Clathrin adaptor AP-1 complex excludes multiple postsynaptic receptors from axons in C. elegans

Abstract

Neurons are highly polarized cells with morphologically and molecularly distinct axonal and dendritic compartments. It is not well understood how postsynaptic receptors are selectively enriched in dendrites in vivo. We investigated the molecular mechanisms of dendritically polarized localization of a glutamate receptor, an acetylcholine receptor, and a ROR-type receptor tyrosine kinase in the interneuron RIA in C. elegans. We found that the clathrin adaptor AP-1 complex mu1 subunit UNC-101 functions cell autonomously to maintain the correct localization of these receptors in a dynamin-dependent manner. In unc-101 mutants, instead of being dendritically enriched, all 3 receptors are evenly distributed in the axonal and dendritic compartments. Surprisingly, UNC-101 predominantly localizes to the axonal compartment, suggesting a possible transcytosis model for the dendritic targeting of neurotransmitter receptors.

Fig. 1.

AP-1 subunit μ1/unc-101 is required for polarized localization of glutamate receptor GLR-1 in RIA neurons. (A) Schematic diagrams of the left RIA neuron as viewed from the left and the anterior side of the worm, respectively. RIA neurite is polarized, with a proximal region that is exclusively postsynaptic (green), followed by an asynaptic isthmus region (black), and a distal, mainly presynaptic region (red). (B and C) Representative wild-type animal expressing synaptic vesicle marker mCherry::RAB-3 (B) and AMPA-type glutamate receptor subunit GLR-1::GFP (C). For this and all subsequent images, asterisk denotes the RIA cell body; arrows point to the presynaptic region of the RIA neurite. Anterior is to the left, ventral is down. (Scale bar: 10 μm.) High-magnification views of RIA presynaptic region are shown in B′ and C′, and of postsynaptic region in B′′ and C′′. (D) A diagram depicting localization of GLR-1::GFP in a wild-type RIA neuron. (E and F) Representative unc-101(m1) animal expressing mCherry::RAB-3 (E) and GLR-1::GFP (F). GLR-1::GFP is mislocalized to the presynaptic region of the RIA neurite in unc-101 mutants (arrows). High-magnification views of RIA presynaptic region are shown in E′ and F′, and of postsynaptic region in E′′ and F′′. (G) A diagram depicting localization of GLR-1::GFP in an unc-101 RIA neuron. (H) Polarity index quantification for mCherry::RAB-3 and GLR-1::GFP in wild-type and unc-101 mutants. ***P < 0.001; error bars, SEM.

Fig. 2.

μ1/unc-101 is required for dendritic localization of 2 additional postsynaptic receptors in RIA neurons, and acts cell autonomously to maintain polarized receptor distribution. (A and B) α7-type nicotinic acetylcholine receptor subunit ACR-16::GFP localizes selectively to the RIA postsynaptic region in wild-type animals (A), but is mislocalized to the presynaptic region (arrows) in unc-101(m1) mutants (B). (C and D) ROR-type receptor tyrosine kinase CAM-1::YFP localizes selectively to the RIA postsynaptic region in wild-type animals (C) but is mislocalized to the presynaptic region (arrows) in unc-101(m1) mutants (D). (E) Polarity index quantification for ACR-16::GFP and CAM-1::YFP in wild-type and unc-101(m1) animals. In addition, polarity indices for 2 presynaptic markers, SNB-1::YFP and SNG-1::YFP are also shown. (F) Polarity indices quantification for GLR-1::GFP, ACR-16::GFP, and CAM-1::YFP in unc-101(m1) mutants expressing unc-101 cDNA under the control of either RIA-specific glr-3 promoter or heat-shock promoter. ***P < 0.001; error bars, SEM.

Fig. 3.

Cytosolic domains of postsynaptic receptors are necessary and sufficient for dendritic localization in RIA neurons, in a manner dependent on unc-101. (A) Schematic representation of GLR-1, CAM-1, and ACR-16. Transmembrane regions are shown in yellow, key cytosolic regions in red. (B) Localization of the truncated GLR-1 lacking its C-terminal cytoplasmic domain, GLR-1_Δ876–962::YFP, in wild-type RIA neurons. (C) Localization of truncated CAM-1 lacking its C-terminal cytoplasmic domain, CAM-1_Δ467–902::YFP, in wild-type RIA neurons. (D) Polarity index quantification. (E) PAT-3(TM)::YFP is unpolarized in RIA neurons. Inset in this and subsequent images shows the schematic representation of the construct. (F and G) RIA localization of PAT-3(TM)::GLR-1_876–962::YFP in wild-type (F) and unc-101(m1) (G) animals. (H and I) RIA localization of PAT-3(TM)::CAM-1_467–902::YFP in wild-type (H) and unc-101 (I) animals. (J and K) RIA localization of PAT-3(TM)::ACR-16_314–472::YFP in wild-type (J) and unc-101 (K) animals. (L) Polarity index quantification for PAT-3 gain-of-function constructs. ***P < 0.001; error bars, SEM.

Fig. 4.

Juxtamembrane 30 aa of GLR-1 are necessary and sufficient for RIA postsynaptic localization. (A) Amino acid sequence of GLR-1 cytoplasmic C terminus and alignment with vertebrate glutamate receptor C termini. Sequence boxed in red is necessary and sufficient for GLR-1 dendritic localization. (B) PAT-3 fused to juxtamembrane 30 aa of GLR-1 (PAT-3(TM)::GLR-1_876–905::YFP) is dendritically enriched in RIA neurons. (C) PAT-3 fused to distal 56 aa of GLR-1 (PAT-3(TM)::GLR-1_906–962::YFP) is more uniformly distributed in RIA neurons. (D) GLR-1 lacking juxtamembrane 30 aa (GLR-1_Δ876–905::YFP) is unpolarized in RIA neurons. (E) GLR-1 lacking the distal 56 aa of its C terminus (GLR-1_Δ906–962::YFP) localizes to the RIA dendrite. (F) Polarity index quantification. ***P < 0.001; error bars, SEM.

Fig. 5.

μ1/UNC-101 localizes to presynaptic sites, where it may be involved in retrieval of postsynaptic receptors. (A and B) Localization of UNC-101::YFP to the presynaptic region of RIA neurons (A), and the accompanying schematic (B). (C–E) DA9 localization of UNC-101::YFP (C), mCherry::RAB-3 (D), and the overlay (E). (F) GLR-1::GFP is mislocalized to the presynaptic region of RIA (arrows) in the presence of dynamin DYN-1(K46A). (G) Polarity index quantification for GLR-1::GFP, ACR-16::GFP, and CAM-1::YFP in the absence and presence of DYN-1(K46A). ***P < 0.001; error bars, SEM. (H) DYN-1(K46A)::YFP localizes to the presynaptic region of RIA neurons.