Continuous endosomes form functional subdomains and orchestrate rapid membrane trafficking in trypanosomes
- Department of Cell & Developmental Biology, Biocentre, University of Würzburg, Germany
- Department of Biotechnology & Biophysics, Biocentre, University of Würzburg, Germany
- Molecular, Cellular & Developmental Biology, University of Colorado Boulder, United States
- Imaging Core Facility, Biocentre, University of Würzburg, Germany
Figures
Figure 1 with 8 supplements
Tomographic reconstructions of the endosomal apparatus of T. brucei after cargo uptake reveal a large and mostly continuous membrane system.
(A) Elongated and highly fenestrated endosomal sheets and palisades. (B) Elongated and slightly fenestrated endosomes. (C) Large circular endosomal cisternae. (D) Two different substructures are …
Figure 1—video 1
z-stack of electron tomogram from Figure 1A.
Scale bar: 500 nm.
Figure 1—video 2
3D model of electron tomogram from Figure 1A.
Scale bar: 500 nm.
Figure 1—video 3
z-stack of electron tomogram from Figure 1B.
Scale bar: 500 nm.
Figure 1—video 4
3D model of electron tomogram from Figure 1B.
Scale bar: 500 nm.
Figure 1—video 5
z-stack of electron tomogram from Figure 1C.
Scale bar: 500 nm.
Figure 1—video 6
3D model of electron tomogram from Figure 1C.
Scale bar: 500 nm.
Figure 1—video 7
z-stack of electron tomogram from Figure 1D.
Scale bar: 500 nm.
Figure 1—video 8
3D model of electron tomogram from Figure 1D.
Scale bar: 500 nm.
Figure 2 with 2 supplements
Super-resolution light microscopy reveals continuous endosomal membranes.
(A) Visualization of the endosomal system in T. brucei using expansion microscopy (ExM). Bloodstream form cells were pulsed with dextran conjugated to Alexa488 and fixed with 4% formaldehyde and …
Figure 2—figure supplement 1
Visualization of the endosomal system in T. brucei by direct stochastic optical reconstruction microscopy (dSTORM).
A transgenic EP1::HaloTag expressing bloodstream form cell line was generated and used to visualize the endosomal system. Cells were labelled with a HaloTag ligand and fixed with 4% formaldehyde. …
Figure 2—figure supplement 2
Validation of the generated EP1::HaloTag cell line.
(A) Schematic of the tagging construct used for the inducible, ectopic expression of the Halo-tagged version of EP1. The bleomycin (Ble) resistance is driven by the T7 promoter while the expression …
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Figure 2—figure supplement 2—source data 1
Integration PCR for generation of EP1::Halo cell line.
- https://cdn.elifesciences.org/articles/91194/elife-91194-fig2-figsupp2-data1-v1.zip
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Figure 2—figure supplement 2—source data 2
Integration PCR for generation of EP1::Halo cell line - labelled.
- https://cdn.elifesciences.org/articles/91194/elife-91194-fig2-figsupp2-data2-v1.zip
Figure 3 with 1 supplement
The endosomal markers TbRab5A, TbRab7, and TbRab11 partly colocalize in the posterior region of the cell.
(A) The experimental workflow, from fluorescence images to object generation and quantitative 3D colocalization analysis, is exemplified. EP1::GFP (I) expressing cells were fixed and labelled with …
Figure 3—figure supplement 1
Validation of different anti-TbRab antibodies.
Antibodies against (A) TbRab5A, (B) TbRab7, and (C) TbRab11 were generated in different animals and validated by immunoblotting using protein samples of RNAi cell lines. Each panel shows the …
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Figure 3—figure supplement 1—source data 1
Antibody validation western blots unlabelled.
- https://cdn.elifesciences.org/articles/91194/elife-91194-fig3-figsupp1-data1-v1.zip
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Figure 3—figure supplement 1—source data 2
Antibody validation western blots labelled.
- https://cdn.elifesciences.org/articles/91194/elife-91194-fig3-figsupp1-data2-v1.zip
Figure 4
The endosomal markers TbRab5A and TbRab7 are present on the same membrane.
(A) Schematic representation of antibody binding to visualize the maximum linkage error of 30 nm between a bound epitope and the gold particle. (B–G) Electron micrographs of cryosections labelled …
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Figure 4—source data 1
Uncropped electron micrographs of Figure 4.
- https://cdn.elifesciences.org/articles/91194/elife-91194-fig4-data1-v1.zip
Figure 5
The endosomal markers TbRab5A and TbRab11 are present on the same membranes.
(A–F) Electron micrographs of cryosections labelled with rat anti-TbRab5A and rabbit anti-TbRab11 antibodies and visualized with 6 or 12 nm gold-coupled secondary antibodies. For each panel, an …
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Figure 5—source data 1
Uncropped electron micrographs of Figure 5.
- https://cdn.elifesciences.org/articles/91194/elife-91194-fig5-data1-v1.zip
Figure 6
The endosomal markers TbRab7 and TbRab11 are present on the same membranes.
(A–G) Electron micrographs of cryosections labelled with guinea pig anti-TbRab7 and rabbit anti-TbRab11 antibodies visualized with 6 or 12 nm gold-coupled secondary antibodies. For each panel, an …
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Figure 6—source data 1
Uncropped electron micrographs of Figure 6.
- https://cdn.elifesciences.org/articles/91194/elife-91194-fig6-data1-v1.zip
Figure 7
Cargo uptake and anti-VSG immunogold labelling confirm the endosomal identity.
(A–I) Electron micrographs of cryosections labelled with anti-TbRab5A (yellow), anti-TbRab7 (cyan), or anti-TbRab11 (magenta) antibodies and 12 nm gold-conjugated secondary antibodies. For each …
Figure 8 with 4 supplements
3D Tokuyasu demonstrated continuous endosomal membranes in cryosections.
(A, B) Tomographic reconstruction of the endosomal apparatus. (I–III) Isolated view of different organelle structures. Cryosections (250 nm) were labelled with anti-TbRab antibodies and 6 or 12 nm …
Figure 8—video 1
z-stack of electron tomogram from Figure 8A.
Scale bar: 500 nm.
Figure 8—video 2
3D model of electron tomogram from Figure 8A.
Scale bar: 500 nm.
Figure 8—video 3
z-stack of electron tomogram from Figure 8B.
Scale bar: 500 nm.
Figure 8—video 4
3D model of electron tomogram from Figure 8B.
Scale bar: 500 nm.
Figure 9
Schematic representation of the endosomal system in T. brucei.
The endosome is marked by the presence of small GTPases of the Rab family: Rab5A (yellow dots), Rab7 (cyan dots), and Rab11 (red dots). Class I clathrin-coated vesicles (CCV I), class II …
Videos
Video 1
Tomographic reconstructions of T. brucei.
The movie is available online and includes annotations for better understanding.
Video 2
Tomographic reconstructions of T. brucei.
Cells were chemically fixed before further processing. Scale bar: 500 nm.
Video 3
Tomographic reconstructions of T. brucei.
Cells were chemically fixed before further processing. Scale bar: 500 nm.
Video 4
Tomographic reconstructions of T. brucei.
Cells were chemically fixed before further processing. Scale bar: 500 nm.
