Axon guidance in vertebrates
Axon Guidance in Vertebrates
The significance of the nervous system is such that its diverse functions range from cognition to movement. In order to carry out these functions the correct wiring of neural circuits to communicate with their targets is essential and this occurs during embryonic development. During development all vertebrate neural cells originate from ventricular zone of the neural tube derived from the ectoderm. Cells of the neural tube undergo proliferation in order to form neural precursors (neuroblasts) or glial precursors (glioblasts). There is radial migration of the ventricular zone cells into the outer mantle zone. The mantle zone later develops to give rise to most of the neurons of the spinal cord and brain. The neural crest cells arise from the dorsal neural tube and give rise to the much of the peripheral nervous system and to the autonomic nervous system. Neuronal migration is of great importance as it is the method by which neurons travel from their origin to their target position in the brain.
In order for neuronal migration to occur, growing axons must navigate through diverse embryonic environments by following specific pathways to reach their targets, this is known as axon guidance. In order for this to occur the growing axon has a highly specialized region known as the growth cone at the leading tip of the neurite. This highly motile structure is able to sense the surrounding environment and direct the axon to the specified target through changes in the cytoskeleton which changes the direction of axon growth. In order to fully understand the mechanisms of growth cone motility, the structure of the growth cone will be examined, This essay will focus on the dynamics of the growth cone and look at how the interactions between actin and the microtubules results in growth cone advance and a change in direction. The mechanisms as to how F-actin retrograde flow results in growth cone advance and the hypothesis behind it will also be considered.
Guidance cues in the environment provide the signalling information for the axon to navigate to its correct destination. The growth cone must distinguish between multiple sources of environmental cues and be able to adapt to these signals. These guidance cues can either be soluble molecules that are secreted from distant sites or they can be present along the substratum which the growth cone travels along or present on the surrounding cells. Over the years it has become evident that in order for the axon to navigate correctly the guidance molecules must be expressed at the right time and place suggesting that they are under tight spatiotemporal control. There is great knowledge about growth cone behaviours from in vitro studies and to date there are four modes of actions of axon guidance molecules. These environmental cues have been divided into attractive or repulsive signals that are either contact mediated or secreted, acting via diffusion gradients. Through genetic and biochemical investigations several families of growth cone guidance molecules have shown to be highly conserved. These include netrins, ephrins, slits and semaphorins. However, this essay will look at the semaphorin family paying particular attention to Semaphorin-3a and how it affects the growth cone resulting in a change in direction.
Birth of the Growth Cone; a historical overview
The extensive investigations into the functions and dynamics of the growth cone began over a hundred years ago and the first observations of the growth cone were made by the Spanish anatomist Ramon y Cajal. Ramon y Cajal (1890) came across the discovery of the growth cone by analysing the dorsal commissural neurons of the embryonic chick spinal cord. In his initial discovery he illustrated the growth cones in the spinal cords of E4 (embryonic day 4) chick embryos. His drawing illustrated the diversity of shapes exhibited by these structures ranging from simple forms to more complex ones. Fig 1. shows a section of the embryonic chick spinal cord stained with Camillo Golgi's method in order to identify nervous tissue. This drawing is a modification of his initial drawing and shows that certain axons of commissural neurons have crossed to midline to form the ventral commissure. However, Ramon y Cajal was only able to observe the growth cones in static images due to limitations in technology in that time. Even so, his observations made still remain valid to this day. He noted that the growth cones are highly motile regions at the tips of growing neurites and are capable of extending a finger-like extension, called filopodia and a sheet-like expansion called lamellipodia which extend between the filopodia [Gordon-Weeks PR, 2000].
The next key step to the fundamental understanding of growth cones was founded by Ross Harrison in 1910. Harrison was the first to demonstrate the motility of the growth cone. The reason for this important breakthrough was due to his successful new technique of tissue culturing. Other scientist were successful in examining cells in vitro but were unsuccessful in culturing and experimenting on them [Witkowski JA, 1983]. Harrison began by isolating pieces of embryonic tissue of either fragments of medullary tube or ectoderm from the branchial region from frog embryos approximately 3mm long by microsurgery using an aseptic technique. The reason for taking the neural tissue from this stage in frog development was because the medullary folds have just closed and there is no apparent differentiation of the nerve elements [del Pino et al., 2007]. He then explanted the neural tissue into a drop of fresh lymph on a sterile cover slip which had been drawn from one of the lymph-sac of an adult frog. The lymph clotted rapidly and this held the tissue in position and then Harrison inverted the cover slip over a hollow slide creating a hanging drop culture (Fig 2). This technique enabled him to observe the development of the nerve fibres from the explanted tissue in vitro and this technique continued to be used until the 1950s. This technique enabled him to observe the tissues for up to a week and up to four weeks with some specimens when taking aseptic precautions.
Harrison noted that the growth cones over time extended from the explants and that the amoeboid-like movements involved elongation from the edge of the growth cone. This region is rich in filamentous actin unlike the microtubular axon which less motile. During this investigation he observed that the growth cones appeared to navigate by feeling their way along the surface by extending and retracting the filamentous filopodia. He also noted that the growth cones bifurcated to form new neurites and that side branches were able to retract [Harrison RG, 1910]. The image below (Fig.3) is a reproduction of the original drawing from original paper. It shows how the axon has grown over a period of time as well as the defasciculation of some axons in the culture. It also shows how the axons from the brainstem penetrate the cylindrical blood clots demonstrating that preformed structures are not required for axon growth. Harrison's experiment was of great importance as it highlighted that growth was not just extrusion of cytoplasm from the cell body but it showed that in order for the axon to grow it needed to be anchored to a substratum. The reason for the growth cone to be anchored to the substratum is so that the growth cone can pull the growth cone forward. There has been much controversy as to whether axon elongation is due to growth cone pull by tension generated from its forward motility or as a result of cytoplasm extrusion from the neurite cell body. However, Lamoureux P et al., (1989) have directly showed that axon growth is a result of growth cone pull. They used glass needles of known compliance to measure axon tension in neurites of cultured chick sensory neurons by measuring the distance covered within a period of time.
So far the examination of growth cones had been made in vitro but Speidel CC (1933) was the first to observe growth cones in living animals. He used the tail fin of the frog tadpole in order to observe the growth cones of sensory nerves for days and even weeks. Speidel was able to examine the behaviour of growth cones when they encountered obstructions as well as how they were able branch, change direction and respond to electrical stimulation. His reports were consistent with observations made from in vitro studies showing consistency in the behaviour of growth cones.
The initial discovery of growth cones occurred over a hundred years ago but true discoveries were not apparent until nearly seventy years later. This is because the development and our understanding of molecular biology has led to key breakthroughs in molecules that govern axon guidance and how the intrinsic machinery operates in order to propel the growth cone in all directions.
Signposting the way home: Guidance Molecules
Prior to discussing the dynamics of the growth cone skeleton one must consider the environmental cues which direct the axon to its designated target. As mentioned previously these guidance cues can have different function and as highlighted by Tessier-Lavigne M and Goodman CS (1996) they suggested that there are four types of these molecular cues. Firstly, contact adhesion - the growth cone may be more attractive to one type of substrate compared to another and this may cause the axon to change direction. Secondly, contact repulsion - the growth cone may come in contact with repulsive signals on cells which may cause the growth cone to change direction or collapse. Thirdly, long-range attraction - there may be a gradient of positive diffusible cues (chemotaxis) from a distant source which causes the growth cone to grow in this path. Lastly, long-range repulsion - a chemotaxis of repulsive diffusible molecules warns the growth cone not to grow in that direction.
When guidance cues are detected by growth cone receptors a cascade of signalling events converge onto the Rho-family GTPases which results in changes in the dynamics of the cytoskeleton to allow for growth cone motility in all directions and the role of these GTPase will be investigated further into the essay. One must appreciate that the growth cone must be able to detect many different guidance cues simultaneously and incorporate these signals in order for the growth cone to navigate correctly. There are several main guidance cues such as ephrins, netrins, slits and adhesion molecules, however, the focus will be directed specifically to Semaphorin3a and its role in axon guidance.
In brief, Netrins are secreted molecules that are structurally similar to laminin and have a chemotropic effect in axon guidance. Dependent on the concentration of netrin the growth cone will either move towards the source of away from it [Manitt C and Kennedy TE, 2002]. Deleted in Colorectal Cancer (DCC) and UNC5 families are receptors of Netrins and in vertebrates it has been shown that DCC has a role in mediating axon growth as well as showing chemoattractant effects in vitro [de la Torre JR et al., 1997]. It has also been shown through studies on Xenopus neurons that Netrin-1 attraction can be reversed to repulsion by direct interaction between DCC and UNC5 receptors [Hong K et al., 1999]. Ephrins (Ephs) are divided into two classes; EphA and EphB and these bind to their respective receptors which are receptor tyrosine kinases. Both of these classes have a role in axon guidance and it has been shown that EphA signalling is involved in topographic mapping of the anterior-posterior tectal/superior collicular axis [Feldheim DA et al., 2000].At the spinal cord midline EphB1 and EphB3 have shown to induce collapse of the commissural growth cones in vitro, suggesting that they acts as chemorepellents [ImondiR et al., 2000]. Slits are glycoproteins which have 3 family members (Slit1-3) that are conserved throughout vertebrates. Studies have demonstrated that both Slit1 and 2 have a central role in the formation of several of the fiber tracts of the nervous systems such as the optic and lateral olfactory tracts [Bagri A et al.,2002]. The canonical receptor for Slit is Robo and it is known to mediate the repellent effects of Slit which prevent longitudinal axons from crossing the midline [Kidd T et al., 1999]. Slit also has a role is preventing of recrossing of axons.
The diagram below (Fig.11) is a Kegg (Kyoto Encyclopaedia of Genes and Genomes) Pathway of the molecules in axon guidance showing their pathways leading to growth cone motility. These pathways are manually drawn from a collection of online databases and according to Kanehisa M et al., (2006) it is a way of linking genomes to biological systems through wiring diagrams of different networks.
Semaphorins constitute a family of secreted and membrane-associated proteins that affect axon guidance through their inhibitory and chemo-repellent effects in vitro whereas they are factors that control fasciculation in vivo [Taniguchi M et al., 1997]. However, research by Wong JTW et al., (1999) showed that the transmembrane Semaphorin-1a was able to function as a chemoattractant when ectopically overexpressed in the developing nervous system of grasshoppers. The semaphorins are a large family of phylogenetically conserved genes that encode for transmembrane, secreted and glycosyl-phosphatidylinositol (GPI) anchored proteins [Mark MD et al., 1997]. To date, the semaphorins are the largest family of guidance cues and are characterized by at least 30 members of which these are divided into 7 classes whereby classes 3 to 7 are found in vertebrates [Fiore R and Püschel AW 2003]. They have a distinctive extracellular domain composed of 500 amino acids at the amino terminal of which there are 16 conserved cysteines as well as containing immunoglobulin-like (Ig) and thrombospondin domains [Zhou Y et al., 2008]. Classes 1, 4, 5, 6 and 7 are membrane-associated semaphorins which are either transmembrane of GPI linked. In contrasts semaphorin classes 2 and 3 are secreted and it has been show by Zhu L et al., (2007) that some of the membrane-associated semaphorins can undergo proteolytic cleavage to form soluble proteins.
The Dynamic Cytoskeleton
As mentioned previously the growth cone has a multifunctional role in detecting environmental cues and guiding their way towards the appropriate target. This is achieved by microspikes of the growth cone which fan out and sample the environment and relay signals back to the soma [Davenport et al., 1993]. It is believed that the environmental cues induce signals that are integrated which in turn leads to a cascade of events which results in changes in shape and motility of the growth cone [Bandtlow C et al., 1990]. This is achieved by bundling and extending actin filaments into the filopodia and microspikes. In order the fully appreciate how the growth cone is so motile it is important to consider the growth cone structure.
There are three key components to the growth structure; the peripheral (P) domain, the transitional (T) domain and the central (C) domain. The C-domain is located in the centre of the growth cone in continuation with the axon and the microtubules (MT) that run along the axon in bundles separate from each other and splay out into this domain as individual microtubules [Sabry JH et al., 1991]. Unipolar actin fibers are particularly concentrated in the P-domain and these fibers form the basic cytoskeleton of the lamellipodia and the filopodia which contains thick bundles of actin. In the filopodia the microfilaments are longitudinally arranged into bundles that run the entire length of the structure whereas in the lamellipodia there is a more random orientation of individual microfilaments which form the dense meshwork [Lewis AK and Bridgman PC, 1992]. In the filopodia the actin filaments are orientated with their plus ends towards the tips so that they are pointing away from the centre and towards the periphery. The T-domain is a thin interface between the P and C domains and contain intradopia or ruffles which are very dynamic F-actin structures [Rochlin MW et al., 1999]. It is also known the actin filaments are recycled in the T-domain by a combination of severing and disassembly [Medeiros et al., 2006]. Also actomyosin contractile structures called actin arcs which lie perpendicular to F-actin bundles are contained within the T-domain. Actin arcs are of importance as they interact with microtubules and transport them into the C-domain during axon growth [Schaefer AW et al., 2002]. The image below (Fig.4) highlights the key components of the growth cone.
They also have the ability to adopt a wide variety of shapes dependent on the type of neuron, its location and its age. Its ability to change shape is due to the dynamic reconstruction of the cytoskeleton by the polymerisation and disassembly of actin filaments and microtubules. The changes in shape tend to occur at choice points or decisions regions and they tend to have complex shapes [Tosney KW and Landmesser LT, 1985].
Turning on the growth cone engine: F-actin
It is evident from the structure of the growth cone that the well-positioned actin filaments at the leading edge of the growth cone play a central part in axon motility. The microtubules also have a key role during growth and navigation of the axon as they reorganize and reorientate in the direction of future growth [Tanaka EM and Kirschner MW, 1991]. Research by Pollard TD and Borisy GG (2003) has shown that the leading edge of the growth cone is formed by the assembly of actin filaments in the P-domain which results in a physical force due to the growth of polymers beneath the plasma membrane. This was shown through in vitro studies using core proteins and mathematical models to predict the range of motion. As mentioned previously the actin filaments have polarity and this is key to the mechanism of actin assembly. One end is known as the barbed end and the other is the pointed end. The barbed end favours growth therefore they are pointed outwards [Small et al., 1978]. Confirmation of this has been shown by Symons MH and Mitchison TJ (1991) whereby they incorporated rhodamine-labeled muscle actin into permeabilized fibroblasts and showed that the actin subunits were added to the to barbed ends at the leading edge of the lamellipodia by using rhodamine/fluorescein ratio-imaging.
It is known that actin monomers are capable of binding to either ATP or ADP, but ATP has a stronger association. The association and dissociation rates of the barbed end of the actin filaments are responsive to ATP. It is believed that actin filament stabilization is a result of an ATP ‘cap' at the end of the filament and this cap forms when the rate of actin polymerization exceeds the rate of intrafilament ATP hydrolysis. Therefore, at low levels of ATP, actin disassembly occurs rapidly [Forscher P, 1989]. In the same piece of literature it was suggested that two actin-binding proteins, profilin and gelsolin may play a role in regulating the turnover of actin. Profilin is a small protein and Lassing I and Lindberg U (1985) showed that profilin-actin complexes were able to interact with polyphosphatidylinositol 4,5-bisphosphate (PIP2) with a high affinity compared to binding with phosphatidylinositol 4-phosphate (PIP) to a lesser extent. They demonstrated through in vitro studies that PIP2 interaction potentiated actin polymerization through dissociation of the profilin-actin complexes. This is as a result of the high competition of PIP2 with actin for its binding site on profilin as shown in Fig.5. They postulated the possibility of a link between increased polyphosphoinositide (PPI) turnover, which has an important role in translating signals from cell surface receptors into appropriate intracellular responses, and the regulation of F-actin assembly [Nahorski SR, 1988]. When there is increased PIP2 synthesis, the dissociation of profilin-actin complexes leads to the inceased levels of actin monomers at the inner plasma membrane near cytoskeleton assembly sites [Forscher P, 1989].
Whereas, gelsolin is a calcium-activated protein that controls the length of actin filaments by utilizing several different mechanisms [Yin HL, 1988]. Gelsolin unlike profilin has three major effects on actin: (1) gelsolin severs actin filaments in the presence of calcium, (2) it caps the barbed ends of F-actin filaments, (3) paradoxically, it can cause rapid assembly of actin under suitable conditions. However, a question remains as to how a microfilament-severing protein can lead to actin assembly. The answer to this is that gelsolin is tightly regulated by PIP2, which is independent of calcium, and it is able to inhibit its severing capabilities as well as modify it actin-binding properties [Matsudaira PT and Janmey P, 1988]. This paradoxical effect on cell motility has been demonstrated by over expressing gelsolin in NIH 3T3 fibroblasts which results in increased cell migration [Cunningham CC et al., 1991]. Even though it is known that actin polymerisation drives plasma membrane protrusion it is still very controversial as to how this leads to cell motility [Kalil K and Dent EW, 2005].
Research by Goldberg DJ and Burmeister (1986) has shown that neurite growth generally follows a stereotypical sequence of events; protrusion, engorgement and consolidation which are influenced by environmental factors (Fig 6). These stages were proposed by using video-enhanced differential interference contrast microscopy which enables the visualization of membranous organelles in the buccal ganglia of juvenile sea hares, Aplysia californica.
Protrusion is where the flat organelle-free lamellipodia elongate either between two filopodia or between a single filopodium and the body of the growth cone. The role of the filopodia is to restrict the lateral spread of the lamellipodia. It was noted in the experiments that the elongation of the lamellipodia was closely associated with the lengthening of the borders of the filopodia. The experiments also highlighted that lamellipodia advance was episodic and that it only occurred in localized areas dependent on the direction of growth based on the guidance cues. The different modes of lamellipodia dynamics in growth cones has been investigated by Mongiu AK et al. (2006) and they have shown that there types of protrusion and retraction of the lamellipodia during axon growth. Their finding are summarised in Fig.7In order for the axon to grow to lamellipodia had to mature and this occurred by investment of the lamellipodia with vesicle and organelles. This was termed the engorgement stage. Because of this filling of organelles by fast axonal transport and Brownian motion the lamellipodia increase in size while retaining growth cone morphology. As with lamellipodia advance, the filling only occurs in certain areas and was often selective amongst neighbouring lamellipodia. Generally, smaller vesicles engorge the lamellipodia first followed by larger membranous organelles such as mitochondria. After this, consolidation takes place as the growth cone assumes a cylindrical shape with bi-directional transport of organelles, thereby adding a new distal section to the axon which leads to its elongation.
In order for this outgrowth to occur there must be regulation of actin polymerisation, cross-linking and F-actin cytoskeleton cross-linkage to the substratum as well as contraction of molecular motors [Verkhovsky AB et al., 1999]. Importantly, in order for growth to occur, the actin filaments at the leading edge are centripetally transported via retrograde F-actin flow. This is where F-actin moves continuously from the leading edge towards the growth cone centre. This form of movement was first proposed by Bray D (1970) who used time-lapse microphotography to observe the effects of glass or carmine particles placed on isolated rat sympathetic neurons. He noted that the inert particles migrated in a retrograde direction which was suggestive of an actin filament flux or underlying centripetal membrane.
Retrograde flow is a continuous process independent of whether the growth cone is rapidly moving or in a stationary phase. Based on this it is reasonable to presume that whether a filopodium protrudes or retracts is dependant on the balance between actin polymerization and disassembly. The rate of polymerization is governed by the number of exposed barbed end and the concentration of actin monomers available. At this point in time it was accepted that actin polymerization was solely responsible for growth cone motility and growth. However, studies carried out by Forscher P and Smith SJ (1988) showed that actin polymerization was not the only cause for retrograde flow. They investigated the effects of cytochalasin, which binds to the barbed ends of actin filaments and blocks polymerization, on Aplysia growth. The experiment resulted in the detachment of actin from the leading edge, however there was no alteration to the retrograde flow.
Leading on from this new discovery research carried out by Turney SG and Bridgman PC (2005) demonstrated that myosin II contractility played a role in F-actin retrograde flow and growth cone retraction. Using a modified turning assay they imaged neurites growing at borders of substrate-bound laminin-1 in culture and concluded that integrin receptor activation is linked to myosin II and that its contractility enables the growth cone to turn, branch, retract, stop or move ahead along a laminin border. Therfore, it can be deduced that F-actin retrograde flow is induced by both F-actin polymerisation in the P-domain and contractility of the protein myosin II. Myosin II contractility leads to the buckling of F-actin bundles which is augmented by actin polymerisation at the leading edge which gives a ‘pushing' effect [Medeiros NA et al., 2006]. The same paper showed that bundle severing towards the proximal end was next to follow and this was most likely to involve the actin-depolymerizing factor (ADF)/cofilin family.
The ADF family has an important role in regulating actin dynamics as well as being a target of several intracellular signaling pathways which affect the actin cytoskeleton [Kuhn TB et al., 2000]. The severing ADF proteins are capable of increasing the turnover of dissociation of the actin from the pointed ends of the actin filaments, as well as having the ability to severe F-actin to increase the number of actin filaments with available pointed ends [Chen H et al., 2000]. Research by Hayden et al. (1993) has shown that the ADF proteins are capable of binding to ADP-actin subunits of the F-actin and its binding results in rotation of the actin subunits. Because of this the actin filament is reduced in length by approximately 20% [McGough A et al., 1997]. This change in F-actin structure explains the severing activity of the ADF proteins as well as their ability to increase the rate of actin dissociation from the pointed ends of actin filaments [Maciver SK, 1998]. Once severing is complete, the actin monomers are transported back to the periphery so that they are available for actin polymerization at the leading edge [Zicha D et al., 2003]. To date there is significant evidence to suggest that axon growth and guidance is dependent on reorganization of the cytoskeleton, however, the molecular events controlling this coordinated event still remain unclear [Schaefer AW et al., 2008].
Engaging the clutch: Mitchison and Kirschner Model
So far, actin polymerisation and F-actin retrograde flow have been discussed, however, the main question of how the growth cone utilizes actin to gain momentum and move forward is still to be answered. The first theory proposed was the ‘clutch hypothesis' by Mitchison T and Kirschner M (1988) where by they realized that retrograde flow was a continuous process whereas forward movement is variable. Therefore, they postulated that a molecular clutch must be present in order for coupling between actin and receptors to occur. It is known that growing growth cones generate tension through binding to the substratum and the attachment is mediated by receptors [Bray D, 1979]. It is through the binding via focal adhesions to the substratum where F-actin is anchored through the ‘clutch' mechanism to halt retrograde flow to allow for the forward driving of actin to enable growth cone protrusion [Lowery LA and Vactor DV, 2009].
For example, the heterodimeric transmembrane receptor Integrin couples with actin through a physical link between the growth cone and the extracellular matrix to provide traction for migration [Beningo KA et al., 2001]. The adhesions act as signalling centres because the integrin extracellular domains bind to the extracellular ligands and the intracellular domain are linked to structural and signalling molecules along with the actin cytoskeleton [Zaidel-Bar R et al., 2007]. More specifically, they are able to regulate Rho GTPases that control the polymerisation and organization of actin during protrusion [Nalbant P et al., 2004]. The role of Rho GTPases will be discussed in more depth later on. With regards to growth cone protrusion, the integrins have a unique property of being able to regulate their level of affinity, due to activation of adaptor and signalling molecules that bring about changes in the integrin structure. Therfore, during growth cone protrusion, the integrins remains in a high affinity state [Hynes RO, 2002]. It has been hypothesised that when the environmental conditions are suitable the efficiency of actin coupling to extracellular substrates increases and also the rate of F-actin retrograde flow reduces with the accumulation of adhesive interactions [Lin C et al., 1994]. The increase in actin assembly and slowing of retrograde flow has also been demonstrated by Lee AC and Suter DM (2008) whereby fluorescent speckle microscopy was used to monitor actin dynamics in Aplysia growth cones. They observed the changes in actin dynamics as the growth cones turned towards beads coated with APCAM, a cell adhesion molecule.
The key role of the ‘clutch' is to regulate the degree of ‘slippage' between the binding of actin cytoskeleton to the extracellular matrix. For example, when the clutch is ‘disengaged' there is inefficient substrate-cytoskeletal coupling which results in fast F-actin retrograde flow rate and slow growth cone filopodium protrusion. Whereas, at the other end of the extreme, when the clutch is ‘engaged' the coupling system becomes very efficient which progressively slows down F-actin retrograde flow resulting in fast protrusion rates. This result is due to the reduction of F-actin removal from the leading edge; therefore more is available for growth cone advance [Lin C et al., 1994]. The diagram below (Fig.8), illustrates this ‘clutch hypothesis' and how the dynamics of F-actin change once a permissive growth cone substrate is encountered. Interesting, the diagram also shows fish keratinocytes as these motile cells migrate purely at a rate of actin assembly as they exhibit virtually no F-actin retrograde flow.
Recent studies have revealed that there may be several proteins associated with the growth cone specific clutch machinery. A study carried out by Bard L et al., (2008) showed that the protein Catenin can couple N-cadherin receptors and F-actin retrograde flow. This was demonstrated by real-time imaging of primary rat hippocampal neurons which were plated on N-cadherin substrates. In another recent study by Shimada T et al., 2008 they showed that Shootin1, a novel protein, mediates coupling between F-actin retrograde flow and the L1 cell adhesion molecule (L1CAM). This was proven by carrying out experiments whereby linkage was impaired resulting in inhibited L1-dependent axon growth. They also over expressed Shootin1, which boosted the linkage which resulted in faster growth cone advance.
Studies have also been carried out on non-neuronal systems and it has been shown that the focal adhesion proteins Vinculin and Talin are able to interact with actin filaments and generate a molecular clutch through integrin receptors during cell migration [Hu K et al., 2007]. Research by Zhang X et al. (2008) has shown that talin is needed to activate integrins and provides mechanical linkage between the integrins and the actin cytoskeleton through knockout experiments. They also showed that talin is required for adhesion stability and that adhesion disassembly is mediated by calpain, a calcium-dependent protease, which acts on talin [Franco SJ et al., 2004]. To enhance the evidence for talins' role in adhesion stability to generate force Jiang G et al. (2003) showed that talin knockout cells (talin1-/- ABS cells - talin1 cDNA deficient in the genomic sequence for the carboxy-terminal actin-binding site) failed to generate sufficient force at adhesion sites to move out of the optical trap.
Vinculin does not appear to have such an important role as Talin in the formation of stable focal adhesions. Research by Volberg T et al. (1995) showed that when the expression of vinculin was deficient in F9 embryonal carcinoma cells they were capable of forming adhesion to fibronectin-coated surfaces identical to the vinculin-containing cells. However, quantitative digitised microscopy was used to observe the focal adhesions it was apparent that the florescent labelling for alpha-actinin, talin and paxillin was more intense in cells deficient in vinculin. From these observations they suggested that despite the absence of vinculin there are several other molecular mechanisms which are able to form the focal adhesions. The image below (Fig.9) shows the fluorescent imaging for the focal adhesion proteins in the vinculin-null cells (γ229) compared to the vinculin-containing cells (F9).
The most important point to consider with the focal adhesion is the integrity of the connection between the actin cytoskeleton and the substratum through the binding proteins. This is essential for growth cone advance. However, the understanding of the molecular principles of the ‘clutch model' is still not fully understood and further research is still required.
Microtubules help steer the growth cone
It is evident that actin plays a major role in cytoskeleton remodelling in response to guidance cues; however, the role of MTs has received less attention and studies by Tanaka EM and Kirschner MW (1991) have demonstrated that MTs play a major role in growth cone steering. It has been shown by Gordon-Weeks PR, 2004 that the MTs assist in growth cone steering in two major ways.
Firstly, the MTs infiltrate and explore the P-domain just prior to filopodia protrusion [Letourneau PC, 1983 and take the role as sensors for environmental cues. It has been postulated that the MTs may act as transporters of signals involved in growth cone navigation, or they may act as structural supports for the recruitment of signalling components required for growth cone steering [Suter DM et al., 2004]. It is known that Src family tyrosine kinases are important in the growth cone and MTs have a role in accumulating these signalling enzymes at sites of adhesion [Tanaka EM and Kirschner MW, 1991]. The exact cellular functions of Src kinases in live growth cone still remain unclear. Research by Wu B et al., (2008) has show that there is a high concentration of Src2 in filopodia tips and that MTs have a role in regulating the level of active Src at the plasma membrane by controlling recycling of endocytosed Src. Their experiments also showed that the expression of active Src2 resulted in filopodial protrusion therefore they postulated that Src2 controls the size of filopodia and lamellipodia. Because of the MTs role in transporting Src kinase it is possible that they might transport other signalling molecules such as Rho-family GTPase regulators. Secondly, during the engorgement phase the majority of MTs in the C-domain migrate into the area of new growth in order to steer growth cone in a fixed direction [Buck KB and Zheng JQ, 2002].
How microtubules interact with actin
It is evident from the literature that MTs interact with actin in order to navigate correctly and it has been shown that MTs are influenced by actin filaments in the P-domain [Rodriguez OC et al., 2003]. It was originally believed that dynamic MTs utilise the trajectories of the F-actin bundles in order to advance into and explore the P-domain [Zhou FQ and Cohan CS, 2004]. However, it has recently been shown by Burnette DT et al., (2007) that the F-actin bundles are not essential for MT advance and that they prevent MT advance into to the P-domain when coupled to the MTs through MT-actin cross linking proteins. From this study it can be deduced that the actin-MT coupling and uncoupling regulates the MTs dynamics and therefore its ability to explore the P-domain. This prediction was demonstrated by Lee AC and Suter DM (2008), whereby they used fluorescent speckle microscopy to investigate P-domain MTs and actin dynamics in Aplysia growth cones at sites of APCAM-mediated adhesion. They showed that there was an increase in number of MTs that explored the sites of adhesion and that the adhesion molecules control MT rearrangements by regulating the MT-actin coupling and actin movement rather than controlling plus-end polymerisation rates. In contrast, the growth cone encounters a repellent cue there is increased MT-actin coupling which results in increased MT-looping due to the growing plus-ends of the MTs being recycled back towards the C-domain as they are linked to the F-actin retrograde flow [Lowery LA and Vactor DV, 2009]. The result of this is that there is decreased MT exploration into the P-domain.
In contrast, MTs in the C-domain are regulated by the actin network and actin arcs during the engorgement phase. It is known that the contractile actin arcs are regulated by the Rho Kinase and myosin II signalling cascade and that when one of these was inhibited it resulted in failure of MT consolidation during axon advancement [Schaefer AW et al., 2008]. Actin arcs act as a barrier surrounding the C-domain which regulates MT advance in the direction of C-domain advance during engorgement. It was also shown by Burnette DT et al. (2008), that myosin II within the actin arcs actively transports MTs into the C-domain from the sides and compresses them into bundles so that they can be cross linked by MT-associated proteins in the neck of the growth cone. Further to this, myosin II has been shown to negatively regulate F-actin polymerisation which underlies protrusion, in order to allow for consolidation of the axon shaft [Loudon RP et al., 2006]. It is clear that different classes of actin structures are capable of regulating MTs in different domains of the growth cone. F-actin bundles have the ability to inhibit MTs in the P-domain through a coupling mechanism whereas the F-actin arcs are able to control the C-domain MTs during engorgement and consolidation in the distal axon.
Rho-family GTPases in the driver seat
Of the many signal transduction molecules that are involved in passing on guidance information, the Rho-family GTPases are the most well understood. Rho GTPases are key regulators of the actin cytoskeleton and act downstream of all known guidance signalling receptors [Koh CG, 2006]. The Rho-family GTPases include; RhoA, Rac1 and Cdc42 and guidance cues belonging to the semaphorin, ephrin, netrin and slit families signal through pathways to these Rho-family GTPases. This cascade of events leads to the direct actin polymerisation or disassembly which results in growth cone protrusion or retraction [Govek EE et al., 2005].
Rho GTPases are regulated by upstream proteins such as guanine nucleotide exchange factors (GEFs) which activate them, whereas GTPase activating proteins (GAPs) inactivate them [Watabe-Uchida M et al., 2006]. The Rho GTPases have different functions and some of them are capable of being bifunctional. In general, Rac and Cdc42 activation by attractive cues results in actin polymerisation leading to growth cone protrusion [Guan KL and Rao Y, 2003]. More specifically, Cdc42 and Rac which are present in lamellipodia activates the proteins of the Wiskott-Aldrich syndrome family (WASp) which cause a conformational change so that the WASp C-terminus binds to and activates the Arp2/3complex. The Arp2/3 complex is a seven subunit protein that nucleates new F-actin resulting in a broad dendritic shape actin network [Jaffe AB and Hall A, 2005]. It has been shown that Arp2/3 is needed for guidance but the question remains as to whether it has a similar role in neuronal and non-neuronal systems [Strasser GA et al., 2004]. However, Korobova F and Svitkina T (2008), have shown that by depleting the Arp2/3 complex in primary neurons and neuroblastoma cells there was inhibition of lamellipodia and filopodia protrusion but enhance Rho A activity. They postulated that Arp2/3 not only has a role in nucleating actin filaments but the complex can manipulate growth cone motility by altering Rho GTPase signalling.
In the filopodia, Cdc42 acts through formins and vasodilator-stimulated phosphoproteins (VASP) in order to promote linear actin polymerisation [Mellor H, 2010]. VASP proteins elongate F-actin by either binding to the barbed ends of the F-actin at the leading edge in order to prevent the binding of capping proteins. They also act by recycling actin to the P-domain so that they can be used for further polymerisation [Drees F and Gertler FB, 2008]. Following this, fascin, monomeric actin filament bundling protein organizes the newly polymerised actin into stiff and parallel filament bundles [Mattila PK and Lappalainen P, 2008].
In contrast, RhoA activation results in either growth cone retraction through myosin II contraction or it can promote axon outgrowth by blocking the actin-depolymerizing factor through phosphorylation [Wen Z et al., 2007]. The confocal images to the left (Fig.10) shows the effects of inhibiting Cdc42 or Rac on the cytoskeleton of Xenopus spinal neuron growth cones.
Not only do Rho GTPases possess the ability to carry out different roles within the growth cone but they can also respond to more than one guidance cue due to the complex signalling network combinations between GEFs, GAPs and their corresponding GTPase. The key principle with the Rho-GTPases is that the balance between the opposing effects dictates the morphology and function of the growth cone [Koh CG, 2006].
One fundamental problem which must be noted with the vast number of studies carried out on the role of Rho GTPases, is that many of the experiments have been carried out on non-neuronal systems. Fibroblasts, neutrophils and many other systems have been used but there are differences in the molecular makeup of these cells in comparison to neuronal growth cones. Therefore, it is safe not to assume that the functions of Rho-family GTPases are not identical between neuronal and non-neuronal cells [Mortimer D et al., 2008].
Regulators of microtubules
The role of Rho-family GTPase signalling in regulating actin dynamics is clear however, the MTs do not appear to be regulated by this same signalling system even though studies in non-neuronal systems suggest there is a link [Rogers SL et al., 2004]. One way of controlling the MT dynamics is through the MT-actin interactions by the MT-associated proteins (MAPs). Recently a group of MAPs known as the family of MT plus-end tracking proteins (+TIPs) has been shown to play a critical role in MT dynamics [Akhmanova A and Steinmetz MO, 2008]. This family includes End-binding (EB) proteins which have a N-terminal domain which is necessary for microtubule binding [Hayashi I and Ikura M, 2003].
+TIPs are cellular factors that accumulate at the plus-ends of the MT and form networks through interactions with proteins in either a negative or positive way [Schuyler SC and Pellman D, 2001]. There are two members of the +TIP family, Lissencephaly1(LIS1) and Adenomatosis Polyposis Coli (APC) which has shown to induce MT-actin uncoupling resulting in reduced MT advance. Research by Grabham et al. (2007) have shown that LIS1 interacts with cytoplasmic Dynein, a MT motor protein, to promote uncoupling of MT-actin complexes. They inhibited both LIS1 and dynein function by injecting antibodies to the proteins prior to axon initiation and this resulted decreased penetration into the P-domain of the growth cone of chick neurons on a laminin substrate. The inhibition of both LIS1 and dynein has no effect on MT assembly but it diminished the ability of the MT to resist F-actin retrograde flow. Because of this the growth cones were unable to steer accurately on the laminin substrate and they concluded that LSI1 plays a significant role in MT advance during axonal growth. However, the signalling pathways leading to LIS1 and dynein activity have not been fully discovered but it has been identified that LIS1 interacts with IQGAP1, an integral protein of cytoskeletal organization and a regulator of Rac1 and Cdc42 [Kholmanskikh SS et al., 2003].
Comparable to LSI1, APC also promotes MT-actin uncoupling and interacts directly with IQGAPI [Watanabe T et al., 2004]. APC is only capable of binding to a certain population of MTs either directly or indirectly via EB1 (+TIP protein) and this affects the assembly of MTs [Munemitsu S et al., 1994]. It is known that when APC associates with MTs there is enhance growth cone steering possibly by MT being prevented from binding to F-actin. Research by Watanabe T et al. (2004) have show that when Vero cells are depleted of APC or IQGAP1 there was inhibition of actin meshwork formation and cell migration. This speculation has been further supported by Purro SA et al. (2008) whereby they demonstrated that APC depletion from the MT plus-ends results in greater MT looping and therefore dampened growth cone progression. They built this finding by examining how Wnts bring about axon terminal remodelling. They demonstrated that Wnt3a is able to positively regulate MT looping and therefore halt the advancement of growth cone by using time-lapse imaging of the MT plus-ends. They concluded that Wnt signalling has an important function in regulating the APC-MT association which affects the direction of MT growth.
While these proteins prevent MT advancement into the P-domain by coupling mechanisms to F-actin retrograde flow, interactions between MTs and F-actin can in turn enhance MTs extension. Drebrin, a F-actin associated protein is capable of binding directly to EB3, a MT-binding protein. Research by Geraldo S et al. (2008) has shown this interaction occurs only when drebrin is positioned on F-actin present in proximal part of the filopodia and when the tips of MTs invading the filopodia are present with EB3. EB3 was the focus of the experiment rather than EB1 as it is expressed in the nervous system in comparison to the ubiquitously expressed EB1 and in part to determine whether they have different functions. They showed that when the interaction between EB3 and drebrin was disrupted, there was impaired growth cone formation and neurite extension suggesting that EB3 is a target of drebrin in order to coordinate MT-F-actin interactions. Even though the mechanisms behind cytoskeleton rearrangements in response to guidance cues are being uncovered there is still much to understand as to the specific roles of MTs and MT-actin networks within the growth cone.
- Receptors - neuropilin
- Sema 3a (collapsing) - first discovered on grasshopper
- How sema3a works - growth cone collapse
- PTEN accumulates rapidly at the growth cone membrane suggestinga mechanism by which PTEN couples Sema3A signalling to growthcone collapse (Chadborn NH et al., 2006)
Chadborn NH, Aminul I. Ahmed, Mark R. Holt, Rabinder Prinjha, Graham A. Dunn, Gareth E. Jones and Britta J. Eickholt, 2006. PTEN couples Sema3A signalling to growth cone collapse. Journal of Cell Science, 119, pp.951-957.
In contrast, vertebrate class 3 secreted semaphorins utilize receptor complexes consisting of neuropilin, serving as the ligand-binding component, and plexin, functioning as the signal-transducing component [111-114]. To date, two neuropilins, Npn-1 and Npn-2, and nine plexins (Plex-A1, -2, -3, -4; Plex-B1, -2, -3; Plex-C1, -D1) have been identified in mammals (reviewed in Ref. ). Neuropilins are also receptors for particular isoforms of the vascular endothelial