Conditional protein tagging methods reveal highly specific subcellular distribution of ion channels in motion-sensing neurons
- Max Planck Institute of Neurobiology, Germany
- Graduate School of Systemic Neurosciences, LMU Munich, Germany
Figures
Figure 1
Overview of the fly optic lobe and anatomy of T4/T5 neurons with their presynaptic partners and distribution of input synapses.
(A) Horizontal view of optic lobe with retina, lamina, medulla, lobula, and lobula plate. T4 dendrites (darker gray) reside in layer 10 of the medulla, T5 dendrites (lighter gray) in layer 1 of the …
Figure 2 with 1 supplement
Subcellular localization of the inhibitory glutamate receptor GluClα in T4/T5 neurons.
(A) Optic lobe with T4/T5 neurons labeled with myr::tdTomato and GluClα::GFP. Left panel: horizontal view on the optic lobe overview (scale bar: 20 μm). Central panel: close-up of medulla layer M10, …
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Figure 2—source data 1
Table with numbers of GluClα puncta quantified for T4 dendrites.
- https://cdn.elifesciences.org/articles/62953/elife-62953-fig2-data1-v3.xlsx
Figure 2—figure supplement 1
Pan-neuronal GluClα levels and distribution in the optic lobe are comparable for MiMIC GFSTF, FlpTag and UAS-line.
Optic lobe with MiMIC GFSTF GluClα (A), pan-neuronal expression of UAS-GluClα::GFP (B) and pan-neuronal expression of FlpTag GluClα (C) (scale bar: 20 μm). (D) Quantification of mean fluorescence …
Figure 3 with 1 supplement
Subcellular localization of the GABA receptor Rdl in T4/T5 neurons.
(A) Optic lobe with T4/T5 neurons labeled with myr::tdTomato and Rdl::GFP. Left panel: horizontal view on the optic lobe overview (scale bar: 20 μm). Right panel: close-up of medulla layer M10, …
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Figure 3—source data 1
Table with numbers of Rdl puncta quantified for T4/T5 dendrites.
- https://cdn.elifesciences.org/articles/62953/elife-62953-fig3-data1-v3.xlsx
Figure 3—figure supplement 1
Rdl is not detectable in the lamina neuron L1.
Optic lobe with L1-splitGal4 combined with UAS-myr::tdTomato and UAS-Rdl::GFP. Overview in left panel, close-up of lamina and part of the medulla in the middle and right panel (scale bar: 20 μm in …
Figure 4 with 1 supplement
Subcellular localization of the ACh receptor subunit Dα7 in T4/T5 neurons.
(A) Optic lobe with T4/T5 neurons labeled with myr::tdTomato and Dα7::GFP. Left panel: horizontal view on the optic lobe overview (scale bar: 20 μm). Right panel: close-up of medulla layer M10, …
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Figure 4—source data 1
Table with numbers of Dα7 puncta quantified for T4/T5 dendrites.
- https://cdn.elifesciences.org/articles/62953/elife-62953-fig4-data1-v3.xlsx
Figure 4—figure supplement 1
Pan-neuronal Dα7 levels and distribution in the optic lobe as seen with UAS-Dα7::GFP line, Dα7 antibody staining and Dα7-Trojan-Gal4 line.
(A) Optic lobe with pan-neuronal UAS-myr::tdTomato and UAS-Dα7::GFP expression. Overview in left panel, close-up of M10 of the medulla and Lo1 of the lobula in right panel (scale bar: 20 μm in …
Figure 5
FlpTag, a new tool for cell-type-specific, endogenous labeling as shown with GluClα.
(A) Scheme of FlpTag cassette (first panel) and integration of FlpTag cassette into target gene (second panel). The FlpTag cassette consists of attB-sites, specific FRT sites which form a …
Figure 6
FlpTag lines for Gaba-b-r1, para and Ih.
Optic lobes with pan-neuronal expression of FlpTag Gaba-b-r1 (A), FlpTag para (C), and FlpTag Ih (E). (B) Expression of FlpTag Gaba-b-r1 in T4/T5 neurons labeled with myr::tdTomato. Left panel: …
Figure 7
Summary of the receptor distributions of GluClα, Rdl and Dα7 in T4 and T5 neurons.
(A) Scheme of EM-reconstructed T4 neuron with distribution of receptors on dendrite and axon terminal (image extracted from Seven medulla column connectome dataset, https://emdata.janelia.org/#/repo/…
Tables
Table 1
Overview of available MiMIC GFSTF and FlpTag lines for investigated genes.
| Gene | MiMIC insertion (coding intron) | MiMIC GFSTF existing | MiMIC GFSTF working | Chromosome | Phase | FlpTag working | Localization in T4/T5 neurons | |
|---|---|---|---|---|---|---|---|---|
| 1 | GluClα | MI02890, MI14426 | MI02890 | Yes | III | 2 | Yes, MI02890 | T4: dendrites + terminals; T5: terminals |
| 2 | Rdl | MI02620, MI02957 | MI02620 | No | III | 0 | No, MI02620 | From UAS line: dendrites |
| 3 | Dα7 | MI12545 | This study (MI12545) | No | X | 1 | No | From UAS line: dendrites |
| 4 | Gaba-b-r1 | MI01930, MI05755 | MI01930 | Yes | II | 0 | Yes, MI01930 | No |
| 5 | para | MI08578 | This study (MI08578) | Yes | X | 0 | Yes, MI08578 | T4/T5 axonal fibers |
| 6 | Ih | MI03196, MI12136 | This study (MI12136) | Yes | II | 2 | Yes, MI12136 | T4/T5 dendrites |
Additional files
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Supplementary file 1
Plasmid map of the full sequence of pJFRC7-20xUAS-GluClα-GFP.
- https://cdn.elifesciences.org/articles/62953/elife-62953-supp1-v3.gbk
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Supplementary file 2
Plasmid map of the full sequence of pJFRC7-20xUAS-Rdl-GFP.
- https://cdn.elifesciences.org/articles/62953/elife-62953-supp2-v3.gbk
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Supplementary file 3
Plasmid map of the full sequence of pUC57-FlpTag-GFP-ph0.
- https://cdn.elifesciences.org/articles/62953/elife-62953-supp3-v3.gbk
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Supplementary file 4
Plasmid map of the full sequence of pUC57-FlpTag-GFP-ph1.
- https://cdn.elifesciences.org/articles/62953/elife-62953-supp4-v3.gbk
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Supplementary file 5
Plasmid map of the full sequence of pUC57-FlpTag-GFP-ph2.
- https://cdn.elifesciences.org/articles/62953/elife-62953-supp5-v3.gbk
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Supplementary file 6
Plasmid map of the full sequence of pHD-FlpTag-DsRed-HDR-ph0.
- https://cdn.elifesciences.org/articles/62953/elife-62953-supp6-v3.gbk
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Supplementary file 7
Plasmid map of the full sequence of pHD-FlpTag-DsRed-HDR--ph1.
- https://cdn.elifesciences.org/articles/62953/elife-62953-supp7-v3.gbk
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Supplementary file 8
Plasmid map of the full sequence of pHD-FlpTag-DsRed-HDR-ph2.
- https://cdn.elifesciences.org/articles/62953/elife-62953-supp8-v3.gbk
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Supplementary file 9
3D-image of a T4 dendrite (subtype d) (magenta) with GluClα::GFP (green).
- https://cdn.elifesciences.org/articles/62953/elife-62953-supp9-v3.avi.zip
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Supplementary file 10
3D-image of a T4 dendrite (subtype d) (magenta) with Rdl::GFP (yellow).
- https://cdn.elifesciences.org/articles/62953/elife-62953-supp10-v3.avi.zip
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Supplementary file 11
3D-image of a T4 dendrite (subtype d) (magenta) with Dα7::GFP (cyan).
- https://cdn.elifesciences.org/articles/62953/elife-62953-supp11-v3.avi.zip
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Transparent reporting form
- https://cdn.elifesciences.org/articles/62953/elife-62953-transrepform-v3.docx
