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Article |
µ2 adaptin facilitates but is not essential for synaptic vesicle recycling in Caenorhabditis elegans
Correspondence to Erik M. Jorgensen: jorgensen{at}biology.utah.edu
Synaptic vesicles must be recycled to sustain neurotransmission, in large part via clathrin-mediated endocytosis. Clathrin is recruited to endocytic sites on the plasma membrane by the AP2 adaptor complex. The medium subunit (µ2) of AP2 binds to cargo proteins and phosphatidylinositol-4,5-bisphosphate on the cell surface. Here, we characterize the apm-2 gene (also called dpy-23), which encodes the only µ2 subunit in the nematode Caenorhabditis elegans. APM-2 is highly expressed in the nervous system and is localized to synapses; yet specific loss of APM-2 in neurons does not affect locomotion. In apm-2 mutants, clathrin is mislocalized at synapses, and synaptic vesicle numbers and evoked responses are reduced to 60 and 65%, respectively. Collectively, these data suggest AP2 µ2 facilitates but is not essential for synaptic vesicle recycling.
© 2008 Gu et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
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Introduction |
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Clathrin is linked to cargo and membranes by the clathrin adaptor complex (Keen, 1987). Four different adaptor omplexes have been identified in mammals: AP1, AP2, AP3, and AP4 (Keen, 1987; Simpson et al., 1997; Dell'Angelica et al., 1999). These adaptor protein complexes localize to different membranes in the cell and coordinate cargo selection and vesicle biogenesis (Lewin and Mellman, 1998; Robinson and Bonifacino, 2001; Robinson, 2004). AP2 is the adaptor complex functioning during endocytosis at the plasma membrane (Mahaffey et al., 1990; Traub, 2003). There are four different subunits in the AP2 complex: 
(large), β2 (large), µ2 (medium), and 
2 (small; Matsui and Kirchhausen, 1990), and each subunit serves a specific function. In particular, the µ2 subunit recruits cargo proteins containing the tyrosine-based Yxx
motif (Owen and Evans, 1998) and mediates in part the association of the AP2 complex to membranes (Gaidarov and Keen, 1999; Rohde et al., 2002; Honing et al., 2005).
Here, we characterize mutants that lack µ2 adaptin, encoded by the apm-2 gene (also called dpy-23), in the nematode Caenorhabditis elegans. We demonstrate that µ2 is partially required for synaptic localization of clathrin and for the stability of the AP2 complex. However, synaptic vesicles are still recycled in the absence of µ2. Our data suggest that despite previous predictions, µ2 is not absolutely required for synaptic vesicle endocytosis. Moreover, the decrease in synaptic vesicle number does not cause a locomotion defect in µ2 knockout mutants, so a smaller reserve pool might be adequate for C. elegans under normal condition.
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Results |
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The dpy-23 mutant phenotype was mapped to the interval between –7.91 to –7.55 on chromosome X (Fig. 1 A). The gene encoding µ2 adaptin, called apm-2, maps to this interval, and RNA interference to this gene gave rise to a variable dumpy phenotype suggesting dpy-23 is likely to encode µ2 (Grant and Hirsh, 1999). We cloned the dpy-23 gene and demonstrated that the mutated gene is apm-2. Two overlapping cosmids, D1079 and C33G6, in the region rescued the dpy-23 mutant phenotype. A 12-kb genomic PCR fragment (5 kb upstream, 5 kb coding sequence of µ2 adaptin, and 2 kb downstream) could fully rescue the dumpy, uncoordinated, and egg-laying defects of dpy-23(e840) and dpy-23(gm17) (Fig. S1 A). Interestingly, overexpression of dpy-23 in a wild-type background causes the same phenotypes as dpy-23 loss-of-function mutations (Fig. S1 A). Because dpy-23 encodes µ2 we will refer to the gene by its alternative name, apm-2 (adaptor protein medium subunit 2), throughout the remainder of the manuscript.
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100 kb that includes the entire coding sequence of µ2 adaptin. To identify the endpoints of the deletion, individual open reading frames (ORFs) were PCR amplified from apm-2(e840) mutant DNA and it was found that the deletion removes 17 additional ORFs from col-165 to F25F6.1 (Fig. 1 B). apm-2(gm17) was found to contain a G to A point mutation in the splice donor site of the last intron (Fig. 1 C). Failure to splice at this intron would introduce a stop codon 27 nt downstream of the splice junction. If translated, 9 amino acids encoded by the intron would replace the 40 amino acids at the C terminus comprising β strands 15, 16, and 17. β16 has been shown to be critical for binding cargo containing the YXX motif (Ohno et al., 1995; Owen and Evans, 1998). Although apm-2(gm17) appears to have a slightly dominant phenotype, the recessive phenotypes of apm-2(e840) and apm-2(gm17) are virtually identical, suggesting that gm17 fully disrupts µ2 function. µ2 is thought to be a critical component of the AP2 complex and should therefore be present in all tissues. To determine where µ2 is expressed, a construct fusing GFP to the APM-2 protein was expressed under the control of the endogenous apm-2 promoter (Fig. S4, pMG4, available at http://www.jcb.org/cgi/content/full/jcb.200806088/DC1). Because the N terminus of µ2 adaptin is involved in assembly of the AP2 complex (Aguilar et al., 1997; Collins et al., 2002), GFP was fused to the C terminus of the APM-2 protein. The APM-2::GFP fusion protein fully rescues apm-2 mutant phenotypes, suggesting the tagged protein is functional and is expressed in tissues that require µ2 function. Fluorescence is observed in the nervous system, coelomocyte, spermatheca, and vulva (Fig. 2). In addition, weaker expression is observed in the intestine and the hypodermis. Although fluorescence is not detected in body muscles of animals expressing the tagged protein, muscle expression is observed in animals expressing a transcriptional reporter (Fig. 2 and Fig. S4, pMG3). Thus, apm-2 is expressed in all tissues examined, which confirms and extends a previous paper claiming that apm-2 is expressed in neurons and some hypodermal cells (Shim and Lee, 2000).
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Because apm-2 is expressed in virtually all tissues, it is not possible to assay synaptic localization with the rescuing GFP construct, the fluorescence signal is simply too high. To look at a small subset of neurons, the apm-2 cDNA (R160.1b) was placed under the control of a GABA neuron–specific promoter and GFP was fused at the C terminus. APM-2::GFP is localized at synapses and colocalizes with the synaptic vesicle protein synaptobrevin/VAMP (Fig. 3 A). In addition, APM-2::GFP colocalizes with C-terminal RFP-tagged clathrin heavy chain (CHC) at the synapse (Fig. 3 B). Interestingly, synaptic localization of APM-2::GFP is not dependent on AP180 (unc-11), synaptojanin (unc-26), synaptotagmin (snt-1), or stonin (unc-41) (Fig. S2 A, available at http://www.jcb.org/cgi/content/full/jcb.200806088/DC1). These data indicate that µ2 associates with synaptic varicosities as predicted, but this localization is independent of other endocytosis proteins.
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Mutants with defects in synaptic vesicle endocytosis exhibit reduced synaptic transmission and are uncoordinated (Nonet et al., 1999; Harris et al., 2000; Schuske et al., 2003). apm-2 mutants are also uncoordinated but the uncoordinated phenotype arises from defects in the hypodermis rather than the nervous system. When APM-2::GFP is expressed under the control of a ubiquitous promoter the tagged µ2 protein rescues all apm-2(e840) mutant phenotypes (Fig. 5, A and B). When µ2 protein is expressed under a pan-neuronal promoter, the apm-2 mutants are still dumpy, uncoordinated, and egg-laying defective, effectively looking the same as the original apm-2(e840) mutants. In contrast, when µ2 protein is expressed under a hypodermal promoter, the apm-2(e840) transgenic animals are not dumpy (Fig. 5 B, skin APM-2); moreover, the jerky uncoordinated phenotype is also rescued. Previous studies have demonstrated that the µ2 functions in the skin rescue developmental defects in the nervous system (Pan et al., 2008); our data suggest that these nonautonomous defects might extend to development or function of motor neurons as well. However, skin-rescued animals are egg-laying defective and the body bends are increased in amplitude, suggesting neuronal function is somewhat altered. To quantify locomotion in the mosaic strains, animals were placed in a drop of liquid and body bends were counted for 90 s for each strain (Fig. 5 C). Thrashing rates of the hypodermal rescued strains are the same as in the wild type; whereas neuronal expression of µ2 does not rescue thrashing. The reduced thrashing is not due to a dominant-negative effect because overexpression of µ2 in the neurons does not impair locomotion in the wild type. Collectively, these data suggest the uncoordinated phenotype of apm-2 mutants is almost exclusively caused by hypodermal defects rather than defects in the nervous system.
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adaptin knockdowns are more severe than µ2 adaptin knockdowns in the yolk uptake assay (Grant and Hirsh, 1999). RNA interference against clathrin, adaptin, or β2 adaptin each abolished uptake of a GFP-tagged vitellogenin (YP170-GFP) into oocytes. In contrast, RNA interference against either µ2 or 2 adaptin did not disrupt uptake, suggesting that not all of the subunits of the AP2 complex are required for this process. Because RNA interference is not always fully penetrant we used our deletion allele to assay µ2 function in yolk uptake. Approximately 120 gonads of each genotype were assayed for the presence of YP170-GFP fluorescence in oocytes. Over 98% of the gonads in both apm-2(e840) and apm-2(gm17) mutants were scored as positive for yolk protein uptake (Fig. 9 A).
However, the number of oocytes within each gonad containing YP170-GFP was reduced in both alleles of apm-2 compared with the wild type (wild type: one oocyte, 7%; two or more, 93%; apm-2(e840): one oocyte, 42%; two or more oocytes, 58%; apm-2(gm17): one oocyte, 36%; two or more oocytes, 64%; Fig. 9 B). In all genotypes yolk accumulation is greatest in the oldest oocyte, which is found adjacent to the spermatheca. The defect is not as severe as that seen when the or β2 subunits of the complex are depleted by RNA interference (Grant and Hirsh, 1999), suggesting that µ2 adaptin subunits are less important than the adaptin or β2 adaptin subunits in this process.
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adaptin remains in a complex with 2 adaptin in the µ2 adaptin mutants. Previous results suggested that each subunit is required for function and stability of the AP2 complex; e.g., RNA interference of the µ2 subunit in HeLaM cells greatly reduced the expression level of the subunit (Motley et al., 2003). In addition, expression of any single AP2 subunit in bacteria produces an insoluble protein; only simultaneous expression of all subunits produces a soluble protein complex (Collins et al., 2002). To determine if the AP2 complex is stable in the absence of the µ2 subunit, we performed quantitative Western blot analysis of adaptin. adaptin is reduced to 60% in apm-2(e840) and to 12% in amp-2(gm17) (Fig. 9 C). Oddly, the reduction in adaptin is more severe in the truncated allele of µ2 rather than in the null mutant; it is possible that incorporation of truncated µ2 leads to a destabilization of the whole complex. Consistent with this result, apm-2(e840)/+ heterozygous animals are wild type, whereas apm-2(gm17)/+ animals are slightly uncoordinated and have an egg-laying defect. The reduction in adaptin levels in both alleles indicates that µ2 is required to stabilize the AP2 complex; however, some residual adaptin remains in the absence of µ2.
The residual adaptin remains bound to 2 adaptin. adaptin tagged with GFP was immunoprecipitated from the wild type and apm-2 mutants. β adaptin coimmunoprecipitated from the wild-type animals but not from µ2 mutant animals (Fig. S5 A, available at http://www.jcb.org/cgi/content/full/jcb.200806088/DC1). In contrast, 2 adaptin (HA tagged) coimmunoprecipitated from both the wild-type and µ2 mutant animals (Fig. S5, B and C). Moreover, residual adaptin can still be localized in the absence of µ2. Coelomocytes are scavenger cells in C. elegans with high levels of endocytosis. N-Terminal tagged adaptin is localized properly to the plasma membrane of mutant coelomocytes (Fig. 9 D). Similarly, tagged adaptin is localized to synapses in apm-2 mutants. These data suggest that the protein is folded and transported correctly in mutant cells. Thus, it is possible that AP2 is at least partially functional in apm-2 mutants.
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Discussion |
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60% of the normal number of vesicles at synaptic varicosities, and synaptic vesicle proteins are properly localized at the synapse. This phenotype is much less severe than worm mutants lacking other recycling proteins such as AP180 (unc-11), synaptojanin (unc-26), and endophilin (unc-57) (Nonet et al., 1999; Harris et al., 2000; Schuske et al., 2003). For example, the number of synaptic vesicles in synaptojanin and endophilin mutants is reduced to 35%, the normal number of synaptic vesicles found at neuromuscular junctions. The conclusion that µ2 is not essential for synaptic vesicle recycling leads to several considerations. (a) Do other proteins recruit cargo? (b) Can other medium subunits stabilize AP2? (c) Do other proteins recruit clathrin?
The specific role of µ2 is in cargo recruitment, in particular, its interactions with synaptotagmin were thought to be essential for synaptic vesicle biogenesis (Zhang et al., 1994; Jorgensen et al., 1995; Haucke et al., 2000). However, our data indicate that µ2 is not required to recruit proteins to synaptic vesicles. The essential synaptic vesicle proteins are synaptobrevin, synaptotagmin, and the neurotransmitter transporters. Other ancillary proteins have been identified that recruit these synaptic vesicle proteins to sites of endocytosis. AP180 is required to recruit synaptobrevin to synaptic vesicles (Zhang et al., 1998; Nonet et al., 1999; Bao et al., 2005). Stonin, which is distantly related to µ2, is required for synaptotagmin recycling (Fergestad and Broadie, 2001; Martina et al., 2001; Walther et al., 2004). The vesicular GABA transporter is recruited by a LAMP-related protein called UNC-46 (Schuske et al., 2007). Because of these defects in cargo recruitment, all of these mutants are severely uncoordinated in worms. In contrast, mutants lacking µ2 in the nervous system are not uncoordinated and evoked responses are at 82% of the levels observed in the wild type, indicating that synaptic transmission is largely intact. Thus, if µ2 recruits cargo to recycling vesicles it is unlikely to be a component essential for neurotransmission.
The medium subunit µ2 is also known to stabilize the AP2 complex (Motley et al., 2003). One could imagine that medium subunits from the AP1 or AP3 complexes (there is no AP4 in C. elegans) could substitute for µ2 and provide AP2 complex function. However, adaptins from different complexes do not appear to be redundant in other organisms. For example, in yeast, overexpression of 2 cannot substitute for the loss of 1 (Phan et al., 1994). Similarly, we found that other medium subunits cannot substitute for µ2 in C. elegans. Mutants lacking µ1 (unc-101) are severely uncoordinated and exhibit defects in anterograde transport of olfactory receptors to olfactory cilia (Dwyer et al., 2001). Overexpression of µ2 cannot rescue the severely uncoordinated phenotype of the µ1 mutant in a thrashing assay (unpublished data). Moreover, apm-2 unc-101 (µ1 µ2) double mutants exhibit an additive dumpy and uncoordinated phenotype rather than a synthetic phenotype, suggesting that these proteins are not acting redundantly. Mutants lacking µ3 (apm-2(tm920)) are outwardly wild type but slightly aldicarb resistant. Again, µ2 and µ3 mutations do not show synthetic interactions: apm-2 apm-3 double mutants (with µ2 rescued in skin) exhibit similar aldicarb sensitivity as the µ3 mutant alone. In addition, our data show that AP2 is destabilized in the absence of µ2, suggesting µ2 is the only medium subunit used by AP2 in C. elegans.
Our data are consistent with a study in other systems suggesting that clathrin can be recruited by alternative adaptors (Traub, 2003). In particular, AP180 is required for normal synaptic vesicle endocytosis and it is likely that AP180 could recruit clathrin and form vesicles in the absence of AP2 (Zhang et al., 1998; Nonet et al., 1999). Our results for µ2 seem to conflict with previous studies that suggest that the AP2 subunit adaptin is essential for synaptic vesicle recycling in Drosophila melanogaster. A weak mutation in the Drosophila adaptin subunit D-Ada1 leads to slowly moving larvae, which die as pupae (Gonzalez-Gaitan and Jackle, 1997). The neurons of these animals are not efficient in taking up FM1-43 dye at boutons upon stimulation. The null mutants die before hatching and the electron microscopy data suggest that these animals are depleted of synaptic vesicles. However, in C. elegans, the behavioral defects of apm-2–null mutants are less pronounced. The hypodermal rescued mutants have almost normal movement and the evoked current upon stimulation is close to the wild type. Thus the neurotransmission defect in µ2 mutants in worms is less severe than that of adaptin mutants in flies. Although it is possible that residual AP2 function accounts for vesicle recycling in µ2 mutants, it is also possible that AP2 is not essential for synaptic vesicle recycling at C. elegans neuromuscular junctions.
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Materials and methods |
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C. elegans strains
The wild strain is Bristol N2. The reference strains for e840 and gm17 were outcrossed twice before phenotypic analysis. The outcrossed strains are EG2988 apm-2(gm17)X and EG3622 apm-2(e840)X.
The strains used in the synaptobrevin and clathrin colocalization assays were EG4052 lin-15(n765ts)X; oxEx761[Punc-47:APM-2(cDNA)::GFP Punc-47:SNB::mCherry lin-15(+)] and EG4051 lin-15(n765ts)X; oxEx759[Punc-47:APM-2(cDNA)::GFP Punc-47:CHC-1::RFP lin-15(+)].
The strains used in the synaptic vesicle protein distribution assays were MT8247 lin-15(n765ts) nIs52[Punc-25::SNB::GFP lin-15(+)]X, EG3733 apm-2(e840) nIs52[Punc-25::SNB::GFP lin-15(+)]X, EG3229 apm-2(gm17) nIs52[Punc-25::SNB::GFP lin-15(+)]X, NM1233 jsIs219[SNG-1::GFP rol-6(su1006)]II, EG3563 jsIs219[SNG-1::GFP rol-6(su1006)]II; apm-2(e840)X, EG3736 jsIs219[SNG-1::GFP rol-6(su1006)]II; apm-2(gm17)X, EG3855 lin-15(n765ts) oxIs224[Punc-47::GFP::SNT-1 lin-15(+)]X, EG3889 apm-2(e840) oxIs224[Punc-47::GFP::SNT-1,lin-15(+)]X, and EG3891 apm-2(gm17) oxIs224[Punc-47:GFP::SNT-1 lin-15(+)]X.
The strains used in the clathrin distribution assays were EG3381 oxIs164[Punc-47::GFP::CHC-1 lin-15(+)]IV; lin-15(n765)X, EG3735 oxIs164[Punc-47:GFP::CHC-1 lin-15(+)]IV; apm-2(e840)X and EG3564 oxIs164[Punc-47::GFP::CHC-1 lin-15(+)]IV; apm-2(gm17)X.
The strains used in APM-2 distribution assays were EG4055 snt-1(n2665)II; oxEx763[Punc-47::APM-2(cDNA)::GFP Pmyo-2::GFP], EG4091 unc-41(n268)V; oxEx767[Punc-47::APm-2(cDNA)::GFP Pmyo-2::GFP], EG4103 unc-11(e47)I; oxEx767[Punc-47::APM-2(cDNA)::GFP Pmyo-2::GFP], and EG4089 unc-26(s1710)IV; oxEx767[Punc-47::APM-2(cDNA)::GFP Pmyo-2::GFP].
The strains used in APM-2 tissue-specific rescue assay were EG4017 lin-15(n765)X; oxEx747[Prab-3::APM-2(cDNA)::GFP (pMG9) lin-15(+)], EG1616 apm-2(e840)X; oxEx730[APM-2::GFP (pMG4) Punc-122::GFP], EG4213 apm-2(e840)X; oxEx789[Prab-3::APM-2(cDNA)::GFP (pMG9) Punc-122::GFP], EG4015 apm-2(e840)X; oxEx745[Pdpy-30::APM-2(cDNA)::GFP (pMG10) Punc-122::GFP], EG4029 apm-2(e840)X; oxEx753[Ppdi-2::APM-2(cDNA)::GFP (pMG8) Punc-122::GFP], EG4030 apm-2(e840)X; oxEx745[Ppdi-2::APM-2(cDNA)::GFP (pMG8) Prab-3::APM-2(cDNA)::GFP (pMG9) Punc-122::GFP], and EG4093 apm-2(e840)X, oxEx773[Ppdi-2::APM-2(cDNA)::GFP (pMG8) Pmyo-2::GFP]. Punc-122 is the coelomocyte promoter.
The strains used in yolk uptake assay were DH1033 sqt-1(sc103)II; bIs1[[vit-2::GFP rol-6(su1006)]X, EG4062 apm-2(e840) bIs1[vit-2::GFP rol-6(su1006)]X, EG4059 apm-2(gm17) bIs1[vit-2::GFP rol-6(su1006)]X.
The strain used in adaptin coelomocyte plasma membrane localization assay were RT490 unc-119(ed3) III; pwIs177[Punc-122:mRFP::apa-2; cb-unc-119(+)], EG4188 apm-2(e840) X; pwIs177[Punc-122:mRFP::apa-2; cb-unc-119(+)], EG4189 apm-2(gm17) X; pwIs177[Punc-122:mRFP::apa-2; cb-unc-119(+)]. They were provided by B.D. Grant (Rutgers University, Piscataway, NJ)
The strains used in adaptin GABA synaptic localization assay were GK275 unc-119(ed3) III; dkIs160[Punc-25:GFP::apa-2,unc-119(+)], EG4203 apm-2(e840) X; dkIs160[Punc-25:GFP::apa-2,unc-119(+)], and EG4204 apm-2(gm17) X; dkIs160[Punc-25:GFP::apa-2,unc-119(+)]. They were provided by K. Sato (Gunma University, Gunma, Japan).
The strains used in AP2 assembly immunoprecipitation were EG5264 oxEx1275[Papa-2::apa-2::GFP Punc-122::GFP], EG5265 apm-2(e840)X, oxEx1275[Papa-2::apa-2::GFP Punc-122::GFP], EG5266 oxEx1276[Papa-2::apa-2::GFP Paps-2::aps-2::HA Punc-122::GFP], and EG5267 apa-2(e840)X oxEx1276 [Papa-2::apa-2::GFP Paps-2::aps-2::HA Punc-122::GFP].
Phylogenetic analysis
The phylogenetic tree of µ adaptin was made by ClustalX (1.83.1 Mac) and Treeview X. The protein accession numbers are as follows: µ3A mouse (Q9JKC8); µ3B mouse (Q8R2R9); µ3 Drosophila (NP_788873); APM-3 (NP_508184); stB Drosophila (Q24212); APT-10 (stB Ce; NP_505566); stB mouse (NP_780576); µ2 Drosophila (NP_732744); µ2 mouse (NP_033809); APM-2 (NP_001024865); APM-1(NP_491572); UNC-101 (NP_001040675); µ1A mouse (AAF61814); µ1B mouse (AF067146); µ1 Drosophila (NP_649906).
GFP constructs
pMG1, apm-2 genomic region.
A 12-kb apm-2 genomic PCR fragment was amplified from cosmid C33G6, including 5 kb upstream of the start codon, 5 kb of coding sequence, and 2 kb downstream of the stop codon, and was cloned between BamHI and PstI restriction sites in pGEM-3zf(+) (Promega). Primers used were 5'-ATTAGGATCCAGGTGGTGGTGGTGAAGA-3' and 5'-AGATCTGCAGTCGGCTAACGGCTAATTCGGCTAA-3'.
pMG2, apm-2 transcriptional GFP.
The construct comprises the apm-2 promoter only driving GFP. This construct does not show expression in neurons, suggesting that a neuronal enhancer is contained within an intron.
pMG3, apm-2 transcriptional GFP.
GFP with the unc-54 3'UTR was PCR amplified from the plasmid pPD95.77 (provided by A. Fire, Carnegie Institution of Washington, Baltimore, MD). The primers used were 5'-GGCTGAAATCACTCACAACGATGG-3' and 5'-TACAGTCGACTACGGCCGACTAGTAGGAAACAGT-3'. This fragment contains SalI restriction sites at both ends and was inserted into the SalI site of pMG1, which is in the second exon of apm-2.
pMG4, apm-2 translational GFP.
apm-2 10-kb genomic PCR fragment, including 5 kb upstream of the start codon and 5 kb of coding sequence without the stop codon, was amplified from pMG1 and cloned between the BamHI and PstI restriction sites in pGEM-3zf(+). Primers used were 5'-ATTAGGATCCAGGTGGTGGTGGTGAAGA-3' and 5'-ACAGCTGCAGGCATCTGGTTTCATACAGTCCCGA-3'. GFP with the unc-54 3'UTR was cloned from the plasmid pPD95.77 with a PstI site at one end and a HindIII site at the other end and was then inserted after the apm-2 coding sequence to make a GFP fusion protein. Primers used were 5'-ACATCTGCAGTTGGCCAAAGGACCCAAAGGTATG-3' and 5'-ACGCAAGCTTCGGCCGACTAGTAGGAAACAGTTA-3'.
Constructs for apm-2 tissue-specific rescue assay
Multisite Gateway three-fragment construction vectors were used (Invitrogen). Promoter entry vectors, including pENTRY4-1 Pdpy-30 and pENTRY4-1 Ppdi-2, were ordered from Open Biosystems. The Ppdi-2 construct (Open Biosystems) lacks 373 nt (–808 to approximately –435 from the start codon of pdi-2) in the promoter region; however, skin expression was still observed using this construct. pENTRY4-1 Prab-3 was generated by BP reaction. ORF entry vector pENTRY1-2 apm-2 (ORF) was ordered from Open Biosystems. 3'UTR entry vector pENTRY2-3 GFP::unc-54 3'UTR was made by a Gateway BP reaction. The final constructs, apm-2::GFP fusion gene driven by different promoters, were generated by Gateway LR reactions.
Constructs for AP2: assembly immunoprecipitation
pMG16, apa-2 translational GFP.
apa-2 6-kb genomic sequence, including 2.3 kb of promoter and 3.7 kb of coding sequence without stop codon, was cloned between KpnI and XbaI sites in pGEM-3zf(+). Primers used were 5'-ACTCGGTACCGCATACTTGATGGAAAACCCGCTC-3' and 5'-AGCCTCTAGAAAATTGGTTGCCCAATAAGTCTAC-3'.
GFP with the unc-54 3'UTR was cloned from Fire laboratory vector pPD 95.77 with an XbaI site at one end and a HindIII site at the other end. This fragment was inserted after the apm-2 coding sequence to make a GFP fusion protein. Primers used were 5'-ACCGTCTAGAGGGGTAGAAAAAATGAGTAAAGGA-3' and 5'-AGCGAAGCTTCGGCCGACTAGTAGGAAACAGTTA-3'.
pMG28, HA-tagged translational APS-2.
aps-2 2-kb genomic sequence, including 1 kb of promoter and 1 kb of coding sequence (stop codon is replaced by HA tag), was cloned between EcoRI and PstI sites in pGEM-3zf(+). Primers used were 5'-AGCGGAATTCCGTTTAGTTCTTGAGTGGCTTG-3' and 5'-ACAGCTGCAGAGCGTAATCTGGAACATCGTATGGGTATTCCAGGGAAGTAAGCATGAGCA-3'.
unc-54 3'UTR was cloned from Fire laboratory vector pPD 95.77 with a PstI site at one end and a HindIII site at the other end. This fragment was inserted after the aps-2::HA coding sequence. Primers used were 5'-AGGCCTGCAGTAGCATTCGTAGAATTCCAACTGA-3' and 5'-AGGCAAGCTTCCCATAGACACTACTCCACTTTC-3'.
Microinjection
The total DNA concentration of injection mix is 100 ng/µl. The injection marker was 50 ng/µl Punc-122::GFP, if not specified. 1-kb DNA ladder (Fermentas) was used to make the injection mix final concentration 100 ng/µl.
Rescue experiment.
10 ng/µl of 12-kb apm-2 genomic PCR fragment was injected into N2 to overexpress APM-2 in wild-type animals. 0.25 ng/µ of 12-kb apm-2 genomic PCR fragment was injected into N2. The extrachromosomal array was then crossed into apm-2(e840) and apm-2(gm17) mutants to evaluate rescue.
Tissue-specific rescue.
1 ng/µl each of pMG10 Pdpy-30::apm-2::GFP, pMG8 Ppdi-2::apm-2::GFP, and pMG9 Prab-3::apm-2::GFP DNAs were injected, respectively, into apm-2(e840). For the skin and nervous system double rescue experiment, 1 ng/µl each of pMG8 and pMG9 were injected together. 10 ng/µl pMG9 and 50 ng/µl lin-15(+) were coinjected into lin-15(n765ts) to overexpress APM-2::GFP only in the nervous system. 1 ng/µl pMG8 and 2 ng/µl Pmyo-2::GFP were coinjected into apm-2(e840) to rescue apm-2 mutant in skin with a different injection marker.
AP2 complex immunoprecipitation.
pMG16 was injected at 10 ng/µl into the wild type. The array was crossed into apm-2(e840). pMG16 and pMG28 were coinjected at 10 ng/µl into the wild type and the same array was then crossed into apm-2(e840).
Western blot analysis
Worm samples were prepared by boiling 1 vol of the worm pellet in 9 vol of 1x loading buffer for 5 min. Samples were run on a 10% SDS-PAGE gel then transferred to polyvinylidene fluoride transfer membrane (Immobilon). Primary antibody for adaptin was a rabbit polyclonal APA-2 antibody at a dilution of 1:500 (provided by B. Grant). Primary antibody incubation was done in 1% milk at room temperature for 4 h. Primary antibody for the standard control tubulin was a 12G10 mouse monoclonal antibody (Developmental Studies Hybridoma Bank) at a dilution of 1:5,000. Primary antibody incubation was done in 1% milk at room temperature for 1 h, and then the membrane was washed three times in 10 ml of 1x PBS plus Tween 20 (PBST). Secondary antibodies were anti-rabbit and mouse IgG fragment conjugated with HRP (GE Healthcare). Secondary incubations were done in 10% milk at room temperature for 45 min, and then the membrane was washed five times in 10 ml of 1x PBST. Detection reagent used was Lumigen PS-3 (GE Healthcare).
AP2 complex immunoprecipitation
250 µl (± 50 µl) of worm pellet was harvested. The pellet was suspended in 2 ml of ice-cold lysis buffer (5% Triton X-100, 50 mM Hepes, pH 7.3, 50 mM NaCl, and 1 tablet of protease inhibitor cocktail [Roche]). The sample was lysed by a bead beater (NMB) for 10 s, three times, and was spun, and the supernatant was recovered. The supernatant was pushed through a 0.22-µm filter. 15 µl of agarose-conjugated rat anti-GFP IgG2a beads (MBL International) were added to the lysate. The mixture was incubated at 4°C for 2 h. Beads were harvested and washed with 1 ml of lysis buffer three times. 100 µl of loading buffer was added on the bead pellet and boiled for 15 min.
Samples were run on 7, 10, or 15% SDS-PAGE and transferred to a polyvinylidene fluoride membrane. Primary antibody for GFP was a mouse monoclonal antibody at a dilution of 1:5,000 (Clontech Laboratories, Inc.). Primary antibody for HA was a mouse monoclonal antibody (12CA5; Santa Cruz Biotechnology, Inc.) at a dilution of 1:5,000. Primary antibody for β adaptin was a mouse monoclonal antibody (Thermo Fisher Scientific) at a dilution of 1:5,000. Primary antibody incubation was performed in 5% BSA at 4°C overnight, and then the membrane was washed three times in 10 ml of 1x PBST. Secondary antibodies were goat anti–mouse IgG fragment conjugated with HRP (GE Healthcare). Secondary incubations were performed in 5% BSA at 22.5°C for 2 h, and then the membrane was washed five times in 10 ml of 1x PBST. Detection reagent was SuperSignal West Dura kit (Thermo Fisher Scientific).
Thrashing assay
A single worm was put into a 50-µl drop of M9 solution. The worm was allowed to adapt to the liquid environment for 2 min. The number of body bends was counted for 90 s for each genotype (n = 7). We analyzed lines of apm-2(e840) rescued by skin APM-2::GFP with two different injection markers (EG4029 Punc-122::GFP and EG4093 Pmyo-2::GFP). The results are the same.
Aldicarb resistance assay
Each agar plate was seeded with bacteria and weighed to top spread plates with the appropriate amount of aldicarb. Plates with six different aldicarb final concentrations were prepared (0.1, 0.3, 0.5, 0.7, 0.9, and 1.1 mM). Aldicarb was allowed to soak in overnight at room temperature. For each genotype, 20 worms were put onto plates containing each different aldicarb concentration. The plates were blinded and the percentage of paralyzed worms were scored after 4 h of exposure to aldicarb. The same experiment was repeated five times for each genotype except apm-2(e840) oxEx755[Prab-3::apm-2::GFP, Punc-122::GFP], which had only 10 worms on each plate and the experiment was repeated four times because of fewer transgenic animals.
Confocal microscopy
Worms are immobilized by using 2% phenoxy propanol and imaged on a confocal microscope (Pascal LSM5; Carl Zeiss, Inc.) with a plan-Neofluar 10x 0.3 NA, 20x 0.5 NA, or 40x 1.3 NA or plan-apochromat 63x 1.4 NA oil objectives (Carl Zeiss, Inc.).
Electron microscopy
Wild-type (N2), apm-2(e840), EG4029 apm-2(e840); oxEx753[Ppdi-2::apm-2::GFP, Punc-122::GFP], and EG4213 apm-2(e840); oxEx789[Prab-3::apm-2::GFP, Punc-122::GFP] adult nematodes were prepared in parallel for transmission electron microscopy as previously described (Hammarlund et al., 2007). In brief, 10 young adult hermaphrodites were placed onto a freeze chamber (100-µm well of type A specimen carrier) containing space-filling bacteria, covered with a type B specimen carrier flat side down, and frozen instantaneously in the HPM 010 (Leica). This step was repeated for animals of all genotypes. The frozen animals were fixed in the EM AFS system (Leica) with 1% osmium tetroxide and 0.1% uranyl acetate in anhydrous acetone for 2 d at –90°C and for 38.9 more hours with gradual temperature increase (6°C/h to –20°C over 11.7 h, constant temperature at –20°C for 16 h, and 10°C/h to 20°C over 4 h). The fixed animals were embedded in araldite resin following the infiltration series (30% araldite/acetone for 4 h, 70% araldite/acetone for 5 h, 90% araldite/acetone overnight, and pure araldite for 8 h). Mutant and control blocks were blinded. Ribbons of ultra-thin (33-nm) serial sections were collected using an Ultracut 6 microtome (Leica) at the level of the anterior reflex of the gonad. Images were obtained on an electron microscope (H-7100; Hitachi) using a digital camera (Gatan). 250 ultra-thin contiguous sections were cut, and the ventral nerve cord was reconstructed from two animals representing each genotype. Image analysis was performed using ImageJ software. The numbers of synaptic vesicles (30 nm), dense-core vesicles (40 nm), and large vesicles (>40 nm) in each synapse were counted and their distance from presynaptic specialization and plasma membrane as well as the diameter of each were measured from acetylcholine neurons VA and VB and the GABA neuron VD. A synapse was defined as the serial sections containing a dense projection as well as sections on either side of that density, which contain synaptic vesicle numbers above the mean number of synaptic vesicles per profile.
Electrophysiology
C. elegans were grown at room temperature (22–24°C) on agar plates with a layer of OP50 Escherichia coli. Adult hermaphrodite animals were used for electrophysiological analysis. Miniature and evoked postsynaptic currents (mPSCs and ePSCs) at the neuromuscular junction were recorded as previously described (Liu et al., 2007) using a technique originally developed by Richmond and Jorgensen (1999). In brief, an animal was immobilized on a sylgard-coated glass coverslip by applying a cyanoacrylate adhesive along the dorsal side. A longitudinal incision was made in the dorsolateral region. After clearing the viscera, the cuticle flap was folded back and glued to the coverslip, exposing the ventral nerve cord and two adjacent muscle quadrants. A microscope (Axioskop; Carl Zeiss, Inc.) equipped with a 40x water immersion lens and 15x eyepieces was used for viewing the preparation. Borosilicate glass pipettes with a tip resistance of 3–5 M
were used as electrodes for voltage clamping. The classical whole-cell configuration was obtained by rupturing the patch membrane of a gigaohm seal formed between the recording electrode and a body wall muscle cell. The cell was voltage clamped at –60 mV to record mPSCs and ePSCs. ePSCs were evoked by applying a 0.5-ms square wave current pulse at a supramaximal voltage (25 V) through a stimulation electrode placed in close apposition to the ventral nerve cord. Postsynaptic currents were amplified (EP10; HEKA) and acquired with Patchmaster software (HEKA). Data were sampled at a rate of 10 kHz after filtering at 2 kHz. The recording pipette solution contained the following: 120 mM KCl, 20 mM KOH, 5 mM TES, 0.25 mM CaCl2, 4 mM MgCl2, 36 mM sucrose, 5 mM EGTA, and 4 mM Na2ATP; pH adjusted to 7.2 with KOH and osmolarity at 310–320 mosM. The standard external solution included the following: 150 mM NaCl, 5 mM KCl, 5 mM CaCl2, 1 mM MgCl2, 5 mM sucrose, 10 mM glucose, and 15 mM Hepes; pH adjusted to 7.35 with NaOH with osmolarity 330–340 mosM.
Amplitude and frequency of mPSCs were analyzed using MiniAnalysis (Synaptosoft). A detection threshold of 10 pA was used in initial automatic analysis, followed by visual inspections to include missed events (
5 pA) and to exclude false events resulting from baseline fluctuations. Amplitudes of ePSCs were measured with Fitmaster (HEKA). The amplitude of the largest peak of ePSCs from each experiment was used for statistical analysis. Data were imported into Origin, version 7.5 (OriginLab), for graphing and statistical analysis. Unpaired t test was used for statistical comparisons. A value of P < 0.05 is considered statistically significant. All values are expressed as mean ± SEM. n is the number of worms that were recorded.
Quantification
ImageJ 1.36b was used for the pixel intensity analysis of adaptin Western blot.
Online supplemental material
Fig. S1 shows rescue of the apm-2 mutant phenotype. Fig. S2 shows that APM-2 is not mislocalized in endocytic mutants. Fig. S3 shows apm-2 aldicarb assay. Fig. S4 is a cartoon structure of apm-2::GFP DNA constructs. Fig. S5 depicts the assembly of AP2 with or without µ2. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200806088/DC1.
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Acknowledgments |
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This research was supported by National Institutes of Health grants 5R37NS034307-14 (to E.M. Jorgensen) and NS32057 (to G. Garriga).
E.M. Jorgensen is a Howard Hughes Medical Institute Investigator.
Submitted: 13 June 2008
Accepted: 30 October 2008
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