Axon injury triggers EFA-6 mediated destabilization of axonal microtubules via TACC and doublecortin like kinase

Axon injury triggers a series of changes in the axonal cytoskeleton that are prerequisites for effective axon regeneration. In Caenorhabditis elegans the signaling protein Exchange Factor for ARF-6 (EFA-6) is a potent intrinsic inhibitor of axon regrowth. Here we show that axon injury triggers rapid EFA-6-dependent inhibition of axonal microtubule (MT) dynamics, concomitant with relocalization of EFA-6. EFA-6 relocalization and axon regrowth inhibition require a conserved 18-aa motif in its otherwise intrinsically disordered N-terminal domain. The EFA-6 N-terminus binds the MT-associated proteins TAC-1/Transforming-Acidic-Coiled-Coil, and ZYG-8/Doublecortin-Like-Kinase, both of which are required for regenerative growth cone formation, and which act downstream of EFA-6. After injury TAC-1 and EFA-6 transiently relocalize to sites marked by the MT minus end binding protein PTRN-1/Patronin. We propose that EFA-6 acts as a bifunctional injury-responsive regulator of axonal MT dynamics, acting at the cell cortex in the steady state and at MT minus ends after injury. DOI: http://dx.doi.org/10.7554/eLife.08695.001


Introduction
In mature nervous systems axons regenerate poorly after injury, leading to permanent functional deficits. Both the nature of the extracellular environment and the intrinsic growth competence of the neuron contribute to the extent of axon regeneration (Case and Tessier-Lavigne, 2005). The mammalian central nervous system (CNS) expresses a variety of environmental regeneration inhibitory factors, including myelin-associated proteins, chondroitin sulfate proteoglycans and glial scar tissue that functions as a physical barrier (Schwab, 2004;Silver and Miller, 2004). However genetic removal of these inhibitory factors results in only limited improvement in regeneration of severed axons (Lee et al., 2009. Recent studies have strongly supported the importance of cell-intrinsic determinants in axon regeneration. Loss of function in cell-intrinsic growth inhibitors such as Phosphatase and Tensin homolog, PTEN, and Suppressor Of Cytokine Signaling-3, SOCS3, can dramatically improve axon regrowth even in the inhibitory CNS environment (Park et al., 2008;Sun et al., 2011). Genetic and pharmacological manipulation of cell autonomous signaling pathways can dramatically improve regrowth of severed axons in various injury paradigms (Moore et al., 2009;Hellal et al., 2011;Sengottuvel et al., 2011;Shin et al., 2012;Watkins et al., 2013;Ruschel et al., 2015).
During developmental axon outgrowth and in regenerative regrowth of mature neurons, the formation and extension of growth cones involve extensive remodeling of the microtubule (MT) cytoskeleton (Bradke et al., 2012;Chisholm, 2013). Cellular compartments undergoing rapid morphological changes, such as axonal growth cones, are enriched in dynamic MTs (Suter et al., 2004), while mature axons or dendrites contain predominantly stabilized MTs (Baas et al., 1993). When an axon is injured, MTs are locally disassembled or severed, potentially creating free plus ends for new MT polymerization. Subsequently, the number of growing MTs increases, followed by more persistent MT growth correlated with formation of regenerative growth cone and axon extension (Erez and Spira, 2008;Ghosh-Roy et al., 2012). Complete removal of an axon also leads to dramatic upregulation of MT dynamics in the soma and dendrites (Stone et al., 2010). MT disorganization contributes to dystrophic end bulb formation after injury in CNS (Ertürk et al., 2007). Moderate stabilization of MT dynamics by Taxol or other MT stabilizers can promote axon regrowth in vitro and in the mammalian CNS (Usher et al., 2010;Hellal et al., 2011;Sengottuvel et al., 2011;Ruschel et al., 2015); the effects of Taxol in vivo have been partly replicated (Popovich et al., 2014;Ruschel et al., 2015). Thus, there is a critical need to define the endogenous regulators of MTs after injury.
In a large-scale screen for genes affecting adult axon regeneration in Caenorhabditis elegans, we identified Exchange Factor for ARF-6 (EFA-6) as an intrinsic inhibitor of axon regrowth . The EFA-6/EFA6 protein family is conserved from yeast to mammals, and is defined by its Sec7 domain, which confers guanine exchange factor (GEF) activity for Arf6 GTPases (Franco et al., 1999). Unexpectedly, the regrowth-inhibitory function of EFA-6 is independent of its GEF activity, and instead is mediated by its N-terminal domain . The EFA-6 N-terminal domain inhibits MT growth at the cell cortex of C. elegans embryos via a conserved motif of 18 amino acids (O'Rourke et al., 2010). Nonetheless, the mechanism by which EFA-6 regulates MT dynamics is unknown.
Here we reveal that axon injury triggers rapid and transient relocalization of EFA-6, concomitant with an initial downregulation of axonal MT dynamics. The N-terminal 18-aa motif is required for injuryinduced relocalization and for inhibition of axonal MT growth. We show that the EFA-6 N-terminal domain interacts with MT associated proteins TAC-1, a member of the transforming acidic coiled-coil (TACC) family, and ZYG-8, an ortholog of doublecortin-like kinase (DCLK). TAC-1 and ZYG-8 are required for initiation of axon regeneration, and their overexpression can promote regrowth. We further show that injury triggers relocalization of EFA-6 and TAC-1 to sites overlapping with the MT minus end binding protein Patronin/PTRN-1. We propose that EFA-6 is a bifunctional injury-responsive regulator of MT dynamics, acting at the cell cortex in the steady state and at MT minus ends after axon injury. eLife digest In the nervous system, cells called neurons carry information around the body.
These cells have long thin projections called axons that allow the information to pass very quickly along the cell to junctions with other neurons. Neurons in adult mammals are limited in their ability to regenerate, so any damage to axons, for example, due to a stroke or a brain injury, tends to be permanent. Therefore, an important goal in neuroscience research is to discover the genes and proteins that are involved in regenerating axons as this may make it possible to develop new therapies.
An internal scaffold called the cytoskeleton supports the three-dimensional shape of the axons. Changes in the cytoskeleton are required to allow neurons to regenerate axons after injury, and drugs that stabilize filaments called microtubules in the cytoskeleton can promote these changes. Chen et al. used a technique called laser microsurgery to sever individual axons in a roundworm known as C. elegans and then observed whether these axons could regenerate. The experiments reveal that a protein called EFA-6 blocks the regeneration of neurons by preventing rearrangements in the cytoskeleton.
EFA-6 is normally found at the membrane that surrounds the neuron. However, Chen et al. show that when the axon is damaged, this protein rapidly moves to areas near the ends of microtubule filaments. EFA-6 interacts with two other proteins that are associated with microtubules and are required for axons to be able to regenerate. Chen et al.'s findings demonstrate that several proteins that regulate microtubule filaments play a key role in regenerating axons. All three of these proteins are found in humans and other animals so they have the potential to be targeted by drug therapies in future. The next challenge is to understand the details of how EFA-6 activity is affected by axon injury, and how this alters the cytoskeleton.

Axon injury triggers redistribution of EFA-6
In the one-cell stage embryo EFA-6 localizes to the plasma membrane via its C-terminal PH (pleckstrin homology) domain, and this plasma membrane localization of EFA-6 is necessary for it to inhibit cortical MT growth (O'Rourke et al., 2010). To determine the subcellular location of EFA-6 in neurons, we expressed a series of GFP-tagged EFA-6 fusion proteins. Full-length EFA-6 tagged with GFP at the N-or C-termini, expressed at a range of concentrations, localized to the plasma membrane of the soma and processes of neurons ( Figure 1A,B, Figure 1-figure supplement 1A); deletion of the PH domain (FLΔPH) resulted in cytosolic localization ( Figure 1C, upper panel). The first 150 residues of the EFA-6 N-terminus (N150), expression of which inhibits axon regrowth , was localized to the cytosol similarly to FLΔPH ( Figure 1C,D). Conversely, EFA-6 proteins lacking the N-terminal 150 amino acids (FLΔN150) showed plasma membrane localization resembling that of full-length EFA-6 ( Figure 1B,E).
We next examined how axon injury affected EFA-6 localization. Within seconds of laser axotomy of the PLM axon, GFP::EFA-6FL redistributed from a generally even plasma membrane localization to more discrete puncta ( Figure 1B, Video 1). This relocalization was also observed when GFP was tagged to the C terminus or in the middle of EFA-6 (Figure 1-figure supplement 1B), and did not require the PH domain ( Figure 1C). GFP::EFA-6N150, although not membrane associated, also became punctate after injury, whereas GFP::EFA-6FLΔN150 did not relocalize after axon injury ( Figure 1D,E). Full-length EFA-6 and the N terminal domain (N150) appear to relocalize to the same puncta in response to injury, as shown by co-expressing EFA-6FL::GFP and EFA-6N150::mKate2 ( Figure 1F, Figure 1-figure supplement 2A). mKate2::EFA-6N150Δ18 (which does not relocalize, see below) did not co-localize with EFA-6FL::GFP after injury, suggesting EFA-6 puncta are not due to non-specific aggregation of proteins after injury (Figure 1-figure supplement 2B). The region of relocalized EFA-6FL or EFA-6N150 expanded bidirectionally from the injury site at ∼8 μm s −1 , into the soma and dendrite (Figure 1-figure supplement 2C, Video 1). The density of injury-triggered puncta of EFA-6FL and EFA-6N150 gradually decreased over the next 20-60 min ( Figure 1B These results indicate that injury-triggered EFA-6 relocalization occurs in multiple neuron types, irrespective of the site of injury and independent of the Sec7 or PH domains. The N-terminal 1-70 aa (N70) was the smallest fragment tested that displayed relocalization after injury ( Figure 1I). We tested a variety of other neuronal proteins, including the MT plus-end binding proteins EBP-1 and EBP-2, KLP-7/kinesin-13 Ghosh-Roy et al., 2012), ARF-6 (the presumed substrate for EFA-6's GEF activity), SAX-3 (transmembrane receptor) and synaptic vesicle proteins (SNB-1/synaptobrevin, RAB-3/GTPase, UNC-57/endophilin), and found that none showed similar relocalization after axon injury (Figure 1-figure supplement 3G, and data not shown). Thus, injury-triggered relocalization is specific to EFA-6.
The punctate distribution of EFA-6 after injury suggested that EFA-6 might become sequestered to a subcellular compartment. To address whether injury alters EFA-6 mobility within the cell we performed FRAP (Fluorescence Recovery After Photobleaching). In uninjured neurons, EFA-6FL::GFP recovered with t ½ = 4 s and an immobile fraction of 25%. In contrast, EFA-6FL::GFP puncta 2 min after injury showed dramatically reduced recovery, with >85% of the protein in the immobile fraction ( Figure 2A,B). We were not able to calculate t ½ due to the extremely low recovery rate. By 1 hr after injury, EFA-6FL::GFP partially returned to its steady state localization ( Figure 1B), and its recovery rate was partially restored, with an immobile fraction of 50% (Figure 2A,B). GFP::EFA-6N150 gave similar FRAP results (not shown). This analysis suggests that after injury EFA-6 is sequestered to subcellular structures.
The ability of EFA-6 fragments to inhibit developmental axon outgrowth and regrowth after axon injury correlates with their localization efa-6(lf) mutants display significantly increased PLM axon regrowth following laser axotomy in adults, as well as impenetrant developmental overshooting of PLM axons; conversely, overexpression of . We therefore tested if the capacity of EFA-6 protein fragments to relocalize correlated with their effects on PLM development and regrowth. We found that overexpression of the EFA-6 N terminal fragments that displayed injury-induced relocalization (N150, N100, N70) all caused axons of PLM neurons to undershoot ( Figure 2C Table 1). We performed axotomy on those axons that exhibited normal morphology in L4 larvae and found that these axons all showed significantly reduced regrowth ( Figure 2D In contrast, overexpression of smaller fragments (N24, N42 or N[43-70 aa]) of the EFA-6 N-terminus that did not relocalize in response to axotomy did not significantly inhibit PLM outgrowth or regrowth ( Figure 2C,D and Table 1). Interestingly, overexpression of EFA-6FLΔN150, but not of EFA-6FL, caused PLM overshooting, suggesting the C-terminal region of EFA-6 may inhibit the activity of the N-terminus. Overexpression of EFA-6FL strongly inhibits axonal MT dynamics, as assessed using the EBP-GFP assay for growing MT plus ends . Using live imaging and quantitative kymograph analysis of EBP-2::GFP comets, we found that only those EFA-6 fragments displaying injury-induced relocalization also inhibited steady-state axonal MT dynamics when overexpressed ( Figure 2E, Figure 2-figure supplement 1C). These observations suggest the ability of EFA-6 protein fragments to inhibit axon growth and MT dynamics closely correlates with their ability to relocalize in response to injury. EFA-6 function and relocalization require a conserved 18 aa motif in the N terminus All EFA6 family members contain extended N-termini of low sequence complexity and lack defined sequence motifs ( Figure 2-figure supplement 2A). The sole known region of sequence conservation among N-termini of some EFA6 family members is a motif of 18 aa (residues 25-42 in C. elegans EFA-6), necessary and partially sufficient for EFA-6's effects on cortical MT growth in the embryo (O'Rourke et al., 2010). Analysis of EFA-6/EFA6 N-termini using algorithms that predict natively disordered protein regions (Xue et al., 2010) indicates that the EFA-6 N-terminus has a high probability of protein disorder, with the exception of the 18 aa motif (Figure 2-figure supplement 2B).
To address the role of the 18 aa motif in EFA-6's function in neurons, we expressed constructs in Figure 1. Continued terminal 150 aa (N150) (Pmec-4-GFP::EFA-6N150, juEx3531), and EFA-6 lacking the N-terminus (FLΔN150) (Pmec-4-GFP::EFA-6FLΔN150, juEx6154). (F) Colocalization of EFA-6FL and EFA-6N150 puncta after injury. Localization of EFA-6FL::GFP and EFA-6N150::mKate2 (juEx6522) in PLM before and after axotomy. EFA-6 full length protein and N terminus relocalize to overlapping puncta. (G, H) Requirement for the 18-aa motif for relocalization of EFA-6FL and EFA-6N150. Localization of GFP::EFA-6FLΔ18aa (juEx6156), and GFP::EFA-6N150Δ18aa (juEx3535) in touch neurons before and 2 min after axotomy. (I) Quantitation of puncta before and 2 min after injury in axons expressing different EFA-6 fragments. Statistics, one-way ANOVA with Bonferroni post test; n = 5 for each bar; **p < 0.01, ns, not significant. Scale, 10 μm. DOI: 10.7554/eLife.08695.003 The following figure supplements are available for figure 1: which the 18 aa motif was deleted or mutated. We found that full length EFA-6 lacking the motif, GFP:: EFA-6FLΔ18, localized to the plasma membrane but did not cause axonal developmental defects ( Figures 1G, 2C). GFP::EFA-6N150 was diffuse in the cytosol and excluded from the nucleus ( Figure 1D).  Deletion of the 18 aa motif from the N-terminus (N150Δ18) abolished this nuclear exclusion, largely resembling free GFP ( Figure 1H). Overexpression of EFA-6N150Δ18 also did not cause developmental abnormalities ( Figure  , suggesting that high levels of EFA-6 N-terminal domain perturb function of many neurons, in a manner dependent on the 18 aa motif. Moreover, mutating 2 or more residues within the 18 aa motif to alanines significantly reduced the activity of the N-terminus in multiple assays ( Table 1), suggesting the sequence of the 18 aa motif is critical for the N-terminus to function. The 18 aa motif was essential for injury-triggered relocalization of EFA-6FL and EFA-6N150 ( Figure 1G,H), as well as for their inhibitory effects in axon regrowth ( Figure 2D). Expression of the 18 aa motif alone did not cause PLM developmental defects or confer injury-induced re-localization ( Figures 1I, 2C), but mildly inhibited axon regrowth ( Figure 2D), suggesting that injured axons are highly sensitive to the activity of this motif, but that the surrounding context is required for full activity of the N terminus.
Two efa-6 loss of function alleles, tm3124 and ok3353, delete genomic sequences that encode the Sec7 domain, and are predicted to cause frameshifts after the N-terminus (Figure 2-figure supplement 2C). Both mutations cause embryonic phenotypes similar to efa-6 RNAi (O'Rourke et al., 2010) and display similarly enhanced axon regrowth . As these mutations do not delete the N-terminus, it is possible that truncated proteins might be produced in these mutants. We therefore generated a targeted deletion, efa-6(ju1200), that removes the genomic sequences encoding the 18 aa motif (Figure 2-figure supplement 2C). The axon developmental and regrowth phenotypes of efa-6(ju1200) mutants were indistinguishable from those of efa-6(tm3124) ( Figure 2C,D). In addition, single copy transgene expression of EFA-6N150 (juSi86) rescued the regeneration defects of both efa-6 (tm3124) and efa-6(ju1200) to similar degrees ( Figure 2-figure supplement 2D), and also rescued developmental axon overgrowth (not shown). Thus, the increased axon regrowth of efa-6 mutants reflects a complete loss of EFA-6 function, and the major activity of EFA-6 in axon growth resides in the N-terminus, dependent on the 18 aa motif. Below, we refer to efa-6(tm3124) as efa-6(0).

EFA-6 is required for the injury-induced downregulation of axonal MT dynamics
Our previous analysis showed that loss of EFA-6 function resulted in elevated axonal MT dynamics several hours after injury . To test whether MT dynamics might be influenced by EFA-6 immediately after injury, we examined the acute effects of axon injury on MT dynamics. In wild type animals, axonal EBP-2::GFP comets were dramatically reduced within 50 s post axotomy, consistent with injury triggering rapid MT destabilization ( Figure 5A). In efa-6(tm3124) or efa-6 (ju1200) mutants we observed slightly increased numbers of EBP-2::GFP comets in uninjured axons, compared to wild type; but these animals did not show a significant reduction in EBP-2::GFP comets immediately after injury ( Figure 5A and Figure 5-figure supplement 1A). The slight reduction in axonal comets after injury in efa-6(0) suggests that some MTs can also be downregulated independent of EFA-6. In efa-6(gf) animals overexpressing EFA-6N150, the total number of growing MT plus ends in uninjured axons was reduced compared to wild type, and was not further reduced after injury ( Figure 5A). tac-1(lf) mutants had slightly reduced axonal MT dynamics in uninjured axons, and displayed injury-dependent downregulation, whereas zyg-8(lf) mutants showed fewer dynamic MTs in uninjured axons, and did not further downregulate MTs after injury ( Figure 5A). The mild phenotype in tac-1(lf) could be due to incomplete loss of function of this ts allele. We further tested the tac-1 (ok3305) deletion allele using Cre-induced tissue-specific knockout ('Materials and methods'). Compared to control, tac-1(0) touch neurons displayed reduced dynamics in uninjured axons similar to zyg-8(lf), and showed no further reduction after injury ( Figure 5-figure supplement 1B). As reported previously, dynamic axonal MTs are significantly increased at 3 hr post axotomy, and this is further enhanced in efa-6(0) mutants (Chen et al., 2011) ( Figure 5B). Neither tac-1(lf) nor zyg-8(lf) mutants upregulated dynamic axonal MTs by 3 hr post injury ( Figure 5B). Moreover, MT dynamics in efa-6(0) tac-1(lf) or efa-6(0) zyg-8(lf) double mutants resembled tac-1(lf) or zyg-8(lf) single mutants ( Figure 5B), consistent with EFA-6 functioning upstream of ZYG-8. Thus, axon injury causes an immediate inhibition in growing MTs, dependent on EFA-6 and correlating with its relocalization, followed by a more prolonged increase in growing MTs, dependent on the function of TAC-1 and ZYG-8.

Discussion
We previously identified EFA-6 as a potent intrinsic inhibitor of axon regeneration .
Here we dissected how EFA-6 regulates MT dynamics in the axonal response to injury, yielding the following insights: (1) injury triggers a rapid relocalization of EFA-6 from the cell membrane to subcellular structures overlapping with the MT minus end binding protein PTRN-1/Patronin; (2) injury also triggers a rapid local downregulation of axonal MT dynamics, a process that requires EFA-6 and which occurs simultaneously with EFA-6 relocalization; (3) a conserved motif in the otherwise unstructured EFA-6 N-terminus is essential for its relocalization and for its effects on axonal MT dynamics; (4) the EFA-6 N-terminus directly interacts with two highly conserved MAPs, TAC-1/TACC and ZYG-8/DCLK, both of which are required for normal regrowth and for the later phase of upregulated axonal MT dynamics. Our studies reveal that axonal injury triggers an intricate sequence of alterations in axonal MT dynamics. Precise control of the axonal MT response by positive and negative regulators of MT dynamics appears critical for the conversion of the mature axon into a regrowing growth cone. Strains were maintained at 15˚C, shifted to 25˚C 20 hr before axotomy. Alleles: efa-6(tm3124), tac-1(or455ts), zyg-8(or484ts). Statistics, one-way ANOVA with Bonferroni post test; ***p < 0.001; **p < 0.01; *p < 0.05; ns, not significant. n ≥ 10 axons per condition. DOI: 10.7554/eLife.08695.016 The following figure supplement is available for figure 5:  . TAC-1 relocalizes in response to injury to become co-localized with EFA-6. (A) Localization of GFP::ZYG-8 in touch neurons before and 2 min after axotomy in wild type and efa-6(tm3124). GFP::ZYG-8 localization is not affected by axon injury or loss of EFA-6. Transgene: Pmec-4-GFP::ZYG-8(juEx5932). (B) GFP::TAC-1 in PLM before and 2 min after axotomy at wild type and efa-6(ju1200) backgrounds. Injury triggered relocalization of TAC-1 was similar to EFA-6 and not dependent on EFA-6. Pmec-4-GFP::TAC-1(juEx5759). (C) GFP::EFA-6N150 (juEx3531) Figure 6. continued on next page EFA-6 functions in the steady state and injured axon EFA-6 induces catastrophe or pausing of growth at MT plus ends at the cortex of embryonic cells (O'Rourke et al., 2010), and our analysis is consistent with this model in steady-state (uninjured) axons. In mature neurons EFA-6 localizes to the cell membrane via its PH domain. efa-6((0)) mutant axons display elevated numbers of growing MTs in the steady state, and display impenetrant developmental overgrowth, indicating that in the absence of injury EFA-6 restrains axonal MT dynamics and mildly inhibits axon outgrowth. These steady-state roles of EFA-6 are mediated by the N-terminus, as they are fully rescued by expression of the N-terminal 150 aa. Overexpression of either full length EFA-6 or the N-terminus causes axons to undershoot, whereas overexpression of EFA-6ΔN causes axons to overshoot. The opposing effects of overexpression of the N-terminus and the C-terminus suggest that in the steady state the EFA-6 N-terminus might be inhibited by the remainder of the protein.
After axon injury, EFA-6 displays a dramatic and specific transient relocalization to punctate structures associated with MT minus ends. Interestingly, this relocalization does not require membrane association, as EFA-6 fragments lacking the PH domain, or containing only the N-terminal 1-70 aa, were not membrane-localized, yet became relocalized to axonal puncta after injury. As relocalized fulllength EFA-6 appears to remain membrane associated, it is possible that after injury EFA-6 localizes to the cytoskeleton via its N terminus while remaining membrane associated via its PH domain. Speculatively, injury signals may increase EFA-6 N-terminus activity by releasing it from inhibition by the EFA-6 C-terminus.

The EFA-6 N-terminus is an intrinsically disordered region
The N-termini of EFA6 family members display low sequence complexity and minimal primary sequence similarity, with the exception of the 18 aa motif found in invertebrate family members (O'Rourke et al., 2010). The N-termini of C. elegans, Drosophila and mammalian EFA6 proteins all have a high probability of intrinsic disorder. Intrinsically disordered proteins (IDPs) and disordered protein regions are increasingly recognized as having important biological activities (Oldfield and Dunker, 2014). Well-studied examples of IDPs in the nervous system include the MT-binding proteins Tau (Schweers et al., 1994) and stathmin (Honnappa et al., 2006). Intrinsic disorder does not imply a lack of structure, but rather allows these regions to function as binding surfaces with multiple interacting partners; IDPs are often hubs in protein-protein interaction networks (Cumberworth et al., 2013). In the EFA-6 N-terminus the 18 aa motif is predicted to have relative structural order, and might act as a molecular recognition feature as in other IDPs. The 18 aa motif is critical for EFA-6 relocalization and for the EFA-6 N-terminus to interact with ZYG-8 and TAC-1. Moreover, as mutation of two or more residues within the 18 aa motif impairs its activity, both the exact sequence of the 18 aa motif and the surrounding extended intrinsically disordered domain appear to be critical for the ability of the EFA-6 N-terminus to regulate MT dynamics. In some assays, the requirement for the 18 aa motif in EFA-6 function was not all-or-none, suggesting it may facilitate the function of the surrounding interacting domain. Moreover, neither ZYG-8 nor TAC-1 may directly bind the 18 aa motif; the motif may be important for correct folding of the larger N-terminal interacting domain. Figure 6. Continued localization in wild-type, tac-1(ok3305) and a putative zyg-8 null allele zyg-8(t1518) (Gönczy et al., 2001). Relocalization of EFA-6N150 was not dependent on TAC-1 or ZYG-8. (D) Localization of EFA-6FL::GFP and mKate2:: TAC-1 before and after axotomy in a touch neuron. Before axotomy, TAC-1 was diffuse in soma and along the axon, and concentrated in a large perinuclear dot. EFA-6 was predominantly localized to the plasma membrane and also in the perinuclear dot marked by TAC-1. After axotomy, both proteins became punctate and the puncta were partially co-localized; enlargements in small boxes below. Graphs of line scans along the axon are shown below the enlarged images. Arrow, injury site; scale, 10 μm. DOI: 10.7554/eLife.08695.018 The following figure supplement is available for figure 6: The EFA-6 N-terminus binds MAPs ZYG-8/DCLK and TAC-1/TACC Our analysis suggests the EFA-6 N-terminus regulates MT dynamics indirectly via the MAPs ZYG-8 and TAC-1. Identification of TAC-1 and ZYG-8 as EFA-6 interactors was unexpected, as in embryonic Figure 7. EFA-6 and TAC-1 re-localize to puncta overlapping with the MT minus end-binding protein Patronin/PTRN-1. (A) Localization of PTRN-1 and EFA-6N150 in PLM before and after axotomy. EFA-6N150::mKate2 was diffuse in soma and axon before injury, and became punctate after injury and these puncta co-localized to GFP::PTRN-1. Enlarged images of the regions in boxes are shown below. Graphs of line scans along the axon and F(in)/F(out) ratio quantitation are shown below. Increased F(in)/F(out) ratio indicates higher degree of colocalization post axotomy; see Figure 7-figure supplement 1A and 'Materials and methods' for calculation of F(in)/F(out). Statistics: Student's t-test. ***p < 0.001. (B) Epistatic interactions between efa-6(0), ptrn-1(0), and tac-1(lf). Normalized PLM regrowth. Strains without or with temperature shift (cultured at 15˚C and upshifted to 25˚C 20 hr before axotomy and kept at 25˚C for 24 hr after axotomy) were quantified separately. Statistics: one-way ANOVA with Bonferroni post test. DOI: 10.7554/eLife.08695.020 The following figure supplements are available for figure 7: cells TAC-1 is predominantly centrosomal or cytoplasmic (Le Bot et al., 2003), and ZYG-8 localizes along MTs (Bellanger et al., 2007), whereas EFA-6 is cortically localized (O'Rourke et al., 2010). TAC-1 and ZYG-8 interact physically, but localize independently (Bellanger et al., 2007). However EFA-6, TAC-1, and ZYG-8 are all present in axons, suggesting these proteins may interact directly in differentiated cells. TAC-1 and EFA-6 partly colocalize after injury, consistent with their interaction being regulated by injury. Although ZYG-8 localization appeared unaffected by injury, it is predicted to associate along the length of MTs, and so could also interact with EFA-6 after injury. Notably, loss of function of TAC-1 or ZYG-8 did not detectably affect developmental axon outgrowth, but strongly blocked axon regeneration, indicating regenerative regrowth is highly dependent on these MAPs.
Neither TACCs nor ZYG-8/DCLK exclusively associate with MT plus ends. TACC proteins can act both at plus and minus ends of centrosomal MTs (Lee et al., 2001;Peset and Vernos, 2008). Localization of human TACC3 to minus ends is regulated by Aurora A dependent phosphorylation (Barros et al., 2005;LeRoy et al., 2007). Conversely, Doublecortin (DCX) decorates the length of the MT lattice and stabilizes it (Moores et al., 2006). DCX can also track MT plus ends, and acts as a nucleation factor, stabilizing polymerization intermediates (Moores et al., 2004;Bechstedt and Brouhard, 2012). Like DCX, DCLK is thought to be able to interact with MTs along their length (Burgess and Reiner, 2000). Our results suggest that after axon injury, TAC-1, EFA-6, and possibly ZYG-8 may interact with one another at specific subregions of MTs at or adjacent to minus ends. As EFA-6 has opposite effects to TAC-1 and ZYG-8, EFA-6 may transiently inhibit the activity of TAC-1 or ZYG-8, resulting in the rapid disruption of axonal MT growth. Following this initial phase, EFA-6 returns to its steady state, relieving the inhibition of TAC-1 and ZYG-8, which are then required for the later upregulation of axonal MT dynamics.

EFA-6 and TAC-1 relocalize to sites of MT minus ends
CAMSAPs/Patronins directly bind MT minus ends and protect them from depolymerization by Kinesin-13 (Tournebize et al., 2000;Meng et al., 2008;Goodwin and Vale, 2010;Hendershott and Vale, 2014;Jiang et al., 2014). The minus end protection activity of CAMSAP family proteins is important for their function in maintaining noncentrosomal MTs. In C. elegans axons PTRN-1 localizes to puncta that are thought to define sites of MT minus end anchoring or stabilization (Marcette et al., 2014;Richardson et al., 2014). Strikingly, EFA-6 and TAC-1 relocalize close to or at these sites after injury, suggesting MT minus ends may be an important site of regulation. As PTRN-1 itself is not required for EFA-6 or TAC-1 relocalization, other proteins may be involved in their targeting. Indeed, the near-normal development and behavior of ptrn-1 null mutants suggests additional factors can stabilize MT minus ends in noncentrosomal arrays. Like TAC-1 and ZYG-8, PTRN-1 is required for axon regrowth. EFA-6 or its interactors might modulate the function of PTRN-1 in axon regrowth.

EFA-6 and injury-triggered MT dynamics
MT dynamic instability, first studied as a function of tubulin concentration in vitro, is influenced by a wide array of positive and negative regulators in vivo. For example, in Xenopus egg extracts, MT dynamics are determined by a balance between the MT growth-promoting XMAP215 and MT-destabilizing XKCMI (Tournebize et al., 2000;Kinoshita et al., 2001). Our findings suggest that the initial stages in axon regeneration are also driven by a sequence of shifts in the balance between opposing activities of MT destabilizers such as EFA-6 and MT growth promoting factors such as TAC-1 and ZYG-8.
Axonal injury in C. elegans triggers a highly regulated sequence of changes in MT dynamics that correlate closely with changes in EFA-6 localization and activity. We did not detect significant down-regulation of MT growth in response to injury in the absence of EFA-6, using two independent alleles. Although a small decrease in MT dynamics was observed in efa-6 mutants, this was not statistically significant, and calculations of statistical power suggest that changes of >15% should be detectable in experiments of the sample size used here. Nevertheless there may be EFA-6-dependent and EFA-6-independent effects on MT dynamics immediately after injury. Changes in MT dynamics within seconds of axon injury have been studied in Aplysia neurons (Erez et al., 2007;Erez and Spira, 2008), which display rapid local MT depolymerization followed by repolymerization over several minutes. Drosophila neurons also display acute and chronic alterations in MT dynamics after injury (Chen et al., 2012;Lu et al., 2015). Recent in vivo imaging of mammalian axons found an acute increase in axonal MT dynamics after laser axotomy, followed by a sustained increase over several days (Kleele et al., 2014). Thus, the exact sequence of MT dynamics changes after injury may vary between cell types and organisms. An important future goal will be to address the role of EFA6 family members in mammalian axon regeneration, and whether manipulation of this MT regulatory pathway can enhance regeneration in therapeutic settings.

Molecular biology and transgenes
We made new plasmids by Gateway recombination (Invitrogen / Life Technologies, Grand Island, NY) or Gibson assembly, as listed in Supplementary file 1A; new transgenes are listed in Supplementary file 1C. We amplified cDNAs from existing clones or from total first-strand cDNA; all clones were sequenced. Mutations were introduced by Quikchange mutagenesis (Agilent Technologies, Santa Clara, CA). We followed standard procedures to generate multicopy extrachromosomal transgenes; plasmids were injected at 1-30 ng/μl, and co-injection markers at 75 ng/μl. We analyzed 3 to 5 lines per construct. We made single copy insertions using Mos-SCI (http://www.wormbuilder.org/), on chromosomes IV (strain EG8081) or V (EG8083).

Live imaging, laser axotomy, and FRAP
We collected fluorescence images on Zeiss LSM710 or LSM510 confocal microscopes. We performed laser axotomy as described . We performed live imaging and analysis of EBP-2::GFP dynamics as described (Ghosh-Roy et al., 2012); in some experiments we immobilized animals in 30 mM muscimol on pads of 10% agarose in M9.
For quantitative analysis of protein localization and colocalization, animals were immobilized with either 0.7% phenoxypropanol or 30 mM muscimol. Confocal z-stacks were collected with 0.5 μm intervals. Typically 3-4 slices span an axon (1-2 μm diameter). Images were analyzed using Zeiss Zen and Metamorph (Molecular Devices, Sunnyvale, CA). Briefly, we drew lines along the axon, starting at the injury site or soma, then used the line scan tool to measure the average fluorescence intensity of 8 pixels (∼0.7 μm) surrounding the lines. For puncta number (or peak #) analysis, we counted any peak with intensity greater than the mean +1 SD as a punctum. To quantitate colocalization, we measured the F(in)/F(out) ratio (see Figure 7-figure supplement 1A) using Metamorph software. A small ROI (region of interest) was drawn to cover one punctum of GFP::PTRN-1. Average intensity of mKate2 (EFA-6N150) within the ROI was measured as F(in). The ROI was then duplicated to cover a small region in the axon with no GFP::PTRN-1 puncta, and average intensity of mKate2 (EFA-6N150) within this ROI was measured as F(out). F(in)/F(out) was then calculated as [F(in) − background intensity]/[F (out) − background intensity]. Before injury, EFA-6N::mKate2 is evenly distributed in the axon, and mKate2 intensity inside and outside of the GFP::PTRN-1 puncta is similar in level, so the F(in)/F(out) ratio is close to 1. 2 min post injury, EFA-6N is relocalized to PTRN-1 puncta, so mKate2 intensity within the PTRN-1 puncta is much higher than outside the puncta, resulting in a significantly higher F(in)/F(out) ratio.
For FRAP we set circular regions of interest (ROIs) for acquisition and photobleaching, using 2% laser power for acquisition and 100% laser power (488 nm, Zeiss LSM710) for photobleaching. We acquired 5 and 25 images before and after photobleaching. We chose ROIs 1 μm diameter from regions of median initial intensity in the soma or axon. In videos of uninjured neurons, we placed ROIs in regions with diffuse or relatively enriched GFP signal; in injured neurons, ROIs were drawn around puncta. Average fluorescence intensity in each ROI at each frame was measured in Zen. To generate FRAP curves we normalized intensity to the average of the five frames prior to photobleaching. We calculated t 1/2 following standard formulas; the immobile fraction was calculated by the Zen program.

Yeast two-hybrid screening and assays
We performed two-hybrid screening as described . We cloned EFA-6 full-length cDNA and fragments into a pMB27-Gal4-BD-gtwy vector, derived from the pPC97-Gal4-BD vector. We transformed baits into yeast strain Y8930, and mated these to a pPC86-Gal4-AD prey library of mixed-stage C. elegans cDNAs in strain Y8800. Plasmids for two-hybrid experiments are listed in Supplementary file 1B. We screened >2 × 10 6 independent colonies per bait, and identified interacting cDNAs by plasmid amplification and sequencing. To test specific interactions we cloned the appropriate full length or fragment cDNAs into the pACT2 (Gal4 activation domain) or pBTM116 (LexA DNA-binding domain) vectors (Clontech, Mountain View, CA) and co-transformed constructs into yeast strain L40. We grew transformed yeasts on agar plates with SD medium (synthetic minimal medium) lacking leucine and tryptophan; interactions were examined on plates with SD medium lacking leucine, tryptophan, and histidine, with or without 3-AT.

CRISPR targeted deletion
We used CRISPR based gene targeting (Dickinson et al., 2013) to delete the genomic region encoding the EFA-6 18 aa motif. Briefly, we obtained Cas9-sgRNA plasmid from the Goldstein lab and inserted an sgRNA sequence targeting efa-6 into the vector using Quikchange mutagenesis. The two sgRNA sequences were GGCGAGGGGCTCCATCAATGG and GATGCAACTGTGGTACCTGG, targeting efa-6 exon 1 and exon 2 respectively. 50 ng/μl of each Cas9-sgRNA plasmid and 20 ng/μl Psur-5-mCherry were co-injected into wild type animals. From 15 F 1 progeny we found one animal heterozygous for efa-6(ju1200), which deletes 500 bp of exon 1, intron 1, and exon 2, and has a 26 bp insertion. mRNAs produced in efa-6(ju1200) encode polypeptides with a premature stop codon after amino acid 15, eliminating the 18 aa motif and the rest of EFA-6.

Locomotion analysis
We measured locomotion velocity using WormTracker 2.0 (Brown et al., 2013) NGM plates were seeded with OP50 bacteria 3 hr before experiments. Individual young adult worms were picked gently from the culture plate to a fresh tracking plate. 1 min later, the plate was placed on the worm tracker platform and locomotion recorded for 1 min at 10 frames per second for each animal.

Statistical analysis
We used Prism (GraphPad Software, La Jolla, CA) for all statistical analysis. A two-tailed Student's t-test was used for comparisons of two groups. For multiple comparisons we used one-way ANOVA with Bonferroni post test. To compare variables such as growth cone percentage we used the Fisher exact test.