---
_id: '12239'
abstract:
- lang: eng
  text: Biological systems are the sum of their dynamic three-dimensional (3D) parts.
    Therefore, it is critical to study biological structures in 3D and at high resolution
    to gain insights into their physiological functions. Electron microscopy of metal
    replicas of unroofed cells and isolated organelles has been a key technique to
    visualize intracellular structures at nanometer resolution. However, many of these
    methods require specialized equipment and personnel to complete them. Here, we
    present novel accessible methods to analyze biological structures in unroofed
    cells and biochemically isolated organelles in 3D and at nanometer resolution,
    focusing on Arabidopsis clathrin-coated vesicles (CCVs). While CCVs are essential
    trafficking organelles, their detailed structural information is lacking due to
    their poor preservation when observed via classical electron microscopy protocols
    experiments. First, we establish a method to visualize CCVs in unroofed cells
    using scanning transmission electron microscopy tomography, providing sufficient
    resolution to define the clathrin coat arrangements. Critically, the samples are
    prepared directly on electron microscopy grids, removing the requirement to use
    extremely corrosive acids, thereby enabling the use of this method in any electron
    microscopy lab. Secondly, we demonstrate that this standardized sample preparation
    allows the direct comparison of isolated CCV samples with those visualized in
    cells. Finally, to facilitate the high-throughput and robust screening of metal
    replicated samples, we provide a deep learning analysis method to screen the “pseudo
    3D” morphologies of CCVs imaged with 2D modalities. Collectively, our work establishes
    accessible ways to examine the 3D structure of biological samples and provide
    novel insights into the structure of plant CCVs.
acknowledged_ssus:
- _id: EM-Fac
- _id: LifeSc
- _id: Bio
acknowledgement: A.J. is supported by funding from the Austrian Science Fund I3630B25
  (to J.F.). This research was supported by the Scientific Service Units of Institute
  of Science and Technology Austria (ISTA) through resources provided by the Electron
  Microscopy Facility, Lab Support Facility, and the Imaging and Optics Facility.
  We acknowledge Prof. David Robinson (Heidelberg) and Prof. Jan Traas (Lyon) for
  making us aware of previously published classical on-grid preparation methods. No
  conflict of interest declared.
article_processing_charge: Yes (via OA deal)
article_type: original
author:
- first_name: Alexander J
  full_name: Johnson, Alexander J
  id: 46A62C3A-F248-11E8-B48F-1D18A9856A87
  last_name: Johnson
  orcid: 0000-0002-2739-8843
- first_name: Walter
  full_name: Kaufmann, Walter
  id: 3F99E422-F248-11E8-B48F-1D18A9856A87
  last_name: Kaufmann
  orcid: 0000-0001-9735-5315
- first_name: Christoph M
  full_name: Sommer, Christoph M
  id: 4DF26D8C-F248-11E8-B48F-1D18A9856A87
  last_name: Sommer
  orcid: 0000-0003-1216-9105
- first_name: Tommaso
  full_name: Costanzo, Tommaso
  id: D93824F4-D9BA-11E9-BB12-F207E6697425
  last_name: Costanzo
  orcid: 0000-0001-9732-3815
- first_name: Dana A.
  full_name: Dahhan, Dana A.
  last_name: Dahhan
- first_name: Sebastian Y.
  full_name: Bednarek, Sebastian Y.
  last_name: Bednarek
- first_name: Jiří
  full_name: Friml, Jiří
  id: 4159519E-F248-11E8-B48F-1D18A9856A87
  last_name: Friml
  orcid: 0000-0002-8302-7596
citation:
  ama: Johnson AJ, Kaufmann W, Sommer CM, et al. Three-dimensional visualization of
    planta clathrin-coated vesicles at ultrastructural resolution. <i>Molecular Plant</i>.
    2022;15(10):1533-1542. doi:<a href="https://doi.org/10.1016/j.molp.2022.09.003">10.1016/j.molp.2022.09.003</a>
  apa: Johnson, A. J., Kaufmann, W., Sommer, C. M., Costanzo, T., Dahhan, D. A., Bednarek,
    S. Y., &#38; Friml, J. (2022). Three-dimensional visualization of planta clathrin-coated
    vesicles at ultrastructural resolution. <i>Molecular Plant</i>. Elsevier. <a href="https://doi.org/10.1016/j.molp.2022.09.003">https://doi.org/10.1016/j.molp.2022.09.003</a>
  chicago: Johnson, Alexander J, Walter Kaufmann, Christoph M Sommer, Tommaso Costanzo,
    Dana A. Dahhan, Sebastian Y. Bednarek, and Jiří Friml. “Three-Dimensional Visualization
    of Planta Clathrin-Coated Vesicles at Ultrastructural Resolution.” <i>Molecular
    Plant</i>. Elsevier, 2022. <a href="https://doi.org/10.1016/j.molp.2022.09.003">https://doi.org/10.1016/j.molp.2022.09.003</a>.
  ieee: A. J. Johnson <i>et al.</i>, “Three-dimensional visualization of planta clathrin-coated
    vesicles at ultrastructural resolution,” <i>Molecular Plant</i>, vol. 15, no.
    10. Elsevier, pp. 1533–1542, 2022.
  ista: Johnson AJ, Kaufmann W, Sommer CM, Costanzo T, Dahhan DA, Bednarek SY, Friml
    J. 2022. Three-dimensional visualization of planta clathrin-coated vesicles at
    ultrastructural resolution. Molecular Plant. 15(10), 1533–1542.
  mla: Johnson, Alexander J., et al. “Three-Dimensional Visualization of Planta Clathrin-Coated
    Vesicles at Ultrastructural Resolution.” <i>Molecular Plant</i>, vol. 15, no.
    10, Elsevier, 2022, pp. 1533–42, doi:<a href="https://doi.org/10.1016/j.molp.2022.09.003">10.1016/j.molp.2022.09.003</a>.
  short: A.J. Johnson, W. Kaufmann, C.M. Sommer, T. Costanzo, D.A. Dahhan, S.Y. Bednarek,
    J. Friml, Molecular Plant 15 (2022) 1533–1542.
date_created: 2023-01-16T09:51:49Z
date_published: 2022-10-03T00:00:00Z
date_updated: 2023-08-04T09:39:24Z
day: '03'
ddc:
- '580'
department:
- _id: JiFr
- _id: EM-Fac
- _id: Bio
doi: 10.1016/j.molp.2022.09.003
external_id:
  isi:
  - '000882769800009'
  pmid:
  - '36081349'
file:
- access_level: open_access
  checksum: 04d5c12490052d03e4dc4412338a43dd
  content_type: application/pdf
  creator: dernst
  date_created: 2023-01-30T07:46:51Z
  date_updated: 2023-01-30T07:46:51Z
  file_id: '12435'
  file_name: 2022_MolecularPlant_Johnson.pdf
  file_size: 2307251
  relation: main_file
  success: 1
file_date_updated: 2023-01-30T07:46:51Z
has_accepted_license: '1'
intvolume: '        15'
isi: 1
issue: '10'
keyword:
- Plant Science
- Molecular Biology
language:
- iso: eng
license: https://creativecommons.org/licenses/by/4.0/
month: '10'
oa: 1
oa_version: Published Version
page: 1533-1542
pmid: 1
project:
- _id: 26538374-B435-11E9-9278-68D0E5697425
  call_identifier: FWF
  grant_number: I03630
  name: Molecular mechanisms of endocytic cargo recognition in plants
publication: Molecular Plant
publication_identifier:
  issn:
  - 1674-2052
publication_status: published
publisher: Elsevier
quality_controlled: '1'
scopus_import: '1'
status: public
title: Three-dimensional visualization of planta clathrin-coated vesicles at ultrastructural
  resolution
tmp:
  image: /images/cc_by.png
  legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode
  name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)
  short: CC BY (4.0)
type: journal_article
user_id: 4359f0d1-fa6c-11eb-b949-802e58b17ae8
volume: 15
year: '2022'
...
---
_id: '15037'
abstract:
- lang: eng
  text: Protein abundance and localization at the plasma membrane (PM) shapes plant
    development and mediates adaptation to changing environmental conditions. It is
    regulated by ubiquitination, a post-translational modification crucial for the
    proper sorting of endocytosed PM proteins to the vacuole for subsequent degradation.
    To understand the significance and the variety of roles played by this reversible
    modification, the function of ubiquitin receptors, which translate the ubiquitin
    signature into a cellular response, needs to be elucidated. In this study, we
    show that TOL (TOM1-like) proteins function in plants as multivalent ubiquitin
    receptors, governing ubiquitinated cargo delivery to the vacuole via the conserved
    Endosomal Sorting Complex Required for Transport (ESCRT) pathway. TOL2 and TOL6
    interact with components of the ESCRT machinery and bind to K63-linked ubiquitin
    via two tandemly arranged conserved ubiquitin-binding domains. Mutation of these
    domains results not only in a loss of ubiquitin binding but also altered localization,
    abolishing TOL6 ubiquitin receptor activity. Function and localization of TOL6
    is itself regulated by ubiquitination, whereby TOL6 ubiquitination potentially
    modulates degradation of PM-localized cargoes, assisting in the fine-tuning of
    the delicate interplay between protein recycling and downregulation. Taken together,
    our findings demonstrate the function and regulation of a ubiquitin receptor that
    mediates vacuolar degradation of PM proteins in higher plants.
article_processing_charge: No
article_type: original
author:
- first_name: Jeanette
  full_name: Moulinier-Anzola, Jeanette
  last_name: Moulinier-Anzola
- first_name: Maximilian
  full_name: Schwihla, Maximilian
  last_name: Schwihla
- first_name: Lucinda
  full_name: De-Araújo, Lucinda
  last_name: De-Araújo
- first_name: Christina
  full_name: Artner, Christina
  id: 45DF286A-F248-11E8-B48F-1D18A9856A87
  last_name: Artner
- first_name: Lisa
  full_name: Jörg, Lisa
  last_name: Jörg
- first_name: Nataliia
  full_name: Konstantinova, Nataliia
  last_name: Konstantinova
- first_name: Christian
  full_name: Luschnig, Christian
  last_name: Luschnig
- first_name: Barbara
  full_name: Korbei, Barbara
  last_name: Korbei
citation:
  ama: Moulinier-Anzola J, Schwihla M, De-Araújo L, et al. TOLs function as ubiquitin
    receptors in the early steps of the ESCRT pathway in higher plants. <i>Molecular
    Plant</i>. 2020;13(5):717-731. doi:<a href="https://doi.org/10.1016/j.molp.2020.02.012">10.1016/j.molp.2020.02.012</a>
  apa: Moulinier-Anzola, J., Schwihla, M., De-Araújo, L., Artner, C., Jörg, L., Konstantinova,
    N., … Korbei, B. (2020). TOLs function as ubiquitin receptors in the early steps
    of the ESCRT pathway in higher plants. <i>Molecular Plant</i>. Elsevier. <a href="https://doi.org/10.1016/j.molp.2020.02.012">https://doi.org/10.1016/j.molp.2020.02.012</a>
  chicago: Moulinier-Anzola, Jeanette, Maximilian Schwihla, Lucinda De-Araújo, Christina
    Artner, Lisa Jörg, Nataliia Konstantinova, Christian Luschnig, and Barbara Korbei.
    “TOLs Function as Ubiquitin Receptors in the Early Steps of the ESCRT Pathway
    in Higher Plants.” <i>Molecular Plant</i>. Elsevier, 2020. <a href="https://doi.org/10.1016/j.molp.2020.02.012">https://doi.org/10.1016/j.molp.2020.02.012</a>.
  ieee: J. Moulinier-Anzola <i>et al.</i>, “TOLs function as ubiquitin receptors in
    the early steps of the ESCRT pathway in higher plants,” <i>Molecular Plant</i>,
    vol. 13, no. 5. Elsevier, pp. 717–731, 2020.
  ista: Moulinier-Anzola J, Schwihla M, De-Araújo L, Artner C, Jörg L, Konstantinova
    N, Luschnig C, Korbei B. 2020. TOLs function as ubiquitin receptors in the early
    steps of the ESCRT pathway in higher plants. Molecular Plant. 13(5), 717–731.
  mla: Moulinier-Anzola, Jeanette, et al. “TOLs Function as Ubiquitin Receptors in
    the Early Steps of the ESCRT Pathway in Higher Plants.” <i>Molecular Plant</i>,
    vol. 13, no. 5, Elsevier, 2020, pp. 717–31, doi:<a href="https://doi.org/10.1016/j.molp.2020.02.012">10.1016/j.molp.2020.02.012</a>.
  short: J. Moulinier-Anzola, M. Schwihla, L. De-Araújo, C. Artner, L. Jörg, N. Konstantinova,
    C. Luschnig, B. Korbei, Molecular Plant 13 (2020) 717–731.
date_created: 2024-02-28T08:55:56Z
date_published: 2020-05-04T00:00:00Z
date_updated: 2024-02-28T12:41:52Z
day: '04'
ddc:
- '580'
department:
- _id: EvBe
doi: 10.1016/j.molp.2020.02.012
external_id:
  pmid:
  - '32087370'
file:
- access_level: open_access
  checksum: c538a5008f7827f62d17d40a3bfabe65
  content_type: application/pdf
  creator: dernst
  date_created: 2024-02-28T12:39:56Z
  date_updated: 2024-02-28T12:39:56Z
  file_id: '15038'
  file_name: 2020_MolecularPlant_MoulinierAnzola.pdf
  file_size: 3089212
  relation: main_file
  success: 1
file_date_updated: 2024-02-28T12:39:56Z
has_accepted_license: '1'
intvolume: '        13'
issue: '5'
keyword:
- Plant Science
- Molecular Biology
language:
- iso: eng
month: '05'
oa: 1
oa_version: Published Version
page: 717-731
pmid: 1
publication: Molecular Plant
publication_identifier:
  issn:
  - 1674-2052
publication_status: published
publisher: Elsevier
quality_controlled: '1'
status: public
title: TOLs function as ubiquitin receptors in the early steps of the ESCRT pathway
  in higher plants
tmp:
  image: /images/cc_by.png
  legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode
  name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)
  short: CC BY (4.0)
type: journal_article
user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87
volume: 13
year: '2020'
...
---
_id: '6920'
article_processing_charge: No
article_type: original
author:
- first_name: Christina
  full_name: Artner, Christina
  id: 45DF286A-F248-11E8-B48F-1D18A9856A87
  last_name: Artner
- first_name: Eva
  full_name: Benková, Eva
  id: 38F4F166-F248-11E8-B48F-1D18A9856A87
  last_name: Benková
  orcid: 0000-0002-8510-9739
citation:
  ama: Artner C, Benková E. Ethylene and cytokinin - partners in root growth regulation.
    <i>Molecular Plant</i>. 2019;12(10):1312-1314. doi:<a href="https://doi.org/10.1016/j.molp.2019.09.003">10.1016/j.molp.2019.09.003</a>
  apa: Artner, C., &#38; Benková, E. (2019). Ethylene and cytokinin - partners in
    root growth regulation. <i>Molecular Plant</i>. Cell Press. <a href="https://doi.org/10.1016/j.molp.2019.09.003">https://doi.org/10.1016/j.molp.2019.09.003</a>
  chicago: Artner, Christina, and Eva Benková. “Ethylene and Cytokinin - Partners
    in Root Growth Regulation.” <i>Molecular Plant</i>. Cell Press, 2019. <a href="https://doi.org/10.1016/j.molp.2019.09.003">https://doi.org/10.1016/j.molp.2019.09.003</a>.
  ieee: C. Artner and E. Benková, “Ethylene and cytokinin - partners in root growth
    regulation,” <i>Molecular Plant</i>, vol. 12, no. 10. Cell Press, pp. 1312–1314,
    2019.
  ista: Artner C, Benková E. 2019. Ethylene and cytokinin - partners in root growth
    regulation. Molecular Plant. 12(10), 1312–1314.
  mla: Artner, Christina, and Eva Benková. “Ethylene and Cytokinin - Partners in Root
    Growth Regulation.” <i>Molecular Plant</i>, vol. 12, no. 10, Cell Press, 2019,
    pp. 1312–14, doi:<a href="https://doi.org/10.1016/j.molp.2019.09.003">10.1016/j.molp.2019.09.003</a>.
  short: C. Artner, E. Benková, Molecular Plant 12 (2019) 1312–1314.
date_created: 2019-09-30T10:00:40Z
date_published: 2019-10-07T00:00:00Z
date_updated: 2023-08-30T06:55:02Z
day: '07'
department:
- _id: EvBe
doi: 10.1016/j.molp.2019.09.003
external_id:
  isi:
  - '000489132500002'
  pmid:
  - '31541740'
intvolume: '        12'
isi: 1
issue: '10'
language:
- iso: eng
month: '10'
oa_version: None
page: 1312-1314
pmid: 1
project:
- _id: 2685A872-B435-11E9-9278-68D0E5697425
  name: Hormonal regulation of plant adaptive responses to environmental signals
publication: Molecular Plant
publication_identifier:
  issn:
  - 1674-2052
  - 1752-9867
publication_status: published
publisher: Cell Press
quality_controlled: '1'
scopus_import: '1'
status: public
title: Ethylene and cytokinin - partners in root growth regulation
type: journal_article
user_id: 4359f0d1-fa6c-11eb-b949-802e58b17ae8
volume: 12
year: '2019'
...