99 results on '"Daniël Van Damme"'
Search Results
2. Nanobody-Dependent Delocalization of Endocytic Machinery in Arabidopsis Root Cells Dampens Their Internalization Capacity
- Author
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Joanna Winkler, Andreas De Meyer, Evelien Mylle, Veronique Storme, Peter Grones, and Daniël Van Damme
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nanobody ,endocytosis ,Arabidopsis ,protein delocalization ,fluorescence microscopy ,TPLATE complex (TPC) ,Plant culture ,SB1-1110 - Abstract
Plant cells perceive and adapt to an ever-changing environment by modifying their plasma membrane (PM) proteome. Whereas secretion deposits new integral membrane proteins, internalization by endocytosis removes membrane proteins and associated ligands, largely with the aid of adaptor protein (AP) complexes and the scaffolding molecule clathrin. Two AP complexes function in clathrin-mediated endocytosis at the PM in plant cells, the heterotetrameric AP-2 complex and the hetero-octameric TPLATE complex (TPC). Whereas single subunit mutants in AP-2 develop into viable plants, genetic mutation of a single TPC subunit causes fully penetrant male sterility and silencing single subunits leads to seedling lethality. To address TPC function in somatic root cells, while minimizing indirect effects on plant growth, we employed nanobody-dependent delocalization of a functional, GFP-tagged TPC subunit, TML, in its respective homozygous genetic mutant background. In order to decrease the amount of functional TPC at the PM, we targeted our nanobody construct to the mitochondria and fused it to TagBFP2 to visualize it independently of its bait. We furthermore limited the effect of our delocalization to those tissues that are easily accessible for live-cell imaging by expressing it from the PIN2 promoter, which is active in root epidermal and cortex cells. With this approach, we successfully delocalized TML from the PM. Moreover, we also show co-recruitment of TML-GFP and AP2A1-TagRFP to the mitochondria, suggesting that our approach delocalized complexes, rather than individual adaptor complex subunits. In line with the specific expression domain, we only observed minor effects on root growth, yet realized a clear reduction of endocytic flux in epidermal root cells. Nanobody-dependent delocalization in plants, here exemplified using a TPC subunit, has the potential to be widely applicable to achieve specific loss-of-function analysis of otherwise lethal mutants.
- Published
- 2021
- Full Text
- View/download PDF
3. Mitochondrial uncouplers inhibit clathrin-mediated endocytosis largely through cytoplasmic acidification
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Wim Dejonghe, Sabine Kuenen, Evelien Mylle, Mina Vasileva, Olivier Keech, Corrado Viotti, Jef Swerts, Matyáš Fendrych, Fausto Andres Ortiz-Morea, Kiril Mishev, Simon Delang, Stefan Scholl, Xavier Zarza, Mareike Heilmann, Jiorgos Kourelis, Jaroslaw Kasprowicz, Le Son Long Nguyen, Andrzej Drozdzecki, Isabelle Van Houtte, Anna-Mária Szatmári, Mateusz Majda, Gary Baisa, Sebastian York Bednarek, Stéphanie Robert, Dominique Audenaert, Christa Testerink, Teun Munnik, Daniël Van Damme, Ingo Heilmann, Karin Schumacher, Johan Winne, Jiří Friml, Patrik Verstreken, and Eugenia Russinova
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Science - Abstract
Plant cells maintain strict proton gradients over different membranes. Here, Dejonghe et al. show that several protonophores, including the known tyrosine kinase inhibitor TyrphostinA23, inhibit clathrin-mediated endocytosis by disturbing these gradients and causing cytoplasmic acidification.
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- 2016
- Full Text
- View/download PDF
4. The ArathEULS3 Lectin Ends up in Stress Granules and Can Follow an Unconventional Route for Secretion
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Malgorzata Dubiel, Tibo De Coninck, Vinicius Jose Silva Osterne, Isabel Verbeke, Daniël Van Damme, Guy Smagghe, and Els J. M. Van Damme
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arabidopsis ,aratheuls3 ,intrinsically disordered regions ,plant lectin ,stress granules ,unconventional protein secretion ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Stress granules are cytoplasmic compartments, which serve as mRNA storage units during stress, therefore regulating translation. The Arabidopsis thaliana lectin ArathEULS3 has been widely described as a stress inducible gene. This study aimed to examine in detail the localization of ArathEULS3 lectin in normal and stressed cells. Colocalization experiments revealed that the nucleo-cytoplasmic lectin ArathEULS3 relocates to stress granules after stress. The ArathEULS3 sequence encodes a protein with a EUL lectin domain and an N-terminal domain with unknown structure and function. Bioinformatics analyses showed that the N-terminal domain sequence contains intrinsically disordered regions and likely does not exhibit a stable protein fold. Plasmolysis experiments indicated that ArathEULS3 also localizes to the apoplast, suggesting that this protein might follow an unconventional route for secretion. As part of our efforts we also investigated the interactome of ArathEULS3 and identified several putative interaction partners important for the protein translation process.
- Published
- 2020
- Full Text
- View/download PDF
5. The transcription factor AtMYB12 is part of a feedback loop regulating cell division orientation in the root meristem vasculature
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Brecht Wybouw, Helena E Arents, Baojun Yang, Jonah Nolf, Wouter Smet, Michael Vandorpe, Max Minne, Xiaopeng Luo, Inge De Clercq, Daniël Van Damme, Matouš Glanc, Bert De Rybel, and Turner, Simon
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EMS screen ,cytokinin ,Arabidopsis thaliana ,root development ,Physiology ,transcription factors ,Biology and Life Sciences ,Plant Science ,vascular development - Abstract
Transcriptional networks are crucial to integrate various internal and external signals into optimal responses during plant growth and development. In Arabidopsis thaliana, primary root vasculature patterning and proliferation are controlled by a network centred around the basic Helix-Loop-Helix transcription factor complex, formed by TARGET OF MONOPTEROS 5 (TMO5) and LONESOME HIGHWAY (LHW), which control cell proliferation and division orientation by modulating the cytokinin response and other downstream factors. Despite recent progress, many aspects of the TMO5/LHW pathway are not fully understood. In particular, the upstream regulators of TMO5/LHW activity remain unknown. Here, using a forward genetics approach to identify new factors of the TMO5/LHW pathway, we discovered a novel function of the MYB-type transcription factor, MYB12. MYB12 physically interacts with TMO5 and dampens the TMO5/LHW-mediated induction of direct target gene expression, as well as the periclinal/radial cell divisions. The expression of MYB12 is activated by the cytokinin response, downstream of TMO5/LHW, resulting in a novel MYB12-mediated negative feedback loop that restricts TMO5/LHW activity, to ensure optimal cell proliferation rates during root vascular development.
- Published
- 2023
6. The endocytic TPLATE complex internalizes ubiquitinated plasma membrane cargo
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Peter Grones, Andreas De Meyer, Roman Pleskot, Evelien Mylle, Michael Kraus, Michael Vandorpe, Klaas Yperman, Dominique Eeckhout, Jonathan Michael Dragwidge, Qihang Jiang, Jonah Nolf, Benjamin Pavie, Geert De Jaeger, Bert De Rybel, and Daniël Van Damme
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POLAR LOCALIZATION ,DEUBIQUITINATING ENZYME AMSH3 ,BORATE TRANSPORTER BOR1 ,BRASSINOSTEROID INSENSITIVE1 ,INTRACELLULAR TRAFFICKING ,Biology and Life Sciences ,RECOGNITION RECEPTOR FLS2 ,SOMATIC CYTOKINESIS ,Plant Science ,DEPENDENT ENDOCYTOSIS ,CLATHRIN-MEDIATED ENDOCYTOSIS ,Article ,CELLULOSE SYNTHASE - Abstract
Endocytosis controls the perception of stimuli by modulating protein abundance at the plasma membrane. In plants, clathrin-mediated endocytosis is the most prominent internalization pathway and relies on two multimeric adaptor complexes, the AP-2 and the TPLATE complex (TPC). Ubiquitination is a well-established modification triggering endocytosis of cargo proteins, but how this modification is recognized to initiate the endocytic event remains elusive. Here, we show that TASH3, one of the large subunits of TPC, recognizes ubiquitinated cargo at the plasma membrane via its SH3 domain-containing appendage. TASH3 lacking this evolutionary specific appendage modification allows TPC formation, but the plants show severely reduced endocytic densities, which correlates with reduced endocytic flux. Moreover, comparative plasma membrane proteomics identified differential accumulation of multiple ubiquitinated cargo proteins for which we confirm altered trafficking. Our findings position TPC as a key player for ubiquitinated cargo internalization, allowing future identification of target proteins under specific stress conditions.
- Published
- 2022
7. BRASSINOSTEROID INSENSITIVE1 internalization can occur independent of ligand binding
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Lucas Alves Neubus Claus, Derui Liu, Ulrich Hohmann, Nemanja Vukašinović, Roman Pleskot, Jing Liu, Alexei Schiffner, Yvon Jaillais, Guang Wu, Sebastian Wolf, Daniël Van Damme, Michael Hothorn, Eugenia Russinova, Universiteit Gent = Ghent University (UGENT), Shandong Agricultural University (SDAU), VIB [Belgium], Université de Genève = University of Geneva (UNIGE), Austrian Academy of Sciences (OeAW), Institute of Experimental Botany of the Czech Academy of Sciences (IEB / CAS), Czech Academy of Sciences [Prague] (CAS), Shaanxi Normal University (SNNU), Universitätsklinikum Tübingen - University Hospital of Tübingen, Eberhard Karls Universität Tübingen = Eberhard Karls University of Tuebingen, Reproduction et développement des plantes (RDP), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Center for Plant Systems Biology (PSB Center), Vlaams Instituut voor Biotechnologie [Ghent, Belgique] (VIB), and École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL)
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PERCEPTION ,ligand-binding ,RECEPTOR ,Physiology ,PROTEINS ,ENDOCYTOSIS ,Biology and Life Sciences ,Plant Science ,[SDV.BV.BOT]Life Sciences [q-bio]/Vegetal Biology/Botanics ,DEGRADATION ,EXTRACELLULAR DOMAIN ,BRI1 ,brassinosteroids ,non-canonical ,[SDE]Environmental Sciences ,Genetics ,KINASE ,endocytosis ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,TRAFFICKING - Abstract
The brassinosteroid (BR) hormone and its plasma membrane (PM) receptor BR INSENSITIVE1 (BRI1) are one of the best-studied receptor–ligand pairs for understanding the interplay between receptor endocytosis and signaling in plants. BR signaling is mainly determined by the PM pool of BRI1, whereas BRI1 endocytosis ensures signal attenuation. As BRs are ubiquitously distributed in the plant, the tools available to study the BRI1 function without interference from endogenous BRs are limited. Here, we designed a BR binding-deficient Arabidopsis (Arabidopsis thaliana) mutant based on protein sequence-structure analysis and homology modeling of members of the BRI1 family. This tool allowed us to re-examine the BRI1 endocytosis and signal attenuation model. We showed that despite impaired phosphorylation and ubiquitination, BR binding-deficient BRI1 internalizes similarly to the wild type form. Our data indicate that BRI1 internalization relies on different endocytic machineries. In addition, the BR binding-deficient mutant provides opportunities to study non-canonical ligand-independent BRI1 functions.
- Published
- 2023
8. Adaptor protein complex interaction map in Arabidopsis identifies P34 as a common stability regulator
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Peng Wang, Wei Siao, Xiuyang Zhao, Deepanksha Arora, Ren Wang, Dominique Eeckhout, Jelle Van Leene, Rahul Kumar, Anaxi Houbaert, Nancy De Winne, Evelien Mylle, Michael Vandorpe, Ruud A. Korver, Christa Testerink, Kris Gevaert, Steffen Vanneste, Geert De Jaeger, Daniël Van Damme, and Eugenia Russinova
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Life Science ,Laboratorium voor Plantenfysiologie ,Plant Science ,Laboratory of Plant Physiology - Abstract
Adaptor protein (AP) complexes are evolutionarily conserved vesicle transport regulators that recruit coat proteins, membrane cargoes and coated vesicle accessory proteins. As in plants endocytic and post-Golgi trafficking intersect at the trans-Golgi network, unique mechanisms for sorting cargoes of overlapping vesicular routes are anticipated. The plant AP complexes are part of the sorting machinery, but despite some functional information, their cargoes, accessory proteins and regulation remain largely unknown. Here, by means of various proteomics approaches, we generated the overall interactome of the five AP and the TPLATE complexes in Arabidopsis thaliana. The interactome converged on a number of hub proteins, including the thus far unknown adaptin binding-like protein, designated P34. P34 interacted with the clathrin-associated AP complexes, controlled their stability and, subsequently, influenced clathrin-mediated endocytosis and various post-Golgi trafficking routes. Altogether, the AP interactome network offers substantial resources for further discoveries of unknown endomembrane trafficking regulators in plant cells.
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- 2023
9. Phase separation to visualize protein-protein interactions and kinase activities in planta
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Alaeddine Safi, Wouter Smagghe, Amanda Goncalves, Benjamin Cappe, Franck Riquet, Evelyn Mylle, Daniël Van Damme, Danny Geelen, Geert De Jaeger, Tom Beeckman, Jelle Van Leene, and Steffen Vanneste
- Abstract
Protein complex formation and dynamic post-translational modifications are notoriously difficult to monitor at cellular resolution. Here, we developed a versatile modular toolbox of fluorescently labelled, artificial homo-oligomerizing peptide-tags (HOTag) that install interaction-dependent liquid-liquid phase-separation upon interaction between two proteins of interest. We deployed our novel toolbox for the in planta visualization of inducible, binary and ternary protein-protein interactions (PPIs), as well as specific phosphorylation, showing its great potential to become a robust standard technique to study PPIs and phosphorylation in plants.
- Published
- 2022
10. Phase separation-based visualization of protein-protein interactions and kinase activities in plants
- Author
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Alaeddine Safi, Wouter Smagghe, Amanda Gonçalves, Ke Xu, Ana Ibis Fernandez, Benjamin Cappe, Franck B. Riquet, Evelien Mylle, Daniël Van Damme, Danny Geelen, Geert De Jaeger, Tom Beeckman, Jelle Van Leene, and Steffen Vanneste
- Abstract
Protein activities depend heavily on protein complex formation and dynamic post-translational modifications, such as phosphorylation. Their dynamic nature is notoriously difficult to monitor in planta at cellular resolution, often requiring extensive optimization and high-end microscopy. Here, we generated and exploited the SYnthetic Multivalency in PLants (SYMPL)-vector set to study protein-protein interactions (PPIs) and kinase activities in planta based on phase separation. This technology enabled easy detection of inducible, binary and ternary protein-protein interactions among cytoplasmic, nuclear and plasma membrane proteins in plant cells via a robust image-based readout. Moreover, we applied the SYMPL toolbox to develop an in vivo reporter for SnRK1 kinase activity, allowing us to visualize tissue-specific, dynamic SnRK1 activation upon energy deprivation in stable transgenic Arabidopsis plants. The applications of the SYMPL cloning toolbox lay the foundation for the exploration of PPIs, phosphorylation and other post-translational modifications with unprecedented ease and sensitivity.
- Published
- 2022
11. Mapping the adaptor protein complex interaction network in Arabidopsis identifies P34 as a common stability regulator
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Peng Wang, Wei Siao, Xiuyang Zhao, Deepanksha Arora, Ren Wang, Dominique Eeckhout, Jelle Van Leene, Rahul Kumar, Anaxi Houbaert, Nancy De Winne, Evelien Mylle, Michael Vandorpe, Ruud A. Korver, Christa Testerink, Kris Gevaert, Steffen Vanneste, Geert De Jaeger, Daniël Van Damme, and Eugenia Russinova
- Abstract
Adaptor protein (AP) complexes are evolutionarily conserved vesicle transport regulators that recruit coat proteins, membrane cargos and coated vesicle accessory proteins. Since in plants endocytic and post-Golgi trafficking intersect at the trans-Golgi network, unique mechanisms for sorting cargos of overlapping vesicular routes are anticipated. The plant AP complexes are part of the sorting machinery, but despite some functional information, their cargoes, accessory proteins, and regulation remain largely unknown. Here, by means of various proteomics approaches, we generated the overall interactome of the five AP and the TPLATE complexes in Arabidopsis thaliana. The interactome converged on a number of hub proteins, including the thus far unknown adaptin binding-like protein, designated P34. P34 interacted with the clathrin-associated AP complexes, controlled their stability and, subsequently, influenced clathrin-mediated endocytosis and various post-Golgi trafficking routes. Altogether, the AP interactome network offers substantial resources for further discoveries of unknown endomembrane trafficking regulators in plant cells.
- Published
- 2022
12. Phosphorylation and ubiquitination independent endocytosis of BRI1
- Author
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Lucas Alves Neubus Claus, Derui Liu, Ulrich Hohmann, Nemanja Vukašinović, Roman Pleskot, Jing Liu, Alexei Schiffner, Yvon Jaillais, Guang Wu, Sebastian Wolf, Daniël Van Damme, Michael Hothorn, and Eugenia Russinova
- Abstract
The brassinosteroid (BR) hormone and its plasma membrane receptor BR INSENSITIVE1 (BRI1) is one of the best-studied receptor-ligand pairs for understanding the interplay between receptor endocytosis and signaling in plants. BR signaling is mainly determined by the plasma membrane pool of BRI1, whereas BRI1 endocytosis ensures signal attenuation. Since BRs are ubiquitously distributed in the plant, the tools available to study BRI1 function without interference from endogenous BRs are limited. Here, we designed a BR-binding-deficient mutant based on protein sequence-structure analysis and homology modeling of BRI1 and its close homologues. This new tool allowed us to re-examine the BRI1 endocytosis and signal attenuation model. We show that despite decreased phosphorylation and ubiquitination, the BR-binding-deficient BRI1 was internalized similar to the wild type form. These results reinforce the hypothesis that BRI1 is internalized via parallel endocytic routes and machineries. In addition, BR-binding-deficient mutant provides opportunities to study non-canonical ligand-independent BRI1 functions.
- Published
- 2022
13. Author Correction: The endocytic TPLATE complex internalizes ubiquitinated plasma membrane cargo
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Peter Grones, Andreas De Meyer, Roman Pleskot, Evelien Mylle, Michael Kraus, Michael Vandorpe, Klaas Yperman, Dominique Eeckhout, Jonathan Michael Dragwidge, Qihang Jiang, Jonah Nolf, Benjamin Pavie, Geert De Jaeger, Bert De Rybel, and Daniël Van Damme
- Subjects
Plant Science - Published
- 2023
14. Biomolecular condensation orchestrates clathrin-mediated endocytosis in plants
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Daniël Van Damme, Jonathan Dragwidge, Yanning Wang, Lysiane Brocard, Andreas De Meyer, Roman Hudeček, Dominique eeckhout, Peter Grones, Matthieu Buridan, Clément Chambaud, Přemysl Pejchar, Martin Potocký, Joanna Winkler, Michael Vandorpe, Nelson Serre, Matyas Fendrych, Amélie Bernard, Geert De Jaeger, Roman Pleskot, and Xiaofeng Fang
- Abstract
SummaryClathrin-mediated endocytosis (CME) is an essential cellular internalisation pathway involving the dynamic assembly of clathrin and accessory proteins to form membrane-bound vesicles. In plants, the evolutionarily ancient TSET/TPLATE complex (TPC) plays an essential, but not well-defined role in CME. 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, early-, and late-stage endocytic accessory proteins. Importantly, clathrin forms ordered assemblies within the condensate environment. Biomolecular condensation therefore acts to promote dynamic protein assemblies throughout clathrin-mediated endocytosis. Furthermore, the disordered region sequence properties of AtEH1 regulate the material properties of the endocytic condensatesin vivoand alteration of these material properties influences endocytosis dynamics, and consequently plant adaptive growth.HighlightsAtEH subunits are endocytic scaffolds which drive condensation of the TPCAtEH1 condensates nucleate on the plasma membrane via lipid interactionsCondensation of AtEH1/TPC facilitates clathrin re-arrangement and assemblyAtEH1 IDR1 composition controls condensate properties to regulate endocytosis
- Published
- 2022
15. MYB12 spatiotemporally represses TMO5/LHW-mediated transcription in the Arabidopsis root meristem
- Author
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Brecht Wybouw, Helena E. Arents, Baojun Yang, Jonah Nolf, Wouter Smet, Michael Vandorpe, Daniël Van Damme, Matouš Glanc, and Bert De Rybel
- Abstract
Transcriptional networks are crucial to integrate various internal and external signals into optimal responses during plant growth and development. Primary root vasculature patterning and proliferation are controlled by a network centred around the basic Helix-Loop-Helix transcription factor complex formed by TARGET OF MONOPTEROS 5 (TMO5) and LONESOME HIGHWAY (LHW), which control cell proliferation and orientation by modulating cytokinin response and other downstream factors. Despite recent progress, many aspects of the TMO5/LHW pathway are not fully understood. In particular, the upstream regulators of TMO5/LHW activity remain unknown. Here, using a forward genetic approach to identify new factors of the TMO5/LHW pathway, we discovered a novel function of the MYB-type transcription factor MYB12. MYB12 physically interacts with TMO5 and dampens the TMO5/LHW-mediated induction of direct target gene expression as well as the periclinal/radial cell divisions. The expression ofMYB12is activated by the cytokinin response, downstream of TMO5/LHW, resulting in a novel MYB12-mediated negative feedback loop that restricts TMO5/LHW activity to ensure optimal cell proliferation rates during root vascular development.
- Published
- 2022
16. Mapping of the plant SnRK1 kinase signalling network reveals a key regulatory role for the class II T6P synthase-like proteins
- Author
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Jelle Van Leene, Dominique Eeckhout, Astrid Gadeyne, Caroline Matthijs, Chao Han, Nancy De Winne, Geert Persiau, Eveline Van De Slijke, Freya Persyn, Toon Mertens, Wouter Smagghe, Nathalie Crepin, Ellen Broucke, Daniël Van Damme, Roman Pleskot, Filip Rolland, and Geert De Jaeger
- Subjects
Arabidopsis Proteins ,METABOLIC PATHWAYS ,Trehalose ,Biology and Life Sciences ,LOCALIZATION ,Plant Science ,Protein Serine-Threonine Kinases ,Plants ,SENSOR SNRK1 ,EVOLUTION ,Gene Expression Regulation, Plant ,ARABIDOPSIS-THALIANA ,NEGATIVE REGULATORS ,STARCH ,Sugar Phosphates ,COMPLEXES ,TREHALOSE 6-PHOSPHATE ,PHOSPHORYLATION ,Signal Transduction - Abstract
The central metabolic regulator SnRK1 controls plant growth and survival upon activation by energy depletion, but detailed molecular insight into its regulation and downstream targets is limited. Here we used phosphoproteomics to infer the sucrose-dependent processes targeted upon starvation by kinases as SnRK1, corroborating the relation of SnRK1 with metabolic enzymes and transcriptional regulators, while also pointing to SnRK1 control of intracellular trafficking. Next, we integrated affinity purification, proximity labelling and crosslinking mass spectrometry to map the protein interaction landscape, composition and structure of the SnRK1 heterotrimer, providing insight in its plant-specific regulation. At the intersection of this multi-dimensional interactome, we discovered a strong association of SnRK1 with class II T6P synthase (TPS)-like proteins. Biochemical and cellular assays show that TPS-like proteins function as negative regulators of SnRK1. Next to stable interactions with the TPS-like proteins, similar intricate connections were found with known regulators, suggesting that plants utilize an extended kinase complex to fine-tune SnRK1 activity for optimal responses to metabolic stress.
- Published
- 2022
17. Auxin analog-induced Ca 2+ signaling is not involved in inhibition of endosomal aggregation in Arabidopsis roots
- Author
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Ren Wang, Ellie Himschoot, Matteo Grenzi, Jian Chen, Alaeddine Safi, Melanie Krebs, Karin Schumacher, Moritz K Nowack, Wolfgang Moeder, Keiko Yoshioka, Daniël Van Damme, Ive De Smet, Danny Geelen, Tom Beeckman, Jiří Friml, Alex Costa, Steffen Vanneste
- Published
- 2022
- Full Text
- View/download PDF
18. Proteomic characterization of isolated Arabidopsis clathrin-coated vesicles reveals evolutionarily conserved and plant-specific components
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Dana A Dahhan, Gregory D Reynolds, Jessica J Cárdenas, Dominique Eeckhout, Alexander Johnson, Klaas Yperman, Walter A Kaufmann, Nou Vang, Xu Yan, Inhwan Hwang, Antje Heese, Geert De Jaeger, Jiří Friml, Daniël Van Damme, Jianwei Pan, and Sebastian Y Bednarek
- Subjects
SECRETORY TRAFFICKING ,ADAPTER COMPLEX ,Biology and Life Sciences ,Cell Biology ,Plant Science ,DOMAIN-CONTAINING PROTEINS ,TRANS-GOLGI NETWORK ,PLASMA-MEMBRANE ,DEPENDENT TRAFFICKING ,VACUOLAR SORTING RECEPTOR ,MEDIATED ENDOCYTOSIS ,SUBCELLULAR-LOCALIZATION ,CELL PLATE - Abstract
Mass spectrometry analyses of Arabidopsis suspension-cultured cell clathrin-coated vesicles delineate the plant clathrin-coated vesicle (CCV) proteome and identify evolutionarily conserved and plant-specific CCV-associated factors. In eukaryotes, clathrin-coated vesicles (CCVs) facilitate the internalization of material from the cell surface as well as the movement of cargo in post-Golgi trafficking pathways. This diversity of functions is partially provided by multiple monomeric and multimeric clathrin adaptor complexes that provide compartment and cargo selectivity. The adaptor-protein assembly polypeptide-1 (AP-1) complex operates as part of the secretory pathway at the trans-Golgi network (TGN), while the AP-2 complex and the TPLATE complex jointly operate at the plasma membrane to execute clathrin-mediated endocytosis. Key to our further understanding of clathrin-mediated trafficking in plants will be the comprehensive identification and characterization of the network of evolutionarily conserved and plant-specific core and accessory machinery involved in the formation and targeting of CCVs. To facilitate these studies, we have analyzed the proteome of enriched TGN/early endosome-derived and endocytic CCVs isolated from dividing and expanding suspension-cultured Arabidopsis (Arabidopsis thaliana) cells. Tandem mass spectrometry analysis results were validated by differential chemical labeling experiments to identify proteins co-enriching with CCVs. Proteins enriched in CCVs included previously characterized CCV components and cargos such as the vacuolar sorting receptors in addition to conserved and plant-specific components whose function in clathrin-mediated trafficking has not been previously defined. Notably, in addition to AP-1 and AP-2, all subunits of the AP-4 complex, but not AP-3 or AP-5, were found to be in high abundance in the CCV proteome. The association of AP-4 with suspension-cultured Arabidopsis CCVs is further supported via additional biochemical data.
- Published
- 2022
19. Establishment of Proximity-Dependent Biotinylation Approaches in Different Plant Model Systems
- Author
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Francis Impens, Petra Van Damme, Thomas Ott, Jelle Van Leene, Daniël Van Damme, Geert De Jaeger, Alain Goossens, Nikolaj B. Abel, Klaas Yperman, Lam Dai Vu, Anna Tornkvist, Chen Liu, Barbara Korbei, Jie Wang, Dominique Eeckhout, Deepanksha Arora, and Panagiotis N. Moschou
- Subjects
0106 biological sciences ,0301 basic medicine ,Green Fluorescent Proteins ,Lotus japonicus ,Endocytic cycle ,Arabidopsis ,Biotin ,Plant Science ,Computational biology ,Breakthrough Report ,Biology ,01 natural sciences ,03 medical and health sciences ,chemistry.chemical_compound ,Solanum lycopersicum ,Affinity chromatography ,Labelling ,Tobacco ,Biotinylation ,Carbon-Nitrogen Ligases ,Protein Interaction Maps ,Plant system ,030304 developmental biology ,Plant Proteins ,2. Zero hunger ,chemistry.chemical_classification ,0303 health sciences ,DNA ligase ,Escherichia coli Proteins ,fungi ,Cell Membrane ,Temperature ,Biology and Life Sciences ,A protein ,Cell Biology ,Plants, Genetically Modified ,biology.organism_classification ,Recombinant Proteins ,Repressor Proteins ,Protein Subunits ,030104 developmental biology ,Regular Content ,chemistry ,Lotus ,010606 plant biology & botany - Abstract
The use of proximity-dependent biotin labelling (PDL) approaches coupled with mass spectrometry recently greatly advanced the identification of protein-protein interactions and study of protein complexation. PDL is based on the expression of a promiscuous biotin ligase (PBL), e.g. BirA* or a peroxidase fused to a bait protein of interest. In the presence of biotin as substrate, PBL enables covalent biotin labelling of proteins in the vicinity of the PBL-fused bait in vivo , allowing the subsequent capture and identification of interacting and neighbouring proteins without the need for the protein complex to remain intact during purification. To date, PDL has not been extensively used in plants. Here we present the results of a systematic multi-lab study applying a variety of PDL approaches in several plant systems under various conditions and bait proteins. We show that TurboID is the most promiscuous variant for PDL in plants and establish protocols for its efficient application. We demonstrate the applicability of TurboID in capturing membrane protein interactomes using the Lotus japonicus symbiotically active receptor kinases RLKs NOD FACTOR RECEPTOR 5 (NFR5) and LRR-RLK SYMBIOTIC RECEPTOR-KINASE (SYMRK) as test-cases. Furthermore, we benchmark the efficiency of various PBLs using the octameric endocytic TPLATE complex and compare PDL with one-step AP-MS approaches. Our results indicate that different PDL approaches in plants may differ in signal-to-noise ratio and robustness. We present a straightforward strategy to identify both non-biotinylated as well as biotinylated proteins in plants in a single experimental setup. Finally, we provide initial evidence that this technique has potential to infer structural information of protein complexes. Our methods, tools and adjustable pipelines provide a useful resource for the plant research community.
- Published
- 2020
20. Conservation of centromeric histone 3 interaction partners in plants
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Burcu Nur Keçeli, Daniël Van Damme, Chunlian Jin, and Danny Geelen
- Subjects
0106 biological sciences ,0301 basic medicine ,FLOWERING TIME ,Cell division ,Physiology ,KINETOCHORE FUNCTION ,Centromere ,Plant Science ,Haploidy ,Biology ,H3 VARIANT CSE4 ,01 natural sciences ,CENH3 ,Histones ,Chromosome segregation ,03 medical and health sciences ,Chromosome instability ,Homologous chromosome ,Animals ,chromosome ,protein interaction ,DNA METHYLATION ,Review Papers ,Genetics ,COMPLEX ,AcademicSubjects/SCI01210 ,haploid induction ,Kinetochore ,Biology and Life Sciences ,food and beverages ,Chromosome ,Plants ,ARABIDOPSIS ,030104 developmental biology ,post-translational modification ,centromere ,E3 UBIQUITIN LIGASE ,N-TERMINUS ,Histone fold ,Pollen ,CHROMOSOME SEGREGATION ,CENP-A ,010606 plant biology & botany - Abstract
Here we have listed 36 human and yeast CENH3 interaction partners and their corn, rice, and Arabidopsis homologs that are potentially interacting with plant CENH3., The loading and maintenance of centromeric histone 3 (CENH3) at the centromere are critical processes ensuring appropriate kinetochore establishment and equivalent segregation of the homologous chromosomes during cell division. CENH3 loss of function is lethal, whereas mutations in the histone fold domain are tolerated and lead to chromosome instability and chromosome elimination in embryos derived from crosses with wild-type pollen. A wide range of proteins in yeast and animals have been reported to interact with CENH3. The histone fold domain-interacting proteins are potentially alternative targets for the engineering of haploid inducer lines, which may be important when CENH3 mutations are not well supported by a given crop. Here, we provide an overview of the corresponding plant orthologs or functional homologs of CENH3-interacting proteins. We also list putative CENH3 post-translational modifications that are also candidate targets for modulating chromosome stability and inheritance.
- Published
- 2020
21. New opportunities and insights into Papaver self-incompatibility by imaging engineered Arabidopsis pollen
- Author
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Vernonica E. Franklin-Tong, Maurice Bosch, Zongcheng Lin, Ludi Wang, J. Carli, Daniël Van Damme, Moritz K. Nowack, Marina Muñoz Triviño, and Deborah J. Eaves
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0106 biological sciences ,0301 basic medicine ,Physiology ,Arabidopsis ,Plant Science ,medicine.disease_cause ,01 natural sciences ,live-cell imaging ,CA2+ ,TPLATE ,TUBES ,Arabidopsis thaliana ,TIP GROWTH ,Pollination ,Plant Proteins ,biology ,pH ,pollen tube growth ,BINDING PROTEINS ,food and beverages ,SOMATIC CYTOKINESIS ,Research Papers ,Cell biology ,Papaver ,Pollen ,Pollen tube ,actin-binding proteins (ABPs) ,self-incompatibility (SI) ,ORGANIZATION ,PROGRAMMED CELL-DEATH ,03 medical and health sciences ,fluorescent probes ,Live cell imaging ,medicine ,endocytosis ,Tip growth ,Actin ,calcium ,AcademicSubjects/SCI01210 ,RHOEAS ,Biology and Life Sciences ,biology.organism_classification ,Actin cytoskeleton ,ACTIN-DEPOLYMERIZING FACTOR ,programmed cell death (PCD) ,030104 developmental biology ,010606 plant biology & botany - Abstract
Use of genetically encoded fluorescent probes to monitor self-incompatibility (SI)-induced changes in pollen of the heterologous Arabidopsis ‘SI’ system has allowed multiparameter imaging and identified the involvement of clathrin-mediated endocytosis., Pollen tube growth is essential for plant reproduction. Their rapid extension using polarized tip growth provides an exciting system for studying this specialized type of growth. Self-incompatibility (SI) is a genetically controlled mechanism to prevent self-fertilization. Mechanistically, one of the best-studied SI systems is that of Papaver rhoeas (poppy). This utilizes two S-determinants: stigma-expressed PrsS and pollen-expressed PrpS. Interaction of cognate PrpS–PrsS triggers a signalling network, causing rapid growth arrest and programmed cell death (PCD) in incompatible pollen. We previously demonstrated that transgenic Arabidopsis thaliana pollen expressing PrpS–green fluorescent protein (GFP) can respond to Papaver PrsS with remarkably similar responses to those observed in incompatible Papaver pollen. Here we describe recent advances using these transgenic plants combined with genetically encoded fluorescent probes to monitor SI-induced cellular alterations, including cytosolic calcium, pH, the actin cytoskeleton, clathrin-mediated endocytosis (CME), and the vacuole. This approach has allowed us to study the SI response in depth, using multiparameter live-cell imaging approaches that were not possible in Papaver. This lays the foundations for new opportunities to elucidate key mechanisms involved in SI. Here we establish that CME is disrupted in self-incompatible pollen. Moreover, we reveal new detailed information about F-actin remodelling in pollen tubes after SI.
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- 2020
22. Plant AtEH/Pan1 proteins drive autophagosome formation at ER-PM contact sites with actin and endocytic machinery
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Klaas Yperman, Jingze Zang, Evelien Mylle, Daniël Van Damme, Kun Wang, Michaël Vandorpe, Jindriska Fiserova, Joanna Winkler, Patrick J. Hussey, Patrick Duckney, Tong Zhang, Jie Wang, Pengwei Wang, Jinli Gong, Christine Richardson, Yajie Guan, and Roman Pleskot
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0106 biological sciences ,0301 basic medicine ,Endocytic cycle ,Arabidopsis ,General Physics and Astronomy ,Plasma protein binding ,CYTOSKELETON ,Endoplasmic Reticulum ,01 natural sciences ,TPLATE ,Protein trafficking in plants ,Cytoskeleton ,lcsh:Science ,Phylogeny ,Multidisciplinary ,biology ,Chemistry ,PHOSPHATIDYLINOSITOL ,Microfilament Proteins ,ARP2/3 ,Endocytosis ,Cell biology ,Actin Cytoskeleton ,Protein Binding ,EXPRESSION ,Saccharomyces cerevisiae Proteins ,Science ,3-PHOSPHATE ,BIOGENESIS ,ENDOPLASMIC-RETICULUM ,macromolecular substances ,Clathrin ,Models, Biological ,General Biochemistry, Genetics and Molecular Biology ,Actin-Related Protein 2-3 Complex ,Article ,03 medical and health sciences ,Autophagy ,Actin ,COMPLEX ,Activator (genetics) ,Arabidopsis Proteins ,Cell Membrane ,Autophagosomes ,Biology and Life Sciences ,General Chemistry ,Actin cytoskeleton ,Actins ,030104 developmental biology ,PLASMA-MEMBRANE ,biology.protein ,lcsh:Q ,010606 plant biology & botany ,GENERATION - Abstract
The Arabidopsis EH proteins (AtEH1/Pan1 and AtEH2/Pan1) are components of the endocytic TPLATE complex (TPC) which is essential for endocytosis. Both proteins are homologues of the yeast ARP2/3 complex activator, Pan1p. Here, we show that these proteins are also involved in actin cytoskeleton regulated autophagy. Both AtEH/Pan1 proteins localise to the plasma membrane and autophagosomes. Upon induction of autophagy, AtEH/Pan1 proteins recruit TPC and AP-2 subunits, clathrin, actin and ARP2/3 proteins to autophagosomes. Increased expression of AtEH/Pan1 proteins boosts autophagosome formation, suggesting independent and redundant pathways for actin-mediated autophagy in plants. Moreover, AtEHs/Pan1-regulated autophagosomes associate with ER-PM contact sites (EPCS) where AtEH1/Pan1 interacts with VAP27-1. Knock-down expression of either AtEH1/Pan1 or VAP27-1 makes plants more susceptible to nutrient depleted conditions, indicating that the autophagy pathway is perturbed. In conclusion, we identify the existence of an autophagy-dependent pathway in plants to degrade endocytic components, starting at the EPCS through the interaction among AtEH/Pan1, actin cytoskeleton and the EPCS resident protein VAP27-1., Arabidopsis EH/Pan1 proteins are part of the TPLATE complex (TPC) that is required for endocytosis in plants. Here, the authors show AtEH/Pan1 proteins also act in actin-mediated autophagy, by interacting with VAP27-1 at ER-PM contact sites and recruiting TPLATE and AP-2 complex subunits, clathrin and ARP2/3/ proteins to autophagosomes.
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- 2019
23. The TPLATE complex mediates membrane bending during plant clathrin–mediated endocytosis
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Alexander Johnson, Dana A. Dahhan, Nataliia Gnyliukh, Walter A. Kaufmann, Vanessa Zheden, Tommaso Costanzo, Pierre Mahou, Mónika Hrtyan, Jie Wang, Juan Aguilera-Servin, Daniël Van Damme, Emmanuel Beaurepaire, Martin Loose, Sebastian Y. Bednarek, and Jiří Friml
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RECRUITMENT ,0106 biological sciences ,Microscopy, Electron, Scanning Transmission ,PROTEINS ,Arabidopsis ,Plant Biology ,clathrin-mediated endocytosis ,ADAPTER ,01 natural sciences ,ACTIN ,03 medical and health sciences ,TPLATE ,Plant Cells ,TRAFFICKING ,030304 developmental biology ,Fluorescent Dyes ,0303 health sciences ,Multidisciplinary ,Arabidopsis Proteins ,Cell Membrane ,Biology and Life Sciences ,Biological Sciences ,Clathrin ,Endocytosis ,Microscopy, Fluorescence ,Seedlings ,CHAIN ,membrane remodeling ,010606 plant biology & botany - Abstract
Significance Endocytosis transports cargos inside the cell by creating spherical vesicles from the plasma membrane. This membrane remodeling requires proteins to generate force to bend the membrane inwards, overcoming the high-turgor pressure in plant cells. However, as plants create clathrin-coated vesicles without actin, the machinery to bend membranes during endocytosis is entirely unknown and appears distinct from other model systems. Here, we refine the physiological role of the plant-specific and essential endocytic TPLATE complex. We find it localizes outside of clathrin-coated vesicles and mediates membrane bending, contrasting with previous predictions. We further demonstrate that the TPLATE complex contains protein domains which have intrinsic membrane-bending activity; thus, we identify a component of the unique endocytosis membrane-bending machinery in plants., Clathrin-mediated endocytosis is the major route of entry of cargos into cells and thus underpins many physiological processes. During endocytosis, an area of flat membrane is remodeled by proteins to create a spherical vesicle against intracellular forces. The protein machinery which mediates this membrane bending in plants is unknown. However, it is known that plant endocytosis is actin independent, thus indicating that plants utilize a unique mechanism to mediate membrane bending against high-turgor pressure compared to other model systems. Here, we investigate the TPLATE complex, a plant-specific endocytosis protein complex. It has been thought to function as a classical adaptor functioning underneath the clathrin coat. However, by using biochemical and advanced live microscopy approaches, we found that TPLATE is peripherally associated with clathrin-coated vesicles and localizes at the rim of endocytosis events. As this localization is more fitting to the protein machinery involved in membrane bending during endocytosis, we examined cells in which the TPLATE complex was disrupted and found that the clathrin structures present as flat patches. This suggests a requirement of the TPLATE complex for membrane bending during plant clathrin–mediated endocytosis. Next, we used in vitro biophysical assays to confirm that the TPLATE complex possesses protein domains with intrinsic membrane remodeling activity. These results redefine the role of the TPLATE complex and implicate it as a key component of the evolutionarily distinct plant endocytosis mechanism, which mediates endocytic membrane bending against the high-turgor pressure in plant cells.
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- 2021
24. Conditional and tissue-specific approaches to dissect essential mechanisms in plant development
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Moritz K. Nowack, Joanna Winkler, Marie L. Pfeiffer, Thomas Jacobs, and Daniël Van Damme
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EXPRESSION ,0106 biological sciences ,Plant Development ,Plant Science ,Computational biology ,Biology ,01 natural sciences ,Genome ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Degron ,Gene silencing ,CRISPR ,Clustered Regularly Interspaced Short Palindromic Repeats ,Gene ,Conditional ,030304 developmental biology ,CRISPR-Activation ,Gene Editing ,0303 health sciences ,Biology and Life Sciences ,RNA ,DNA ,ARABIDOPSIS ,Reverse genetics ,chemistry ,Nanobodies ,CRISPR-Cas Systems ,Tissue-specific ,Function (biology) ,010606 plant biology & botany - Abstract
Reverse genetics approaches are routinely used to investigate gene function. However, mutations, especially in critical genes, can lead to pleiotropic effects as severe as lethality, thus limiting functional studies in specific contexts. Approaches that allow for modifications of genes or gene products in a specific spatial or temporal setting can overcome these limitations. The advent of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technologies has not only revolutionized targeted genome modification in plants but also enabled new possibilities for inducible and tissue-specific manipulation of gene functions at the DNA and RNA levels. In addition, novel approaches for the direct manipulation of target proteins have been introduced in plant systems. Here, we review the current development in tissue-specific and conditional manipulation approaches at the DNA, RNA, and protein levels.
- Published
- 2021
25. Chromatin attachment to the nuclear matrix represses hypocotyl elongation in Arabidopsis thaliana
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Linhao Xu, Shiwei Zheng, Katja Witzel, Eveline Van De Slijke, Alexandra Baekelandt, Evelien Mylle, Daniel Van Damme, Jinping Cheng, Geert De Jaeger, Dirk Inzé, and Hua Jiang
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Science - Abstract
Abstract The nuclear matrix is a nuclear compartment that has diverse functions in chromatin regulation and transcription. However, how this structure influences epigenetic modifications and gene expression in plants is largely unknown. In this study, we show that a nuclear matrix binding protein, AHL22, together with the two transcriptional repressors FRS7 and FRS12, regulates hypocotyl elongation by suppressing the expression of a group of genes known as SMALL AUXIN UP RNAs (SAURs) in Arabidopsis thaliana. The transcriptional repression of SAURs depends on their attachment to the nuclear matrix. The AHL22 complex not only brings these SAURs, which contain matrix attachment regions (MARs), to the nuclear matrix, but it also recruits the histone deacetylase HDA15 to the SAUR loci. This leads to the removal of H3 acetylation at the SAUR loci and the suppression of hypocotyl elongation. Taken together, our results indicate that MAR-binding proteins act as a hub for chromatin and epigenetic regulators. Moreover, we present a mechanism by which nuclear matrix attachment to chromatin regulates histone modifications, transcription, and hypocotyl elongation.
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- 2024
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26. The TPLATE complex mediates membrane bending during plant clathrin-mediated endocytosis
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Pierre Mahou, Emmanuel Beaurepaire, Dana A Dahhan, Juan Aguilera-Servin, Mónika Hrtyan, Martin Loose, Nataliia Gnyliukh, Jie Wang, Tommaso Costanzo, Walter A. Kaufmann, Sebastian Y. Bednarek, Jiri Friml, Alexander W. Johnson, Daniël Van Damme, and Vanessa Zheden
- Subjects
Membrane bending ,Endocytic vesicle ,Chemistry ,Vesicle ,Turgor pressure ,Biophysics ,Receptor-mediated endocytosis ,Endocytosis ,Actin ,Intracellular - Abstract
Clathrin-mediated endocytosis in plants is an essential process but the underlying mechanisms are poorly understood, not least because of the extreme intracellular turgor pressure acting against the formation of endocytic vesicles. In contrast to other models, plant endocytosis is independent of actin, indicating a mechanistically distinct solution. Here, by using biochemical and advanced microscopy approaches, we show that the plant-specific TPLATE complex acts outside of endocytic vesicles as a mediator of membrane bending. Cells with disrupted TPLATE fail to generate spherical vesicles, and in vitro biophysical assays identified protein domains with membrane bending capability. These results redefine the role of the TPLATE complex as a key component of the evolutionarily distinct mechanism mediating membrane bending against high turgor pressure to drive endocytosis in plant cells.One Sentence SummaryWhile plant CME is actin independent, we identify that the evolutionarily ancient octameric TPLATE complex mediates membrane bending against high turgor pressure in plant clathrin-mediated endocytosis.
- Published
- 2021
27. Visualizing protein-protein interactions in plants by rapamycin-dependent delocalization
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Daniël Van Damme, Andreas De Meyer, Julie Merchie, Peter Grones, Benjamin Pavie, Evelien Mylle, and Joanna Winkler
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0106 biological sciences ,Nicotiana benthamiana ,Plant Science ,01 natural sciences ,Plant science ,Arabidopsis ,Protein Interaction Mapping ,LIVING CELLS ,Plant Proteins ,0303 health sciences ,biology ,Chemistry ,food and beverages ,LOCALIZATION ,ARABIDOPSIS ,Recombinant Proteins ,Mitochondria ,TRANSLOCATION ,FKBP ,GROWTH ,VECTORS ,Life Sciences & Biomedicine ,Biochemistry & Molecular Biology ,Green Fluorescent Proteins ,Context (language use) ,Computational biology ,Article ,Protein–protein interaction ,Tacrolimus Binding Proteins ,03 medical and health sciences ,Plant Cells ,Tobacco ,REVEALS ,030304 developmental biology ,Sirolimus ,Science & Technology ,Plant Sciences ,fungi ,Biology and Life Sciences ,Cell Biology ,biology.organism_classification ,Plant Leaves ,Luminescent Proteins ,CYTOKINESIS ,ESTABLISHMENT ,Protein Multimerization ,Cytokinesis ,Function (biology) ,SYSTEM ,010606 plant biology & botany - Abstract
Identifying protein-protein interactions (PPI) is crucial for understanding biological processes. Many PPI tools are available, yet only some function within the context of a plant cell. Narrowing down even further, only a few tools allow complex multi-protein interactions to be visualized. Here, we present a conditionalin vivoPPI tool for plant research that meets these criteria. Knocksideways in plants (KSP) is based on the ability of rapamycin to alter the localization of a bait protein and its interactors via the heterodimerization of FKBP and FRB domains. KSP is inherently free from many limitations of other PPI systems. Thisin vivotool does not require spatial proximity of the bait and prey fluorophores and it is compatible with a broad range of fluorophores. KSP is also a conditional tool and therefore the visualization of the proteins in the absence of rapamycin acts as an internal control. We used KSP to confirm previously identified interactions inNicotiana benthamianaleaf epidermal cells. Furthermore, the scripts that we generated allow the interactions to be quantified at high throughput. Finally, we demonstrate that KSP can easily be used to visualize complex multi-protein interactions. KSP is therefore a versatile tool with unique characteristics and applications that complements other plant PPI methods.
- Published
- 2021
28. Distinct EH domains of the endocytic TPLATE complex confer lipid and protein binding
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Thomas Evangelidis, Yehudi Bloch, Dominique Eeckhout, Martin Potocký, Rosa Grigoryan, Qihang Jiang, Jelle Van Leene, Geert De Jaeger, Konstantinos Tripsianes, Daniël Van Damme, Savvas N. Savvides, Frank Vanhaecke, Peter Vandenabeele, Romain Merceron, Steven De Munck, Klaas Yperman, Pieter Tack, Laszlo Vincze, Anna C. Papageorgiou, and Roman Pleskot
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0106 biological sciences ,General Physics and Astronomy ,Plasma protein binding ,Membrane trafficking ,Crystallography, X-Ray ,01 natural sciences ,MOTIFS ,Protein trafficking in plants ,Integral membrane protein ,Plant Proteins ,0303 health sciences ,Multidisciplinary ,Chemistry ,Signal transducing adaptor protein ,food and beverages ,Plants, Genetically Modified ,Endocytosis ,Transport protein ,Protein Transport ,ddc:500 ,Molecular modelling ,Protein Binding ,NPF ,EXPRESSION ,Protein subunit ,Science ,Protein domain ,Sequence alignment ,Molecular Dynamics Simulation ,General Biochemistry, Genetics and Molecular Biology ,Article ,ACTIN ,03 medical and health sciences ,Protein Domains ,Tobacco ,030304 developmental biology ,Adaptor Proteins, Signal Transducing ,X-ray crystallography ,PHOSPHATIDIC-ACID ,IDENTIFICATION ,Arabidopsis Proteins ,Calcium-Binding Proteins ,Cell Membrane ,PAN1P ,Membrane Proteins ,Biology and Life Sciences ,General Chemistry ,Membrane protein ,Biophysics ,Sequence Alignment ,Solution-state NMR ,010606 plant biology & botany - Abstract
Nature Communications 12(1), 3050 (2021). doi:10.1038/s41467-021-23314-6, Clathrin-mediated endocytosis (CME) is the gatekeeper of the plasma membrane. In contrast to animals and yeasts, CME in plants depends on the TPLATE complex (TPC), an evolutionary ancient adaptor complex. However, the mechanistic contribution of the individual TPC subunits to plant CME remains elusive. In this study, we used a multidisciplinary approach to elucidate the structural and functional roles of the evolutionary conserved N-terminal Eps15 homology (EH) domains of the TPC subunit AtEH1/Pan1. By integrating high-resolution structural information obtained by X-ray crystallography and NMR spectroscopy with all-atom molecular dynamics simulations, we provide structural insight into the function of both EH domains. Both domains bind phosphatidic acid with a different strength, and only the second domain binds phosphatidylinositol 4,5-bisphosphate. Unbiased peptidome profiling by mass-spectrometry revealed that the first EH domain preferentially interacts with the double N-terminal NPF motif of a previously unidentified TPC interactor, the integral membrane protein Secretory Carrier Membrane Protein 5 (SCAMP5). Furthermore, we show that AtEH/Pan1 proteins control the internalization of SCAMP5 via this double NPF peptide interaction motif. Collectively, our structural and functional studies reveal distinct but complementary roles of the EH domains of AtEH/Pan1 in plant CME and connect the internalization of SCAMP5 to the TPLATE complex., Published by Nature Publishing Group UK, [London]
- Published
- 2021
29. Endocytosis of BRASSINOSTEROID INSENSITIVE1 Is Partly Driven by a Canonical Tyr-Based Motif
- Author
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Isabelle Vanhoutte, Eugenia Russinova, Lucas Alves Neubus Claus, Rajesh Kumar, Peng Wang, Wei Siao, Daniël Van Damme, Jiří Friml, Derui Liu, Xiuyang Zhao, Grégory Vert, Alexander W. Johnson, Klaas Yperman, Sara Martins, Kyle W. Bender, Universiteit Gent = Ghent University (UGENT), Center for Plant Systems Biology (PSB Center), Vlaams Instituut voor Biotechnologie [Ghent, Belgique] (VIB), Institute of Science and Technology [Klosterneuburg, Austria] (IST Austria), University of Illinois at Urbana-Champaign [Urbana], University of Illinois System, Laboratoire de Recherche en Sciences Végétales (LRSV), Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Signalisation Cellulaire et Ubiquitination, Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), European Project: 1009584(2010), Universiteit Gent = Ghent University [Belgium] (UGENT), Institute of Science and Technology [Austria] (IST Austria), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), and Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3)
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POLAR LOCALIZATION ,0106 biological sciences ,0301 basic medicine ,ADAPTER COMPLEX ,Endocytic cycle ,Amino Acid Motifs ,MESH: Cell Membrane / metabolism ,Arabidopsis ,Plant Science ,01 natural sciences ,chemistry.chemical_compound ,MESH: Amino Acid Motifs ,MESH: Endocytosis / physiology ,Internalization ,media_common ,MESH: Arabidopsis / metabolism ,TYROSINE PHOSPHORYLATION ,biology ,Signal transducing adaptor protein ,CLATHRIN-MEDIATED ENDOCYTOSIS ,ARABIDOPSIS ,Plants, Genetically Modified ,Endocytosis ,Cell biology ,MESH: Arabidopsis Proteins / genetics ,FACTOR RECEPTOR ,MESH: Protein Domains ,MESH: Arabidopsis / genetics ,EPIDERMAL-GROWTH-FACTOR ,CELLULOSE SYNTHASE ,MESH: Mutation ,SORTING SIGNALS ,media_common.quotation_subject ,Green Fluorescent Proteins ,[SDV.BC]Life Sciences [q-bio]/Cellular Biology ,Clathrin ,In Brief ,03 medical and health sciences ,MESH: Arabidopsis Proteins / metabolism ,Protein Domains ,MESH: Arabidopsis Proteins / chemistry ,Kinase activity ,MESH: Protein Kinases / chemistry ,Arabidopsis Proteins ,Cell Membrane ,fungi ,Biology and Life Sciences ,Tyrosine phosphorylation ,Cell Biology ,Receptor-mediated endocytosis ,MESH: Protein Kinases / genetics ,MESH: Protein Kinases / metabolism ,030104 developmental biology ,chemistry ,MESH: Plants, Genetically Modified ,MESH: Tyrosine / chemistry ,Mutation ,biology.protein ,Tyrosine ,MESH: Green Fluorescent Proteins / genetics ,Protein Kinases ,STRUCTURAL EXPLANATION ,010606 plant biology & botany - Abstract
Clathrin-mediated endocytosis (CME) and its core endocytic machinery are evolutionarily conserved across all eukaryotes. In mammals, the heterotetrameric adaptor protein complex-2 (AP-2) sorts plasma membrane (PM) cargoes into vesicles via the recognition of motifs based on Tyr or di-Leu in their cytoplasmic tails. However, in plants, very little is known about how PM proteins are sorted for CME and whether similar motifs are required. In Arabidopsis (Arabidopsis thaliana), the brassinosteroid (BR) receptor BR INSENSITIVE1 (BRI1) undergoes endocytosis, which depends on clathrin and AP-2. Here, we demonstrate that BRI1 binds directly to the medium AP-2 subunit (AP2M). The cytoplasmic domain of BRI1 contains five putative canonical surface-exposed Tyr-based endocytic motifs. The Tyr-to-Phe substitution in Y898KAI reduced BRI1 internalization without affecting its kinase activity. Consistently, plants carrying the BRI1Y898F mutation were hypersensitive to BRs. Our study demonstrates that AP-2–dependent internalization of PM proteins via the recognition of functional Tyr motifs also operates in plants.
- Published
- 2020
30. Synaptotagmins at the endoplasmic reticulum-plasma membrane contact sites maintain diacylglycerol homeostasis during abiotic stress
- Author
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Johnathan A. Napier, Armando Albert, Miguel A. Botella, Abel Rosado, Selene García-Hernández, Jessica Pérez-Sancho, Julio Salinas, Vitor Amorim-Silva, Daniël Van Damme, Jinxing Lin, Rafael Catalá, Carlos Perea-Resa, Noemi Ruiz-Lopez, Alicia Esteban del Valle, Alberto P. Macho, Richard P. Haslam, Steffen Vanneste, Jiří Friml, José G. Vallarino, Ministerio de Economía y Competitividad (España), Ministerio de Ciencia, Innovación y Universidades (España), Universidad de Málaga, European Commission, Biotechnology and Biological Sciences Research Council (UK), Chinese Academy of Sciences, National Thousand Young Talents program of China, and Natural Sciences and Engineering Research Council of Canada
- Subjects
0106 biological sciences ,0301 basic medicine ,STRUCTURAL BASIS ,Osmotic shock ,Arabidopsis ,Plant Science ,COLD-ACCLIMATION ,SYT1 ,Endoplasmic Reticulum ,01 natural sciences ,In Brief ,Synaptotagmins ,Diglycerides ,03 medical and health sciences ,FREEZING TOLERANCE ,Phosphatidylinositol Phosphates ,HYPEROSMOTIC STRESS ,Arabidopsis thaliana ,EXTENDED SYNAPTOTAGMINS ,Diacylglycerol kinase ,Abiotic component ,PHOSPHATIDIC-ACID ,biology ,Abiotic stress ,Arabidopsis Proteins ,Endoplasmic reticulum ,Cell Membrane ,Biology and Life Sciences ,Cell Biology ,ABSCISIC-ACID ,biology.organism_classification ,ARABIDOPSIS ,PHOSPHOLIPASE-C ,3. Good health ,Cell biology ,SMP DOMAINS ,030104 developmental biology ,010606 plant biology & botany - Abstract
23 pags., 6 figs., Endoplasmic reticulum–plasma membrane contact sites (ER–PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis thaliana mutants lacking the ER–PM protein tether synaptotagmin1 (SYT1) exhibit decreased PM integrity under multiple abiotic stresses, such as freezing, high salt, osmotic stress, and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER–PM tether that also functions in maintaining PM integrity. The ER–PM CS localization of SYT1 and SYT3 is dependent on PM phosphatidylinositol-4-phosphate and is regulated by abiotic stress. Lipidomic analysis revealed that cold stress increased the accumulation of diacylglycerol at the PM in a syt1/3 double mutant relative to wild-type while the levels of most glycerolipid species remain unchanged. In addition, the SYT1-green fluorescent protein fusion preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work, This work was supported by the Ministerio de Economıa y Competitividad, co-financed by the European Regional Development Fund (grant no. BIO2017-82609-R to M.A.B.), the Ministerio de Ciencia, Innovacion y Universidades (grant no. PGC2018-098789-B-I00 to N.R.-L.) UMA-FEDER (grant UMA18-FEDERJA-154 to N.R.-L.), and the Marie SkłodowskaCurie actions (grant no. H2020-655366-IIF- PLICO to M.A.B. and N.R.-L.). N.R.L. was supported by the Ramon y Cajal program RYC-2013-12699 (MINECO, Spain). J.P.-S. and S.G.-H. were funded by the Ministerio de Economıa y Competitividad in Formacion del Personal Investigador Fellowship (grant no. BES-2012-052324) and (PRE2018- 085284), respectively. R.P.H. and J.A.N. received support from the Biotechnology and Biological Sciences Research Council (BBSRC, UK) in the form of an Institute Strategic Programme Grant (grant no. BBS/E/C/000I0420). J.L. is supported by the Program of Introducing Talents of Discipline to Universities (111 Project, grant no. B13007). A.P.M. and J.P.-S. were supported by the Shanghai Center for Plant Stress Biology (Chinese Academy of Sciences), Chinese 1000 Talents Program. A.R. was supported by the Natural Sciences and Engineering Research Council of Canada (NSERCDiscovery Grant no. RGPIN-2019-05568). Support was also provided by AEI/FEDER, UE (grant nos. BIO2016-79187-R and PID2019-106987RB-I00 to J.P.-S.) and by the European Research Council under the European Union’s Seventh Framework Programme (grant no. FP7/2007-2013)/ERC grant agreement no. 742985 to J.F. and T-Rex (project number 682436 to D.V.D.).
- Published
- 2020
31. Correction to: Establishment of Proximity-Dependent Biotinylation Approaches in Different Plant Model Systems
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Deepanksha Arora, Nikolaj B Abel, Chen Liu, Petra Van Damme, Klaas Yperman, Dominique Eeckhout, Lam Dai Vu, Jie Wang, Anna Tornkvist, Francis Impens, Barbara Korbei, Jelle Van Leene, Alain Goossens, Geert De Jaeger, Thomas Ott, Panagiotis Nikolaou Moschou, and Daniël Van Damme
- Subjects
Cell Biology ,Plant Science - Published
- 2022
32. Molecular architecture of the endocytic TPLATE complex
- Author
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Daniël Van Damme, Roman Pleskot, Klaas Yperman, Michael Kraus, Pieter De Bruyn, Bert De Rybel, Jie Wang, Geert De Jaeger, Evelien Mylle, Jonah Nolf, Dominique Eeckhout, Peter Grones, Romain Merceron, Michaël Vandorpe, Remy Loris, Martin Potocký, Savvas N. Savvides, Lam Dai Vu, Eliana Mor, Joanna Winkler, Faculty of Sciences and Bioengineering Sciences, Department of Bio-engineering Sciences, and Structural Biology Brussels
- Subjects
0106 biological sciences ,Plant molecular biology ,Endocytic cycle ,Plant Science ,Endocytosis ,Biochemistry ,01 natural sciences ,03 medical and health sciences ,Membrane interaction ,structural biology ,endocytosis ,Research Articles ,030304 developmental biology ,0303 health sciences ,Multidisciplinary ,biology ,Chemistry ,Plant Sciences ,CD spectroscopy ,Biology and Life Sciences ,food and beverages ,SciAdv r-articles ,Lipidome ,biology.organism_classification ,Dictyostelium ,Cell biology ,Structural biology ,Coatomer ,Membrane proteome ,010606 plant biology & botany ,Research Article - Abstract
An integrative structural approach reveals crucial roles of specific subunits in the plant TPLATE/TSET complex., Eukaryotic cells rely on endocytosis to regulate their plasma membrane proteome and lipidome. Most eukaryotic groups, except fungi and animals, have retained the evolutionary ancient TSET complex as an endocytic regulator. Unlike other coatomer complexes, structural insight into TSET is lacking. Here, we reveal the molecular architecture of plant TSET [TPLATE complex (TPC)] using an integrative structural approach. We identify crucial roles for specific TSET subunits in complex assembly and membrane interaction. Our data therefore generate fresh insight into the differences between the hexameric TSET in Dictyostelium and the octameric TPC in plants. Structural elucidation of this ancient adaptor complex represents the missing piece in the coatomer puzzle and vastly advances our functional as well as evolutionary insight into the process of endocytosis.
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- 2020
33. Pharmacological and genetic manipulations of Ca2+ signaling have contrasting effects on auxin-regulated trafficking
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Jian Chen, Melanie Krebs, Alex Costa, Karin Schumacher, Daniël Van Damme, Steffen Vanneste, Ive De Smet, Jiří Friml, Moritz K. Nowack, Matteo Grenzi, Tom Beeckman, Ellie Himschoot, and Ren Wang
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chemistry.chemical_classification ,Chemistry ,Mechanism (biology) ,media_common.quotation_subject ,fungi ,Mutant ,Endocytic cycle ,food and beverages ,Cell biology ,Auxin ,Developmental plasticity ,heterocyclic compounds ,Polar auxin transport ,Internalization ,Intracellular ,media_common - Abstract
A large part of a plants’ developmental plasticity relies on the activities of the phytohormone auxin and the regulation of its own distribution. This process involves a cohort of transcriptional and non-transcriptional effects of auxin on polar auxin transport, regulating the abundancy, biochemical activity and polar localization of the molecular components, predominantly PIN auxin exporters. While the transcriptional auxin signaling cascade has been well characterized, the mechanism and role of non-transcriptional auxin signaling remains largely elusive. Here, we addressed the potential involvement of auxin-induced Ca2+ signaling in auxin’s inhibitory effect on PIN endocytic trafficking. On the one hand, exogenous manipulations of Ca2+ availability and signaling effectively antagonized auxin effects suggesting that auxin-induced Ca2+ signaling is required for inhibition of internalization. On the other hand, we addressed the auxin-mediated inhibition of PIN internalization in the auxin signaling (tir1afb2,3) or Ca2+ channel (cngc14) mutants. These mutants were strongly defective in auxin-triggered Ca2+ signaling, but not in auxin-inhibited internalization. These data imply that, while Ca2+ signaling may be required for normal PIN trafficking, auxin-mediated increase in Ca2+ signaling is not a direct part of a downstream mechanism that mediates auxin effects on Brefeldin A-visualized PIN intercellular aggregation. These contrasting results obtained by comparing the mutant analysis versus the exogenous manipulations of Ca2+ availability and signaling illustrate the critical importance of genetics to unravel the role of Ca2+ in a process of interest.
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- 2020
34. The TPLATE subunit is essential for structural assembly of the endocytic TSET complex
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Pieter De Bruyn, Daniël Van Damme, Klaas Yperman, Roman Pleskot, Evelien Mylle, Joanna Winkler, Martin Potocký, Jie Wang, Romain Merceron, Michael Kraus, Jonah Nolf, Remy Loris, Savvas N. Savvides, Bert De Rybel, Michaël Vandorpe, Geert De Jaeger, Lam Dai Vu, Eliana Mor, Dominique Eeckhout, and Peter Grones
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biology ,Membrane interaction ,Coatomer ,Chemistry ,Endocytic cycle ,Computational biology ,Membrane proteome ,Lipidome ,Endocytosis ,biology.organism_classification ,Dictyostelium ,Structural approach - Abstract
SummaryAll eukaryotic cells rely on endocytosis to regulate the plasma membrane proteome and lipidome. Most eukaryotic groups, with the exception of fungi and animals, have retained the evolutionary ancient TSET complex as a regulator of endocytosis. Despite the presence of similar building blocks in TSET, compared to other coatomer complexes, structural insight into this adaptor complex is lacking. Here, we elucidate the molecular architecture of the octameric plant TSET complex (TPLATE complex/TPC) using an integrative structural approach. This allowed us to describe a plant-specific connection between the TML subunit and the AtEH/Pan1 proteins and show a direct interaction between the complex and the plasma membrane without the need for any additional protein factors. Furthermore, we identify the appendage of TPLATE as crucial for complex assembly. Structural elucidation of this ancient adaptor complex vastly advances our functional as well as evolutionary insight into the process of endocytosis.Graphical abstract
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- 2020
35. Synaptotagmins Maintain Diacylglycerol Homeostasis at Endoplasmic Reticulum-Plasma Membrane Contact Sites during Abiotic Stress
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Julio Salinas, Rafael Catalá, Alicia Esteban del Valle, Jir□í Friml, Miguel A. Botella, Daniël Van Damme, José G. Vallarino, Armando Albert, Noemi Ruiz-Lopez, Abel Rosado, Richard P. Haslam, Selene García-Hernández, Jessica Pérez-Sancho, Alberto P. Macho, Carlos Perea-Resa, Vitor Amorim-Silva, Steffen Vanneste, Jinxing Lin, and Johnathan A. Napier
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Synaptotagmins ,Abiotic component ,biology ,Osmotic shock ,Abiotic stress ,Chemistry ,Arabidopsis ,Endoplasmic reticulum ,biology.organism_classification ,SYT1 ,Diacylglycerol kinase ,Cell biology - Abstract
SUMMARYEndoplasmic Reticulum-Plasma Membrane contact sites (ER-PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis mutants lacking the ER-PM protein tether synaptotagmin1 (SYT1) exhibit decreased plasma membrane (PM) integrity under multiple abiotic stresses such as freezing, high salt, osmotic stress and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER-PM tether that also functions in maintaining PM integrity. The ER-PM CS localization of SYT1 and SYT3 is dependent on PM phosphatidylinositol-4-phosphate and is regulated by abiotic stress. Lipidomic analysis revealed that cold stress increased the accumulation of diacylglycerol at the PM in a syt1/3 double mutant relative to WT while the levels of most glycerolipid species remain unchanged. Additionally, SYT1-GFP preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work uncovers a crucial SYT-dependent mechanism of stress adaptation counteracting the detrimental accumulation of diacylglycerol at the PM produced during episodes of abiotic stress.
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- 2020
36. Nanobody-Dependent Delocalization of Endocytic Machinery in
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Joanna, Winkler, Andreas, De Meyer, Evelien, Mylle, Veronique, Storme, Peter, Grones, and Daniël, Van Damme
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nanobody ,Arabidopsis ,endocytosis ,protein delocalization ,Plant Science ,fluorescence microscopy ,Original Research ,TPLATE complex (TPC) - Abstract
Plant cells perceive and adapt to an ever-changing environment by modifying their plasma membrane (PM) proteome. Whereas secretion deposits new integral membrane proteins, internalization by endocytosis removes membrane proteins and associated ligands, largely with the aid of adaptor protein (AP) complexes and the scaffolding molecule clathrin. Two AP complexes function in clathrin-mediated endocytosis at the PM in plant cells, the heterotetrameric AP-2 complex and the hetero-octameric TPLATE complex (TPC). Whereas single subunit mutants in AP-2 develop into viable plants, genetic mutation of a single TPC subunit causes fully penetrant male sterility and silencing single subunits leads to seedling lethality. To address TPC function in somatic root cells, while minimizing indirect effects on plant growth, we employed nanobody-dependent delocalization of a functional, GFP-tagged TPC subunit, TML, in its respective homozygous genetic mutant background. In order to decrease the amount of functional TPC at the PM, we targeted our nanobody construct to the mitochondria and fused it to TagBFP2 to visualize it independently of its bait. We furthermore limited the effect of our delocalization to those tissues that are easily accessible for live-cell imaging by expressing it from the PIN2 promoter, which is active in root epidermal and cortex cells. With this approach, we successfully delocalized TML from the PM. Moreover, we also show co-recruitment of TML-GFP and AP2A1-TagRFP to the mitochondria, suggesting that our approach delocalized complexes, rather than individual adaptor complex subunits. In line with the specific expression domain, we only observed minor effects on root growth, yet realized a clear reduction of endocytic flux in epidermal root cells. Nanobody-dependent delocalization in plants, here exemplified using a TPC subunit, has the potential to be widely applicable to achieve specific loss-of-function analysis of otherwise lethal mutants.
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- 2020
37. Fluctuating auxin response gradients determine pavement cell-shape acquisition
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Daniël Van Damme, Siamsa M. Doyle, Mateusz Majda, Peter Grones, and Stéphanie Robert
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EFFLUX ,POLARIZATION ,PIN PROTEINS ,Arabidopsis ,Plant Biology ,pavement cells ,Plant Epidermis ,auxin response gradient ,ROOT ,INTERDIGITATION ,Auxin ,medicine ,TRAFFICKING ,PIN proteins ,Cell shape ,Cell Shape ,auxin transporter ,PHYLLOTAXIS ,GENE-EXPRESSION ,chemistry.chemical_classification ,Pavement cells ,Multidisciplinary ,biology ,Epidermis (botany) ,Indoleacetic Acids ,Arabidopsis Proteins ,fungi ,Biology and Life Sciences ,food and beverages ,Biological Transport ,Phyllotaxis ,Biological Sciences ,biology.organism_classification ,TRANSPORT ,Lobe ,Plant Leaves ,medicine.anatomical_structure ,chemistry ,Plant Stomata ,Biophysics ,lobe formation - Abstract
Significance As the outermost cell layer of an organism, the epidermis plays a key role in controlling morphogenesis. In this work, we investigated cell-shape regulation in young, lobing pavement cells of the Arabidopsis leaf epidermis. By taking advantage of their developmental synchrony, we showed that the establishment of a local auxin gradient is necessary for the initiation of first-lobe formation. However, the auxin gradient is not stable over time but rather fluctuates according to the particular developmental stage of the cells. These changes are established by the specific distribution of auxin transporters at the different membranes of these young pavement cells. This work reports an observation of auxin fluctuation during cell-shape determination in plants., Puzzle-shaped pavement cells provide a powerful model system to investigate the cellular and subcellular processes underlying complex cell-shape determination in plants. To better understand pavement cell-shape acquisition and the role of auxin in this process, we focused on the spirals of young stomatal lineage ground cells of Arabidopsis leaf epidermis. The predictability of lobe formation in these cells allowed us to demonstrate that the auxin response gradient forms within the cells of the spiral and fluctuates based on the particular stage of lobe development. We revealed that specific localization of auxin transporters at the different membranes of these young cells changes during the course of lobe formation, suggesting that these fluctuating auxin response gradients are orchestrated via auxin transport to control lobe formation and determine pavement cell shape.
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- 2020
38. Distinct EH domains of the endocytic TPLATE complex confer lipid and protein binding
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Daniël Van Damme, Laszlo Vincze, Thomas Evangelidis, Roman Pleskot, Kostantinos Tripsianes, Klaas Yperman, Romain Merceron, Anna C. Papageorgiou, Dominique Eeckhout, Steven De Munck, Yehudi Bloch, Geert De Jaeger, Jelle Van Leene, Martin Potocky, Savvas N. Savvides, Peter Vandenabeele, and Pieter Tack
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Membrane protein ,Chemistry ,media_common.quotation_subject ,Protein subunit ,Endocytic cycle ,Interactor ,Plasma protein binding ,Endocytosis ,Internalization ,Integral membrane protein ,Cell biology ,media_common - Abstract
Clathrin-mediated endocytosis (CME) is the gatekeeper of the plasma membrane. In contrast to animals and yeasts, CME in plants depends on the TPLATE complex (TPC), an evolutionary ancient adaptor complex. The mechanistic contribution of the individual TPC subunits to plant CME remains however elusive. In this study, we used a multidisciplinary approach to elucidate the structural and functional roles of the evolutionary conserved N-terminal Eps15 homology (EH) domains of the TPC subunit AtEH1/Pan1. By integrating high-resolution structural information obtained by X-ray crystallography and NMR spectroscopy with all-atom molecular dynamics simulations, we provide structural insight into the function of both EH domains. Whereas one EH domain binds negatively charged PI(4,5)P2lipids, unbiased peptidome profiling by mass-spectrometry revealed that the other EH domain interacts with the double N-terminal NPF motif of a novel TPC interactor, the integral membrane protein Secretory Carrier Membrane Protein 5 (SCAMP5). Furthermore, we show that AtEH/Pan1 proteins control the internalization of SCAMP5 via this double NPF peptide interaction motif. Collectively, our structural and functional studies reveal distinct but complementary roles of the EH domains of AtEH/Pan1 have in plant CME and connect the internalization of SCAMP5 to the TPLATE complex.
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- 2020
39. High temporal resolution reveals simultaneous plasma membrane recruitment of the TPLATE complex subunits
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Evelien Mylle, Daniël Van Damme, Roman Pleskot, Nienke Besbrugge, Geert De Jaeger, Jie Wang, Jiří Friml, and Alexander W. Johnson
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Membrane ,Live cell imaging ,Cytoplasm ,Chemistry ,Protein subunit ,Endocytic cycle ,Biophysics ,Signal transducing adaptor protein ,Endocytosis ,Plant cell - Abstract
The TPLATE complex (TPC) is a key endocytic adaptor protein complex in plants. TPC contains six evolutionary conserved subunits and two plant specific subunits, AtEH1/Pan1 and AtEH2/Pan1, which are not associated with the hexameric subcomplex in the cytoplasm. To investigate the dynamic assembly of the octameric TPC at the plasma membrane (PM), we performed state-of-the-art dual-color live cell imaging at physiological and a lowered temperature. Our data show that lowering the temperature slows down endocytosis and thereby enhances the temporal resolution of the differential recruitment of endocytic components. Under both normal and lowered temperature conditions, the core TPC subunit TPLATE, and the AtEH/Pan1 proteins, exhibited simultaneous recruitment at the PM. These results, together with our co-localization analysis of different TPC subunits, allow us to conclude that in plant cells, TPC is not recruited to the PM sequentially but as an octameric complex.One sentence summaryLowering the temperature increases spatiotemporal resolution of protein recruitment at the plasma membrane.
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- 2020
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40. High Temporal Resolution Reveals Simultaneous Plasma Membrane Recruitment of TPLATE Complex Subunits
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Roman Pleskot, Jie Wang, Geert De Jaeger, Daniël Van Damme, Nienke Besbrugge, Jiří Friml, Evelien Mylle, and Alexander W. Johnson
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0106 biological sciences ,DYNAMICS ,COATED VESICLE FORMATION ,Physiology ,Protein subunit ,Endocytic cycle ,PROTEIN ,Plant Science ,Endocytosis ,ADAPTER ,01 natural sciences ,Live cell imaging ,Arabidopsis ,Genetics ,TEMPERATURE ,AP2 ,biology ,Chemistry ,Signal transducing adaptor protein ,Biology and Life Sciences ,SOMATIC CYTOKINESIS ,Receptor-mediated endocytosis ,biology.organism_classification ,CLATHRIN-MEDIATED ENDOCYTOSIS ,ARABIDOPSIS ,Cytoplasm ,Biophysics ,CELLULOSE SYNTHASE ,010606 plant biology & botany - Abstract
The TPLATE complex (TPC) is a key endocytic adaptor protein complex in plants. TPC in Arabidopsis (Arabidopsis thaliana) contains six evolutionarily conserved subunits and two plant-specific subunits, AtEH1/Pan1 and AtEH2/Pan1, although cytoplasmic proteins are not associated with the hexameric subcomplex in the cytoplasm. To investigate the dynamic assembly of the octameric TPC at the plasma membrane (PM), we performed state-of-the-art dual-color live cell imaging at physiological and lowered temperatures. Lowering the temperature slowed down endocytosis, thereby enhancing the temporal resolution of the differential recruitment of endocytic components. Under both normal and lowered temperature conditions, the core TPC subunit TPLATE and the AtEH/Pan1 proteins exhibited simultaneous recruitment at the PM. These results, together with co-localization analysis of different TPC subunits, allow us to conclude that TPC in plant cells is not recruited to the PM sequentially but as an octameric complex.
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- 2020
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41. Functional divergence of TPX2 family members in Arabidopsis thaliana
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Beáta Petrovská, Andreas Houben, Daniël Van Damme, Twan Rutten, Dmitri Demidov, Alisa Tugai, Klara Ptosková, Jaroslav Dolezel, Petr Dvorak, and Eva Dvořák Tomaštíková
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Genetics ,biology ,Arabidopsis thaliana ,biology.organism_classification ,Functional divergence - Abstract
Background TPX2 (Targeting protein for Xklp2) is an evolutionary conserved microtubule-associated protein important for microtubule nucleation and proper mitotic spindle assembly. The protein was also described as an activator of the mitotic kinase Aurora A in humans and the Arabidopsis AURORA 1 (AUR1) kinase. In contrast to animal genomes that encode only one TPX2 gene in their genome, higher plants genomes encode a family with several TPX2-LIKE gene members (TPXL). Results Arabidopsis genome comprise of 21 TPXL genes. To better understand the functional divergence within the TPXL gene family in plants, we characterized eight genes most closely related to the canonical TPX2 Arabidopsis. TPXL genes of Arabidopsis can be divided into two groups. Group A proteins (TPXL2, 3, 4 and 8) contain Aurora binding and TPX2_importin domains, while group B proteins (TPXL1, 5, 6 and 7) harbor an Xklp2 domain. Canonical Arabidopsis TPX2 contains all above mentioned domains. We confirmed using in vitro kinase assays that the group A proteins contain a functional Aurora kinase binding domain and are able to at least double the activity of recombinant AUR1 kinase. Transient expression of GFP-tagged Arabidopsis TPX2-like proteins in Nicotiana benthamiana revealed preferential localization to microtubules and nuclei, except TPXL7, which localized mainly to the nuclear envelope. Co-expression of AUR1 together with TPX2-like proteins changed the localization of AUR1, indicating that these proteins serve as targeting factors for Aurora kinases. Conclusion Taken together, we visualize the various localizations of the TPX2-LIKE family in Arabidopsis as proxy to their functional divergence and provide evidence of their role in the targeted regulation of AUR1 kinase activity.
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- 2019
42. Disruption of endocytosis through chemical inhibition of clathrin heavy chain function
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Eugenia Russinova, Wim Dejonghe, Andrzej Drozdzecki, Daniel Valentin Savatin, An Staes, Kiril Mishev, Volker Haucke, Riet De Rycke, Isha Sharma, Kris Gevaert, Annemieke Madder, Evelien Mylle, Steven De Munck, Bram Denoo, Daniël Van Damme, Johan M. Winne, Klaas Yperman, Wim Nerinckx, Savvas N. Savvides, Haydar Bulut, Jiří Friml, Qing Lu, Mina Vasileva, and Dominique Audenaert
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Models, Molecular ,Arabidopsis ,Thiophenes ,Endocytosis ,Clathrin ,03 medical and health sciences ,symbols.namesake ,Benzene Derivatives ,Molecule ,Humans ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,Heavy chain ,biology ,Molecular Structure ,Chemistry ,030302 biochemistry & molecular biology ,Cell Biology ,Golgi apparatus ,biology.organism_classification ,Small molecule ,Clathrin Heavy Chains ,biology.protein ,Biophysics ,symbols ,Function (biology) - Abstract
Clathrin-mediated endocytosis (CME) is a highly conserved and essential cellular process in eukaryotic cells, but its dynamic and vital nature makes it challenging to study using classical genetics tools. In contrast, although small molecules can acutely and reversibly perturb CME, the few chemical CME inhibitors that have been applied to plants are either ineffective or show undesirable side effects. Here, we identify the previously described endosidin9 (ES9) as an inhibitor of clathrin heavy chain (CHC) function in both Arabidopsis and human cells through affinity-based target isolation, in vitro binding studies and X-ray crystallography. Moreover, we present a chemically improved ES9 analog, ES9-17, which lacks the undesirable side effects of ES9 while retaining the ability to target CHC. ES9 and ES9-17 have expanded the chemical toolbox used to probe CHC function, and present chemical scaffolds for further design of more specific and potent CHC inhibitors across different systems.
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- 2019
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43. TPX2-LIKE PROTEIN 3 is the primary activator of α-Aurora kinases and is essential for embryogenesis
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Daniël Van Damme, Bo Liu, Matthias Van Durme, Moritz K. Nowack, Geert De Jaeger, Evelien Mylle, Joanna Boruc, Dominique Eeckhout, Michaël Vandorpe, Dmitri Demidov, Xingguang Deng, Tom Beeckman, Eva Dvořák Tomaštíková, Honghui Lin, Tong-Reen Connie Tan, and Nienke Besbrugge
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Proteomics ,0106 biological sciences ,Physiology ,Arabidopsis ,Cell Cycle Proteins ,Plant Science ,Plasma protein binding ,Protein Serine-Threonine Kinases ,Biology ,01 natural sciences ,Gene Expression Regulation, Plant ,Microtubule ,Genetics ,Amino Acid Sequence ,Mitosis ,Microscopy, Confocal ,Sequence Homology, Amino Acid ,Arabidopsis Proteins ,Kinase ,Gene Expression Regulation, Developmental ,Biology and Life Sciences ,Plants, Genetically Modified ,biology.organism_classification ,Cell biology ,Enzyme Activation ,Seeds ,Kinesin ,Microtubule-Associated Proteins ,Protein Binding ,Research Article ,010606 plant biology & botany ,Binding domain - Abstract
Aurora kinases are key regulators of mitosis. Multicellular eukaryotes generally possess two functionally diverged types of Aurora kinases. In plants, including Arabidopsis (Arabidopsis thaliana), these are termed α- and β-Auroras. As the functional specification of Aurora kinases is determined by their specific interaction partners, we initiated interactomics analyses using both Arabidopsis α-Aurora kinases (AUR1 and AUR2). Proteomics results revealed that TPX2-LIKE PROTEINS2 and 3 (TPXL2/3) prominently associated with α-Auroras, as did the conserved TPX2 to a lower degree. Like TPX2, TPXL2 and TPXL3 strongly activated the AUR1 kinase but exhibited cell-cycle–dependent localization differences on microtubule arrays. The separate functions of TPX2 and TPXL2/3 were also suggested by their different influences on AUR1 localization upon ectopic expressions. Furthermore, genetic analyses showed that TPXL3, but not TPX2 and TPXL2, acts nonredundantly to enable proper embryo development. In contrast to vertebrates, plants have an expanded TPX2 family and these family members have both redundant and unique functions. Moreover, as neither TPXL2 nor TPXL3 contains the C-terminal Kinesin-5 binding domain present in the canonical TPX2, the targeting and activity of this kinesin must be organized differently in plants.
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- 2019
44. Ectopic activation of cortical cell division during the accommodation of arbuscular mycorrhizal fungi
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Daniël Van Damme, Giulia Russo, Marco Chiapello, Andrea Genre, Gennaro Carotenuto, Veronica Volpe, and Valentina Fiorilli
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0106 biological sciences ,0301 basic medicine ,cell division ,Cell division ,Physiology ,Cell ,Context (language use) ,Plant Science ,Root hair ,01 natural sciences ,Plant Roots ,03 medical and health sciences ,live confocal imaging ,Periarbuscular membrane ,Gene Expression Regulation, Plant ,Mycorrhizae ,Gene expression ,Medicago truncatula ,medicine ,endocytosis ,Plant Proteins ,biology ,arbuscular mycorrhiza ,Cell plate ,biology.organism_classification ,symbiosis ,Cell biology ,Daucus carota ,030104 developmental biology ,medicine.anatomical_structure ,Lotus japonicus ,010606 plant biology & botany ,Signal Transduction - Abstract
Arbuscular mycorrhizas (AMs) between plants and soil fungi are widespread symbioses with a major role in soil nutrient uptake. In this study we investigated the induction of root cortical cell division during AM colonization by combining morphometric and gene expression analyses with promoter activation and protein localization studies of the cell-plate-associated exocytic marker TPLATE. Our results show that TPLATE promoter is activated in colonized cells of the root cortex where we also observed the appearance of cells that are half the size of the surrounding cells. Furthermore, TPLATE-green fluorescent protein recruitment to developing cell plates highlighted ectopic cell division events in the inner root cortex during early AM colonization. Lastly, transcripts of TPLATE, KNOLLE and Cyclinlike 1 (CYC1) are all upregulated in the same context, alongside endocytic markers Adaptor-Related Protein complex 2 alpha 1 subunit (AP2A1) and Clathrin Heavy Chain 2 (CHC2), known to be active during cell plate formation. This pattern of gene expression was recorded in wild-type Medicago truncatula roots, but not in a common symbiotic signalling pathway mutant where fungal colonization is blocked at the epidermal level. Altogether, these results suggest the activation of cell-division-related mechanisms by AM hosts during the accommodation of the symbiotic fungus.
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- 2019
45. The TPX-Like protein TPXL3, but not TPX2, is the primary activator of α Aurora kinases and is essential for embryogenesis in Arabidopsis
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Michaël Vandorpe, Tong-Reen Connie Tan, Nienke Besbrugge, Eva Dvořák Tomaštíková, Daniël Van Damme, Geert De Jaeger, Honghui Lin, Xingguang Deng, Evelien Mylle, Bo Liu, Matthias Van Durme, Dominique Eeckhout, Dmitri Demidov, Moritz K. Nowack, Joanna Boruc, and Tom Beeckman
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Multicellular organism ,medicine.anatomical_structure ,biology ,Microtubule ,Kinase ,Activator (genetics) ,Arabidopsis ,Cell ,medicine ,biology.organism_classification ,Proteomics ,Mitosis ,Cell biology - Abstract
Aurora kinases are key regulators of mitosis. Multicellular eukaryotes generally possess two functionally diverged types. In plants like Arabidopsis, these are termed α versus β Auroras. As the functional specification of Aurora kinases is determined by their specific interaction partners, we initiated interactomics analyses using both α Aurora kinases (AUR1 and AUR2). Proteomics results revealed the TPX2-Like proteins 2 and 3 (TPXL2/3) prominently associating with α Auroras, as did the conserved TPX2 to a lower degree. Like TPX2, TPXL2 and TPXL3 strongly activated AUR1 kinase but exhibited cell cycle-dependent localization differences on microtubule arrays. The separate functions of TPX2 and TPXL2/3 were also suggested by their different influences on AUR1 localization upon ectopic expressions. Furthermore, genetic analyses disclosed that TPXL3, but not TPX2 and TPXL2, acts non-redundantly to secure proper embryo development. In contrast to vertebrates, plants expanded the TPX2 family for both redundant and unique functions among its members.
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- 2018
46. Mitochondrial uncouplers inhibit clathrin-mediated endocytosis largely through cytoplasmic acidification
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Stéphanie Robert, Kiril Mishev, Le Son Long Nguyen, Fausto Andres Ortiz-Morea, Eugenia Russinova, Corrado Viotti, Sebastian Y. Bednarek, Jiorgos Kourelis, Jaroslaw Kasprowicz, Simon Delang, Mareike Heilmann, Olivier Keech, Matyáš Fendrych, Wim Dejonghe, Gary A. Baisa, Mateusz Majda, Johan M. Winne, Sabine Kuenen, Teun Munnik, Jef Swerts, Xavier Zarza, Andrzej Drozdzecki, Ingo Heilmann, Stefan Scholl, Karin Schumacher, Isabelle Van Houtte, Evelien Mylle, Jiří Friml, Patrik Verstreken, Daniël Van Damme, Mina Vasileva, Dominique Audenaert, Christa Testerink, Anna-Mária Szatmári, and SILS (FNWI)
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0301 basic medicine ,Protonophore ,ADAPTER COMPLEX ,Arabidopsis ,General Physics and Astronomy ,Mitochondrion ,Quinolones ,COATED PITS ,Adenosine Triphosphate ,OXIDATIVE-PHOSPHORYLATION ,Inner mitochondrial membrane ,Multidisciplinary ,biology ,Biochemistry and Molecular Biology ,Endocytosis ,Cell biology ,Mitochondria ,Protein Transport ,TERMINAL DOMAIN ,Uncoupling Agents ,Science ,Clathrin ,General Biochemistry, Genetics and Molecular Biology ,Article ,03 medical and health sciences ,PHOSPHATIDYLINOSITOL 4 ,5-BISPHOSPHATE ,Humans ,Life Science ,Electrochemical gradient ,Institut für Biochemie und Biologie ,Organelles ,ALPHA-ADAPTIN ,ANTIMYCIN-A ,Cell Membrane ,Biology and Life Sciences ,General Chemistry ,Receptor-mediated endocytosis ,030104 developmental biology ,PLASMA-MEMBRANE ,biology.protein ,ARABIDOPSIS-THALIANA ,570 Life sciences ,ATPASE ACTIVITY ,Energy Metabolism ,Acids ,Biokemi och molekylärbiologi ,HeLa Cells - Abstract
ATP production requires the establishment of an electrochemical proton gradient across the inner mitochondrial membrane. Mitochondrial uncouplers dissipate this proton gradient and disrupt numerous cellular processes, including vesicular trafficking, mainly through energy depletion. Here we show that Endosidin9 (ES9), a novel mitochondrial uncoupler, is a potent inhibitor of clathrin-mediated endocytosis (CME) in different systems and that ES9 induces inhibition of CME not because of its effect on cellular ATP, but rather due to its protonophore activity that leads to cytoplasm acidification. We show that the known tyrosine kinase inhibitor tyrphostinA23, which is routinely used to block CME, displays similar properties, thus questioning its use as a specific inhibitor of cargo recognition by the AP-2 adaptor complex via tyrosine motif-based endocytosis signals. Furthermore, we show that cytoplasm acidification dramatically affects the dynamics and recruitment of clathrin and associated adaptors, and leads to reduction of phosphatidylinositol 4,5-biphosphate from the plasma membrane., Plant cells maintain strict proton gradients over different membranes. Here, Dejonghe et al. show that several protonophores, including the known tyrosine kinase inhibitor TyrphostinA23, inhibit clathrin-mediated endocytosis by disturbing these gradients and causing cytoplasmic acidification.
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- 2016
47. Aurora Kinases Throughout Plant Development
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Daniël Van Damme, Inna Lermontova, Annika K. Weimer, Dmitri Demidov, and Tom Beeckman
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0301 basic medicine ,Cell division ,Kinase ,Aurora inhibitor ,Plant Development ,macromolecular substances ,Plant Science ,Plants ,Biology ,Substrate Specificity ,Chromatin ,Cell biology ,Protein Transport ,enzymes and coenzymes (carbohydrates) ,03 medical and health sciences ,Multicellular organism ,030104 developmental biology ,Aurora kinase ,Aurora Kinases ,embryonic structures ,Asymmetric cell division ,biological phenomena, cell phenomena, and immunity ,Mitosis ,Protein Binding - Abstract
Aurora kinases are evolutionarily conserved key mitotic determinants in all eukaryotes. Yeasts contain a single Aurora kinase, whereas multicellular eukaryotes have at least two functionally diverged members. The involvement of Aurora kinases in human cancers has provided an in-depth mechanistic understanding of their roles throughout cell division in animal and yeast models. By contrast, understanding Aurora kinase function in plants is only starting to emerge. Nevertheless, genetic, cell biological, and biochemical approaches have revealed functional diversification between the plant Aurora kinases and suggest a role in formative (asymmetric) divisions, chromatin modification, and genome stability. This review provides an overview of the accumulated knowledge on the function of plant Aurora kinases as well as some major challenges for the future.
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- 2016
48. Endomembrane trafficking overarching cell plate formation
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Daniël Van Damme and Joanna Boruc
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Vesicle fusion ,Cell Membrane ,Endocytic cycle ,Coated vesicle ,Plant Science ,Cell plate ,Biology ,Cell biology ,Cell membrane ,Protein Transport ,medicine.anatomical_structure ,Cell Wall ,Plant Cells ,medicine ,Endomembrane system ,Cytoskeleton ,Cytokinesis - Abstract
By contrast to other eukaryotic kingdoms, plant cytokinesis is an inside-out process. A coordinated action of cytoskeletal transitions and endomembrane trafficking events builds a novel membrane compartment, the cell plate. Deposition of cell wall polymers transforms the lumen of this membrane compartment into a new cross wall, physically separating the daughter cells. The characterization of tethering complexes acting at discrete phases during cell plate formation and upstream of vesicle fusion events, the presence of modulators directing secretion and recycling during cytokinesis, as well as the identification and temporal recruitment of the endocytic machinery, provides a starting point to dissect the transitions in endomembrane trafficking which shape this process. This review aims to integrate recent findings on endomembrane trafficking events which spatio-temporally act to construct the cell plate.
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- 2015
49. Functional Divergence of Microtubule-Associated TPX2 Family Members in Arabidopsis thaliana
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Andreas Houben, Eva Dvořák Tomaštíková, Dmitri Demidov, Petr Dvořák, Jaroslav Doležel, Klara Ptosková, Beáta Petrovská, Alisa Tugai, Twan Rutten, and Daniël Van Damme
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0106 biological sciences ,0301 basic medicine ,Arabidopsis thaliana ,kinase assay ,Targeting Protein for Xklp2 ,Arabidopsis ,PROTEIN ,phylogeny ,Microtubules ,01 natural sciences ,lcsh:Chemistry ,Aurora kinase ,Aurora Kinases ,PHOSPHORYLATION ,aurora kinase 1 ,lcsh:QH301-705.5 ,Spectroscopy ,targeting protein for xklp2 ,General Medicine ,PLANT AURORA KINASES ,Computer Science Applications ,Cell biology ,ALIGNMENT ,Microtubule-Associated Proteins ,Protein Binding ,Binding domain ,arabidopsis thaliana ,Biology ,Genes, Plant ,in vivo co-localization ,Article ,Catalysis ,Inorganic Chemistry ,03 medical and health sciences ,Protein Domains ,Microtubule ,Amino Acid Sequence ,Physical and Theoretical Chemistry ,Kinase activity ,Molecular Biology ,Microtubule nucleation ,targeting protein for Xklp2 ,Arabidopsis Proteins ,Organic Chemistry ,Biology and Life Sciences ,WVD2 ,biology.organism_classification ,030104 developmental biology ,lcsh:Biology (General) ,lcsh:QD1-999 ,SPINDLE ,Functional divergence ,010606 plant biology & botany - Abstract
TPX2 (Targeting Protein for Xklp2) is an evolutionary conserved microtubule-associated protein important for microtubule nucleation and mitotic spindle assembly. The protein was described as an activator of the mitotic kinase Aurora A in humans and the Arabidopsis AURORA1 (AUR1) kinase. In contrast to animal genomes that encode only one TPX2 gene, higher plant genomes encode a family with several TPX2-LIKE gene members (TPXL). TPXL genes of Arabidopsis can be divided into two groups. Group A proteins (TPXL2, 3, 4, and 8) contain Aurora binding and TPX2_importin domains, while group B proteins (TPXL1, 5, 6, and 7) harbor an Xklp2 domain. Canonical TPX2 contains all the above-mentioned domains. We confirmed using in vitro kinase assays that the group A proteins contain a functional Aurora kinase binding domain. Transient expression of Arabidopsis TPX2-like proteins in Nicotiana benthamiana revealed preferential localization to microtubules and nuclei. Co-expression of AUR1 together with TPX2-like proteins changed the localization of AUR1, indicating that these proteins serve as targeting factors for Aurora kinases. Taken together, we visualize the various localizations of the TPX2-LIKE family in Arabidopsis as a proxy to their functional divergence and provide evidence of their role in the targeted regulation of AUR1 kinase activity.
- Published
- 2020
50. The ArathEULS3 Lectin Ends up in Stress Granules and Can Follow an Unconventional Route for Secretion
- Author
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Daniël Van Damme, Guy Smagghe, Els J.M. Van Damme, Isabel Verbeke, Tibo De Coninck, Malgorzata Dubiel, and Vinicius Jose Silva Osterne
- Subjects
0106 biological sciences ,0301 basic medicine ,PREDICTION ,Arabidopsis ,PROTEIN ,aratheuls3 ,01 natural sciences ,Interactome ,lcsh:Chemistry ,Lectins ,Arabidopsis thaliana ,lcsh:QH301-705.5 ,Spectroscopy ,arabidopsis ,Unconventional protein secretion ,biology ,Chemistry ,Translation (biology) ,ArathEULS3 ,General Medicine ,Computer Science Applications ,Cell biology ,unconventional protein secretion ,plant lectin ,GROWTH ,Protein Binding ,stress granules ,intrinsically disordered regions ,Green Fluorescent Proteins ,DISTINCT ,Cytoplasmic Granules ,Article ,Catalysis ,Inorganic Chemistry ,03 medical and health sciences ,Stress granule ,Stress, Physiological ,Amino Acid Sequence ,Physical and Theoretical Chemistry ,Molecular Biology ,Cell Nucleus ,PLANT-LECTINS ,Arabidopsis Proteins ,COMPONENTS ,Organic Chemistry ,EXTRACELLULAR VESICLES ,Biology and Life Sciences ,Lectin ,PROCESSING BODIES ,biology.organism_classification ,030104 developmental biology ,lcsh:Biology (General) ,lcsh:QD1-999 ,Cytoplasm ,biology.protein ,010606 plant biology & botany - Abstract
Stress granules are cytoplasmic compartments, which serve as mRNA storage units during stress, therefore regulating translation. The Arabidopsis thaliana lectin ArathEULS3 has been widely described as a stress inducible gene. This study aimed to examine in detail the localization of ArathEULS3 lectin in normal and stressed cells. Colocalization experiments revealed that the nucleo-cytoplasmic lectin ArathEULS3 relocates to stress granules after stress. The ArathEULS3 sequence encodes a protein with a EUL lectin domain and an N-terminal domain with unknown structure and function. Bioinformatics analyses showed that the N-terminal domain sequence contains intrinsically disordered regions and likely does not exhibit a stable protein fold. Plasmolysis experiments indicated that ArathEULS3 also localizes to the apoplast, suggesting that this protein might follow an unconventional route for secretion. As part of our efforts we also investigated the interactome of ArathEULS3 and identified several putative interaction partners important for the protein translation process.
- Published
- 2020
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