Signal-mediated, AP-1/clathrin-dependent sorting of transmembrane receptors to the somatodendritic domain of hippocampal neurons

Abstract

Plasma membranes of the somatodendritic and axonal domains of neurons are known to have different protein compositions, but the molecular mechanisms that determine this polarized protein distribution remain poorly understood. Herein we show that somatodendritic sorting of various transmembrane receptors in rat hippocampal neurons is mediated by recognition of signals within the cytosolic domains of the proteins by the μ1A subunit of the adaptor protein-1 (AP-1) complex. This complex, in conjunction with clathrin, functions in the neuronal soma to exclude somatodendritic proteins from axonal transport carriers. Perturbation of this process affects dendritic spine morphology and decreases the number of synapses. These findings highlight the primary recognition event that underlies somatodendritic sorting and contribute to the evolving view of AP-1 as a global regulator of cell polarity.

Figure 1. A tyrosine-based motif in the cytosolic tail of TfR is required for its somatodendritic sorting

(A) Amino-acid sequence of the TfR tail indicating residue numbers and a tyrosine-based YXXØ motif (red). TMD: transmembrane domain. (B) Neurons (DIV10) expressing monomeric-GFP-tagged TfR-WT, TfR-Y20A or TfRF23A (grayscale negative, top panels) and mCherry-tagged tubulin (Tub, grayscale, middle panels) were double-immunostained for MAP2 (grayscale, bottom panels) and ankyrin G (Ank-G, cyan, bottom panels). In this figure as well as other figures, MAP2 was used as a marker for dendrites and Ank-G as a marker for the axon initial segment (AIS). mCherry-tagged tubulin was a marker for both dendrites and axons. Arrows point to the AIS and arrowheads indicate the axon in each neuron. Scale bars: 20 µm. (C) Images of axons (top panels) and dendrites (bottom panels) magnified 5X from orange and magenta boxes, respectively, in B. TfR-GFP (green), mCherry-tubulin (red), MAP2 (blue), and merged images are shown. White and yellow in the merged images indicate co-localization. Experiments testing the effects of several amino acid substitutions in the TfR cytosolic tail on the polarized distribution of TfR-YFP and interactions of the TfR cytosolic tail with the µ1A subunit of AP-1 and with the AP-1 core are shown in Figure S1. Surface staining and internalization of YFP-tagged TfR-WT and TfR-Y20A are shown in Figure S2. The effects of amino acid substitutions in the cytosolic tail of CAR on the polarized distribution of GFP-tagged CAR in neurons and on its interaction with the µ1A subunit of AP-1 are shown in Figure S3.

Figure 2. Identification of residues on µ1A that are critical for interactions with tyrosine-based motifs in the cytosolic tails of TfR and CAR

(A) Surface representation of the C-terminal domain of mouse µ1A (light green) (PDB id: 1W63, Heldwein et al., 2004) with ball and stick representation of the DYQRLN peptide from TGN38 (brown) modeled after the structure of the C-terminal domain of rat µ2 in complex with this peptide (PDB id: 1BXX, Owen and Evans, 1998). Residues targeted by mutagenesis are highlighted in green. Model built with PyMOL (DeLano, W.L. The PyMOL Molecular Graphics System (2002) DeLano Scientific, San Carlos, CA). (B) Y2H analysis of the interaction of the cytosolic tails of TfR (residues 1–67), CAR (residues 261–365) or TGN38 (residues 324–353) with WT and mutant forms of µ1A. Growth on plates lacking histidine (-His) is indicative of interactions. Co-transformations of cytosolic tail constructs with SV40 T-Ag, and of µ1A constructs with p53, w ere used as negative controls, and p53-SV40 T-Ag co-transformation was used as a positive control. Results are representative of three experiment s with similar results. (C) Extracts from neurons (DIV-7) expressing transgenic HA-epitope-tagged µ1A-WT, µ1A-D174A or µ1A-W408S were subjected to immunoprecipitation (IP) with antibody to the HA epitope followed by immunoblotting (IB) with antibody to the HA epitope or to the γ-adaptin subunit of AP-1. Five percent of the input extract was run alongside for comparison. (D, E) Immunofluorescence microscopy of neurons (DIV10) expressing transgenic GFP-tagged µ1A-WT or µ1A-W408S (top panels) immunostained for endogenous γ-adaptin (D), TGN38 (E) (middle panels) and MAP2 (bottom panels, blue). Merged images are shown in the bottom panels. Yellow and white in the merged images indicate co-localization. Diamonds indicate dendritic Golgi outposts. Scale bar: 10 µm.

Figure 3. Recognition of a tyrosine-based motif in the TfR tail by µ1A is required for somatodendritic sorting of TfR

(A) Neurons (DIV10) co-expressing transgenic GFP-tagged TfR-WT (grayscale negative, top panels) and HA-tagged µ1A-WT or µ1A-W408S were immunostained for the HA epitope, MAP2 and Ank-G. GFP fluorescence is shown in these panels and the corresponding immunostaining for HA, MAP2 and Ank-G is shown in Figure S4A. Arrow points to the AIS and arrowheads indicate the axon in each neuron. Scale bars: 20 µm. (B) Images of axons (top panels) and dendrites (bottom panels) magnified 5X from orange and magenta boxes, respectively, in A. TfR-GFP (green), MAP2 (red), and merged images are shown. Yellow in the merged images indicates co-localization. Similar experiments for CAR-GFP are shown in Figure S3E. The effects of AP-1 and AP-2 subunits knock-down on the polarized distribution of TfR-YFP are shown in Figure S5.

Figure 4. Clathrin is required for somatodendritic sorting of TfR

(A) Neurons (DIV10) co-transfected with plasmids encoding GFP-tagged TfR-WT (grayscale negative, top panels) together with empty vector (control) or vector encoding T7-epitope-tagged clathrin heavy chain hub were immunostained for the T7 epitope, MAP2 and Ank-G. GFP fluorescence is shown in these panels and the corresponding immunostaining for T7, MAP2 and Ank-G is shown in Figure S4B. Arrow points to AIS and arrowheads indicate the axon in each neuron. Scale bars: 20 µm. (B) Images of axons (top panels) and dendrites (bottom panels) magnified 5X from orange and magenta boxes, respectively, in A. TfR-GFP (green), MAP2 (red), and merged images are shown. Yellow in the merged images indicates co-localization.

Figure 5. AP-1-mediated somatodendritic sorting of TfR involves exclusion from axonal carriers at the level of the soma

(A) Still image of a neuron (DIV8) expressing transgenic GFP-tagged µ1A (grayscale) taken from time-lapse, live-cell imaging shown in Movie S1. Arrow points to the AIS and arrowheads indicate the axon. Box highlights a bifurcation to a dendrite (left) and an axon (right). Scale bar: 10 µm. (B) Orthogonal analysis from box in A (X plane crossing the proximal segment of dendrite and axon, and Z corresponding to 600 s of recording) shows µ1A-GFP-containing particles crossing the X plane of the dendrite but not the axon over the time course of the experiment. See also Movie S1. Movie S2 shows axonal exclusion of TfRYFP. (C, D) Single frames from Movie S3 (top) and kymographs (bottom) of TfR-GFP-containing particles moving along 50 µm of Tau-CFP-positive axons in neurons (DIV10) co-expressing mCherry-tagged µ1A-WT or µ1A-W408S. Lines with negative (blue) and positive (red) slopes in the kymographs rep resent particles moving in anterograde and retrograde directions, respectively. Vertical lines represent particles that are stationary during 30 s of recording. (E) Quantification of the number of TfR-GFP-containing particles per 100 µm of axon in 30 s of recording time. Ant: anterograde, Ret: retrograde, Stat: stationary. Values are mean ± SD of the number of particles (n_p) indicated in the figure. (*) p<0.01.

Figure 6. Interaction with µ1A is required for somatodendritic sorting of neuronal glutamate receptor proteins

(A) Y2H analysis of the interaction of the cytosolic domains of mGluR1 (residues 841–1199), NR2A (1304–1464), NR2B (1315–1484), GluR1 (827–906), GluR2 (834–883) or TGN38 (324–353) (positive control) with WT or W408S mutant forms of µ1A, performed as described in the legend to Figure 2B. (B) Neurons (DIV10) co-expressing combinations of GFP-tagged mGluR1, NR2A, NR2B or GluR1, super-ecliptic pHluorin (SEP)-tagged GluR2 or untagged NgCAM with HA-tagged µ1A-WT or µ1A-W408S were immunostained for the HA epitope and Ank-G. The GFP or SEP signal was enhanced by immunostaining with antibody to GFP. NgCAM was also detected by immunostaining. GFP, SEP and NgCAM immunostaining is shown in these panels and the corresponding immunostaining for HA and Ank-G is shown in Figure S4C. The distribution of all glutamate receptor proteins and NgCAM is shown in grayscale negative images. Arrow points to the AIS and arrowheads to the axon in each neuron. Scale bars: 20 µm.

Figure 7. Axonal missorting of endogenous glutamate receptor proteins upon disruption of interactions with µ1A

(A–D) Neurons (DIV10) co-expressing transgenic GFP with HA-tagged µ1A-WT or µ1A-W408S were immunostained for the HA epitope, Ank-G and the endogenous glutamate receptor proteins NR2A (A), NR2B (B), GluR1 (C) and GluR2 (D). Top panels show merged images of GFP (green) and glutamate receptor proteins (red) in cells expressing HA-tagged µ1A constructs. Arrow points to the AIS and arrowheads indicate the axon of each transfected neuron. Images of axons (middle panels) and dendrites (bottom panels) magnified 5X from boxes in the top panels are also shown. Yellow in the merged images indicates co-localization. Scale bars: 10 µm.

Figure 8. Impaired spine maturation and decreased number of synapses caused by disruption of signal-recognition by µ1A

(A) Z-stack reconstruction of GFP-positive dendrites (grayscale negative) of neurons (DIV18) co-expressing GFP with HA-tagged µ1A-WT or µ1A-W408S. (B) Quantification of the protrusion density per 10 µm of GFP-positive dendrite length. (C) Dendrites from neurons (DIV18) co-expressing transgenic GFP (grayscale and green in merges) with HA-tagged µ1A-WT or µ1A-W408S were stained the excitatory postsynaptic PSD-95 (grayscale and red in merges) and the presynaptic synapsin-1 (Syp-1) (grayscale and blue in merges) markers. Scale bars: 5 µm. (D, E) Quantification of the protrusions immunoreactive for PSD-95 and synaptic contacts per 10 µm of GFP-positive dendrite length. Images in E (left) show higher magnification of synaptic contacts, observed as apposition of PSD95 (red) and synapsin-1 (blue) in GFP-positive dendrite. In all bar graphs (*) p<0.01.