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Biomolecular condensation orchestrates clathrin-mediated endocytosis in plants

Nature Cell Biology volume 26, pages 438–449 (2024)Cite this article

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Abstract

Clathrin-mediated endocytosis is an essential cellular internalization pathway involving the dynamic assembly of clathrin and accessory proteins to form membrane-bound vesicles. The evolutionarily ancient TSET–TPLATE complex (TPC) plays an essential, but ill-defined role in endocytosis in plants. Here we show that two highly disordered TPC subunits, AtEH1 and AtEH2, function as scaffolds to drive biomolecular condensation of the complex. These condensates specifically nucleate on the plasma membrane through interactions with anionic phospholipids, and facilitate the dynamic recruitment and assembly of clathrin, as well as early- and late-stage endocytic accessory proteins. Importantly, condensation promotes ordered clathrin assemblies. TPC-driven biomolecular condensation thereby facilitates dynamic protein assemblies throughout clathrin-mediated endocytosis. Furthermore, we show that a disordered region of AtEH1 controls the material properties of endocytic condensates in vivo. Alteration of these material properties disturbs the recruitment of accessory proteins, influences endocytosis dynamics and impairs plant responsiveness. Our findings reveal how collective interactions shape endocytosis.

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Fig. 1: AtEH proteins phase separate in vivo and in vitro.
Fig. 2: Condensation of AtEH1 is controlled by multiple regions, including IDR1.
Fig. 3: AtEH1 condensates nucleate on the plasma membrane via phospholipid binding domains.
Fig. 4: AtEH1 condensates selectively sequester endocytic machinery.
Fig. 5: Condensation of AtEH1 facilitates clathrin recruitment and assembly.
Fig. 6: Chemical induced re-localization of TPC to mitochondria is sufficient for condensate formation and clathrin assembly.
Fig. 7: The material properties of TPC-driven condensates are important for the progression of endocytosis.

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Data availability

The A. thaliana proteome (NCBI taxon ID 3702) was analysed for prediction of disordered residues using the MobiDB database. The Phytozome v13 database was used to identify AtEH1 homologues. The mass spectrometry data have been deposited with the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD045068. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

Code availability

Codes used in the manuscript are available online: ImageJ script detect_membrane_and_cytoplasm_from_a_roi https://github.com/pegro-psb/Cyto-PM-signal-quantification; ImageJ script MitoTally https://github.com/winkler-joanna/MitTally_NucTally; PlantCMEanalysis https://doi.org/10.5281/zenodo.3888519.

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Acknowledgements

This work was supported by the European Research Council Grant T-REX (682436 (D.V.D.), 852136 (A.B.) and 803048 (M.F.)); the Research Foundation – Flanders (FWO) (1226420N (P.G.), 12S7222N (J.M.D.) and 1124621 N (A.D.M.)); a European Molecular Biology Organization scientific exchange grant (9253 (J.M.D)); the Czech Science Foundation (22-35680M (R.P.) and 19-21758S (M.P.)); a National Natural Science Foundation of China grant (32161133001 (X.F.)); and a Beijing Natural Science Foundation grant (JQ21020 (X.F.)). We acknowledge the Imaging Facility of the Institute of Experimental Botany of the Czech Academy of Sciences (IEB AS CR) supported by the Ministry of Education, Youth and Sports of the Czech Republic (LM2023050 Czech-BioImaging) and IEB AS CR. We acknowledge the Flemisch Institute for Biotechnology (VIB) BioImaging Core and A. Kremer for microscopy support and E. Mylle for technical support with confocal microscopy. We thank the VIB proteomics core for performing the AtEH1-linker-TurboID mass spectrometry data collection. Illustrations (Figs. 1b and 6a,b) were created with BioRender.com.

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Authors and Affiliations

  1. Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium

    Jonathan Michael Dragwidge, Andreas De Meyer, Dominique Eeckhout, Peter Grones, Joanna Winkler, Michaël Vandorpe, Geert De Jaeger & Daniël Van Damme

  2. VIB Center for Plant Systems Biology, Ghent, Belgium

    Jonathan Michael Dragwidge, Andreas De Meyer, Dominique Eeckhout, Peter Grones, Joanna Winkler, Michaël Vandorpe, Geert De Jaeger & Daniël Van Damme

  3. Center for Plant Biology, School of Life Sciences, Tsinghua University, Beijing, China

    Yanning Wang & Xiaofeng Fang

  4. Bordeaux Imaging Center, INSERM, CNRS, Université de Bordeaux, Bordeaux, France

    Lysiane Brocard & Matthieu Buridan

  5. Institute of Experimental Botany of the Czech Academy of Sciences, Prague, Czech Republic

    Roman Hudeček, Přemysl Pejchar, Martin Potocký & Roman Pleskot

  6. Laboratoire de Biogenèse Membranaire, CNRS, Université de Bordeaux, Bordeaux, France

    Clément Chambaud & Amelie Bernard

  7. Department of Experimental Plant Biology, Faculty of Sciences, Charles University, Prague, Czech Republic

    Nelson Serre & Matyáš Fendrych

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  1. Jonathan Michael Dragwidge
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Contributions

J.M.D. initiated the project and designed and performed all experiments unless otherwise indicated. Y.W. performed in vitro assays and yeast localization. L.B., C.C. and J.M.D. performed the CLEM-ET experiments. A.D.M. performed tobacco partitioning assays. R.H., R.P. and M.P. performed pollen tube assays. R.P. performed phylogenetic analysis. M.V. and P.P. cloned constructs. P.G. performed immunolocalization experiments. D.E. analysed proteomics data. R.H. and M.B. performed image analysis. J.W. generated the rapamycin plant lines. N.S. performed rapamycin time-lapse imaging. M.F., A.B., G.D.J., R.P., X.F. and D.V.D. supervised research. J.M.D. and D.V.D. wrote the paper. All authors contributed to finalizing the text.

Corresponding authors

Correspondence to Jonathan Michael Dragwidge or Daniël Van Damme.

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Nature Cell Biology thanks Markus Grebe and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 TPC disorder prediction, and AtEH1 in vitro FRAP assay.

a, Plot of the proportion of disordered residues for TPC subunits. b, Plot of TPC subunits showing prediction of disordered (MobiDB, blue), and prion-like (PLAAC, red) residues. Regions with values >0.5 are considered disordered, or prion-like respectively. Prion-like residues were only identified in TML and TWD40-2 subunits. (c,d) In vitro FRAP assay of purified GFP-AtEH1FL protein. Data is mean ± SD, n indicates the number of puncta from an individual experiment that were used in the quantification shown in panel d. Scale bar = 2 μm. The experiment in c-d was performed two times with similar results.

Extended Data Fig. 2 AtEH1 truncation construct expression in yeast, and evolutionary comparison of EH/Pan1 IDR1 amino acid composition across Archaeplastida.

a, Expression and condensate formation capacities of AtEH1 domain constructs in S. pombe. Scale bars = 5 μm. b, Conservation of amino acids at the single residue level based on Consurf analysis calculated using 128 AtEH homologous sequences throughout plant evolution. The average conservation score is indicated for each region (1 = 0% conservation, 9 = 100% conservation). IDR1 is highly variable at the individual amino acid level. c, Phylogenetic tree representing the maximum likelihood phylogeny of selected EH proteins. The phylogenetic tree was arbitrarily rooted to reflect phylogenetic relationships between chlorophyte and streptophyte lineages. The scale bar represents 0.1 amino acid substitution per site. The composition of the IDR1 for EH/Pan1 proteins is shown, with each amino acid plotted with the total number of each residue indicated. EH/Pan1 homologs from yeast (ScPan1, ScEde1), human (ITSN1), and the A. thaliana proteome average IDR are also indicated. d, NARDINI+ analysis of IDR patterning and compositional features from the IDR1 of AtEH1, AtEH2, and scrambled AtEH1 variants, homologous IDRs from liverwort (MpEH1), yeast (ScPan1, ScEde1), and human (HsITSN1) (used in Fig. 2c), IDRs from the TPLATE complex subunits TWD40-1/2, and the equivalent IDR from selected AtEH homologs throughout the plant kingdom. Z-score matrices are shown, with positive z-scores indicating non-random segregation between two types of residues, a blocky distribution of one type of residue, or the enrichment of the given sequence feature. Negative z-scores indicate nonrandom mixing between two types of residues, a uniform distribution of one type of residue, or the depletion of the given sequence feature. Red boxes indicate notable compositional features that differ between the two clusters including charged residue sequence features. Z-scores ≤ −1.5 and ≥ 1.5 are considered significant. μ, polar; h, hydrophobic; +, basic; ̶, acidic; π, aromatic; pI, isoelectric point; NCPR, net charge per residue; PPII, polyproline II propensity. The sequences used to construct the alignment in panel b and the phylogenetic tree in panel c are provided as Source Data. The experiment in a was performed two times with similar results.

Source data

Extended Data Fig. 3 Condensate motility and phospholipid specificity experiments.

a, Representative time-lapse and tracking of AtEH1 and AtEH2 condensates in Latrunculin B treated (4 μM, 30 minutes) N. benthamiana epidermal cells. b, Schematic representation of the phospholipid localisation domains in Tobacco pollen tubes which maintain a distinctive lipid signature. Images show transient co-expression of pLAT52:AtEH1-YFP with lipid biosensors (pLAT52-biosensor-mCherry) in N. tabacum pollen tubes. The chart shows the quantification of the plasma membrane signal from AtEH1 and lipid biosensors from the tip of the pollen tube. Data represent mean ± SD, quantified from n = 10 time points pooled from two individual pollen tubes (5 timepoints per pollen tube) for each lipid biosensor. Scale bars = 5μm. For a-b the experiments were performed two times with similar results.

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Extended Data Fig. 4 AtEH1 recruits proteins with prionlike domains.

a, Plot of Prion-like proteins identified in the AtEH1 and TPLATE TurboID datasets, n = number of proteins with prion-like domains from the top 100 enriched proteins from each TurboID dataset (n = 33 proteins in the AtEH1 dataset and n = 12 in the TPLATE dataset). b, (Left) Plot of top 100 enriched proteins based on disorder content (MobiDB), x ̄ (mean) disorder score is indicated. (Right) plot showing abundance and disorder content of selected endocytic proteins. c, Partitioning assay of selected nuclear proteins with prion-like domains identified in the AtEH1-TurboID dataset. UBQ10:AtEH1FL-GFP was co-expressed with mScarlet tagged client proteins in N. benthamiana epidermal cells. Partitioning was observed in the cytosol (Med15a, Med25) and in the nucleus (TAF12b). Scale bars = 5 μm (c). The TurboID experiments in a and b are the result of 3 separate MS runs that were performed for each bait protein. Individual TurboID experiments were performed once. For c the experiment was performed two times with similar results.

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Extended Data Fig. 5 CLEM-ET workflow.

a, Overview of the sample preparation procedure. b, Expanded workflow of the Correlative Light and Electron Microscopy (CLEM) imaging of hypocotyl sections. Yellow arrowheads indicate chloroplasts used as natural landmarks for correlation. c, Zoomed images of regions selected for electron tomography (ET) reconstruction. The condensates have similar electron density to CCVs.

Extended Data Fig. 6 B-isox enrichment and TPLATE re-localisation additional data.

a, Enrichment of TPC subunits and endocytic proteins after B-isox treatment. Data shown is from Zhang et al., 2022. Proteins with enrichment values ≥ 2 are considered enriched, and < 2 not enriched. Enrichment values below 2 were not represented in Zhang et al., 2022. b,c, Validation of TPLATE-mCh-FKBP tplate A. thaliana lines. Genotyping PCR indicate TPLATE-mCh-FKBP complements the tplate mutant (b). Primers; Top row: tplate TDNA, bottom row: WT TPLATE. TPLATE-GFP tplate(−/−) (positive control). Endocytic foci from A. thaliana root cells expressing TPLATE-mCh-FKBP (c). d, Whole mount immunolocalisation of AtEH1 (Anti-AtEH1), CHC (Anti-CHC), and TPLATE-FKBP-mCherry (Anti-RFP) in rapamycin treated A. thaliana root cells. Arrows indicate mitochondria clusters. e, CLEM localisation and ET of mitochondria clusters from untreated A. thaliana root cells. No ribosome exclusion zone between mitochondria clusters was observed. f, Quantification of CCV recruitment to mitochondria clusters from tomograms from control (n = 11) and rapamycin treated (n = 12) root cells pooled from resin blocks from two biologically independent seedling samples. Bars indicate mean ± SEM. ** p < 0.01; unpaired two-tailed t-test. For b-d the experiments were performed two times with similar results. Scale bars = 5 μm (c), 10 μm (d), 200 nm (e), or as indicated.

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Extended Data Fig. 7 pAtEH1:AtEH1-GFP reporter validation and functionality.

a, Schematic of the generation of pAtEH1:AtEH1-GFP rescue lines. b, Western blot of A. thaliana protein extracts using an antibody directed against AtEH1, indicating absence of endogenous AtEH1 in the recued lines. Lower molecular weight bands are degradation products. Strain free gel is shown as a loading control. c, Representative plant rosette image of WT Col-0 and AtEH1 rescued lines. d-f, Inhibition of endocytosis in pAtEH1:AtEH1-GFP seedlings using the chemical inhibitor ES9 (3 min, 10 µM). Kymograph shows immobile endocytic foci with stable fluorescence intensity over time (d). Kymograph (e) and fluorescence intensity plot (f) from FRAP experiments of individual endocytic foci from pAtEH1:AtEH1WT-GFP, pAtEH1:AtEH1YF>S-GFP, and pAtEH1:AtEH1YF>W-GFP seedlings. Plotted values indicate mean ± SD from n = 6 endocytic foci. Half recovery times (t1/2) are 22.08s, 11.95s, 28.81s, respectively. g,h, Full intensity profiles from kymograph analysis of endocytic events from root cells of A. thaliana AtEH1:AtEH1GFP/eh1-1 lines in a DRP1a-mRFP/drp1a background (i; see also Fig. 7g, h). Plotted values indicate mean ± SEM. h, Fluorescence intensity ratio of DRP1a compared to AtEH1 from data in (g). Plotted values indicate median ± 95% CI. Data in g and h, is from n = 31 (WT), 44 (YFS), 40 (YFW) endocytic foci pooled from 5 biologically independent samples. i,j, Endocytic flux experiment. Seedlings were treated with the lipophilic dye FM4-64 (2µM, 10min) and root cells were imaged (i). j, Quantification of FM4-64 internalisation. Bars indicate mean ± 95% CI. Statistics indicate significance to WT; n.s. not significant, * p < 0.05, ** p < 0.01, **** p < 0.0001, unpaired ttest. k, Gravitropism assay. Roots grown on ½ MS agar plates were gravistimulated by turning them 90°. The gravitropism plot indicates the angle of the root tip in 22.5° bins. n = 20 roots for each genotype. Scale bar = 10 μm (d). Data in i-j represents pooled data from three independent repeats. The experiment in b, f and k was performed two, two and three times, respectively with similar results. Sample sizes (j, k) are indicated in the Source Data.

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Supplementary information

Reporting Summary

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Supplementary Table 1

List of materials used in the study.

Supplementary Table 2

Proteomics data.

Supplementary Video 1

Related to Fig. 3a. Time-lapse imaging of UBQ10:AtEH1-GFP in N. benthamiana epidermal cells. Condensates appear as immobile plasma-membrane-associated puncta which gradually increase in intensity, before dissociating from their original location (yellow arrows).

Supplementary Video 2

Related to Fig. 5a. ET of hypocotyl sections from A. thaliana seedlings overexpressing AtEH1 (35S:AtEH1-GFP). Clathrin-like lattices and triskelia and electron-dense accumulations are observed within condensates. A CCV is also observed near the condensate.

Supplementary Video 3

Related to Fig. 6c. Time-lapse imaging of A. thaliana root cells expressing Tom70pMTSTagBFP2-FRB (Mito) and TPLATE-mCherry-FKBP (TPLATE) after rapamycin treatment.

Supplementary Video 4

Related to Fig. 6d. Segmented ET reconstruction of a mitochondria cluster from a rapamycin-treated A. thaliana root cell after localization by CLEM. The interior of the mitochondria cluster is devoid of ribosomes, indicating a liquid-like environment. Segmentation of ribosomes (red), mitochondria clusters (yellow) and CCVs (blue) is shown.

Supplementary Video 5

Related to Fig. 7b. 3D surface rendering of condensates from N. benthamiana epidermal cells expressing UBQ10:AtEH1WT/mutGFP constructs. Condensates are coloured by sphericity. AtEH1YF>W condensates have high mobility and reduced sphericity.

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Dragwidge, J.M., Wang, Y., Brocard, L. et al. Biomolecular condensation orchestrates clathrin-mediated endocytosis in plants. Nat Cell Biol 26, 438–449 (2024). https://doi.org/10.1038/s41556-024-01354-6

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