Growth Cones form Adhesions to ECM Proteins that Resemble Fibroblast Focal Contacts
Integrin receptors physically link cells to their immediate environment at distinct contact points containing a large number of linkage and regulatory proteins. Structures such as focal contacts (FCs) and FAs are highly complex and dynamic macromolecular assemblies that link the actin cytoskeleton to ECM proteins through integrin receptors (Fig. 1D). Studies that have largely focused on fibroblasts and epithelial cells have shown that individual cells form distinct adhesion sites that serve unique functions. For example, FCs are nascent adhesions that form first just behind the leading edge of lammelipodial and filopodial protrusions and support transient sampling of the environment. In fibroblasts, some FCs mature into larger and more stable FAs that support traction forces from cell to ECM. FCs and adhesions may contain upward of 150 different proteins that are interchanged and post-translationally modified during their lifetime within the integrin adhesome (Zaidel-Bar et al.,2007).
Neuronal growth cones assemble dynamic adhesion complexes on ECM substrata similar to fibroblast FCs. Live imaging of neurons expressing paxillin-mCherry and GFP-dSH2, a fluorescent phosphotyrosine (PY) reporter, show that adhesions form within nascent filopodial and lamellipodial protrusions, which remain fixed in position during growth cone advance (Fig. 2). Although a subset of growth cone adhesions do stabilize and expand rearward similar to fibroblast FAs (Woo and Gomez,2006), distinct growth cone adhesions have not been systematically classified, because their morphological and kinetic features are not as clearly distinguishable. Therefore, growth cone adhesions are collectively referred to as PCs (Gomez et al.,1996; Renaudin et al.,1999; Robles and Gomez,2006). Retrospective immunofluorescent staining confirms that positionally stable growth cone PCs colocalize with classic FA components such as β1-integrin, vinculin, and paxillin (Robles and Gomez,2006 and unpublished observations). Vinculin and paxillin are adaptor proteins that are involved in direct or indirect linking of actin filaments to the cytoplasmic tail of integrin receptors (Fig. 1). Consistent with this notion, adhesions often cluster along actin filament bundles (Robles and Gomez,2006), suggesting that PCs may mediate traction force generation in growth cones. It is important to note that PCs have only been observed in growth cones on ECM substrata and not in growth cones on non-biological substrata [glass or poly-D-lysine (PDL)] or neural cell adhesion molecule (unpublished observations).
Figure 2. Two-channel total internal reflection fluorescence (TIRF) microscopy of PY and paxillin in a living growth cone. This neuron is co-expressing GFP-dSH2 (green), which brightly labels regions containing concentrated tyrosine-phosphorylated proteins and paxillin-mCherry (red), which labels integrin-dependent adhesion sites. Note that only PY is concentrated at the tips of elongating filopodia (solid white arrowheads mark one extending filopodium and open white arrowheads mark a filopodium that extends then retracts), which appear green due to the absence of paxillin. On the other hand, stable adhesions that contain both paxillin and PY appear yellow throughout this time period (solid arrows). Stable adhesions likely contain many other signaling and adaptor proteins, such as FAK and a-actinin. New adhesions often form from retracting PY-positive filopodia that stabilize and cluster with paxillin (at open arrowhead at 3 min) or by apparent simultaneous clustering of paxillin and PY-proteins within nascent protrusions (open arrows). However, in some instances, paxillin appears to cluster at adhesion sites independent of PY-containing proteins (red arrowheads). Scale, 10 lm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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Growth cone PCs also contain signaling proteins that modulate adhesion assembly and disassembly and likely regulate a number of other important cellular functions. Two crucial proteins are the NTKs, FAK, and Src, which localize to PCs and are known to control the molecular composition of adhesion complexes in motile cells by regulating protein–protein interactions (Robles and Gomez,2006; Woo et al.,2009). Importantly, these kinases are modulated downstream of a variety of receptors (Knoll and Drescher,2004; Li et al.,2004; Bechara et al.,2008) and interact with multiple signaling pathways, suggesting their localization to PCs controls additional cellular functions (Huveneers and Danen,2009). The Rho family GTPases Rac1 and RhoA also regulate the assembly and maturation of growth cone PCs downstream of integrin signaling (Fig. 3). In migrating fibroblasts, FC formation at the leading edge of lamellipodia and filopodia are promoted by Rac1 activity (Rottner et al.,1999), whereas maturation into FAs requires RhoA activity and actomyosin contraction (Chrzanowska-Wodnicka and Burridge,1996). Consistent with the presence of distinct adhesion complexes in growth cones, Rac1 was found to promote the assembly of transient PCs, whereas RhoA was necessary to stabilize existing PCs (Woo and Gomez,2006). It is important to note that although PCs have not yet been observed in neurons in vivo, loss of function of proteins involved in PC assembly and turnover interferes with many aspects of normal neural development (Beggs et al.,2003; Clegg et al.,2003; Rico et al.,2004; Robles and Gomez,2006; Woo et al.,2009), suggesting these adhesion complexes also play crucial roles in developing neurons in vivo. For example, we find that midline crossing by spinal commissural interneurons and retinotopic mapping by RGC is disrupted in FAK loss of function conditions (Robles and Gomez,2006; Woo et al.,2009).
Figure 3. Cooperation between integrin-dependent signaling pathways and modulatory receptors regulate growth cone motility by balancing Rho GTPase signaling. Integrins signal through a Src–FAK complex to promote membrane protrusion by elevating Rac1 and inhibiting RhoA. However, shifting this balance to more RhoA and less Rac1 activity leads to less actin polymerization and more actomyosin contractility. Modest RhoA signaling will stabilize adhesions to promote growth cone stalling or turning. Strong RhoA signaling will cause growth cone collapse and axon retraction. Shifting the balance of Rho GTPase signaling may occur through growth-promoting and inhibiting extracellular ligands that directly signal through Src–FAK. Alternatively, modulation of Src–FAK signals may occur as a result of re-association of FAK-associated protein complexes with modulating receptors. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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As it appears that fibroblast and growth cone adhesions share many similarities, it follows that many of the activities associated with fibroblast FAs and FCs also occur at growth cone PCs. In fibroblasts and other non-neuronal cells, integrin-dependent adhesions have been shown to not simply serve as anchorage points between the ECM and the cytoskeleton. Rather, they function as active signaling centers, where a wide variety of proteins are regulated to control diverse cellular functions (Zaidel-Bar et al.,2007). For example, PCs may locally control actin polymerization by targeting factors such as Arp2/3, p130cas and various guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) for Rho GTPases (Fig. 3; Cary et al.,1998; Huang et al.,2007; Liu et al.,2007; Serrels et al.,2007; Tomar and Schlaepfer,2009). FAs have also been shown to colocalize with and organize caveoli-containing lipid rafts (Gaus et al.,2006), which serve as a platform for diverse signaling events and are known to be necessary for neurite outgrowth and growth cone chemotropism (Guirland et al.,2004; Langhorst et al.,2008). In addition, vinculin, a fundamental component of FAs and growth cone PCs, associates with proteins that target mRNA and protein translation machinery to FAs (Chicurel et al.,1998; Lee et al.,2009; Willett et al.,2009). As local protein translation was found to be necessary for growth cone chemotropism (Campbell and Holt,2001), it is possible that PCs function to locally modulate protein translation in growth cones. Although PCs of growth cones share many similar functions as adhesion sites of motile non-neuronal cells, it is likely that growth cone PCs will have many additional upstream modulators and downstream activities given the profoundly complex roles growth cones serve in neural network assembly and synapse formation.
Modulation of Integrin-dependent Adhesion by Axon Guidance Cues
Several studies have demonstrated that axon guidance factors such as netrins, Semaphorins, Slits, and ephrins can promote or inhibit axon outgrowth by modulating integrin receptors or their associated adhesion complexes (Nakamoto et al.,2004). Some guidance cues act directly on integrin receptors, whereas others indirectly influence integrin-associated adhesion complex proteins. As examples of direct effects on integrin receptors, both Semaphorin 7a (Sema7a) and MAG contain integrin-binding RGD sequences and function as ligands to activate integrin receptors (Pasterkamp et al.,2003; Goh et al.,2008). Interestingly, both MAG and Sema7a activate FAK downstream of integrin binding, yet have opposite effects on axon outgrowth. Activation of FAK (and Src) downstream of both growth promoting and inhibiting axon guidance cues is not uncommon and is discussed later. Briefly, activation of FAK by MAG may lead to adhesion disassembly and subsequent integrin endocytosis, whereas Sema7a may promote adhesion cycling without receptor endocytosis. A second myelin-associated protein, Nogo-A, inhibits axon outgrowth and regeneration by directly inhibiting integrin receptors through an unknown mechanism (Hu and Strittmatter,2008). Several other guidance cues have been shown to bind various integrin heterodimers in non-neuronal cells and neurons. For example, neurotrophins (Staniszewska et al.,2008), Wnt5a (Kawasaki et al.,2007), and netrin (Yebra et al.,2003) each can bind specific integrin heterodimers.
Netrin is particularly intriguing, as it is an important secreted axon guidance cue that works in cooperation with integrin–ECM adhesion and signaling in several ways (Baker et al.,2006). First, netrin was found to support cell adhesion by directly and independently binding integrin and deleted in colorectal cancer (DCC) receptors (Yebra et al.,2003; Shekarabi et al.,2005). Interestingly, netrin colocalizes with the basal lamina ECM in vivo and binds purified ECM proteins in vitro, implying that secreted netrin may be immobilized within the ECM (Yebra et al.,2003). Moreover, cortical GABAergic interneurons expressing α3/β1 integrin receptors bind directly to netrin, which is necessary for proper migration of these neurons into the cortex (Stanco et al.,2009). However, integrin signaling also appears to modulate netrin function downstream of DCC receptors, as chemotropic turning toward netrin is sensitive to the ECM substratum (Hopker et al.,1999). Specifically, the study by Höpker et al. found that attractive turning on FN or PDL was switched to repulsion when neurons were plated on LN. Although substratum-dependent differences in cAMP/PKA/Epac signaling within growth cones have been implicated (Ming etal.,1997; Nishiyama et al.,2003; Murray et al.,2009), the exact mechanisms leading to bidirectional turning responses are unknown.
Another intriguing link between integrin and DCC receptors is their common activation of FAK and Src. The cytoplasmic tails of β1-containing integrin receptors interact with FAK in combination with several other adhesion-related adaptor proteins (Legate and Fassler,2009), leading to FAK auto-phosphorylation at tyrosine 397 (Y397) and recruitment of Src (Fig. 3). Similarly, three articles reported that netrin activates FAK and Src to promote DCC tyrosine phosphorylation and FAK binding of DCC (Li et al.,2004; Liu et al.,2004; Ren et al.,2004). It is noteworthy that both integrin adhesion proteins and DCC receptors bind within the FAT domain of FAK. However, it remains unclear whether integrin and DCC receptors utilize FAK in parallel, or if these receptors cooperate with or compete for FAK function. Future studies should consider the growth substratum when testing the effects of netrin. Lastly, integrin receptors were recently found to facilitate cell migration toward netrin in C. elegans by targeting UNC-40 (DCC) receptors to the plasma membrane (Hagedorn et al.,2009). This result suggests that integrins may associate with DCC in cis.
Semaphorins comprise a large family of secreted and membrane associated guidance cues that influence both axon extension and cell motility by regulating integrin receptors and their associated adhesion complexes. Semaphorins have diverse effects on cell motility, likely owing to the wide variety of receptors and co-receptors that bind semaphorins. The primary ligand-binding receptors for semaphorins are the plexins and neuropilins. However, a number of associated receptors appear necessary for signal transduction and specific cellular responses (Franco and Tamagnone,2008). For example, Sema3A stimulates integrin-dependent extension of hippocampal neuron dendrites by activating FAK (Schlomann et al.,2009). FAK is not activated by Sema3A if integrins are not engaged, suggesting that active FAK is associated with and may influence integrin adhesion complexes. Conversely, growth cone collapse and the repulsive effects of semaphorins on hippocampal axons do not appear to require integrin engagement, because the effects of semaphorins could occur on non-integrin-binding substrata (Song et al.,1998; Schlomann et al.,2009). Although modulation of integrin receptors may not be necessary for the collapsing or repulsive effects of semaphorins, it is still possible that integrins are involved in the normal inhibitory effects of semaphorins that occur within cells on ECM proteins or in vivo. This possibility is supported by observations that paxillin-containing PCs that form within growth cones on LN are rapidly disassembled by soluble Sema3A (Woo and Gomez,2006). Moreover, FAK is necessary for Sema3A-induced collapse of cortical neurons, which depends on cis interactions between the cell adhesion molecule L1 and neuropilin receptors and disassembly of paxillin-containing adhesions (Bechara et al.,2008).
The Eph/ephrins are probably the best characterized and most complex system of receptors/ligands that regulate a number of important developmental functions through the control of cell adhesion. Ephs and ephrins are cell surface molecules that function as both signaling ligands and receptors and work cooperatively with integrin receptors. Eph proteins are transmembrane receptor tyrosine kinases activated by ephrin ligands in forward signaling, whereas ephrins are either transmembrane or GPI-anchored receptors reciprocally activated by Eph ligands in reverse signaling (Pasquale,2005). In non-neuronal cells, forward signaling through Eph receptors has been shown to both activate and inhibit integrin-mediated cell spreading and adhesion, suggesting that complex cellular conditions regulate Eph receptor output (Miao et al.,2000; de Saint-Vis et al.,2003; Deroanne et al.,2003; Miao et al.,2005; Prevost etal.,2005; Sharfe et al.,2008; Noren et al.,2009). The diversity of cellular effects of ephrins likely result from activation of multiple possible signaling pathways affecting the actin cytoskeleton and adhesion (Pasquale,2008). Many Eph receptors activate Ras/Rho family GTPases. For example, EphA receptors activate RhoA signaling through the Rho GEF Ephexin (Shamah et al.,2001). More recently, NTK signaling has been identified as a common pathway of both forward and reverse signaling. Src family kinases interact with EphA receptors, phosphorylate Ephexin (Sahin et al.,2005) and are required for retinal axons to respond to ephrin-A (Knoll and Drescher,2004). In addition, several studies have found that FAK can have diverse effects downstream of ephrins. Ephrin-A was shown to increase FAK phosphorylation and promote cell migration or retraction in certain cell types (Carter et al.,2002; Parri et al.,2007) but reduce FAK phosphorylation and decrease cell adhesion and motility in other cells (Miao et al.,2000; Bourgin et al.,2007). In RGC growth cones, ephrin-A1 activates FAK and Src (Woo et al.,2009) and results in stabilization of integrin adhesions and stalled outgrowth. Interestingly, by comparing active FAK in nasal versus temporal RGC growth cones, it appears that moderate levels of active FAK correlate with maximal rates of outgrowth and changes above or below this optimal set-point inhibit outgrowth. This may explain how ephrin-A inhibits outgrowth by temporal RGC axons, yet promotes outgrowth by nasal RGCs (Hansen et al.,2004; Woo et al.,2009). It is also noteworthy that Eph receptors can physically interact with FAK and other known integrin-associated adaptor proteins and localize to cellular FAs (Miao et al.,2000), suggesting these receptors may associate within a common protein complex (Fig. 3). Adding to this molecular complexity, forward and reverse Eph/ephrin signaling within individual cells may function antagonistically toward integrin adhesion (Sharfe et al.,2008).
The NTKs, FAK, and Src are necessary downstream of many growth promoting and inhibiting axon guidance cues. How can these signaling kinases transduce opposing cellular behaviors from different axon guidance cues? In the context of modulation of integrin-dependent adhesion, several possible mechanisms could explain varying effects on cell motility. First, the molecular targets of FAK/Src may depend on receptor activation. FAK/Src normally target to and regulate integrin-based adhesion sites of cells on ECM substrata. However, on stimulation with guidance cues, FAK and Src may associate with guidance cue receptors (Li et al.,2004; Liu et al.,2004; Ren et al.,2004; Bechara et al.,2008; Shi et al.,2009). As NTKs can activate a number of different targets, including RhoA GEF and GAP (Tomar and Schlaepfer,2009), NTK target specificity could be determined by protein associations driven by receptor binding. In support of this possibility, treatment of hippocampal neurons with ephrin-B2 led to FAK dissociation from β3-containing integrins and association with EphB2 receptors in dendritic spines (Shi et al.,2009). In this model, activation of guidance cue receptors could further affect cell motility by drawing FAK/Src away from integrin receptors. Alternatively, parallel signals activated by guidance cue receptors may modulate permissive integrin-based adhesion dynamics. For example, increased RhoA signaling by Ephexin may activate actomyosin contractility, which stabilizes integrin adhesions (Lim et al.,2008).