AP2 hemicomplexes contribute independently to synaptic vesicle endocytosis

Suggested experiments to improve the manuscript:

One could imagine that different endocytic cargos could have differential sensitivity to AP2 perturbations. An experiment would be to test cargos that are solely trafficked by either hemi-complex. This could be done using chimeric CD4 proteins containing either di-leucine (as in Figure 4) or tyrosine-based endocytic motifs (which mediate binding to alpha2 and mu2 respectively). If hemi-complexes function independently, di-leucine substrate recycling would be disrupted in apa-2 (as indicated in Figure 4D) but unaffected in apm-2 (which was not tested). The converse pattern would be expected for cargos containing a tyrosine endocytic motif. In principle, these experiments could persuasively show that in mutants lacking specific subunits, the remaining AP2 subunits have the capacity to function as hemi-complexes.

As suggested by the reviewers, we engineered CD4 with the di-leucine α-adaptin recognition motif and tested its localization in α or μ2 mutants. Consistent with the model, the substrate was strongly mislocalized in the apa-2 α mutant, and weakly mislocalized in the apm-2 μ2 mutant. Images are shown in Figure 5D and the quantification is shown in Figure 5E. For a μ2 substrate, we used MIG-14 / wntless, which we have previously shown requires μ2 for endocytosis from the surface. In reciprocal experiments we found that MIG-14 endocytosis is more defective in μ2 mutants than α mutants. Images are in Figure 5F and quantification in Figure 5G.

Required textual revisions:

1) Please revise the text to make it as clear as possible how much residual AP2 function may exist in the hemi knockouts. In response to this request, we added two experiments to describe residual AP2 function in the mutants. First, we quantified the levels of remaining AP2 subunits by western blot in each of the mutants (depending on the availability of functional antibodies; Figure 6—figure supplements 1&2). Second, we quantified the fluorescence intensity of all four AP2 subunits at the nerve ring in α and μ2 mutants Figure 6 and Table 1).

2) Please discuss the contribution of hemi-complex function to the wild type animal, when all subunits are present. For example, AP2 binding to cargo endocytic motifs is activated by PIP2 binding, which is mediated by residues in both hemi-complexes. Clathrin recruitment by AP2 is mediated by clathrin binding sequences in both hemi-complexes. Native cargoes may contain motifs that bind both alpha and mu subunits. For these reasons, the avidity of the AP2 holo-complex for substrates and clathrin may be much stronger than for either hemi-complex. In addition, the authors should comment on the observation that protein abundance for the residual hemi-complex is relatively low (e.g., in the apm-2 mutants), suggesting that it would be a minor contributor to wild-type endocytic activity. This is particularly the case if recruitment to the membrane is compromised for the hemi-complex.

It is true, the holocomplex is likely to provide some inseparable functions, and even if completely stable, the function of a hemicomplex might be compromised. It is also important to note that the hemicomplexes are not fully stable. We now emphasize these two points in the new version of the Discussion.

3) Please revise the text to include p-values for all data upon which arguments or conclusions are based. This is particularly important for the diverse double mutant phenotypes that appear to trend toward significance, but which may or may not reach statistical significance. This is also the case when discussing comparisons being made regarding electrophysiological data.

We double-checked our comparisons and have included all p-values in the legends of the figures or the figure supplements. To address the electrophysiological comparisons more precisely, we added the following text:

“In apa-2(ox422) mutants, the amplitude from miniature spontaneously released vesicles (minis) is increased by 40% (Figure 9C). The mini amplitudes in the skin-rescued single and double mutants are also larger, although they do not reach statistical significance.”

4) It is unclear why the authors did not report on the electrophysiological signature of the double (alpha/mu) mutant in the “skin” rescued animals. This is worth at least mentioning.

The data for the skin-rescued double mutants are included in Figure 9. In the text we report the results as follows: “The double mutants exhibit a more severe, 42% reduction in the amplitude of the evoked responses (Figure 9E).”

“There is also a more severe reduction in the rates of tonic synaptic vesicle fusion. Skin-rescued apa-2 animals exhibit a 50% reduction in mini frequency, and the skin-rescued apa-2 apm-2 double mutants exhibit a 68% reduction in mini frequency (Figure 9D).”

5) The authors should reference the mammalian work showing that loss of mu2 leads to loss of alpha and point out that this is in contrast to the findings in this system.

We now note this difference in the Discussion.

6) It is not possible, at the light level, to know whether an altered axon/synapse ratio of protein distribution has anything to do with the recycling of individual proteins. This should be discussed. In C. elegans, stonin / UNC-41 (Mullen et al., 2012) and AP180 / UNC-11 (Nonet et al., 1999) are two characterized adaptors for synaptotagmin and synaptobrevin recycling at synapses. In the absence of these adaptors, the tagged cargo protein is severely diffused into axons that make the fluorescence at the synaptic varicosities decrease or even out with that in axons. So we still believe that the axon/synapse ratio of protein distribution could reflect the efficiency of the cargo recycling to some extent. To make this point clear in the paper, we have made the following change:

“In C. elegans mutants lacking particular adaptor proteins, the cognate cargo protein diffuses into axons. For example in AP180 mutants, synaptobrevin is no longer concentrated at synapses but is diffuse in axons (Nonet et al., 1999). By contrast, in AP2 adaptin mutants, synaptic vesicle proteins are not grossly mislocalized.”

7) The changes in vesicle size and number are similar to that observed in synaptotagmin 1 mutants and following acute inactivation of synaptotagmin 1. This is worth citing since synaptic vesicle endocytosis is never directly assayed in the current study.

That is correct: acute inactivation of synaptotagmin at the Drosophila neuromuscular junction as well as chronic inactivation in C. elegans exhibits a similar phenotype as that found here for AP2 knockouts. Moreover, we previously found that stonin shares overlapping functions with AP2. We now discuss the relationship among synaptotagmin, AP2 and stoning in the Discussion.

8) Figure 8C: “These data suggest that the AP2 complex plays a role in regulating the size of synaptic vesicles.” Does the defect in vesicle size imply a role for determining the size of the vesicle? Many endocytic mutations alter vesicle size and it is unlikely that all participate in the control of vesicle diameter. An alternative conclusion is that any mutation that disrupts the fidelity of endocytosis may well disrupt the ability to reliably generate small vesicles of a consistent size.

We have amended the text to state that the change in vesicle diameter might be indirect. In addition, to try to pinpoint where the defect in AP2 mutants might lie, we analyzed more sections and performed a 3D reconstruction of a synapse from the apa-2 apm-2 double mutant. Defects are observed at the cell membrane in some cases, but the most prominent defects are large vacuoles present in the synaptic varicosity. We speculate that AP2 might have a late role in the regeneration of synaptic vesicles from endosomes. The changes are as follows: Results, “These data suggest that the AP2 complex may play a role in regulating the size of synaptic vesicles. Alternatively, the effect on vesicle size may be indirect due to pleiotropic defects in endocytosis.”

Discussion, “The most prominent defect observed in AP2 mutants is the presence of large diameter vesicles and vacuoles in the synapses of the α-μ2 adaptin double mutants. […] These data suggest that AP2 has a late function and possibly AP2 is required to regenerate synaptic vesicles from endosomes.”