Evolutionarily unique mechanistic framework of clathrin-mediated endocytosis in plants
- Institute of Science and Technology Austria, Austria
- Karlsruhe Institute of Technology, Germany
Figures
Figure 1 with 1 supplement
Ultrastructural characterization of clathrin-coated structures in unroofed protoplasts by SEM.
(A) SEM image showing CCSs found associated to the PM (orange) and intracellular membrane structures (blue). See also Figure 1—figure supplement 1A, B. (B) Illustration of the en face surface-view …
Figure 1—figure supplement 1
Ultrastructural characterization of clathrin-coated structures.
(A) A representative SEM image of metal replica of protoplasts shows CCPs in varying stages of development at the PM. Scale bar: 200 nm. (B) An example SEM image depicting the cytoskeletal and …
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Figure 1—figure supplement 1—source data 1
Source data for the quantification in Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/52067/elife-52067-fig1-figsupp1-data1-v2.zip
Figure 2 with 3 supplements
Characterization of clathrin kinetics at the cell surface.
(A) TIRF-M image of clathrin foci in the root epidermal cell expressing CLC2-GFP (left) and the automated tracking results (right). (B) Intensity-time course of the combined mean fluorescence …
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Figure 2—source data 1
Source data and code for the quantification for Figure 2.
- https://cdn.elifesciences.org/articles/52067/elife-52067-fig2-data1-v2.zip
Figure 2—figure supplement 1
Co-localization of CHC1 and CLC2.
(Top row) Example TIRF-M image of a root epidermal cell expressing CHC1-GFP and CLC2-tagRFP. (Bottom row) A median max Z projection of 10 frames containing the automated significant detections for …
Figure 2—figure supplement 2
Goodness of fit analysis of clathrin sub-populations.
(A and B) AIC and BIC projecting the goodness of fit for the clathrin lifetime histogram subjected to fitting 1 to 5 sub-populations with Weibull distributions (A) or exponential distributions (B). …
Figure 2—figure supplement 3
Example TPLATE and CLC2 positive events.
(A) Fluorescent quantification of the example TPLATE x CLC spot in Figure 2E. (B) A further example of a TPLATE x CLC events on the PM of root epidermal cells using TIRF-M. The black arrows note the …
Figure 3 with 3 supplements
Localization and functional importance of actin during endocytosis.
(A) SEM image of the unroofed protoplasts showing the main and subsidiary filaments of actin in close proximity to the PM. (B) Dual channel TIRF image of hypocotyl epidermal cell expressing ABD2-GFP …
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Figure 3—source data 1
Source data and code for the quantification for Figure 3.
- https://cdn.elifesciences.org/articles/52067/elife-52067-fig3-data1-v2.zip
Figure 3—figure supplement 1
Localization and functional characterization of actin during endocytosis.
(A) Example SEM image of unroofed protoplast showing the cytoskeletal and intracellular membrane tightly linked with other cellular components. Scale bar: 0.5 µm. (B) Representative TIRF-M image …
Figure 3—video 1
Dynamics of CCVs after actin perturbation.
Dual channel TIRF-M imaging of hypocotyl epidermal cells expressing ABD2-GFP and CLC2-mKO after treatment with Jasp – 10 µM for 1.5 hr. Movie shows the arrested actin network and the CCVs still …
Figure 3—video 2
Dynamics of CCVs after actin perturbation.
Dual channel TIRF-M imaging of hypocotyl epidermal cells expressing ABD2-GFP and CLC2-mKO after treatment with LatB – 10 µM for 1 hr. Movie shows the depolymerized actin network and the CCVs still …
Figure 4 with 2 supplements
Role of actin in post-endocytic trafficking.
(A) Representative confocal microcopy images of EE/TGN (marked by CLC2-GFP) aggregated after mock or LatB (10 µM, 1 hr) treatment in hypocotyl epidermal cells. Observations were made from five …
Figure 4—video 1
Actin mediated dynamics of the EE/TGN system.
Dual channel TIRF-M imaging of hypocotyl epidermal cells expressing ABD2-GFP and CLC2-mKO. The EE/TGN could be observed to be constantly moving along the actin filaments. Time label is in seconds. …
Figure 4—video 2
Dynamics of the Golgi- EE/TGN after actin perturbation.
Dual channel TIRF-M imaging of hypocotyl epidermal cells expressing ST-RFP and CLC2-GFP. The dynamics of the Golgi together with the EE/TGN after mock and LatB treatment (10 µM; 1 hr) are shown. The …
Figure 5 with 3 supplements
Role of actin and early endosome movement in CME.
(A) TIRF-M images of hypocotyl epidermal cells expressing ST-RFP and CLC2-GFP. The Golgi apparatus (marked by ST-RFP) which move towards the endocytosed CCVs (marked by CLC2-GFP) are marked by …
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Figure 5—source data 1
Source data for the quantification in Figure 5.
- https://cdn.elifesciences.org/articles/52067/elife-52067-fig5-data1-v2.zip
Figure 5—video 1
EE/TGN moving along actin picking up an endocytosed CCV.
Dual channel TIRF-M imaging of hypocotyl epidermal cells expressing ABD2-GFP and CLC2-mKO. The CCV (arrowed) could be observed being collected by the EE/TGN (boxed) moving along the actin filament. …
Figure 5—video 2
Collection of endocytosed CCVs by the EE/TGN.
Dual channel TIRF-M imaging of hypocotyl epidermal cells expressing ST-RFP and CLC2-GFP. Aggregation of the endocytosed CCVs (arrowed) on the trans-side of the Golgi could be observed as the Golgi …
Figure 5—video 3
Dynamic endosomes enhancing the local rate of CME termination.
An example TIRF-M time lapse movie of root epidermal cells expression TPLATE-GFP and CLC-tagRFP, but only the CLC channel is shown. The left shows the mobile experimental endosome tracks (red …
Figure 6 with 2 supplements
Uncoating of CCVs.
(A and B) TIRF-M images of hypocotyl epidermal cells expressing TPLATE-GFP and CLC2-tagRFP. CLC2 marks both endocytic foci (smaller foci) and the EE/TGN (larger structures). (A) The boxed CCV is …
Figure 6—video 1
Uncoating process as the CCVs reach the stationary EE/TGN.
Dual channel TIRF imaging of hypocotyl epidermal cells expressing TPLATE-GFP x CLC2-RFP. CCVs (arrowed) moving to the stationary EE/TGN lose TPLATE signal by the time of reaching the EE/TGN …
Figure 6—video 2
Uncoating process of a fully developed CCV.
Dual channel TIRF imaging of hypocotyl epidermal cells expressing TPLATE-GFP x CLC2-RFP. CCV moving away from the PM after scission loses TPLATE signal before that of clathrin. Time label is in …
Figure 7
Model for post-endocytic trafficking of CCVs.
Trafficking of the fully developed CCVs and the EE/TGN compartments along actin represented in different scenarios 1) Delayed sequential uncoating of the CCV during the transport to the EE/TGN 2) …
Videos
Video 1
Actin whipping away the CCVs.
Dual channel TIRF-M imaging of hypocotyl epidermal cells expressing ABD2-GFP and CLC2-mKO. Spatial and temporal correlation between appearance of actin subsidiary filaments and the disappearance of …
Video 2
CCVs losing the coat.
TIRF imaging of hypocotyl epidermal cells expressing ST-RFP and CLC2-GFP. CCVs (arrowed) that leave the membrane lose the clathrin coat before reaching EE/TGN. Time label is in seconds. Scale bar: 2 …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Arabidopsis thaliana, Col-0) | Suspension cultured root protoplasts | |||
| Biological sample (Arabidopsis thaliana, Col-0) | Seedlings | |||
| Gene (Arabidopsis thaliana) | AP2A1 | The Arabidopsis Information Resource | AT5G22770 | |
| Gene (Arabidopsis thaliana) | Axl2 | The Arabidopsis Information Resource | AT4G12770 | |
| Gene (Arabidopsis thaliana) | CHC1 | The Arabidopsis Information Resource | AT3G11130 | |
| Gene (Arabidopsis thaliana) | CLC2 | The Arabidopsis Information Resource | AT2G40060 | |
| Gene (Arabidopsis thaliana) | Fim1 | The Arabidopsis Information Resource | AT4G26700 | |
| Gene (Arabidopsis thaliana) | FLS2 | The Arabidopsis Information Resource | AT5G46330 | |
| Gene (Arabidopsis thaliana) | PIN2 | The Arabidopsis Information Resource | AT5G57090 | |
| Gene (Arabidopsis thaliana) | Tplate | The Arabidopsis Information Resource | AT3G01780 | |
| Gene (Arabidopsis thaliana) | VHA-a1 | The Arabidopsis Information Resource | At2g28520 | |
| Genetic reagent (Arabidopsis thaliana) | pCLC2::CLC2-GFP | (Konopka et al., 2008) | ||
| Genetic reagent (Arabidopsis thaliana) | pLAT52::TPLATE-GFP x pRPS5::CLC2-RFP | (Gadeyne et al., 2014) | ||
| Genetic reagent (Arabidopsis thaliana) | 35S::ABD2-GFP | (Kost et al., 1998) | ||
| Genetic reagent (Arabidopsis thaliana) | p35S::CLC2-mKO | (Naramoto et al., 2010) | ||
| Genetic reagent (Arabidopsis thaliana) | pFLS2::FLS2-GFP x pUBQ10::mRFP-ARA7 | (Beck et al., 2012) | ||
| Genetic reagent (Arabidopsis thaliana) | XVE >> Axl2 x pPIN2::PIN2-Dendra | (Adamowski et al., 2018) | ||
| Genetic reagent (Arabidopsis thaliana) | pVHA-a1::VHA-a1-GFP | (Dettmer et al., 2006) | ||
| Genetic reagent (Arabidopsis thaliana) | pPIN2::PIN2-GFP | (Xu and Scheres, 2005) | ||
| Genetic reagent (Arabidopsis thaliana) | p35S::ARA7-mRFP | (Ueda et al., 2004) | ||
| Genetic reagent (Arabidopsis thaliana) | p35S::ST-RFP x pCLC2::CLC2-GFP | (Ito et al., 2012) | ||
| Chemical compound | LatrunculinB | Sigma Aldrich | L5288 | |
| Chemical compound | Jasplakinolide | Santa Cruz Biotechnology | sc-202191 | |
| Chemical compound | FM4-64 | ThermoFisher Scientific | T3166 |
Additional files
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Supplementary file 1
Supplementary tables.
- https://cdn.elifesciences.org/articles/52067/elife-52067-supp1-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/52067/elife-52067-transrepform-v2.docx
