InteractiveFly: GeneBrief

p130CAS: Biological Overview | References

Members of the Cas family of Src homology 3 (SH3)-domain-containing cytosolic signaling proteins are crucial regulators of actin cytoskeletal dynamics in non-neuronal cells; however, their neuronal functions are poorly understood. This study identified Drosophila p130CAS (DCas, CG1212, CAS; not to be confused with the Drosophila genes CAS/CSE1 segregation protein and castor), found that Cas proteins are highly expressed in neurons and showed that DCas is required for correct axon guidance during development. Functional analyses reveal that Cas specifies axon guidance by regulating the degree of fasciculation among axons. These guidance defects are similar to those observed in integrin mutants, and genetic analysis shows that integrins function together with Cas to facilitate axonal defasciculation. These results strongly support Cas proteins working together with integrins in vivo to direct axon guidance events (Huang, 2007).

In mammals, Cas proteins function downstream of several different receptors in non-neuronal cells, including growth factor receptors, G-protein-coupled receptors, T-cell receptors, B-cell receptors and integrins. Interestingly, integrin receptor subunit mutations in Drosophila give rise to CNS and motor axon guidance defects that are strikingly similar to those observed in DCas mutants, suggesting that Cas might function together with integrin receptors to guide axons (Huang, 2007).

Drosophila integrins, like vertebrate integrins, are composed of an α-subunit and a ß-subunit. In Drosophila, there is one gene encoding a typical laminin-binding-type α-subunit (α1, called mew), one encoding an RGD-binding-type α-subunit (α2, called if), and a single ß-subunit gene (ß1, called mys) very similar to the prototype vertebrate ß1 receptor. To investigate the connection between integrin and Cas signaling, the role of integrin receptor function in embryonic motor axon pathfinding was revisited and it was found that integrin-null mutant embryos exhibit defects that are qualitatively and quantitatively similar to DCas mutants. Embryos harboring null alleles for either α1 (mew^M6) or α2 (if^K27E) integrin genes exhibited ISNb and SNa axon guidance defects very similar to those observed in DCas mutants, including increased fasciculation resulting in the absence of muscle innervation. CNS axon guidance defects in were also observed both α1 and α2 integrin mutants that were similar to those observed in DCas mutants (Huang, 2007).

To further address DCas involvement in integrin-mediated axon guidance, dominant genetic interactions were sought between DCas and integrin subunit LOF mutations. Such transheterozygous interactions provide genetic support for two proteins functioning together in the same signaling pathway. It was asked whether removal of a single copy of DCas dominantly enhances heterozygosity at the α1, α2 or ß1 integrin loci. It was found that in α1, α2, ß1 or DCas heterozygotes, motor axon trajectories were not significantly different from wild type. However, removal of a single copy of DCas together with a single copy of α1, α2 or ß1 integrin resulted in highly penetrant axon guidance defects, suggesting that these three genes function in the same signaling pathway. Importantly, the phenotypes resulting from dominant enhancement by DCas are indicative of increased fasciculation, similar to those observed in DCas, α1 or α2 integrin LOF embryos (Huang, 2007).

To further assess the role integrins play in DCas-mediated axon guidance, it was asked whether ß1integrin LOF mutants dominantly suppress DCas GOF motor axon guidance phenotypes. The Drosophila ß1 integrin (encoded by mys¹) is the predominant neuronal ß1 integrin and therefore likely to mediate most, if not all, nervous system integrin signaling. If integrins are indeed necessary for activating DCas signaling, removing a single copy of ß1 integrin should suppress DCas GOF phenotypes. When low levels of DCas were expressed in all neurons in an otherwise wild-type background, moderate guidance defects were observed involving axons of the ISNb, SNa and CNS third longitudinal. Removing a single copy of the ß1 integrin gene in this same neuronal DCas GOF genetic background significantly rescued axon guidance phenotypes resulting from DCas GOF. Importantly, these results also show that neuronal overexpression of DCas does not simply function in a dominant-negative fashion to block integrin, or other, signaling pathways (Huang, 2007).

Supporting a model for how integrins and Cas regulate axonal fasciculation and pathfinding is extensive work on neuronal integrin functions in vitro. Growing axons and migrating cells preferentially elongate on surfaces to which they adhere most strongly, including integrin ligands. How might adhesive interactions influence axonal guidance decisions in vivo? Neuronal growth cones tend to form extensive lamellae, which are indicative of strong adhesive interactions, when cultured on highly adhesive substrata containing integrin ligands. These adhesive interactions stabilize elongating nerve fibers by promoting filopodial extension and expansion of growth cone surfaces. Disruption of axon-substrate attachment in vitro with integrin function-blocking antibodies encourages axon-axon adhesive interactions (fasciculation) in place of axon-substrate adhesion. Furthermore, contact with integrin ligands can slow axon elongation, as axons encountering an increasing gradient of laminin peptide exhibit reduced velocity, but growth cone velocity returns to previous rates when axons turn down the gradient. This in vitro observation resembles in vivo situations in which growth cones slow at a choice point, exhibit increased morphological complexity and then extend along distinct pathways. Drosophila motor axon growth cones also exhibit similar changes in morphological complexity upon contacting different substrates in vivo, suggesting that similar processes function to generate motor axon trajectories. Different combinations of integrin ligands might be responsible for these effects. When vertebrate growth cones in vitro contact either the α1 or α2 integrin ligands, laminin and fibronectin respectively, they decelerate, pause and exhibit short-term growth arrest. Interestingly, in vivo observations show that DCas functions with both α1 (laminin-binding) and α2 [RGD (e.g., Tiggrin)-binding] integrins to mediate correct axon navigation by regulating motor axon fasciculation at choice points, suggesting that integrin/Cas-mediated spatial regulation of growth cone extension underlies correct navigation at these choice points (Huang, 2007 and references therein).

The molecular mechanisms underlying integrin-mediated axon guidance remain to be completely defined. However, results derived from analysis of integrin/Cas signaling on cell migration shed light on how Cas and integrins might specify axonal defasciculation events in vivo. During cell migration, Cas proteins serve to establish linkage between migrating cells and the ECM. Cas plays an important role in regulating cytoskeletal organization, cell adhesion and force sensing, and fibroblasts isolated from p130Cas-null mutant mouse embryos exhibit disorganized and short actin filaments and decreased cell migration. In non-neuronal cells, Cas becomes phosphorylated in response to integrin engagement by many ECM components, including fibronectin and laminin (Defilippi, 2006). FAK and Src family kinases have been implicated in integrin-dependent phosphorylation of Cas. Interestingly, recent in vitro observations reveal that FAK signaling at sites of integrin-mediated adhesion controls axon pathfinding. Furthermore, pharmacological inhibitors of Src family kinases decrease the level of neuronal phosphorylated Cas in vitro, supporting a role for Src kinases in regulating Cas proteins in neurons. Finally, the activity of Rho-family small GTPases is also regulated by Cas interactions with the guanine nucleotide exchange factor Dock180 (Dock1) (Defilippi, 2006). Taken together, these links between Cas signaling components and cytoskeletal reorganization suggest that some of these signaling proteins might also influence axon guidance in vivo during development (Huang, 2007 and references therein).

The results demonstrate that integrin/Cas-mediated signaling is necessary but not sufficient for axonal defasciculation, revealing that integrin/Cas-mediated axon guidance must be integrated with other axon guidance signaling cascades to regulate axon defasciculation events during development. The identity of these other axon guidance pathways is not known. The attractive/permissive guidance cue Netrin binds to integrins, and functions with integrins in non-neuronal cells. The Netrin receptor Deleted in colorectal cancer (DCC) has been found to utilize the integrin effector FAK and recently p130Cas, to mediate Netrin-dependent attractive growth cone steering. Ephrins, best known for their role as repulsive axon guidance cues, also induce cell adhesion and actin cytoskeletal changes in fibroblasts in a p130Cas-dependent manner. Repulsive axon guidance cues may also regulate integrin/Cas-dependent axon guidance during development. The axonal repellent Slit genetically interacts with integrins and their ligands to guide commissural axons in Drosophila. Semaphorin and Ephrin-mediated repulsive effects on non-neuronal cells also appear to involve inhibition of integrin signaling events. Interestingly, a crucial component of semaphorin-dependent repulsive axon guidance, a member of the molecule interacting with Cas-L (MICAL) family, physically associates with Cas-L and preliminary data suggest that these interactions are functionally important for axon guidance. The observation that Cas functions with integrins to mediate axon guidance during development suggests new directions to better understand how integrin/Cas signaling modulates neuronal guidance through interactions with distinct axon guidance signaling pathways (Huang, 2007).

Defilippi, P., Di Stefano, P. and Cabodi, S. (2006). p130Cas: a versatile scaffold in signaling networks. Trends Cell Biol. 16: 257-263. Medline abstract: 16581250

Huang, Z., et al. (2007). Crk-associated substrate (Cas) signaling protein functions with integrins to specify axon guidance during development. Development 134(12): 2337-47. Medline abstract: 17537798

date revised: 26 September 2007

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