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SRC-1 controls growth cone polarity and protrusion with the UNC-6/Netrin receptor UNC-5 in Caenorhabditis elegans
Abstract:
In the Polarity/Protusion model of growth cone migration away from the guidance cue UNC-6/Netrin, the UNC-5 receptor polarizes the VD growth cone such that filopodial protrusions are biased to the dorsal leading edge of the growth cone. UNC-5 also inhibits growth cone protrusion ventrally based upon this polarity. The SRC-1 tyrosine kinase has been previously shown to physically interact with and phosphorylate UNC-5, and to act with UNC-5 in axon guidance and cell migration. Here, the role of SRC-1 in VD growth cone polarity and protrusion is investigated. A precise deletion of src-1 was generated, and mutants displayed unpolarized growth cones with increased size, similar to unc-5 mutants. Transgenic expression of src-1(+) in VD/DD neurons resulted in smaller growth cones, and rescued growth cone polarity defects of src-1 mutants, indicating cell-autonomous function. Transgenic expression of a putative kinase-dead src-1(D831A) mutant caused a phenotype similar to src-1 loss-of-function, suggesting that this is a dominant negative mutation. The D381A mutation was introduced into the endogenous src-1 gene by genome editing, which also had a dominant-negative effect. Genetic interactions of src-1 and unc-5 suggest they act in the same pathway on growth cone polarity and protrusion, but might have overlapping, parallel functions in other aspects of axon guidance. src-1 function was not required for the effects of activated myr::unc-5 , suggesting that SRC-1 might be involved in UNC-5 dimerization and activation by UNC-6, of which myr::unc-5 is independent. In sum, these results show that SRC-1 acts with UNC-5 in growth cone polarity and inhibition of protrusion.
Insights
SRC-1 tyrosine kinase works with UNC-5 receptor to control growth cone polarity and protrusion during axon guidance. Loss of SRC-1 disrupts this process, impacting cell migration.
Area of Science:
- Neuroscience
- Cell Biology
- Molecular Biology
Background:
- Growth cone migration is crucial for neural development and relies on precise polarity and protrusion.
- The UNC-5 receptor, in conjunction with guidance cue UNC-6/Netrin, establishes growth cone polarity, directing filopodial protrusions dorsally and inhibiting ventral protrusion.
- SRC-1 tyrosine kinase is known to interact with and phosphorylate UNC-5, suggesting a role in UNC-5-mediated axon guidance.
Approach:
- Generated a precise deletion mutant of src-1 to investigate its function in VD growth cone polarity and protrusion.
- Utilized transgenic expression of wild-type and kinase-dead SRC-1 to assess cell-autonomous function and dominant-negative effects.
- Employed genome editing to introduce a specific mutation (D381A) into the endogenous src-1 gene.
- Analyzed genetic interactions between src-1 and unc-5 mutants to elucidate their functional relationship.
- Investigated the interaction of SRC-1 with activated myristoylated UNC-5 (myr::unc-5) to understand its role in UNC-5 activation.
Key Points:
- src-1 deletion mutants exhibit unpolarized, enlarged growth cones, phenocopying unc-5 mutants.
- Cell-autonomous function of SRC-1 in restoring growth cone polarity and reducing size in src-1 mutants was confirmed.
- A kinase-dead mutant of SRC-1 (D831A) and an endogenously mutated form (D381A) displayed dominant-negative effects.
- Genetic interactions suggest SRC-1 and UNC-5 function in the same pathway for growth cone polarity and protrusion.
- SRC-1 is not required for myr::unc-5-mediated effects, indicating a role upstream of or in UNC-5 activation.
Conclusions:
- SRC-1 is essential for establishing VD growth cone polarity and regulating filopodial protrusion.
- SRC-1 functions in the same pathway as UNC-5 to control growth cone guidance.
- SRC-1's role in UNC-5 activation may involve dimerization or be independent of UNC-6 binding.
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