MIG-10 functions with ABI-1 to mediate the UNC-6 and SLT-1 axon guidance signaling pathways

Abstract

Extracellular guidance cues steer axons towards their targets by eliciting morphological changes in the growth cone. A key part of this process is the asymmetric recruitment of the cytoplasmic scaffolding protein MIG-10 (lamellipodin). MIG-10 is thought to asymmetrically promote outgrowth by inducing actin polymerization. However, the mechanism that links MIG-10 to actin polymerization is not known. We have identified the actin regulatory protein ABI-1 as a partner for MIG-10 that can mediate its outgrowth-promoting activity. The SH3 domain of ABI-1 binds to MIG-10, and loss of function of either of these proteins causes similar axon guidance defects. Like MIG-10, ABI-1 functions in both the attractive UNC-6 (netrin) pathway and the repulsive SLT-1 (slit) pathway. Dosage sensitive genetic interactions indicate that MIG-10 functions with ABI-1 and WVE-1 to mediate axon guidance. Epistasis analysis reveals that ABI-1 and WVE-1 function downstream of MIG-10 to mediate its outgrowth-promoting activity. Moreover, experiments with cultured mammalian cells suggest that the interaction between MIG-10 and ABI-1 mediates a conserved mechanism that promotes formation of lamellipodia. Together, these observations suggest that MIG-10 interacts with ABI-1 and WVE-1 to mediate the UNC-6 and SLT-1 guidance pathways.

Figure 1. MIG-10 interacts physically with ABI-1.

(A) Diagram of the RNAi screening strategy used to identify ABI-1. A sublibrary of RNAi clones encoding proline-binding domains (SH3, WW, EVH1) was created. Each clone was screened for the ability to phenocopy the HSN ventral guidance defect observed in mig-10 loss of function mutants. This strategy led to the identification of ABI-1 as a potential interaction partner for MIG-10. In this study we have utilized the abi-1(tm494) allele, which is predicted to truncate the ABI-1 protein as indicated by the bracket. (B) Example of HSN axon in wild-type animals. The axon makes a direct ventral migration. (C) Example of HSN ventral guidance defect observed in mig-10(RNAi) animals. The axon migrates laterally prior to turning ventrally. (D) Example of HSN ventral guidance defect observed in abi-1(RNAi) animals. The HSN axon was observed with an unc-86::myrGFP transgene. Arrowheads mark approximate position of the vulva. Note that the migration of the HSN cell body was also affected by mig-10(RNAi) and abi-1(RNAi). However, previous analysis has indicated that HSN axon guidance defects are not secondary consequences of defects in cell body migration , . Scale bars represent 5 µm. (E) MIG-10 binds to the SH3 domain of ABI-1. MIG-10::GFP was incubated with the SH3 domain of ABI-1 fused to GST (GST::ABI-1-SH3) or GST as a control. Bound material was detected by western blotting with an antibody to GFP. For reference, an amount equivalent to 5% of the MIG-10::GFP starting material was run on a gel (5% SM).

Figure 2. ABI-1 is involved in both UNC-6 and SLT-1 signaling pathways.

(A) Example of AVM neuron with normal ventral guidance. (B) Example of AVM neuron with defective ventral guidance. (C–D) Genetic interactions between unc-6 and abi-1 as well as between slt-1 and abi-1 in the AVM (C) and PVM (D), indicate that ABI-1 functions in both the UNC-6 and SLT-1 signaling pathways. (E) Loss of abi-1 function does not enhance defects in the unc-6; slt-1 mutant background. (F) ABI-1 functions cell autonomously to mediate axon guidance. A mec-4::abi-1 transgene suppresses PVM ventral axon guidance defects in slt-1; abi-1 double mutants. mec-4::abi-1(−) represents animals that have lost the mec-4::abi-1 transgene during mitosis. These animals serve as controls and were scored simultaneously on the same slides as the mec-4::abi-1(+) animals, which carry the transgene. Data was combined from 2 independently derived transgenic lines, cueEx1 and cueEx2, which showed similar results. (G) The abi-1(tm494) loss of function mutation suppresses guidance defects in AVM neurons overexpressing UNC-40. Scale bars are 5 µM. Error bars represent standard error of the proportion. Brackets indicate statistically significant difference, Z test for proportions (*p<0.05, **p<0.005). The AVM axon was visualized with the zdIs5 transgene (mec-4::gfp). Scale bars are 10 µM. Alleles used were unc-6(ev400) null, slt-1(eh15) null, and abi-1(tm494) loss of function.

Figure 3. Dosage-sensitive genetic interactions indicate that MIG-10, ABI-1, and WVE-1 function together to mediate axon guidance.

(A) Transheterozygous genetic interaction between abi-1(tm494) and mig-10(ct41). HSN ventral guidance in animals heterozygous for either abi-1(tm494) or mig-10(ct41) was comparable to those in wild-type animals. Animals transheterozygous for abi-1(tm494) and mig-10(ct41) had significantly greater ventral guidance errors compared to wild-type animals. Heterozygous animals were constructed by crossing unc-86::myrgfp males with abi-1(tm494) or mig-10(ct41) hermaphrodites and scoring F1 cross progeny. Transheterozygous animals were constructed by crossing abi-1(tm494); unc-86::myrgfp males with mig-10(ct41) hermaphrodites and scoring F1 cross progeny. (B) Transheterozygous genetic interaction between wve-1(ok3308) and mig-10(ct41). HSN ventral guidance was comparable to wild-type in animals heterozygous for wve-1(ok3308) or mig-10(ct41). HSN ventral guidance in animals heterozygous for either wve-1(ok3308) or mig-10(ct41) was comparable to those in wild-type animals. Animals transheterozygous for wve-1(ok3308) and mig-10(ct41) had significantly greater ventral guidance errors compared to wild-type animals. Similar results were obtained using the wve-1(ne350) allele instead of the wve-1(ok3308) allele. The hT2 balancer covers both wve-1 and mig-10 and was used to construct wve-1 heterozygotes, mig-10 heterozygotes, and the wve-1; mig-10 transheterozygotes. The kyIs262 transgene (unc-86::myrgfp) was used for observing the HSN axon. For the labels in both (A) and (B), “het.” means heterozygous for the mutant or for the unc-86::myrgfp transgene. Whereas, “+” means homozygous for the wild-type gene or homozygous for the unc-86::myrgfp transgene. Brackets indicate statistically significant difference between transheterozygotes and single heterozygotes, Z test for proportions (*p<0.0005).

Figure 4. ABI-1 and WVE-1 mediate outgrowth-promoting activity downstream of MIG-10.

(A) Example of normal ALM neuron with a single anterior axon. (B) Example of ALM multipolar defect caused by transgenic expression of MIG-10A by the mec-4::mig-10a transgene. (C) Loss of function mutations abi-1(tm494) and wve-1(ok3308) suppress MIG-10 transgenic expression phenotype. The max-2(nv162) mutation, a likely null, does not suppress the MIG-10 transgenic expression phenotype. The wve-1(ok3308) mutants were maternally rescued. The AVM axon was visualized with a zdIs5 transgene (mec-4::gfp). (D) The abi-1(tm494) loss of function mutation suppresses the AVM multipolar phenotype that results from transgenic expression of MIG-10 in the unc-6; slt-1 double null mutant background. *Statistically significant difference compared to wild-type or unc-6; slt-1 double mutant, z-test for proportions (p<0.005).

Figure 5. ABI-1 mediates the lamellipodia-forming activity of MIG-10 in cultured HEK293 cells.

(A) Example of cell transfected with GFP and control shRNA. (B) Example of cell transfected with MIG-10::GFP and control shRNA. (C) Example of cell transfected with MIG-10::GFP and Abi1 shRNA. (D) Knockdown of Abi1 suppresses the lamellipodia-forming activity of MIG-10. Graph shows the average cell perimeter with lamellipodia. “Ctr.” means cells transfected with scrambled control shRNA. “−” means cells were not transfected with any shRNA. “Abi1” means cells were transfected with the PAV197 shRNA against Abi1. Error bars represent the standard error of the mean. *Bracket indicates statistically significant difference, t-test (p<0.0001). Scale bars are 5 µm.