trip
The growth cone encounters different types of cues in the environmental terrain. It travels upon a roadway, made up of adhesive molecules presented directly on a neighbouring cell surface (such as transmembrane cell adhesion molecules (CAMs)1) or assembled into a dense and complex extracellular matrix (ECM) (including laminin and fibronectin2). Additionally, anti-adhesive surface-bound molecules (such as Slits, Ephrins, and Chondroitin sulphate proteoglycans) can prohibit growth cone advance and thus provide the roadway `guardrails' that determine roadway boundaries. Finally, diffusible chemotropic cues represent the `road signs' that present further steering instructions to the growth cone, and include various diffusible chemotropic molecules (including Netrins and Semaphorins3,4), as well as morphogens (Wnt, Shh, BMP) and growth/neurotrophic factors like BDNF,, secreted transcription factors, and neurotransmitters. Whereas it was originally thought that some cues function as attractive `go' signals (for example, Netrins) and others as repulsive `stop' signals (for example, Ephrins), it is now clear that the response of attraction versus repulsion is not due to the intrinsic property of the particular cue, but rather to the specific growth cone receptors engaged and the internal signalling of the growth cone. Green circles are attractive cues and red circles are repulsive cues.
Figure 2
Figure 2. The growth cone `vehicle'
A. Boxed regions of the growth cone are shown in subsequent panels. B | Together, F-actin treadmilling (consisting of F-actin polymerization at leading edge, F-actin severing at transition (T)-zone, and recycling of these subunits back to leading edge) and F-actin retrograde flow (F-actin moving backwards towards T-zone) keep the growth cone engine idling. When the retrograde flow and polymerization forces are balanced, no protrusion occurs. When filopodia encounters an adhesive substrate, growth cone receptors bind to substrate and are coupled to F-actin through `clutch' proteins. This engages the clutch, anchoring F-actin with respect to the substrate and attenuating F-actin retrograde flow. Further F-actin polymerization pushes the membrane forward. This results in growth cone protrusion. C | Peripheral (P)-domain microtubules (MTs) explore filopodia along F-actin bundles and might be acting as guidance sensors. As a filopodium encounters a guidance cue, exploratory MTs might be acting as scaffolding for further signalling, and additional MTs are recruited to the region. D | Actin has a role in determining MT localization within the growth cone. Actin arcs constrain and guide C-domain MTs (simple arrows), and F-actin bundles inhibit and guide P-domain MTs (double arrows).
Figure 3
Figure 3. The growth cone as a `navigator'
Rho-family GTPases act as key navigation signalling nodes to integrate upstream directional cues and coordinate downstream cytoskeletal rearrangements. Activation of receptors by guidance cues leads to activation of Rho-GTPase regulators. These include proteins that activate Rho-family GTPases, guanine nucleotide exchange factors (GEFs), and those that inactivate them, GTPase activating proteins (GAPs). Rho-GTPases integrate the responses of upstream pathways and coordinate downstream effects by modifying the function of cytoskeletal effectors. Activation or inactivation of cytoskeletal effectors leads to responses such as actomyosin contraction, F-actin disassembly or F-actin polymerization. The resulting growth cone turning response depends upon the localization of the guidance signalling within the growth cone. Only some examples of guidance cues and receptors, GEFs and GAPs, and cytoskeletal effectors downstream Rho-family GTPases are shown in this figure. Arrows do not necessarily denote direct interaction. Boxed inset shows the Rho-GTPase activation/inactivation cycle, in which GAPs lead to the hydrolysis of GTP to GDP, whereas GEFs catalyze the exchange of GDP for GTP. Actin-Related Protein(Arp)2/3, Enabled/vasolidator-stimulated phosphoprotein (Ena/VASP), LIM domain kinase (LIMK), myosin light chain kinase (MLCK) and Rho kinase (ROCK).
Box 1
Box 1. The structure of the growth cone
The structure of the growth cone is fundamental to its function. The leading edge consists of dynamic, finger-like filopodia that explore the road ahead, separated by lamellipodia-like veils, sheets of membrane between the filopodia (see the figure). The cytoskeletal elements within the growth cone underlie its shape, and the growth cone can be separated into three domains based on cytoskeletal distribution. The peripheral (P)-domain contains long bundled actin filaments (F-actin bundles), which form the filopodia, as well as mesh-like branched F-actin networks, which give structure to lamellipodia-like veils. Additionally, individual dynamic `pioneer' microtubules (MTs) explore this region, usually along F-actin bundles. The central (C)-domain encloses stable, bundled MTs that enter the growth cone from the axon shaft, in addition to numerous organelles, vesicles and central actin bundles. Finally, the transition (T)-zone (also called T-domain) sits at the interface between the P- and C-domains, where actomyosin contractile structures termed actin arcs lie perpendicular to F-actin bundles, forming a hemicircumferential ring within the T-zone. The dynamics of these cytoskeletal players determine growth cone shape and movement during its journey.
Box 2
Box 2. Stages of axon outgrowth
One traditional description of the axon outgrowth process separates it into three stages,: Protrusion, Engorgement and Consolidation, which occur upon encountering attractive, adhesive substrates. This sequence during growth cone progression provides a framework for understanding detailed molecular mechanisms, and we assume that some of the same mechanistic events are utilized in response to diffusible chemotropic cues. The distal end of the growth cone contacts adhesive substrate (see the figure, panel a). Binding of growth cone receptors activates intracellular signalling cascades and begins formation of a molecular `clutch' that links the substrate with the actin cytoskeleton. During protrusion, the `clutch' strengthens, resulting in regional attenuation of F-actin retrograde flow (see the figure, panel b). This anchors the actin with respect to the substrate, so that as F-actin polymerization continues in front of the clutch site, the filopodia and lamellipodia-like veils of the peripheral (P)-domain move forward to extend the leading edge (see for discussion of molecular ratchet model for membrane protrusion). Engorgement occurs after actin clears from the corridor between the adhesion and the central (C)-domain, perhaps as F-actin behind the clutch is severed and removed (see the figure, panel c). F-actin arcs reorient from the C-domain towards the site of new growth,,, followed by C-domain microtubules (MTs) invading this region, guided by T-zone actin arcs and C-domain actin bundles. Finally, consolidation of the recently advanced C-domain occurs as the proximal part of growth cone compacts at the growth cone neck to form a new segment of axon shaft (see the figure, panel d). The myosin II-containing actin arcs function to compress the MTs into the newly localized C-domain (followed by MT-associated protein stabilization). Retraction of filopodia away from the area of new growth occurs as F-actin protrusive activity is suppressed in these regions (also promoted by myosin II activity52), further promoting axon shaft consolidation. These three continuous and overlapping stages occur during formation of nascent axons, and also when new growth cones form from an axon shaft during axon branching,.
Box 3
Box 3. Cytoskeletal dynamics
Actin filaments are polarized polymers composed of actin monomers and their formation, stability and destruction are carefully regulated at every stage in the growth cone. Actin monomers can be added to either end, but changes in equilibria of polymerization dynamics depend on whether ATP or ADP is associated with actin (see the figure). In the growth cone, ATP-actin is usually added to the `plus' (or barbed) end pointing towards the cell membrane, ATP hydrolyzes to form ADP-actin, and ADP-actin disassembled at the `minus' (or pointed) end facing the transition (T) zone. Monomer-binding proteins then transport the actin back to the leading edge to support further growth. Other actin-binding proteins include nucleation factors that create new actin `plus-ends' for new growth, capping proteins that block growth or disassembly, antagonists of capping proteins, filament severing proteins and filament stabilization proteins such as those that assemble F-actin into higher-order structures such as bundles and networks, and those that anchor F-actin to specific regions of the membrane (reviewed in126). Microtubules (MTs) are polarized structures composed of tubulin α/β dimers assembled into linear arrays. A linear array of alternating α- and β-tubulin subunits form a protofilament, and between 11-15 protofilaments form the wall of the MT. GTP-tubulin dimers are added to plus end, GTP hydrolysis can occur, and GDP-tubulin dimers dissociate from the minus end (see the figure). In growth cones, MT `plus' ends, which face outward towards the periphery, exhibit `dynamic instability', where they cycle through periods of growth, shrinkage and occasional pausing. Numerous proteins bind to MTs; some stabilize MTs (such as microtubule associated protein 1B (MAP1B)111), some act as MT motors (for example, dynein and kinesin128) and others are part of a family called `plus-end tracking proteins' (`+TIPs'), which have been implicated in plus-end MT dynamic control and linking MTs with actin- or membrane-associated structures (for example, End-binding proteins (EBs), Adenomatosis Polyposis Coli (APC) and Cytoplasmic Linker Protein (CLIP)-Associated Protein (CLASP)97,98).

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