@article{12239,
  abstract     = {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.},
  author       = {Johnson, Alexander J and Kaufmann, Walter and Sommer, Christoph M and Costanzo, Tommaso and Dahhan, Dana A. and Bednarek, Sebastian Y. and Friml, Jiří},
  issn         = {1674-2052},
  journal      = {Molecular Plant},
  keywords     = {Plant Science, Molecular Biology},
  number       = {10},
  pages        = {1533--1542},
  publisher    = {Elsevier},
  title        = {{Three-dimensional visualization of planta clathrin-coated vesicles at ultrastructural resolution}},
  doi          = {10.1016/j.molp.2022.09.003},
  volume       = {15},
  year         = {2022},
}

@article{15037,
  abstract     = {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.},
  author       = {Moulinier-Anzola, Jeanette and Schwihla, Maximilian and De-Araújo, Lucinda and Artner, Christina and Jörg, Lisa and Konstantinova, Nataliia and Luschnig, Christian and Korbei, Barbara},
  issn         = {1674-2052},
  journal      = {Molecular Plant},
  keywords     = {Plant Science, Molecular Biology},
  number       = {5},
  pages        = {717--731},
  publisher    = {Elsevier},
  title        = {{TOLs function as ubiquitin receptors in the early steps of the ESCRT pathway in higher plants}},
  doi          = {10.1016/j.molp.2020.02.012},
  volume       = {13},
  year         = {2020},
}

@article{6920,
  author       = {Artner, Christina and Benková, Eva},
  issn         = {1674-2052},
  journal      = {Molecular Plant},
  number       = {10},
  pages        = {1312--1314},
  publisher    = {Cell Press},
  title        = {{Ethylene and cytokinin - partners in root growth regulation}},
  doi          = {10.1016/j.molp.2019.09.003},
  volume       = {12},
  year         = {2019},
}