ABSTRACT
Following a transection injury to the axon, neurons from a number of species have the ability to undergo spontaneous repair via fusion of the two separated axonal fragments. In the nematode C. elegans, this highly efficient regenerative axonal fusion is mediated by Epithelial Fusion Failure-1 (EFF-1), a fusogenic protein that functions at the membrane to merge the two axonal fragments. Identifying modulators of axonal fusion and EFF-1 is the next step towards harnessing this process for clinical applications. Here, we present evidence that the small GTPase RAB-5 acts to inhibit axonal fusion, a function achieved via endocytosis of EFF-1 within the injured neuron. Consequently, we find that perturbing RAB-5 activity increases the capacity of the neuron to undergo axonal fusion, through enhanced membranous localization of EFF-1 and the production of extracellular EFF-1-containing vesicles. These findings identify RAB-5 as a novel regulator of axonal fusion and the first regulator of EFF-1 in neurons.
Treating nerve injuries is of great interest in the clinical setting. Significant research efforts have been dedicated to developing new methods to repair an axon following transection, as current outcomes are less than optimal. A novel approach to this problem has come from work in invertebrate systems, where some neurons are capable of spontaneously rejoining the two separated axonal fragments after transection. This regenerative mechanism, called axonal fusion, has been observed in a number of species1–5, but has been best characterized in the mechanosensory neurons of the nematode C. elegans6–10. To undergo axonal fusion, the proximal axon (still attached to the cell body) must regrow, reconnect, and then fuse with its separated axonal fragment. We and others have shown in C. elegans that this fusion not only restores continuity of the axon following UV laser axotomy7, but also restores neuronal function9,10.
The key molecular effector of axonal fusion is the C. elegans membrane fusogen EFF-1. EFF-1, a nematode-specific transmembrane glycoprotein with structural and functional similarity to class II viral fusion proteins11, functions to merge closely apposed plasma membranes. EFF-1 activity in the injured neuron is crucial for successful axonal fusion; eff-1 mutant animals exhibit severe axonal fusion defects6,8, which can be rescued by expression of wild-type EFF-1 selectively in the injured neuron, revealing its cell-autonomous function8. There is strong evidence that the activity of EFF-1 is controlled via its dynamic subcellular localization. To mediate fusion, EFF-1 must be inserted into the membrane, and it is inactive when sequestered in intracellular compartments. This was first demonstrated in other cell types in C. elegans, including the hypodermis, where EFF-1 mediates cell-cell fusion for the formation of syncytia during development12. In these cells, EFF-1 is mobilized from intracellular compartments to the plasma membrane where it mediates fusion13,14. Similarly, in the Posterior Lateral Microtubule (PLM) mechanosensory neurons, EFF-1 exists largely within puncta in the steady state, but following injury is mobilized to the regenerating axonal membrane8. How these changes in EFF-1 localization and activity are regulated within the neuron is currently unknown, but they likely represent a critical step in the control of axonal fusion.
To date, the only other molecules implicated in C. elegans axonal fusion are components of the apoptotic clearance machinery8 and the conserved microRNA let-7, which functions to regulate this pathway10. The clearance signalling pathways were first described as a mechanism for apoptotic cell corpse engulfment15–19, but appear to have been repurposed for axonal fusion, and function upstream of EFF-1 to promote recognition of the severed axonal fragment. Axonal injury triggers exposure of the phospholipid phosphatidylserine (PS) on the surface of the severed axon. PS acts as a ‘save me’ signal by recruiting both the PS receptor PSR-1, present on the growth cone of the regrowing fragment, and the secreted PS-binding protein TTR-52, which initiate signalling to improve the efficiency of axonal fusion8,9. Animals with mutated psr-1 or ttr-52 exhibit axonal fusion defects, which can be rescued with overexpression of EFF-1, indicating that EFF-1 acts genetically downstream of these recognition molecules8.
However, axonal fusion must also involve additional, undiscovered molecules. In the absence of the apoptotic genes, axonal fusion can still occur at a low rate, and EFF-1 is still mobilized to the membrane, indicating the existence of mechanisms which can bypass the apoptotic recognition machinery to enable fusion. Here, we identify a novel regulator of axonal fusion. We reveal that the GTPase RAB-5 can negatively regulate axonal fusion functioning within the injured neuron, and present evidence that this protein controls the level of EFF-1 on the neuronal membrane via endocytosis. Thus, we propose a model in which recycling of EFF-1 via RAB-5 is a critical mechanism for the control of fusogen function and axonal fusion as a mechanism of repair.
RESULTS
RAB-5 regulates the rate of regenerative axonal fusion
To identify molecules with a role in axonal fusion, we used mutations in specific genes and tested their effect on the rate of axonal fusion in a psr-1 mutant background. This sensitized background allowed us to assess whether a candidate gene could effectively bypass the apoptotic recognition machinery and modulate axonal fusion rates through an alternative pathway.
Our primary candidate was the endocytic GTPase RAB-5, as this protein has been shown to modulate the rates of fusion in other C. elegans tissues. Specifically, loss-of-function or depletion of RAB-5 leads to a hyperfusion phenotype in the C. elegans hypodermis14. We first investigated the effect of perturbing RAB-5 activity by performing axotomies on the mechanosensory neurons of psr-1 mutant animals expressing a dominant negative version of RAB-5 (RAB-5(DN)). Axotomies were performed on the PLM neurons, approximately 50 μm anterior to the cell body, as previously described7,8. The rate of axonal fusion was calculated from axons that regrew and visibly reconnected to their distal fragment. Successful axonal fusion occurred if the distal fragment was maintained 48 h post-axotomy (Fig. 1a); fusion was considered unsuccessful if the distal fragment instead underwent degeneration (Fig. 1b).
As previously shown, psr-1 mutant animals displayed a defective rate of axonal fusion compared with wild-type animals8. Remarkably, we found that expression of RAB-5(DN) in the PLM neuron was sufficient to rescue this fusion rate to wild-type levels (Fig. 1c, d). We also performed axotomies on wild-type animals expressing constitutively active RAB-5 (RAB-5(CA)) in the mechanosensory neurons, hypothesizing that this increased RAB-5 activity might generate the opposite phenotype and reduce the rate of axonal fusion. However, although there was a trend towards reduction, there was no significant change in the axonal fusion rates in these animals (Fig. 1e).
Given that EFF-1 is the key molecular effector of this process, we next asked whether RAB-5 modulates axonal fusion through an interaction with EFF-1. To address this, we performed axotomies on double mutant animals of eff-1 and psr-1 expressing RAB-5(DN). We found that loss of eff-1 suppressed the increase in axonal fusion mediated by RAB-5(DN) (Fig. 1d), indicating that RAB-5(DN) increases the axonal fusion rate in an eff-1-dependent manner.
RAB-5 controls EFF-1 localization to the plasma membrane
We next asked how RAB-5 was regulating the activity of EFF-1 in mediating axonal fusion. We suspected that perturbing RAB-5 prevented the endocytosis of EFF-1, which would increase the amount of EFF-1 available at the membrane and enhance its activity. RAB-5 and its mammalian orthologue Rab5 are known to localize to early endosomes and play important roles in endocytosis. They facilitate the transport of clathrin-coated vesicles to early endosomes, fusion between endosomes, and cargo trafficking from endosomes into lysosomes for degradation20–24. Correspondingly, altering RAB-5 activity produces specific cellular phenotypes: perturbing RAB-5 activity using RAB-5(DN) inhibits endocytosis and causes membranous accumulation of proteins25, whereas expression of constitutively active RAB-5(CA) leads to excessive early endosome fusion and the presence of enlarged early endosomes25,26. In the C. elegans hypodermis, depletion or loss-of-function of RAB-5 results in mis-localization of EFF-1 to the plasma membrane, which in turn is associated with excessive hypodermal cell-cell fusion14. We therefore predicted that perturbing RAB-5 activity in the PLM neurons would result in similar changes to EFF-1 localization.
To visualize EFF-1 within the PLM neuron, we used a transgenic strain in which eff-1 null mutant animals express cytoplasmic mCherry in the PLM mechanosensory neurons, as well as GFP-tagged EFF-1. We previously demonstrated that this EFF-1::GFP transgene is functional and sufficient to rescue axonal fusion defects in eff-1 mutants8. Using confocal microscopy, we characterized EFF-1 localization in the cell body and proximal axon of PLM (Fig. 2a-d). In the uninjured, wild-type axon, EFF-1 formed an indiscriminate punctate pattern as we previously reported8 (Fig. 2b). In the cell body, it was also present as intracellular puncta, with no clear localization to the cell membrane (Fig. 2d). We chose to perform subsequent localization studies specifically in the cell body, as this wider structure allows for any mobilization of EFF-1 to the membrane to be clearly identified.
To test if perturbing RAB-5 activity led to EFF-1 localization at the membrane, we coexpressed RAB-5(DN). In these animals, EFF-1::GFP formed a more continuous pattern along the axon (Fig. 2c), and accumulated at the membrane of the PLM cell body (Fig. 2d), a process that was commonly associated with membranous protrusions of EFF-1 (Fig. 2d, arrowhead). Line scan profiles through these protrusions clearly demonstrated that EFF-1 was present on the membrane of the cell body, a phenomenon that we never observed in control animals (Fig. 2e). To quantify EFF-1 localization, we measured the average intensity of GFP in the membrane of the cell body, normalized to the average intensity for the whole cell body (see Methods section). This revealed a significant increase in the relative amount of EFF-1::GFP at the membrane in the presence of RAB-5(DN) (Fig. 2f). These results indicate that neuronal-specific loss of RAB-5 activity leads to EFF-1 accumulation at the membrane of the neuronal cell body.
As expected, enhancing RAB-5 activity, through either overexpression of wild-type RAB-5 or expression of RAB-5(CA), had no effect on the relative amount of EFF-1::GFP at the cell membrane (Fig. 2f). This was consistent with our axotomy results which indicated that increased RAB-5 activity did not significantly alter EFF-1 function. Taken together, these findings suggest that RAB-5 activity functions in the endocytosis of EFF-1 molecules which have been mobilized to the membrane.
Plasma membrane accumulation of EFF-1 forms extracellular vesicles
Interestingly, we observed that animals expressing RAB-5(DN) not only had protrusions of the transmembrane EFF-1::GFP from the PLM cell body, but also generated what appeared to be extracellular EFF-1::GFP-positive vesicles. These extracellular vesicles, ranging in number from 2 - 20 per neuron, were present around the PLM cell body and proximal axon, and were reproducible in multiple independent transgenic strains (Fig. 3a). Intriguingly, previous work has shown the presence of EFF-1 vesicles in cultured medium of baby hamster kidney cells transfected with EFF-1 (ref. 27), and vesicles containing AFF-1, a second C. elegans fusogen, have been described both in vitro from mammalian cells28, and in vivo from seam cells29 (see Discussion).
To determine the composition of these vesicles, we generated transgenic strains that coexpress a cytoplasmic marker (mCherry) and a membrane-bound marker (MYR::mCherry) in addition to EFF-1::GFP and RAB-5(DN). Vesicles observed in these animals presented no detectable mCherry signal (Fig. 3b), indicating that membrane-bound forms of mCherry were excluded from the vesicles, and that they contained a highly reduced volume of cytoplasm (based on the resolution of confocal imaging). This suggests that these vesicles may be selective in their composition, and contain mostly EFF-1, similar to those vesicles reported in cell culture27.
To characterize the dynamics of the vesicles, we undertook time-lapse imaging of the PLM cell body. Confocal imaging of the vesicles at 2 min intervals for up to 60 min revealed that they fell into two categories of mobility: ‘immobile’ vesicles, representing the majority of vesicles which exhibited no movement in this time period, and ‘mobile’ vesicles, which instead exhibited linear or disorganized, oscillatory movements around the cell body (Fig. 3d, e). This occurred at an average rate of 0.2 ± 0.05 μm/min (n = 22). We observed that these vesicles are present from the first and second larval stages. Budding of the vesicles from the cell body was a rare event, but was captured on one occasion at the fourth larval stage (Fig. 3c). EFF-1 retained at the membrane also underwent dynamic changes, and the formation of de novo protrusions of EFF-1 from the cell body was captured within a period of minutes (Fig. 3d).
To determine whether the vesicle dynamics were altered following neuronal injury, we performed axotomies and visualized the axon and cell body at 3 h and 6 h post-axotomy. However, we observed no clear change in vesicle number or localization at these time points (Supplementary Fig. 1). Overall, our results are consistent with a model in which these structures are likely generated via accumulation of EFF-1 at the membrane, such that EFF-1 is ‘pinched off’ in the form of a vesicle.
RAB-5 controls EFF-1 localization to intracellular compartments
We next asked whether RAB-5 also controls EFF-1 localization to intracellular puncta. Given that EFF-1 co-localizes with RAB-5 in other cell types14, we hypothesized that the EFF-1::GFP puncta in PLM neurons represent EFF-1 contained in early endosomes, and are formed through RAB-5-mediated endocytosis and endosome fusion. Changes in RAB-5 activity were therefore expected to alter the morphology of these puncta.
To test this, we expressed the three different versions of RAB-5 described above and characterized the size of the EFF-1::GFP puncta present in the PLM cell body. We used the Squassh imaging tool (ImageJ)30 to automatically select and measure these puncta. Our results revealed no significant difference in the average size of the puncta detected in RAB-5(DN) cell bodies (Fig. 4a). However, overexpression of RAB-5(WT) caused accumulation of EFF-1 within enlarged intracellular puncta (Fig. 4b). This effect was even more pronounced when RAB-5 activity was increased by RAB-5(CA) (Fig. 4c). This result is consistent with the known role of RAB-5 in endosome fusion and the enlarged endosome phenotype generated by RAB-5 overactivity25,26.
To confirm that EFF-1 was accumulating in RAB-5-positive compartments, we performed co-localization studies of EFF-1 with RAB-5 in the context of RAB-5 overexpression. We co-expressed BFP::RAB-5 with EFF-1::GFP in the mechanosensory neurons (Fig. 4d), which reproduced the enlarged EFF-1 puncta phenotype, leading to a significant increase in average EFF-1 puncta size (Fig. 4d). In these enlarged puncta, the average co-localization of EFF-1 with RAB-5 was 85% (range 67 - 98%; n = 12). Taken together, these results strongly support the notion that overactivity of RAB-5 results in EFF-1 accumulation in enlarged early endosomes.
RAB-5 controls the amount of EFF-1 in the PLM neuron
Our results indicated that altering RAB-5 activity causes EFF-1 mis-localization to specific subcellular compartments (either the membrane with decreased RAB-5 activity, or enlarged endosomes with increased RAB-5 activity). We next asked whether this mis-localization affected the ability of the neuron to mediate recycling or degradation of the EFF-1 protein. We hypothesized that defects in such processes would result in a buildup of EFF-1::GFP in the neuron. To test this, we measured the average EFF-1::GFP intensity in the PLM axon and cell body of animals expressing either RAB-5(DN), RAB-5(CA) or RAB-5(WT). We also used an alternative approach to perturb RAB-5 activity using cell-specific RNAi31, whereby animals expressed rab-5(sas) in the mechanosensory neurons for cell-specific silencing of rab-5. We found that the average EFF-1::GFP intensity in both the axon and cell body was significantly greater in animals expressing either RAB-5(DN), RAB-5(CA) or rab-5(sas) (Supplementary Fig. 2a-d, g, h). This may represent defects in recycling and/or degradation of EFF-1 due to altered RAB-5 activity. In contrast, the overexpression of RAB-5(WT) had no significant effect on EFF-1::GFP intensity (Supplementary Fig. 2e, f). This indicates that the RAB-5(WT) molecule may be modified by endogenous regulators, and hence regulated to minimize changes in EFF-1 levels. Thus, only unregulated alterations in RAB-5 activity (generated with expression of RAB-5(DN), RAB-5(CA) or rab-5(sas)) disrupt EFF-1 protein levels.
Other endocytic molecules do not control EFF-1 localization
A number of molecules function in the endocytosis of cargo from the plasma membrane. We sought to determine whether other endocytic regulators might act in conjunction with RAB-5 to regulate neuronal EFF-1. One important candidate for this role is DYN-1, the C. elegans orthologue of dynamin, as it has been demonstrated to act in this fashion alongside RAB-5 to regulate EFF-1 in the hypodermis14. We tested whether loss of DYN-1 function, induced using the temperature-sensitive allele dyn-1(ky51), influenced EFF-1::GFP localization in the PLM neurons. Surprisingly, we found no significant effect of DYN-1 on EFF-1::GFP puncta size, membrane localization or average intensity in the neuron (Supplementary Fig. 3a, c, e).
We also tested EHS-1, a clathrin adaptor involved in endocytosis that localizes to the plasma membrane. However, loss-of-function ehs-1 mutant animals showed no significant change in the same measurements of EFF-1::GFP localization (Supplementary Fig. 3b, d, f). We believe that this is consistent with studies of ehs-1 in other systems which indicate it can act redundantly with other clathrin adaptors32. As such, identifying its potential role in this process requires further investigation.
From early endosomes, the endocytic pathway allows for transport of cargo to downstream compartments, potentially for membrane recycling or degradation. We therefore asked whether molecules localizing to these compartments also participated in EFF-1 regulation. A number of RAB proteins presented good candidates; these included RAB-7, which functions in endosome-to-lysosome trafficking, RAB-10, an endocytic recycling regulator that localizes to endosomes and Golgi, and RAB-11, which controls transport between recycling endosomes and the plasma membrane. We perturbed the function of these rab genes in the PLM neurons, either through expression of dominant negative versions, or by using loss-of-function alleles. However, our results indicated that these RAB molecules were not involved in EFF-1 localization in the PLM neurons. Neither expression of dominant negative RAB-7 or RAB-11, nor the presence of the rab-10(dx2) loss-of-function allele, significantly altered our measurements of EFF-1::GFP intensity or localization (Supplementary Fig. 4). Overall, these results support the specificity of our findings with RAB-5, and suggest that this molecule is a key regulator of neuronal EFF-1 and of axonal fusion.
DISCUSSION
Our data demonstrate that perturbing RAB-5 activity in the PLM neurons has a clear functional effect on axonal repair. Specifically, the presence of RAB-5(DN) increased the capacity for EFF-1 to mediate axonal fusion, as it phenocopied EFF-1 overexpression by rescuing the psr-1 axonal fusion defect. By visualizing EFF-1, we determined that this change in activity reflected mobilization of EFF-1 to the plasma membrane, which also occurs with reduced RAB-5 function in the C. elegans hypodermis14.
Interestingly, the presence of RAB-5(CA) did not produce the opposite phenotype, as it did not significantly reduce axonal fusion rates (Fig. 1e) or remove greater amounts of EFF-1 from the neuronal membrane (Fig. 2). It is possible that there was insufficient overactivity of RAB-5 in the strains tested to fully remove EFF-1 from the membrane, or that only a small number of EFF-1 molecules is required at the membrane for fusion, and sufficient amounts were present even in the presence of RAB-5(CA). Alternatively, it is possible that a RAB-5-independent mechanism exists for the mobilization of EFF-1 to the membrane after injury. Recruitment of EFF-1 to fusion sites in larval hypodermal cells has been shown to be mediated at least in part by the actin regulator VAB-10 (ref. 33), although other pathways may also exist. Much has been characterized about the molecular cascades that are activated in regenerating axons34–36 and it is plausible that some of these molecules play a currently uncharacterized role in EFF-1 recruitment.
However, overactivity of RAB-5 did lead to accumulation of EFF-1 in enlarged RAB-5-positive compartments (Fig. 4c, d). Previous studies have documented 45 - 69% colocalization of endogenous EFF-1 with RAB-5 in hypodermal cells14. As our model visualizes overexpression of RAB-5, our results are not directly comparable and we are unable to conclude to what extent EFF-1 co-localizes with wild-type RAB-5 levels in the neuron. However, our findings are consistent with a role for RAB-5 in determining steady-state EFF-1 localization to early endosomes.
Altering RAB-5 activity also increased the intracellular amount of EFF-1, as reflected by increases in EFF-1::GFP intensity. This occurred in both the axon and the cell body, indicating that it likely represents protein accumulation rather than an axonal transport defect. It was also observed specifically in the presence of RAB-5 molecules with locked activity states, which were associated with abnormal accumulation of EFF-1 either at the membrane or in early endosomes. There is good precedence for RAB-5 functioning in protein degradation and homeostasis. In the C. elegans embryo, this molecule is required for both endocytosis and degradation of the C. elegans caveolin CAV-1 (ref. 37). In HeLa cells, either increasing or decreasing RAB-5 activity (achieved indirectly using a regulator of RAB-5) was found to perturb endocytic trafficking and lead to cargo build-up in specific compartments38. We propose that the accumulation of EFF-1, either at the membrane with RAB-5(DN) or in abnormally enlarged endosomes with RAB-5(CA), occurred due to defects in the trafficking of EFF-1 from these structures. Interestingly, greater intracellular levels of EFF-1 per se did not guarantee an improvement in axonal fusion rates. Rather, our results indicate that EFF-1 must specifically accumulate at the membrane to improve fusion capacity.
Our findings are consistent with a model in which RAB-5 mediates the endocytosis of EFF-1 from the PLM cell membrane, similar to that described in the hypodermis14. In our model (Fig. 5), EFF-1 is transiently inserted into the plasma membrane following synthesis. Active RAB-5 is responsible for subsequent transport of this EFF-1 into early endosomes, where EFF-1 largely resides in the steady state. Decreasing RAB-5 activity allows EFF-1 accumulation at the membrane, whereas overactivity of RAB-5 leads to accumulation in early endosomes. We also propose that, in neurons, RAB-5-mediated endocytosis occurs upstream of pathways for EFF-1 recycling/degradation. Because altering RAB-5 activity sequesters EFF-1 in specific compartments, it prevents downstream trafficking and creates a build-up of EFF-1 in the neuron.
A particularly fascinating aspect of this study was the generation of extracellular EFF-1::GFP-positive vesicles when RAB-5 activity was perturbed. Extracellular vesicles were observed incidentally in the earliest studies of EFF-1-mediated cell-cell fusion using electron microscopy39. Vesicles that specifically contain fusogens have now been documented in vitro27,28, and more recently in vivo in C. elegans29, but their exact characteristics and functionality are yet to be elucidated. The absence of both membrane and cytoplasmic markers in the EFF-1::GFP vesicles suggests that these vesicles may exclude some membrane proteins, such as fluorophores, or contain insufficient amounts for visualization with confocal microscopy. However, it appears unlikely that they contain purely EFF-1::GFP, as fusogen-containing vesicles in vitro have been shown to contain other proteins27, as well as membrane proteins in some cases28. We therefore postulate that these vesicles may be selective in their uptake of membrane proteins, and that their content could differ from the original composition of the PLM plasma membrane. Given that extracellular vesicles are known to deliver cargo in diverse systems40, fusogen-containing vesicles represent an attractive vehicle for imparting fusion competence to surrounding tissues, and potentially to mammalian neurons, as previously postulated29. The evidence that these vesicles may be selective, and largely contain fusogen, suggests that they could be very efficient in delivering fusogenicity, although whether the EFF-1::GFP vesicles have fusogenic activity is currently unclear.
Another matter of speculation is the mechanism through which the EFF-1::GFP vesicles are generated. It remains to be determined whether they are passively extruded, or are instead actively secreted. Given that they occur in the presence of increased intracellular and membranous EFF-1, it is plausible that they are created through excessive build-up of EFF-1 at the membrane. With its known function in membrane sculpting41, EFF-1 could potentially ‘pinch off’ a section of membrane. Consistent with this, the vesicles localize in the vicinity of the PLM cell body, and it may be that the higher volume-to-surface-area ratio in this part of the neuron allows for sufficient build-up of EFF-1::GFP behind the membrane. In support of this, most protrusions, and the observed event of vesicle budding, originated from the cell body. However, if there is instead molecular machinery for active secretion of these vesicles, one strong candidate is the ABC transporter CED-7, which is known to generate extracellular vesicles containing phosphatidylserine during apoptotic cell clearance42. We have shown that CED-7 functions in regeneration of the PLM neuron8 as well as its degeneration43, possibly through vesicle generation. It is therefore possible that CED-7 activity in the neuron is required for the secretion of EFF-1 vesicles.
RAB proteins have well-established roles in intracellular trafficking, and we tested a suite of molecules other than RAB-5 that could potentially regulate such transport of EFF-1 in PLM. As reported for the hypodermis14, RAB-7, RAB-10 and RAB-11 did not influence EFF-1 localization. Similarly, we did not find a role for DYN-1, although this molecule has been demonstrated to negatively regulate EFF-1 and cell-cell fusion in the hypodermis14. However, the literature on dynamin suggests that it can have varying roles in fusion events in different systems, related to its multiple functions in endocytosis as well as actin cytoskeletal rearrangements. In some mammalian cell-cell fusion, including in osteoclasts and myoblasts, dynamin activity appears to instead promote fusion44. Our result is in keeping with an alternative role for DYN-1 in the neuron, potentially in endocytic regulation of molecules other than EFF-1.
In summary, our study identifies RAB-5 as a key regulator of EFF-1 in the nervous system. By controlling the levels of EFF-1 in endosomal compartments, RAB-5 modulates the amount of this protein available on the membrane, thereby regulating its fusogenic capacity. Thus, manipulating the activity of RAB-5 activity provides a means to promote highly efficient neuronal repair through axonal fusion.
METHODS
Strains and genetics
Standard techniques were used for C. elegans strain maintenance and genetic manipulations45. All experiments were performed at 22 °C (room temperature) on L4 animals unless otherwise specified. The following mutations were used: rab-10(dx2) I, eff-1(ok1021) II, ehs-1(ok146) II, psr-1(ok714) IV, dyn-1(ky51) X. The integrated transgenic strain QH3135 [zdIs5(Pmec-4::GFP) I] was used as a background strain for performing axotomies. The transgenic strain QH4748 [eff-1(ok1021) II; vdEx662[Pmec-4::eff-1::gfp; Pmec-4::mCherry; Podr-1::DsRed]]8 was used as a background strain for all experiments involving EFF-1 confocal imaging. To generate extrachromosomal arrays, microinjections were performed into the germline using standard methods46. The following transgenes were generated (concentrations used for the microinjection mix are indicated in brackets; all injection mixes had a total concentration made up to 100 ng/μl using empty pSM plasmid): vdEx1192/vdEx1193/vdEx1197[Pmec-3::rab-5(S33N) (5 ng/μl); Podr-1::gfp (60 ng/μl)], vdEx1450/vdEx1451/vdEx1452[Pmec-3::rab-5(Q78L) (5 ng/μl); Podr-1::gfp (60 ng/μl)], vdEx1194 [Pmec-3::rab-5(WT) (5 ng/μl); Podr-1::gfp (60 ng/μl)], vdEx1237[Pmec-3::rab-5(WT) (10 ng/μl); Podr-1::gfp (60 ng/μl)], vdEx1051/vdEx1055/vdEx1084[Pmec-3s::rab-5(s) (5 ng/μl); Pmec-3::rab-5(as) (5 ng/μl); Podr-1::gfp (60 ng/μl)], vdEx1301/vdEx1302/vdEx1303[Pmec-3::rab-7(T23N) (5 ng/μl); Podr-1::gfp (60 ng/μl)], vdEx1375/vdEx1390/vdEx1443[Pmec-3::rab-11(S25N) (5 ng/μl); Podr-1::gfp (60 ng/μl)], vdEx1389[Pmec-3::bfp::tev-s::rab-5 (5 ng/μl); Pmyo-2::mCherry (2.5 ng/μl)], vdEx1566[Pmec-4::myr::mCherry (5 ng/μl); Pmyo-2::mCherry (2.5 ng/μl)], vdEx1576 [Pmec-4::myr::mCherry (15 ng/μl); Pmyo-2::mCherry (2.5 ng/μl)]. It should be noted that a TEV-S signal exists between the BFP and RAB-5 sequences in vdEx1566; this signal allows for protein cleavage with addition of TEV protease and is therefore highly unlikely to have affected the outcome of the current study.
Molecular biology
Standard molecular biology techniques were used47. To generate the plasmid Pmec-3::rab-5(WT), the rab-5 gene was amplified from the pCL206 plasmid using 5’-gcTCTAGAatggccgcccgaaacgcagg-3’ and 5’-gggaCCCGGGttatttacagcatgaaccc-3’. These primers introduced Xba I and Xma I restriction sites respectively, which were used to clone the amplicon into the plasmid L1026 (Pmec-3, from Fire Vector Kit 1995). The QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies) was then used to generate rab-5(Q78L) (CA) and rab-5(S33N (DN) variants of this plasmid, using the primers 5’-aaatctgggatactgcaggaaaagaaagatatcattattgg-3’, 5’- ccaatgaatgatatctttcttttcctgcagtatcccgattt-3’ and 5’ -ctatcatttcaggcaaaaactctctcgtattgcgattc-3’, 5’-gaatcgcaatacgagagagtttttgcctgaaatgatag-3’ respectively.
To generate Pmec-3s::rab-5(sas), a sense-antisense PCR fusion technique was used31. rab-5 was amplified from genomic C. elegans DNA using standard primers (5’-cgtgccttcaatctttttcg-3’ and 5’-acaatgacgacgatcacaggc-3’). Pmec-3s was amplified from the L3784 plasmid (Pmec-3::gfp, from Fire Vector Kit 1997) with a standard forward primer (5’-aggtacccggagtagttggc-3’) and two different reverse primers with sequences complementary to the extremities of rab-5 at the 5’ ends (5’-atgttgcatttttctttccagaatctataacttgatagcgata-3’ and 5’-cttcccaactaccatgtacaaaatctataacttgatagcgata-3’). The rab-5 and Pmec-3s reactions products were then fused using nested primers 5’-ggcagtaatgaagacgtccat-3’ and 5’-gaagggttgatggtacatgaaa-3’ or 5’-ttctggaaagaaaaatgcaacat’-3’.
Pmec-3::rab-7(T23N) (DN) and Pmec-3::rab-11(S25N) (DN) were generated using the QuikChange II Site-Directed Mutagenesis Kit on Pmec-3::rab-7 and Pmec-3::rab-11 plasmids respectively, with primers 5’-cgggcgttggaaagaattctttgatgaatcaatatg-3’, 5’-catattgattcatcaaagaattctttccaacgcccg-3’ and 5’-gagactcaggcgtcggaaagaataatctcctgtctcgtttcac-3’, 5’-gtgaaacgagacaggagattattctttccgacgcctgagtctc-3’ respectively. The construction of Pmec-3::rab-7 and Pmec-3::rab-11 involved amplification of each gene from C. elegans genomic DNA with primers to introduce Xba I and Xma I restriction sites, followed by cloning into the L1026 plasmid downstream of Pmec-3.
To generate Pmec-3::bfp::tev-s::rab-5, the bfp::tev-s::rab-5 insert was amplified from pOG172, a kind gift from Prof. Guangshuo Ou (Tsinghua University, Beijing). Primers were used to introduce BamH I and Msc I restriction sites for cloning into L1026 downstream of Pmec-3. To generate the Pmec-4::myr::mCherry plasmid, myr::mCherry from the PNV::myr::mCherry plasmid was cloned into Pmec-4::GFP using Msc I and EcoR I restriction enzymes which remove the GFP sequence.
Laser axotomy
We performed UV laser axotomy of PLM in animals at the L4 larval stage as previously described7,8. Animals were anaesthetized using 0.05% tetramizole hydrochloride on 4% agarose pads. The axotomy was performed approximately 50 μm from the cell body using a MicroPoint Laser System Basic Unit attached to a Zeiss Axio Imager A1. At 48 h post-axotomy, animals were analyzed on a Zeiss Axio Imager Z1 equipped with a Photometrics Cool Snap HQ2 camera with MetaMorph software for the presence of reconnection and fusion. If it was unclear whether a distal fragment had been maintained, the animal was scored again at 72 h post-axotomy.
Confocal microscopy
Localization studies of EFF-1 and RAB-5 were performed on a LSM 710 META confocal microscope, equipped with a GaAsP detector and Zen 2012 software. L4 animals were mounted on 3% agarose pads in 25 mM sodium azide. Separate Z-stacks were performed of the PLM cell body and proximal axon. For imaging of EFF-1::GFP and cytoplasmic mCherry, green fluorescence was visualized with a 488 nm laser (5% power for the axon, 2% power for the cell body; gain of 600 and 4x averaging for both) and red fluorescence was visualized with a 543 nm laser (1% power for the axon, 0.2% power for the cell body; gain of 500 and 4x averaging for both). For imaging of BFP::TEV-S::RAB-5, blue fluorescence was visualized with a 405 nm laser (0.5% power, gain of 500, 4x averaging). To image MYR::mCherry, red fluorescence was again visualized with a 543 nm laser (up to 10% power, gain of 500, 4x averaging).
To image EFF-1::GFP post-axotomy, animals were mounted for axotomies in tetramizole as described above. They were then recovered in drops of M9 buffer onto seeded NGM plates for either 3 h or 6 h, after which they were mounted in sodium azide for confocal imaging.
Vesicle time lapse imaging
For characterization of EFF-1::GFP vesicle dynamics, z-stacks of the PLM cell body were acquired at 30 s or 2 min intervals for a total of 6 - 60 min depending on the dynamics observed. Vesicles were classified as immobile or mobile based on the presence of any movement in this timeframe. Mobile vesicles (22 out of 105) were measured for movement in the XY axis per frame by drawing a linear region of interest (ROI) from the center of the vesicle to the center at its subsequent location; the total distance was averaged over the time imaged to be expressed in μm/min.
dyn-1 heat-shock
To test the temperature-sensitive allele dyn-1(ky51), heat-shocks were performed for either 30 min or 2 hr. L4 animals were placed on NGM plates in a 25 °C incubator. Mutant and control animals were heat-shocked concurrently on separate plates and mounted on the same slide for subsequent confocal imaging. dyn-1(ky51) animals raised at 25 °C did not lay viable eggs, whereas those laid at 15 °C and then transferred to 25 °C developed to the L4 stage. However, the animals raised at 25 °C demonstrated non-specific increases in EFF-1::GFP intensity in both mutant and control groups (data not shown). Heat-shocks used in this assay were therefore limited to a maximum of 2 h.
Confocal image analysis
Image analysis was performed using Fiji for Mac OS X (ImageJ). To score for the presence of EFF-1::GFP on the membrane of the cell body, fluorescence profiles of line scans were obtained using the ‘Plot Profile’ tool in ImageJ. EFF-1::GFP was scored as localizing to the membrane if the peak of green intensity (EFF-1::GFP) and the peak of red intensity (cytoplasmic mCherry) did not overlap.
For EFF-1::GFP intensity calculations, average projections of z-stacks were analysed. For the axon, a line scan was performed along the axon for the initial ~50 μm anterior to the cell body, and the mean intensity in the green and red channels was measured. Background fluorescence was calculated using the same line scan moved to three different positions around the axon in the image; the mean background fluorescence from these three readings was subtracted from the average intensity value of the axon. The ratio of the GFP intensity to the mCherry intensity was then calculated to control for differences in transgene expression between animals. For intensity calculations of the cell body, a region of interest was drawn following the boundary of the cell body, and calculations were performed as for the axon.
For measurements of EFF-1::GFP border localization, average projections of z-stacks were analysed. A region of interest was drawn along the border of the PLM cell body. The average GFP intensity was first measured along this line (border intensity), after which the average GFP intensity was measured within the full area (cell body intensity). The final measurement was expressed as a ratio of the average border intensity to cell body intensity. Background subtraction was performed as described for intensity measurements.
To quantify EFF-1::GFP puncta size and number, maximum projection confocal images of z-stacks were analysed. Puncta were identified automatically using the ImageJ plugin Squassh30. The following settings were used: background removal, rolling ball window size 10, regularization 0.1, minimum object intensity 0.3, subpixel segmentation, automatic local intensity estimation, Poisson noise model, and Gaussian psf approximation as for confocal microscopy. The circularity of the resulting objects was calculated using the ‘Analyse Particles’ function on ImageJ. The following exclusion criteria were applied to all objects: circularity <0.7, and object located in the proximal axon or outside the neuron. If none of the puncta selected in a cell body met these criteria, the cell body was discarded from the analysis.
For co-localization studies of EFF-1 and RAB-5, only enlarged puncta were analysed; these were defined as puncta >125 units, as puncta of this size were specific to RAB-5 overactivity and never observed in non-transgenic controls. Squassh analysis was applied to both the green and blue channels, providing co-localization values for the selected objects. Identical settings (described above) were used for the two channels.
Statistical testing
Statistical analysis was performed using GraphPad Prism and Microsoft Excel. Error of proportions was used to assess variation across a single population. Two-way comparison was performed using either the t-test or t-test with Welch correction (the latter was performed if the standard deviations of two compared groups were significantly different).
AUTHOR CONTRIBUTIONS
C.L. designed and performed experiments and wrote the paper; B.N. provided reagents and interpreted experiments; R.G.S. and M.A.H. designed and interpreted experiments and wrote the paper.
ACKNOWLEDGMENTS
We thank Luke Hammond and Rumelo Amor for help with microscopy, as well as Rowan Tweedale and members of the Hilliard lab for helpful discussion and comments. We also thank Guangshuo Ou for the pOG172 plasmid, Nick Valmas for the PNVMYR::mCherry plasmid, and Elia Di Schiavi for advice regarding cell-specific RNAi. This research was supported by a University of Queensland Research Scholarship to C.L and a HFSP Fellowship LT000762/2012 to R.G.S. Some strains were provided by the Caenorhabditis Genetic Center (CGC), which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). This work was supported by NHMRC project grants (1068871 and 1129367), an ARC discovery project (160104359) and an NHMRC Senior Research Fellowship (1111042) to M.A.H.; NHMRC project grants (1101974 and 1099690) to B.N. The LSM 710 META confocal microscope used in this study was supported by an ARC Grant (LIEF LE130100078).