The tetraspanin TSPAN5 regulates AMPAR exocytosis by interacting with the AP4 complex
- Institute of Neuroscience, CNR, Italy
- NeuroMI Milan Center for Neuroscience, University of Milano-Bicocca, Italy
- University of Bordeaux, Interdisciplinary Institute for Neuroscience, France
- Department of Life Sciences, University of Trieste, Italy
- IRCCS Ospedale Policlinico San Martino, Italy
- Center for Synaptic Neuroscience and Technology (NSYN), Istituto Italiano di Tecnologia (IIT), Italy
Figures
Figure 1
TSPAN5 intracellular levels increase with neuronal maturation.
(A) Bis(sulfosuccinimidyl)suberate (BS3) crosslinking experiment on cultured rat hippocampal neurons at DIV12 and -19 blotted for TSPAN5, transferrin receptor (TfR), and tubulin. Arrows indicate the …
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Figure 1—source data 1
individual data values for the bar graphs and pie charts in panels A and B.
- https://cdn.elifesciences.org/articles/76425/elife-76425-fig1-data1-v1.xlsx
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Figure 1—source data 2
Raw images and images with cropped areas highlighted of the blots in panel A.
- https://cdn.elifesciences.org/articles/76425/elife-76425-fig1-data2-v1.zip
Figure 2 with 1 supplement
TSPAN5 interacts with AP4 and forms a complex with GluA2 and Stargazin.
(A) GST-pulldown experiment on adult rat hippocampus and cortex lysates using empty GST or GST fused to TSPAN5 C-terminus (GST-Ct). Input: 2.5% of pulldown volume. Blots probed for AP4ε. Red Ponceau …
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Figure 2—source data 1
List of the proteins identified with the yeast two-hybrid screening performed with full-length or C-terminal tail of TSPAN5 and individual data values for the bar graphs in panel D.
- https://cdn.elifesciences.org/articles/76425/elife-76425-fig2-data1-v1.xlsx
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Figure 2—source data 2
Raw images and images with cropped areas highlighted of the blots in panels A, B, C, and E.
- https://cdn.elifesciences.org/articles/76425/elife-76425-fig2-data2-v1.zip
Figure 2—figure supplement 1
Immunoprecipitation experiment on Hela cells, transfected with TSPAN5-GFP, Stargazin-HA, and GluA2-myc.
NT, non-transfected control. Input: 5% of the immunoprecipitated volume. Immunoprecipitation: α-rabbit IgG, α-GluA2, α-GFP, α-HA. Blots probed for GluA2, GFP, and HA.
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Figure 2—figure supplement 1—source data 1
Raw images and images with cropped areas highlighted of the blots presented.
- https://cdn.elifesciences.org/articles/76425/elife-76425-fig2-figsupp1-data1-v1.zip
Figure 3 with 1 supplement
TSPAN5 complex with AP4, Stargazin, and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) localises in recycling endosomes.
(A) Vesicles fractionation from synaptosomes obtained from adult rat hippocampus and cortex. Ten isovolumetric fractions were isolated. Blots were probed for: EEA1 for early endosomes, Rab7 for late …
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Figure 3—source data 1
Individual data values for the graphs in panels B, C, and D.
- https://cdn.elifesciences.org/articles/76425/elife-76425-fig3-data1-v1.xlsx
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Figure 3—source data 2
Raw images and images with cropped areas highlighted of the blots in panel A.
- https://cdn.elifesciences.org/articles/76425/elife-76425-fig3-data2-v1.zip
Figure 3—figure supplement 1
Control experiments for the proximity ligation assay (PLA).
The experiment was performed as described in the Methods section in the presence of both TSPAN5 and GluA2 antibodies or with TSPAN5 antibody only or with GluA2 antibody only and by incubation with …
Figure 4 with 1 supplement
TSPAN5 depletion affects surface and total levels of the AMPAR subunits GluA2 and GluA1.
(A) Top panel: Confocal images of dendrites from cultured rat hippocampal neurons transfected at DIV12 with either scrambled, Sh-TSPAN5, or rescue constructs, all co-expressing GFP and immunostained …
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Figure 4—source data 1
Individual data values for the graphs in panels A, B, and D.
- https://cdn.elifesciences.org/articles/76425/elife-76425-fig4-data1-v1.xlsx
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Figure 4—source data 2
Raw images and images with cropped areas highlighted of the blots in panel C.
- https://cdn.elifesciences.org/articles/76425/elife-76425-fig4-data2-v1.zip
Figure 4—figure supplement 1
Full images and blots related to Figure 4.
(A) Confocal images of dendrites from cultured rat hippocampal neurons transfected at DIV12 with either scrambled, Sh-TSPAN5, or rescue constructs, all co-expressing GFP (green) and immunostained at …
Figure 5 with 2 supplements
TSPAN5 and AP4 regulate surface GluA2 levels without affecting its internalisation.
(A) Confocal images of DIV20 cultured rat hippocampal neurons transfected with vectors coding for GFP (green) and either a scrambled ShRNA, the Sh-TSPAN5, and a construct carrying the ShTSPAN5 and …
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Figure 5—source data 1
Individual data values for the graphs in panels B, C, and D.
- https://cdn.elifesciences.org/articles/76425/elife-76425-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
CRISPR/Cas9 knockdown of AP-4 subunits in hippocampal neurons.
(A) Real-time PCR quantification of the relative AP4B (left panel) and AP4E (right panel) mRNA levels in DIV20 cultured rat hippocampal neurons transduced at DIV12 with lentiviral particles coding …
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Figure 5—figure supplement 1—source data 1
Individual data values for the graphs presented in panels A and B.
- https://cdn.elifesciences.org/articles/76425/elife-76425-fig5-figsupp1-data1-v1.xlsx
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Figure 5—figure supplement 1—source data 2
Raw images and images with cropped areas highlighted of the blots in panel B.
- https://cdn.elifesciences.org/articles/76425/elife-76425-fig5-figsupp1-data2-v1.zip
Figure 5—figure supplement 2
Left panel: Confocal images of secondary dendrites from DIV20 cultured rat hippocampal neurons transfected at DIV12 with either scrambled or Sh-TSPAN5 constructs, both co-expressing GFP.
After binding with α-GluA2 antibody, neurons were incubated for 0, 5, or 10 min. Secondary antibody was applied in non-permeabilising conditions for the extracellularly retained antibody (extra, …
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Figure 5—figure supplement 2—source data 1
Individual data values for the graphs presented.
- https://cdn.elifesciences.org/articles/76425/elife-76425-fig5-figsupp2-data1-v1.xlsx
Figure 6 with 1 supplement
TSPAN5 regulates exocytosis of GluA2-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs).
(A) Schematic of the FRAP-FLIP experiment presented in panel B. SEP-GluA2 in pre-bleaching condition is either fluorescent (green) if exposed to the extracellular media or quenched (light green) if …
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Figure 6—source data 1
Individual data values for the graphs presented in panels C and G.
- https://cdn.elifesciences.org/articles/76425/elife-76425-fig6-data1-v1.xlsx
Figure 6—figure supplement 1
Control experiments for SEP-GluA2 sensitivity to pH.
(A) Live confocal images of DIV20 culture rat hippocampal neurons transfected with SEP-GluA2 (green) and either scrambled, Sh-TSPAN5, or rescue construct co-expressing mCherry (magenta). Neurons …
Figure 7 with 1 supplement
TSPAN5 regulates exocytosis of newly synthesised α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), possibly by preventing their degradation via the lysosomal pathway.
(A) Schematic of the ARIAD-GluA2 construct. (B) In basal conditions (Berditchevski, 2001), ARIAD-GluA2 is retained in the endoplasmic reticulum (ER) due to the self-assembly properties of the …
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Figure 7—source data 1
Individual data values for the graphs presented in panels C, E, and F.
- https://cdn.elifesciences.org/articles/76425/elife-76425-fig7-data1-v1.zip
Figure 7—figure supplement 1
TSPAN5 knockdown does not affect intracellular transport speed or number of vesicles containing GluA2.
(A) Quantification of the average speed of tdTomato-positive organelles moving from the soma outwards along dendrites after application of the ARIAD ligand in DIV20 rat cultured hippocampal neurons …
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Figure 7—figure supplement 1—source data 1
Individual data values for the graphs presented in panels A, B, and C.
- https://cdn.elifesciences.org/articles/76425/elife-76425-fig7-figsupp1-data1-v1.xlsx
Figure 8
TSPAN5 regulates α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) exocytosis through recycling endosomes by the formation of a tetrameric complex with AP4 and Stargazin.
Working model of TSPAN5 function in mature neurons (left) and TSPAN5 silencing effects (right). TSPAN5 forms a complex with Stargazin and AMPARs in the endoplasmic reticulum or in endoplasmic …
