, 2007). In our screen we found that the MT plus-end binding protein EBP-1 ( Srayko et al., 2005) was required for regrowth ( Figure 5A). The reduced regrowth of ebp-1 mutants could not be bypassed by efa-6(lf) and was not further decreased by EFA-6 overexpression ( Figure 5A). Notably, the morphology of the axon stumps in ebp-1 mutants resembled those in EFA-6 overexpressors

( Figure 5B), suggesting http://www.selleckchem.com/products/Bafilomycin-A1.html the increased regrowth in efa-6 mutants might reflect altered axonal MT dynamics. To test whether EFA-6 affected axonal MT dynamics, we expressed the MT plus-end binding protein EBP-2 fused to GFP (see Experimental Procedures). End binding protein GFP fusions are established markers of growing ends of MTs in vertebrate neurons (Stepanova et al., 2003) and in C. elegans embryos ( Srayko et al., 2005). In wild-type axons within 3 hr of axotomy, before overt regrowth, axonal MTs (defined as motile EBP-2::GFP puncta) became highly dynamic close to the severed end of the axon (arrows, Figure 5D). In contrast, in efa-6(lf) mutants axonal MTs were more abundant Selleckchem Talazoparib and regrew for longer times and distances than in the wild-type ( Figures 5C and 5D). Conversely, in EFA-6 overexpressing axons the number of dynamic axonal MTs was significantly reduced ( Figures 5C and 5D). Axonal MT dynamics were normal in arf-6 mutants (data not shown), suggesting enhanced regrowth in efa-6 mutants is mainly due to the microtubule destabilizing role of EFA-6.

To directly address whether the reduced regrowth in EFA-6 overexpressors is due to destabilization of the MT cytoskeleton, we tested whether the MT stabilizing Rolziracetam drug taxol could overcome regrowth inhibition. Delivery of taxol by microinjection into the body cavity did not affect regrowth in the wild-type, yet significantly rescued regrowth of EFA-6-overexpressing axons (Figure 5E). Conversely, incubation in colchicine blocked axonal regrowth (data not shown). These findings show that MT polymerization is critical for C. elegans axon regrowth and support a specific role for EFA-6 promoting catastrophe of axonal MTs. Our screen identified many genes with positive and negative influences on PLM axonal regrowth. To address

how these pathways interact, we analyzed regrowth in double and triple mutants. Genetic backgrounds that elevate cAMP signaling (kin-2) display enhanced PLM axon regeneration but do not overcome the block in regrowth in dlk-1 mutants ( Ghosh-Roy et al., 2010) ( Figure 6A). Examination of double mutants between dlk-1 and other enhanced-regrowth mutants revealed similar dependence on dlk-1 ( Figure 6A). In contrast, elevated Ca2+ or cAMP signaling in egl-19(gf)/VGCC or pde-4(lf)/Phosphodiesterase mutants enhanced axon regrowth in unc-57/Endophilin mutants ( Figure 6B), suggesting Ca2+ and cAMP act in parallel to UNC-57 and upstream of DLK-1. However, lack of SLT-1 did not promote regrowth in unc-51/Atg1 or unc-57/Endophilin mutants ( Figure 6C).