Previous work from the Lundquist lab showed that the UNC-6/Netrin receptors UNC-40 and UNC-5 regulate growth cone protrusion. UNC-40 stimulates protrusion whereas UNC-5 inhibits protrusion, and asymmetric distribution of protrusive activity across the growth cone results in directed growth cone migration away from UNC-6/Netrin (the Polarity/Protrusion model).
unc-5 mutant VD growth cones display unpolarized and excessive protrusion. UNC-5 inhibits protrusion using the FMO flavin monoxygenases, likely via actin inhibition, and by restricting growth cone microtubule entry via UNC-33/CRMP, which might in turn regulate vesicle entry which drives protrusion. To explore the role of vesicle fusion in growth cone protrusion, we analyzed
tom-1/tomosyn mutants. Tomosyn normally occludes formation of the SNARE complex by interacting with and inhibiting syntaxin-1. VD growth cones of
tom-1 null mutants were similar to wild-type. However,
tom-1 null mutants suppressed the effects of constitutively-activated MYR::UNC-5, which alone causes small growth cones with little protrusion. This suggests that TOM-1 is normally required for the inhibitory effects of MYR::UNC-5 on growth cone protrusion. Mutations specifically affecting
tom-1 long isoforms showed small and non-protrusive growth cones, and did not suppress MYR::UNC-5. This suggests that TOM-1 short and long isoforms might have opposing roles, with TOM-1 short normally inhibiting protrusion, and TOM-1 long stimulating protrusion. Short isoform specific mutations suppressed MYR::UNC-5, and long isoform specific mutations suppressed excess protrusion in
unc-5 loss-of-function mutants, consistent with this idea. Finally, transgenic expression of full-length
tom-1(+) resulted in small and non-protrusive growth cones in both wild-type and
unc-5 mutant backgrounds, consistent with a role of TOM-1 in inhibiting protrusion downstream of UNC-5. In the polarity/protrusion model of growth cone outgrowth, UNC-6/Netrin inhibits growth cone protrusion via the UNC-5 receptor. Previous studies showed that UNC-5 inhibits protrusion via the FMOs and possible actin destabilization, and by preventing MT entry via UNC-33/CRMP. These results suggest that UNC-5 inhibits protrusion via a third pathway, employing TOM-1/tomosyn to prevent vesicle fusion and growth cone protrusion.