Changing receptor abundance at synapses is an important mechanism for regulating synaptic strength. Synapses contain two pools of receptors, immobilized and diffusing receptors, both of which are confined to post-synaptic elements. Here we show that immobile and diffusing GABAA receptors are stabilized by distinct synaptic scaffolds at C. elegans neuromuscular junctions. Immobilized GABAA receptors are stabilized by binding to FRM-3/EPB4.1 and LIN-2A/CASK. Diffusing GABAA receptors are stabilized by the synaptic adhesion molecules Neurexin and Neuroligin. Inhibitory post-synaptic currents are eliminated in double mutants lacking both scaffolds. Neurexin, Neuroligin, and CASK mutations are all linked to Autism Spectrum Disorders (ASD). Our results suggest that these mutations may directly alter inhibitory transmission, which could contribute to the developmental and cognitive deficits observed in ASD.https://doi.org/10.7554/eLife.09648.001
Behaviors ranging from movement to memory are dependent on the activity of extensive networks of cells called neurons. Within these networks, neurons communicate across junctions called synapses. The arrival of an electrical signal called an action potential at the ‘presynaptic’ neuron on one side of the synapse triggers the neuron to release chemical neurotransmitter molecules into the synapse. These molecules then bind to receptors on the ‘postsynaptic’ cell on the other side of the synapse.
At excitatory synapses, the binding of neurotransmitter to postsynaptic receptors increases the likelihood that the postsynaptic cell will fire its own action potential. By contrast, at inhibitory synapses the binding of neurotransmitters reduces the chances of the postsynaptic cell firing. Most inhibitory synapses use a type of neurotransmitter called GABA, which exerts its effects mainly by binding to a class of receptors called GABA-activated chloride channels (also known as GABAA receptors).
GABAA receptors at inhibitory synapses can themselves be divided into two groups: ‘mobile’ receptors, which can move within the cell membrane that surrounds the postsynaptic cell; and ‘immobilized’ receptors that form clusters and cannot move. Recent work in mammalian cells identified a protein complex that anchors GABAA receptors to the cell's internal skeleton to immobilize the receptors. However, there is evidence to suggest that these are not the only proteins that control the location of the receptors.
By studying the inhibitory synapses formed between neurons and body muscles in the roundworm species Caenorhabditis elegans, Tong, Hu et al. now show that different groups of proteins maintain the positioning of immobilized and mobile receptors. Specifically, proteins called LIN-2A (a component of the cell's internal skeleton) and FRM-3 (which joins receptors to the cell's skeleton) immobilize GABAA receptors, whilst the proteins Neuroligin and Neurexin ensure that mobile GABAA receptors remain within the synapse.
Disturbances to the activity of inhibitory synapses are often seen in autism spectrum disorders, and so too are mutations in the genes that encode the mammalian equivalents of Neuroligin, Neurexin and LIN-2A. The work of Tong, Hu et al. thus suggests a mechanism by which these mutations might contribute to information processing impairments in people with autism. Further research could now investigate if (and how) other genes linked to autism spectrum disorders alter inhibitory synapses.https://doi.org/10.7554/eLife.09648.002
Fast synaptic inhibition is primarily mediated by the neurotransmitter GABA and GABA-activated chloride channels (GABAA receptors). Several studies suggest that an important mechanism for modulating inhibitory transmission is altered abundance of synaptic GABAA receptors. In mammalian neurons, variation in the amplitude of miniature inhibitory post-synaptic currents (mIPSCs) is caused by corresponding differences in the abundance of GABAA receptors at synapses (Nusser et al., 1997). Long term potentiation of GABAergic transmission is associated with increased mIPSC amplitudes and increased GABAA abundance at synapses (Petrini et al., 2014) while the converse effects are associated with long term depression (Bannai et al., 2009).
GABAA receptors on the cell surface are mobile, undergoing lateral diffusion in the plasma membrane (Jacob et al., 2005). Like all synaptic receptors, GABAA diffusion is significantly reduced at synapses, resulting in accumulation of receptors at the synapse (Jacob et al., 2005; Thomas et al., 2005; Bannai et al., 2009; Petrini et al., 2014). Local confinement of receptors at synapses is termed diffusional trapping and is mediated by binding to cytoplasmic scaffolds (Choquet and Triller, 2013). The post-synaptic scaffold that immobilizes GABAA receptors is proposed to consist of a ternary complex of Gephyrin, Neuroligin-2 (NL2), and collybistin (Jacob et al., 2005; Poulopoulos et al., 2009). Gephyrin binds directly to the large cytoplasmic loop between the third and fourth transmembrane domains (TM3-4 loop) of GABRA1 and 2 subunits (Tretter et al., 2008), thereby confining these receptors at synapses (Jacob et al., 2005; Mukherjee et al., 2011; Saliba et al., 2012). Genetic manipulations impairing the Gephyrin/NL2/Collybistin complex invariably decrease but fail to eliminate synaptic GABAA receptors (Kneussel et al., 1999; Papadopoulos et al., 2007; Poulopoulos et al., 2009). Thus, it is likely that additional proteins are involved in this process.
Within a post-synaptic element, receptors exhibit heterogenous behavior (Choquet and Triller, 2013). At both excitatory and inhibitory synapses, super-resolution imaging suggests that a subset of receptors are localized in immobile nanoclusters (∼75 nm in diameter) (Nair et al., 2013; Specht et al., 2013). These immobile receptors undergo dynamic exchange with diffusing receptors that are confined to synapses. These studies highlight several important questions. Do immobile and diffusing receptors both contribute to IPSCs? Current models propose that post-synaptic currents are mediated by immobilized receptors and synaptic plasticity is mediated by the dynamic exchange of receptors between the diffusing and immobile pools (Choquet and Triller, 2013). It has not been possible to genetically test these models because mutations that selectively disrupt the two receptor pools are not available. What are the synaptic scaffolds that stabilize immobilized and diffusing GABAA receptors? What controls the exchange between the two receptor pools?
Here we utilize the C. elegans neuromuscular junction (NMJ) as a model to address these questions. We show that immobilized and diffusing GABAA receptors are stabilized by two distinct post-synaptic scaffolds both of which contain subunits encoded by genes linked to ASD.
C. elegans body muscles receive direct inhibitory input from GABAergic motor neurons (White et al., 1986). The GABAA receptors found at these NMJs contain two subunits (UNC-49B and C) both encoded by the unc-49 gene (Bamber et al., 1999). Mutants lacking UNC-49 receptors have defects in GABA-activated muscle currents, as assessed by recording miniature inhibitory post-synaptic currents (mIPSCs) and muscle currents evoked by an exogenous GABA agonist (muscimol) (Figure 1A–E). An mIPSC corresponds to the current evoked by the fusion of a single synaptic vesicle at a GABAergic NMJ and, consequently, measures the function of synaptic UNC-49 receptors. Muscimol activates all surface UNC-49 receptors (including non-synaptic receptors in the nerve cord and muscle cell bodies). The mIPSC (Figure 1A–C) and muscimol-evoked current (Figure 1D,E) defects of unc-49 mutants were rescued by a transgene expressing GFP-tagged UNC-49B receptor in body muscles. Muscimol-evoked currents were significantly larger in GFP-UNC-49B transgenic animals (Figure 1E), presumably because this transgene is expressed at higher levels than the endogenous unc-49 gene. These results demonstrate that the GFP-tag (inserted into the TM3-4 loop) did not impair UNC-49B receptor function.
In wild type animals, GFP-UNC-49B fluorescence exhibits a punctate distribution where each punctum is closely apposed to GABAergic motor neuron nerve terminals (labelled with mCherry-tagged RAB-3) (Figure 1F), consistent with prior studies (Thompson-Peer et al., 2012). To assess the mobility of synaptic UNC-49B receptors, we measured fluorescence recovery after photobleaching (FRAP) of GFP-UNC-49B puncta in the dorsal nerve cord (Figure 1G–I). FRAP assesses the mobility of proteins in vivo whereby increased mobility is indicated by increased FRAP. UNC-49B puncta that were co-localized with mCherry-tagged RAB-3 expressed in GABAergic motor neurons were considered synaptic. In wild type controls, 40% of UNC-49B puncta fluorescence was mobile in FRAP experiments, with recovery occurring several minutes after photobleaching (Figure 1G,H). GFP-UNC-49B puncta fluorescence could comprise receptors on the cell surface and those in intracellular organelles. To more accurately assess the mobility of surface receptors, we analyzed UNC-49B receptors containing a pH-sensitive GFP (pHluorin) tag in the ecto-domain (pH-UNC-49B). pHluorin fluorescence is quenched in intracellular acidic compartments (e.g., endosomes); consequently, pHluorin fluorescence primarily results from molecules in the plasma membrane (Miesenbock et al., 1998). In wild type animals, 46% of pH-UNC-49B synaptic fluorescence recovered following photobleaching (Figure 1I). Because similar mobile fractions were observed with the GFP and pHluorin tagged receptors, these results suggest that the majority of UNC-49B puncta fluorescence results from receptors in the plasma membrane. Collectively, these results suggest that UNC-49B synaptic puncta comprise a mixture of mobile (∼40% total) and immobilized (∼60% total) receptors on the cell surface. Prior studies utilizing both FRAP and single molecule tracking techniques reported similar proportions of mobile and immobilized receptors at both excitatory and inhibitory synapses in cultured mammalian neurons (Jacob et al., 2005; Ashby et al., 2006; Heine et al., 2008).
ERM (Ezrin/Radixin/Moesin) domain containing proteins couple cell surface receptors to the actin cytoskeleton (Tepass, 2009) and are implicated in targeting synaptic glutamate receptors and extra-synaptic GABAA receptors in neurons (Biederer and Sudhof, 2001; Loebrich et al., 2006). To identify ERM proteins that could play a role in UNC-49B targeting, we screened all C. elegans ERM proteins and found that FRM-3 binds the UNC-49B TM3-4 loop in yeast 2-hybrid assays (Figure 2A). FRM-3 is a band 4.1 (EPB4.1) paralog. We did three additional experiments to determine if FRM-3 binds UNC-49B in vivo. First, we showed that a frm-3 promoter construct expressed GFP in body muscles (Figure 2—figure supplement 1A), consistent with FRM-3 function in muscles. Second, we showed that GFP-tagged FRM-3 expressed in body muscles formed puncta in the nerve cord that were co-localized with a post-synaptic marker for GABAergic NMJs (mCherry-tagged NLG-1/Neuroligin) (Pearson's correlation R = 0.80 ± 0.028, p = 0.02, n = 8) (Figure 2B) (Maro et al., 2015; Tu et al., 2015). Third, we showed that FLAG-tagged FRM-3 and GFP-UNC-49B co-immunoprecipitated from worm extracts, when both were expressed in body muscles (Figure 2—figure supplement 1B). Collectively, these results suggest that FRM-3 is localized to GABAergic synapses where it may directly bind UNC-49B receptors.
To estimate the abundance of synaptic GABAA receptors, we measured the intensity GFP-UNC-49B puncta in the dorsal nerve cord. In frm-3 mutants, UNC-49B puncta fluorescence was significantly reduced and this defect was rescued by transgenes restoring FRM-3 expression in body muscles but not by those expressed in GABAergic motor neurons (Figure 2C,D).
To determine if expression of endogenous UNC-49 receptors was also altered, we patch clamped body muscles and recorded mIPSCs and muscimol-evoked currents. mIPSC rate was unaffected in frm-3 mutants, implying that pre-synaptic GABA release was not significantly altered (Figure 2E,G). mIPSC amplitude was significantly reduced in frm-3 mutants (Figure 2E,F), consistent with decreased UNC-49 abundance at synapses. The frm-3 mIPSC amplitude defect was rescued by transgenes restoring FRM-3 expression in body muscles (Figure 2E,F), implying that FRM-3 acts in muscles to promote the function of synaptic UNC-49 receptors. Muscimol-evoked currents were unaltered in frm-3 mutants (Figure 2H); consequently, the frm-3 mutant mIPSC defect is unlikely to result from decreased bulk expression and surface delivery of UNC-49 receptors. Collectively, these results suggest that muscle FRM-3 promotes the localization and function of synaptic UNC-49 receptors but is not required for the function or trafficking of non-synaptic UNC-49 receptors.
CASK is a synaptic scaffolding protein that binds directly to EPB4.1 (Biederer and Sudhof, 2001). C. elegans has two predicted CASK isoforms (LIN-2A and B), both encoded by the lin-2 gene (Hoskins et al., 1996). LIN-2A and B share the PDZ, SH3, and GK domains while only LIN-2A contains the CaMK homology domain (Hoskins et al., 1996). LIN-2A is required for targeting epidermal growth factor receptors (EGFRs) to the basolateral domain of epithelial cells (Simske et al., 1996). The impact of LIN-2/CASK on GABAA receptors has not been determined.
Like their mammalian counterparts (Biederer and Sudhof, 2001), LIN-2A/CASK interacted with FRM-3/EPB4.1 in yeast 2-hybrid assays (Figure 3A). We did several experiments to determine if LIN-2A acts in muscles to promote UNC-49 targeting to NMJs. A lin-2 promoter construct expressed GFP in body muscles, indicating that LIN-2A may function in muscles (Figure 3—figure supplement 1A). mCherry-tagged LIN-2A expressed in muscles formed puncta in the nerve cords and these LIN-2A puncta were co-localized with GFP-UNC-49B at NMJs, consistent with LIN-2A binding to FRM-3 at NMJs (Pearson's correlation R = 0.68 ± 0.057, p = 0.04, n = 5) (Figure 3B). Like frm-3 mutants, lin-2 mutants had decreased UNC-49B puncta fluorescence (Figure 3C,D) and decreased mIPSC amplitudes (Figure 3E,F), both implying that synaptic UNC-49 levels were decreased. mIPSC rates were unaltered in lin-2 mutants (Figure 3G), indicating that pre-synaptic GABA release was unaffected. Muscimol-activated current was unaffected in lin-2 mutants (Figure 3H), indicating that the lin-2 puncta and mIPSC defects were not caused by decreased bulk expression or surface delivery of UNC-49 receptors. The lin-2 puncta and mIPSC defects were rescued by transgenes restoring LIN-2A expression in body muscles but not by those expressed in GABAergic motor neurons (Figure 3D,F). If LIN-2A and FRM-3 function together to localize UNC-49 receptors, lin-2 and frm-3 mutations should not have additive effects in double mutants. Consistent with this idea, UNC-49B puncta fluorescence and mIPSC amplitudes in frm-3 lin-2 double mutants were not significantly different from those in either single mutant (Figure 3D,F). Collectively, these results suggest that LIN-2A/CASK and FRM-3/EPB4.1 function together in body muscles to localize UNC-49B at NMJs but are not required for the expression or function of non-synaptic UNC-49 receptors.
LIN-2/CASK associates with another scaffolding complex that contains LIN-7/Velis and LIN-10/Mint subunits (Butz et al., 1998; Kaech et al., 1998). We found that mIPSC amplitudes were unaltered in lin-7 and lin-10 mutants (Figure 3—figure supplement 1B,C), indicating that this complex is not required for UNC-49 synaptic targeting.
The decreased UNC-49B synaptic abundance in lin-2 and frm-3 mutants could reflect a loss of either mobile or immobilized receptors. To distinguish between these possibilities, we measured FRAP of UNC-49B puncta in the dorsal nerve cord (Figure 4). In both frm-3 (Figure 4A) and lin-2 (Figure 4B) mutants, GFP-UNC-49B FRAP was significantly increased (Figure 4C). The frm-3 FRAP defect was rescued by transgenes restoring FRM-3 expression in body muscles (Figure 4C). A similar increase in FRAP was observed for pH-UNC-49B receptors in frm-3 mutants (Figure 4—figure supplement 1A,B). The GFP-UNC-49B transgene is likely to be expressed at higher levels than endogenous UNC-49. Over-expression could alter UNC-49B mobility at synapses. To address this possibility, we repeated the FRAP measurements using a single copy RFP-tagged UNC-49B transgene (krSi2) (Pinan-Lucarre et al., 2014). Using this single copy transgene, a similar increase in FRAP of RFP-UNC-49B was observed in frm-3 mutants (Figure 4—figure supplement 1C,D). Increased FRAP suggests that synaptic UNC-49B receptors had increased ability to undergo exchange in the nerve cord, most likely due to increased diffusional mobility in the plasma membrane. These results suggest that LIN-2A and FRM-3 function as a scaffold that stabilizes an immobile pool of UNC-49 receptors in the plasma membrane at post-synaptic elements.
Synaptic UNC-49 receptor levels and mIPSC amplitudes were decreased but not eliminated in frm-3 and lin-2 mutants; consequently, other proteins must also play a role in confining UNC-49 receptors to these synapses. In mammals, Neuroligin 2 binds gephyrin and collybistin and is required to recruit GABAA receptors to synapses (Jacob et al., 2005; Poulopoulos et al., 2009). Prompted by these results, we tested the idea that post-synaptic Neuroligin also plays a role in confining UNC-49 receptors at synapses. Several results support this idea. First, an nlg-1 promoter construct expressed GFP in body muscles (Hunter et al., 2010), consistent with NLG-1 function in muscles. Second, mCherry-tagged NLG-1 expressed in body muscles was co-localized with GFP-tagged UNC-49B receptors at NMJs (Pearson's correlation R = 0.81 ± 0.02, p = 0.01, n = 8) (Figure 5A). Third, GFP-UNC-49B puncta fluorescence was significantly reduced in nlg-1 mutants (Figure 5B,C), consistent with a decrease in total synaptic receptors. Fourth, FRAP of GFP-UNC-49B (Figure 5D,E), pH-UNC-49B (Figure 5—figure supplement 1A,B), and single copy RFP-UNC-49B (Figure 5—figure supplement 1C,D) were all significantly reduced in nlg-1 mutants, indicating that the residual synaptic UNC-49B receptors were largely immobile. These results suggest that NLG-1 stabilizes a mobile pool of surface UNC-49B receptors at synapses.
To determine if the function of endogenously expressed UNC-49 was altered, we measured GABA activated currents in body muscles. The mIPSC rate was significantly reduced in nlg-1 mutants (Figure 5F,G). The mean mIPSC amplitude (Figure 5H) was also significantly reduced, consistent with decreased abundance of synaptic UNC-49. The nlg-1 mIPSC rate and amplitude defects were both rescued by a transgene restoring NLG-1 expression in body muscles (Figure 5G,H). The decreased mIPSC rate in nlg-1 mutants could be a secondary consequence of the smaller mIPSC amplitudes (i.e. due to decreased detection of mIPSCs). Alternatively, the decreased mIPSC rate could result from decreased pre-synaptic GABA release. The muscimol-evoked current was unaffected in nlg-1 mutants (Figure 5I), indicating that the nlg-1 puncta and mIPSC defects were not caused by decreased bulk expression or surface delivery of UNC-49 receptors. Collectively, these results suggest that NLG-1 stabilizes a mobile pool of UNC-49 receptors at synapses, and that this receptor pool contributes to post-synaptic currents. These results are consistent with two recent studies, which also showed that NLG-1 promotes UNC-49 targeting to synapses (Maro et al., 2015; Tu et al., 2015).
C. elegans expresses long (NRX-1α) and short (NRX-1β) Neurexin isoforms, both encoded by the nrx-1 gene. NLG-1 binds to the sixth LNS repeat of NRX-1 (Hu et al., 2012) and, consequently, could bind to both NRX-1α and β. To test the impact of NRX-1 on UNC-49B localization, we isolated an nrx-1 null allele (nu485) that inactivates both NRX-1α and β. Mean mIPSC amplitude was significantly increased in nrx-1 null mutants (Figure 6A,B), indicating an increased number of functional UNC-49 receptors at synapses. The mIPSC rate was unaltered in nrx-1 mutants (Figure 6C), suggesting that presynaptic GABA release occurs normally. The amplitude of muscimol-activated current was also unaltered in nrx-1 mutants (Figure 6D); consequently, the increased mIPSC amplitude was unlikely to be caused by increased bulk expression or surface delivery of UNC-49 receptors. GFP-UNC-49B puncta fluorescence was also unaltered in nrx-1 null mutants (Figure 6E), suggesting that the mIPSC amplitude increase was not caused by increased abundance of synaptic UNC-49B receptors. Although UNC-49B puncta fluorescence was unaltered, FRAP of synaptic GFP-UNC-49B was significantly reduced in nrx-1 mutants (Figure 6F,G). The nrx-1 mutant defects in mIPSC amplitude (Figure 6B) and UNC-49B FRAP (Figure 6G) were both rescued by transgenes expressing NRX-1α in GABAergic motor neurons but not by those expressing NRX-1β. NRX-1α transgenes expressed in body muscles lacked rescuing activity (Figure 6B,G). Collectively, these results suggest that the total number of synaptic UNC-49B receptors was unaltered in nrx-1 mutants; however, there was a shift in receptor mobility whereby the pool of immobilized synaptic UNC-49B was enlarged (resulting in increased mIPSC amplitude) while the mobile pool was diminished. These results support the idea that pre-synaptic NRX-1α inhibits the immobilization of mobile UNC-49B receptors at synapses.
Which pool of UNC-49 receptors is required for the increased mIPSC amplitudes in nrx-1 mutants? To address this question, we recorded mIPSCs in double mutants. We found that the effect of nrx-1 mutations on mIPSC amplitude was eliminated in nrx-1; nlg-1 double mutants (Figure 6B). By contrast, the nrx-1 mIPSC amplitude defect was not blocked in nrx-1; frm-3 double mutants (Figure 6B). These results suggest that presynaptic NRX-1α inhibits diffusional trapping of NLG-1-stabilized UNC-49 receptors but has little effect on the mobility of FRM-3-associated receptors.
The preceding results suggest that UNC-49B synaptic puncta comprise two pools of receptors that are stabilized by different scaffolds. Immobilized UNC-49B receptors fail to undergo diffusional exchange in FRAP experiments and are stabilized by FRM-3 and LIN-2A. Mobile UNC-49B receptors mediate fluorescence recovery in FRAP experiments and are stabilized by NLG-1 and NRX-1α. In this scenario, we expect that double mutants lacking both scaffolds would have additive defects, lacking both receptor pools. Consistent with this idea, UNC-49B puncta fluorescence was significantly reduced (Figure 5B,C) while GFP-UNC-49B FRAP was significantly increased (Figure 7A,B) in frm-3 nlg-1 double mutants compared to the corresponding single mutants. In frm-3 nlg-1 double mutants, both the immobile pool of synaptic UNC-49B (Figure 7A,B) and mIPSCs (Figure 5F) were completely eliminated. Given the absence of mIPSCs, we could not measure quantal size in double mutants. As an alternative, we measured mIPSC rate and found that it was dramatically reduced in frm-3 nlg-1 double mutants compared to either single mutant (Figure 5G). Muscimol-activated muscle current in frm-3 nlg-1 double mutants did not significantly differ from wild type controls (Figure 5I), suggesting that the puncta, FRAP, and mIPSC defects were not caused by decreased bulk expression or surface delivery of UNC-49 receptors. These results support the idea that FRM-3 and NLG-1 stabilized UNC-49B receptors represent two distinct pools of synaptic receptors, which together account for all synaptic UNC-49 receptors.
Analysis of excitatory and inhibitory synapses in mammals suggests that differences in synaptic receptor abundance contribute significantly to variation in quantal sizes (Nusser et al., 1997, 1998). The coefficient of variation (CV) of the mIPSC amplitudes was ∼0.6–7 in wild type animals (Figure 7C,D), which is similar to values reported for some mammalian CNS synapses (Bekkers et al., 1990; Hanse and Gustafsson, 2001). If quantal variability is dominated by diffusion of UNC-49 receptors in and out of the postsynaptic site, the mIPSC amplitude CV should scale inversely with the square root of receptor number. In this scenario, we expect that mutations decreasing UNC-49B punta fluorescence (e.g., frm-3, lin-2, and nlg-1) would produce corresponding increases in the CV of mIPSC amplitudes. Contrary to this idea, CV was significantly reduced in frm-3 and nlg-1 mutants and was not significantly altered in lin-2 mutants (Figure 7C,D). The frm-3 and nlg-1 mIPSC amplitude CV defects were rescued by transgenes that restore expression of the corresponding genes in body muscles (Figure 7C,D). Changes in CV were also not correlated with changes in mean mIPSC amplitude. Mean mIPSC amplitude was increased in nrx-1 mutants (Figure 6B) and decreased in lin-2 mutants (Figure 3F) while CV was unaltered in both cases (Figure 7C). Collectively, these results suggest that the FRM-3 and NLG-1 scaffolds increase the diversity of quantal responses, and that this effect cannot be explained by changes in synaptic UNC-49 levels nor by changes in mean mIPSC amplitude.
We have analyzed mechanisms that spatially confine UNC-49/GABAA receptors at the C. elegans NMJ. Our results lead to seven conclusions. First, UNC-49 synaptic puncta comprise both mobile and immobilized receptors, consistent with prior studies analysing mammalian synapses. Second, immobilized UNC-49B is stabilized at synapses via binding to FRM-3/EPB4.1 and LIN-2A/CASK. Third, mobile UNC-49B is stabilized at synapses by post-synaptic NLG-1/Neuroligin. Fourth, pre-synaptic NRX-1α/Neurexin inhibits the immobilization of NLG-1 stabilized UNC-49B. Fifth, the FRM-3 and NLG-1 stabilized receptor pools both contribute to inhibitory synaptic transmission. Sixth, both scaffolds increase the variability of quantal responses. And seventh, all synaptic UNC-49 receptors can be explained by these two scaffolds. Below we discuss the significance of these findings.
Synaptic UNC-49/GABAA receptors are confined by two scaffolds that function in parallel. The FRM-3/EPB4.1 and LIN-2A/CASK scaffold accounts for ∼40% of synaptic receptors while the NLG-1 scaffold accounts for ∼30% of synaptic UNC-49 (assessed by puncta fluorescence). These scaffolds define two pools of synaptic GABAA receptors that function independently. In double mutants lacking both pools, immobilized synaptic UNC-49 receptors and mIPSCs are eliminated. Mobile receptors detected by FRAP in frm-3 nlg-1 double mutants likely correspond to extra-synaptic receptors on the cell surface (which mediate muscimol-activated currents). Thus, the FRM-3 and NLG-1 scaffolds together account for all GABAA receptors at the body wall NMJ. Consequently, the dynamics of GABAA levels at this synapse will be determined by the detailed biochemistry of these two scaffolds. Our results also suggest that synaptic receptors represent a small fraction of total surface UNC-49 receptors. Mutants lacking both scaffolds have no defect in total surface receptors (assessed by muscimol-activated currents). Thus, these scaffolds have no effect on the assembly, anterograde trafficking, and surface delivery of UNC-49 receptors. Instead, these scaffolds play a specific role in concentrating UNC-49 surface receptors at synapses.
Our results strongly support the idea that both GABAA pools are required for inhibitory synaptic transmission. Mutations inactivating the FRM-3/LIN-2A scaffold caused similar decreases in immobilized UNC-49B (40% decrease) and mIPSC amplitude (30% decrease). Inactivating NLG-1 caused corresponding decreases in total synaptic UNC-49B (30% decrease), mobile UNC-49B (40% decrease), and mIPSC amplitudes (48% decrease). Thus, analysis of single mutants suggests that FRM-3 and NLG-1 stabilized receptors contribute equally to post-synaptic currents.
We show that FRM-3/EPB4.1 and LIN-2A/CASK together comprise a scaffold that immobilizes synaptic GABAA receptors. Other ERM domain containing proteins were previously implicated in neurotransmitter receptor localization. The ERM protein Radixin stabilizes extra-synaptic GABRA5 receptors in mammalian neurons (Loebrich et al., 2006), suggesting that different populations of GABA receptors are stabilized by distinct ERM proteins. Mammalian EPB4.1 and its Drosophila counterpart (coracle) were previously implicated in synaptic localization of glutamate receptors (Shen et al., 2000; Chen et al., 2005). Excitatory and inhibitory synaptic transmission were not significantly altered in mouse CASK knockouts (Atasoy et al., 2007); however, subtle defects (e.g., the ∼20% decrease in quantal size reported here) could have been missed in this study. Drosophila CASK mutants have decreased targeting of glutamate receptors to larval NMJs (Chen and Featherstone, 2011). A role for CASK in synaptic targeting of GABAA receptors has not been described.
We found that frm-3 EPB4.1 and lin-2 CASK mutants have decreased synaptic GABAA levels, decreased immobilization of synaptic GABAA receptors, and corresponding decreases in mIPSC amplitudes. Both mutants had significant residual levels of immobile synaptic GABAA receptors and post-synaptic currents. Thus, FRM-3 and LIN-2 define a sub-population of immobilized synaptic GABAA receptors, accounting for ∼40% of total synaptic receptors. For all synaptic phenotypes, lin-2 defects were less severe than those observed in frm-3 mutants, perhaps because LIN-2/CASK regulates UNC-49 targeting by modulating FRM-3 function. For example, prior studies suggest that ERM proteins equilibrate between active (open) and inactive (closed) conformations (Pearson et al., 2000). It is possible that LIN-2/CASK binding stabilizes the active conformation of FRM-3. Collectively, these results suggest that ERM and CASK proteins play a conserved role in targeting neurotransmitter receptors. In each case, CASK and ERM proteins account for a subset of receptors confined to a synapse.
In mammals, Neuroligin 2 works in conjunction with gephyrin and collybistin to localize GABAA receptors to synapses. Knockout mutations in each of these genes reduce but do not eliminate synaptic GABAA receptors (Mukherjee et al., 2011; Nair et al., 2013; Specht et al., 2013). Gephyrin binding to the TM3-4 loop has been shown to immobilize GABAA receptors at synapses (Jacob et al., 2005; Mukherjee et al., 2011). The impact of Neuroligin 2 mutations on GABAA receptor mobility has not been determined. Our results are largely consistent with these prior studies. Mutations inactivating NLG-1 decreased but did not eliminate synaptic UNC-49 and caused a corresponding decrease in mIPSC amplitudes. Two recent studies reported similar effects of nlg-1 mutations on synaptic UNC-49 levels (Maro et al., 2015; Tu et al., 2015). Thus, as in mammalian neurons, NLG-1 represents one of multiple mechanisms for confining GABAA receptors at synapses. NLG-1 association with UNC-49 could be mediated by binding to an intermediary Gephyrin-like molecule. The C. elegans ortholog of gephyrin is MOC-1; however, its role in synaptic targeting of UNC-49 has not been determined.
Our studies provide further insights into how Neurexin and Neuroligin function to target synaptic GABAA receptors. Two results suggest that the NLG-1 stabilized pool consists of both mobile and immobilized UNC-49 receptors. First, in double mutants lacking both scaffolds (i.e. frm-3 nlg-1 double mutants), immobilized UNC-49B receptors and mIPSCs were both eliminated. This result implies that immobile UNC-49B receptors observed in frm-3 single mutants are derived from the NLG-1-stabilized pool and that immobilization of these receptors does not require FRM-3. Second, mIPSC amplitudes and immobile synaptic UNC-49B levels are significantly increased in mutants lacking presynaptic NRX-1α. Both of these effects were abolished in nrx-1; nlg-1 double mutants. These results suggest that pre-synaptic NRX-1α inhibits immobilization of diffusing NLG-1-stabilized UNC-49 receptors. Trans-synaptic NRX-1α/NLG-1 complexes may confine mobile synaptic receptors via low affinity binding interactions with UNC-49B, or by sterically interfering with UNC-49B lateral diffusion (Santamaria et al., 2010). Because NLG-1 stabilizes both forms of synaptic receptors, the contribution of the NLG-1 pool to post-synaptic currents could be mediated by immobilized or diffusing UNC-49 receptors (or a mixture of the two).
Synaptic responses are intrinsically noisy, varying considerably even among inputs to the same cell. This noise arises from variability in both pre- and post-synaptic processes (Lisman et al., 2007). Prior studies suggested that an important contributor to post-synaptic noise is variation in receptor levels between synapses (Nusser et al., 1997), which can be adjusted bidirectionally by activity (Bannai et al., 2009; Petrini et al., 2014).
How do FRM-3 and NLG-1 alter quantal size? Prior modelling studies suggest that the non-homogeneous distribution of synaptic receptors into nanoclusters has profound effects on synaptic transmission (MacGillavry et al., 2013; Nair et al., 2013). Although these studies analyzed glutamatergic synapses, in the following we assume that similar principles apply to GABAergic synapses. Within post-synaptic elements, ∼60% of receptors are localized in immobile nanoclusters (mean diameter 75 nm, mean receptor number 25) while the remaining ∼40% are mobile and diffusely distributed (MacGillavry et al., 2013; Nair et al., 2013). The effect of receptor nanoclusters on transmission results from the fact that a single synaptic vesicle (SV) fusion activates receptors in a subsynaptic domain. For glutamate, the domain of activated receptors is estimated to have diameter ∼200 nm (Raghavachari and Lisman, 2004). The size of the GABA domain has not been calculated, but is likely to be larger (due to the higher affinity of GABAA receptors). SV fusions at C. elegans cholinergic and GABAergic NMJs occur in an active zone with a diameter of 700 nm (Hammarlund et al., 2007; Watanabe et al., 2013). Thus, the spatial extent of GABA in the synaptic cleft will vary depending on the location of the SV fusion event within the active zone (Barberis et al., 2011). Consequently, quantal amplitude will vary depending on the proximity of the vesicle fusion site to the receptor nanocluster and the density of receptors in each nanocluster (Franks et al., 2003; MacGillavry et al., 2013; Nair et al., 2013). By contrast, mobile (unclustered) receptors have lower but uniform surface density; consequently, mobile receptors are predicted to mediate smaller quantal responses that have lower CV (MacGillavry et al., 2013). Our results are largely consistent with these modelling studies. The FRM-3 and NLG-1 scaffolds increase mIPSC amplitude and CV, presumably due to an increase in the number of UNC-49 nanoclusters at synapses or an increase in the number of receptors in each nanocluster.
Changes in synaptic inhibition are proposed to play an important role in the pathophysiology of several neuropsychiatric disorders. Decreased inhibition is implicated in autism spectrum disorders (ASD) (Rubenstein and Merzenich, 2003), whereas excess inhibition has been proposed to occur in mental retardation syndromes, such as Down and Rett Syndromes (Kleschevnikov et al., 2004; Dani et al., 2005). Recurrent mutations in Neurexin, Neuroligin, and CASK are found in ASD (Sanders et al., 2012; O'Roak et al., 2012). We propose that these mutations may directly alter inhibitory transmission by altering the synaptic confinement of GABAA receptors. These results provide additional biochemical links between ASD associated genes and inhibitory transmission. As all of these molecules are conserved, the mechanisms we describe for confining synaptic GABAA receptors are likely to be conserved in other systems, including humans.
Strains were maintained at 20° C under standard conditions. OP50 Escherichia coli was used as a food source for all experiments except electrophysiology where HB101 E. coli was utilized. A description of all alleles can be found at www.wormbase.org. The following strains were utilized in this study:
KP7532 nrx-1(nu485); nlg-1(ok259)
KP7367 frm-3(gk585) nlg-1(ok259)
KP7534 nrx-1(nu485); frm-3(gk585)
KP7514 frm-3(gk585) lin-2(e1309)
KP5931 nuIs283 [Pmyo-3::unc-49::gfp::unc-54 3′UTR; Punc-25::snb-1::mcherry::unc-54 3′UTR]
KP7341 nuIs283; frm-3(gk585)
KP7478 nuIs283; nrx-1(nu485)
KP7133 nuIs283; lin-2(e1309)
KP7596 nuIs283; nlg-1(ok259)
KP7474 nuIs283; frm-3(gk585) nlg-1(ok259)
KP7340 nuIs283; frm-3(gk585) lin-2(e1309)
KP7597 nuIs283; unc-49(e407);
KP7545 nuIs522 [Pmyo-3::lin-2::mcherry::unc-54 3′UTR];
KP7552 nuIs523 [Pmyo-3::pHluorin::unc-49::unc-54 3′UTR];
KP7614 nuIs523; nlg-1(ok259);
KP7615 nuIs523; frm-3(gk585);
KP7553 nuIs524 [Pmyo-3::gfp::frm-3::unc-54 3′UTR];
KP7364 nuEx490 [Pfrm-3::gfp::unc-54 3′UTR];
KP7363 nuEx489 [Plin-2::gfp::unc-54 3′UTR];
KP7631 nuIs532 [Pmyo-3::NLG-1::mcherry::unc-54 3′UTR];
EN2630 LGII, krSi2 [Punc-49::unc-49B-RFP; unc-49(e407)
EN3224 LGII, krSi2 [Punc-49::unc-49B-RFP; unc-49(e407); nlg-1(ok259)
KP7894 LGII, krSi2 [Punc-49::unc-49B-RFP; unc-49(e407); frm-3(gk585)
KP7893 nuIs531 [Pmyo-3::frm-3::2flag::unc-54 3′UTR]; nuIs283
The nrx-1(nu485) null allele was isolated using the mosDEL protocol (Frokjaer-Jensen et al., 2010). The C. elegans transposon insertion line ttTi26330 was obtained from the NemaGENETAG consortium. 10.416 kb of the nrx-1 locus was replaced with a Cb-unc-119(+) selectable marker (Frokjaer-Jensen et al., 2010). The engineered deletion included 982bp of upstream sequence and the first 9434bp of reference transcript C29A12.4a. The nu485 mutation deletes exons 1–20, or 85%, of the nrx-1 coding sequence. Exons 21–27, downstream of the ttTi26330 insertion site, were left intact. These could potentially encode a 234-residue C-terminal NRX-1 fragment. The EN2630 and EN3224 strains were kindly provided by Dr. Jean-Louis Bessereau.
All expression vectors are based on the pPD49.26 backbone (A. Fire). Standard methods were utilized to construct all plasmids. A 3 kb myo-3 myosin promoter was used for expression in body muscles, a 1.2 kb unc-25 GAD promoter was used for expression in GABAergic neurons. The transcriptional reporter for frm-3 and lin-2 used 5 kb of 5′ flanking sequence. nlg-1 (C40C9.5c), nrx-1α (C29A12.4a), nrx-1β (C29A12.4f), frm-3 (H05G16.1), lin-2 (F17E5.1a), and unc-49B (T21C12.1b) cDNAs were cloned from a cDNA library using primers corresponding to the predicted start and stop codons of the indicated EST. The ERM domain of frm-1, frm-2, frm-3, frm-4, frm-5.1, frm-8, frm-9, frm-10, max-1, kin-32, kri-1, erm-1, nfm-1 cDNAs were cloned from a cDNA library using primers corresponding to the predicted start and stop codons of ERM domain. nuIs524 contains GFP tagged FRM-3 cDNA constructs in which GFP was inserted in frame in the C-terminus. nuIs523 contains pHluorin tagged UNC-49B cDNA constructs in which super-ecliptic pHluorin was inserted in-frame immediately after the signal peptide sequence. nuIs522 contains an mCherry-tagged LIN-2 cDNA construct, in which mCherry is inserted in the N-terminus. nuIs532 contains an mCherry-tagged NLG-1 cDNA construct, in which mCherry was inserted 13 residues from the carboxy-terminus (leaving the predicted PDZ ligand intact). nuIs531 contains an FLAG-tagged FRM-3 cDNA construct, containing two copies of the FLAG epitope at the C-terminus. Full descriptions of all plasmids are available upon request. Transgenic animals were prepared by microinjection, and integrated transgenes were isolated following UV irradiation.
For yeast two-hybrid screens all the ERM domain containing proteins, Y2HGold yeast cells were co-transformed with pGADT7-UNC-49-TM3-4 loop and all the pGBKT7-ERM constructs respectively (BD clontech). Transformants selected from the SD-Leu-Trp plates were restreaked onto SD-Leu-Trp-His-Ade plates to test interactions. False positive from autoactivation was ruled out by co-transformation of pGBKT7-ERM construct with pGADT7 empty vector alone.
Strains for electrophysiology were maintained at 20°C on plates seeded with HB101. Cyanoacrylate glue was used to immobilize adult worms on the Sylgard-coated coverslip. The dissected adult worms were superfused in an extracellular solution containing 127 mM NaCl, 5 mM KCl, 26 mM NaHCO3, 1.25 mM NaH2PO4, 20 mM glucose, 1 mM CaCl2, and 4 mM MgCl2, bubbled with 5% CO2, 95% O2 at 20°C. Endogenous GABA IPSC recordings were carried out at 0 mV using an internal solution containing 120 mM CsCH3SO3, 4 mM CsCl, 15 mM CsF, 4 mM MgCl2, 5 mM EGTA, 0.25 mM CaCl2, 10 mM HEPES, and 4 mM Na2ATP, adjusted to pH 7.2 using CsOH. For muscimol-activated current recordings, 100 μM muscimol was pressure ejected for 1.0 s onto body muscles of adult worms.
For colocalization studies, images were captured using a 60x objective (NA 1.45) on an Olympus FV-1000 confocal microscope at 5× digital zoom. Worms were immobilized with 30 mg/ml 2,3-Butanedione monoxamine (Sigma, St. Louis, MO, United States). UNC-49B puncta fluorescence was quantified by wide field fluorescence microscopy in, the dorsal nerve cord of young adults (midway between the vulva and the tail). Images were acquired using a Zeiss Axioskop I, Olympus PlanAPO 100× 1.4 NA objective, and a CoolSnap HQ CCD camera (Roper Scientific, Tuscon, AZ, United States). Maximum intensity projections of Z-series stacks were made using Metamorph 7.1 software (Molecular Devices, Sunnyvale, CA, United States). Line scans of dorsal cord fluorescence were analyzed in Igor Pro (WaveMetrics, Lake Oswego, OR, United States) using custom-written software. Mean fluorescence of 0.5 μm FluoSphere beads (Thermo Fisher, Waltham, MA, United States), which was measured during each experiment, was used to control the illumination intensity. All fluorescence values are normalized to wild type controls to facilitate comparison. All p-values indicated were based on ONE-way ANOVA or student t-tests.
For FRAP studies, images were captured using a 60x objective (NA 1.45) on an Olympus FV-1000 confocal microscope at 5× digital zoom. Worms were immobilized with 0.1 μm polystyrene microspheres (Polysciences), and pads composed of 10% agarose in M9. To image GFP-UNC-49B, pH-UNC-49B, and RAB-3-mCherry, we used 0.5% power from a 473 nm (GFP) and 559 nm (mCherry) solid state diode laser. Five frames of GFP and mCherry signals were recorded prior to photobleaching. Photobleaching was achieved by one scan (90% power from the 473 nm laser) on a square region of interest (ROI, about 1.5 × 1.5 μm) that covered a single GFP punctum. GFP and mCherry signals were further recorded for 5 min at 0.2 frames/second. To eliminate motion artifacts, traces with more than 10% changes in mCherry signals were discarded. UNC-49B mobile fractions were calculated in MATLAB by fitting the data with a single exponential function, Ifrap(t) = A(1-e-τt), where A is reported as the mobile fraction. Statistical significance was determined using ONE-way ANOVA or Student's t test and all values reported are means ±SEM.
Extracts were prepared from mixed staged worms expressing GFP-UNC-49 or GFP-UNC-49 and FLAG-FRM3 using a microfluidizer in buffer A (50 mM HEPES PH7.7, 50 mM KAc, 2mMgAc2, 250 mM sucrose, 1 mM EDTA and proteinase inhibitors). Worm extracts were clarified by centrifugation (15 min, 7000g). Membranes were isolated from the resulting supernatant by high speed centrifugation (40 min, 45,000g). Membrane proteins were solubilized with a dounce homogenizer (5 times) in IP buffer (20 mM HEPES PH7.4, 150 mM NaCl, 2 mM MgCl2, 0,1 mM EDTA, 1% Triton and proteinase inhibitors), and incubated with FLAG M2 affinity gel (A2220) overnight at 4o. Flag affinity gel were washed three times with IP buffer, and eluted with loading buffer for Western blot.
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Eunjoon KimReviewing Editor; Korea Advanced Institute of Science and Technology, Republic of Korea
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Thank you for submitting your work entitled “A network of Autism linked genes stabilizes two pools of synaptic GABAA receptors” for peer review at eLife. Your submission has been favorably evaluated by Gary Westbrook (Senior Editor) and three reviewers, one of whom is a member of our Board of Reviewing Editors. One of the three reviewers, Kang Shen (Reviewer 2), has agreed to reveal his identity.
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Previous single molecule-receptor tracking experiments have identified mobile and immobile pools of postsynaptic receptors at excitatory and inhibitory synapses, although these results rely on mostly in vitro results. Tong et al. employ in vivo approaches to demonstrate that there are mobile and immobile pools of GABAA receptors (UNC-49) at C. elegans neuromuscular junctions. In addition, they explore molecular mechanisms that control UNC-49 localization and report distinct phenotypes for two different scaffolds, one consisting of the trans-synaptic neurexin-neuroligin complex and one consisting of a cytoplasmic scaffold containing FRM-3 and LIN-2. Mutation of each scaffold individually produces specific phenotypes with respect to receptor mobility (assessed by FRAP) and synaptic transmission. Importantly, the authors find that FRM-3 and NLG-1, function in parallel to recruit GABAA receptors to synapses, and that simultaneous inactivation of both scaffolds results in a near complete loss of GABAergic transmission and receptor stabilization at synapses.
This work complements two very recent studies (Shen and Bessereau labs, Neuron, 2015) that reported the importance of NLG-1 in synaptic recruitment of UNC-49. The identification of a function for FRM-3 for synaptic UNC-49 concentration is novel and important. Moreover, an important concept advanced by this work is the presence of multiple systems that act in parallel to stabilize functionally different pools of postsynaptic neurotransmitter receptors at the same class of synapse. This concept is backed up by strong in vivo data, and is accompanied by specific molecular mechanisms that stabilize the two different pools of receptors.
1) The authors show that the UNC-49 fluorescence is reduced in the nlg-1 mutant. They also show that the FRAP rate is modified by the nlg-1 mutation and that the frm-3 mutant phenotype is greatly enhanced by the nlg-1 mutation. These data argue strongly that nlg-1 and frm-3 function in parallel, both contributing to the synaptic targeting of UNC-49. The one piece of data that does not support this model is the lack of changes of either mIPSC frequency or amplitude. Nevertheless, two recent papers (Tu et al. and Maro et al.) did report electrophysiological phenotypes in nlg-1 mutant. Both papers showed a strong reduction of mIPSC frequency and a subtle reduction of mIPSC amplitude. All these results seem to be consistent with the parallel model. Based on the lack of observed functional phenotypes, the authors propose that the nlg-1 anchored UNC-49 receptors do not contribute to the GABAergic synaptic transmission. The authors have several options to deal with this discrepancy. First, the authors can provide reasons why the recording conditions that they used are better than the two published reports. Or, they can provide additional evidence to show that the NLG-1-dependent UNC-49 receptors are inactive and do not contribute to synaptic currents. Or, the authors can modify their conclusions.
2) The data from the different genetic manipulations strongly suggests that there are different pools of UNC-49 defined by their differentially dependence on NLG-1 and FRM-3. What is not entirely clear is whether “mobile” and “immobile” is an accurate classification. If the NLG-1-dependent pool is “mobile” then why does mobility in frm-3, nlg-1 double mutants increase compared to frm-3 mutants alone? The authors suggest that this “immobilization of the mobile pool” is correlated to these receptors participating in synaptic transmission in the mutant context but a mechanism to explain this is not offered. Additional data or indications to clarify this point this would greatly improve the paper.
3) One potential weakness of the receptor mobility (FRAP) experiments is the use of transgenically overexpressed UNC-49 receptors. Thus, the size of mobile and immobile pools may differ between conditions. What supports the validity of the transgenically expressed receptors is that the mIPSC amplitudes and rates are similar to wild-type. Also, the magnitude of loss of UNC-49-GFP fluorescence in nlg-1 mutants is similar to what was reported by Bessereau and colleagues who used a tag introduced into the endogenous nlg-1. As the mobility measurements are a central part of this study this issue needs to be addressed somewhere in the text.
4) This study does not detect a significant reduction in mIPSC amplitude in nlg-1 mutants, which is different from the Bessereau and Shen work. In the Discussion the authors point out that nlg-1 mutant animals are behaviorally normal and suggest that this supports their observation that GABAergic transmission is not severely impaired. This important point could be made much more clearly if the authors included data on this observation for nlg-1, frm-3 and nlg-1:frm-3 double mutants.
5) The authors state in Discussion that “mobile NLG-1-stabilized UNC-49 receptors can be actively recruited to the receptor field that mediates synaptic responses, and that this recruitment is inhibited by pre-synaptic NRX-1α”. Given that NLG-1 mutation suppresses UNC-49B puncta (Figure 9C), perhaps NRX-1α is inhibiting the translocation of NLG-stabilized UNC-49B from a subsynaptic region that is not affected by GABA to an adjacent subsynaptic region that mediates GABA synaptic transmission in the same synapse? If this is the case, how is it possible that GABA released from the nerve terminal acts so specifically on a subsynaptic region but not on another in the same inhibitory synapse? This should be clarified.https://doi.org/10.7554/eLife.09648.014
- Xia-Jing Tong
- Zhitao Hu
- Dorian Anderson
- Joshua M Kaplan
- Xia-Jing Tong
- Zhitao Hu
- Yu Liu
- Dorian Anderson
- Joshua M Kaplan
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
We thank the following for strains and reagents: the C. elegans Genetics Stock Center and Jean-Louis Bessereau. We thank members of the Kaplan lab, Peter Juo, Jihong Bai, and Jeremy Dittman for helpful discussions and critical comments on the manuscript.
- Eunjoon Kim, Reviewing Editor, Korea Advanced Institute of Science and Technology, Republic of Korea
- Received: June 24, 2015
- Accepted: October 15, 2015
- Version of Record published: November 17, 2015 (version 1)
© 2015, Tong et al.
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.