Dokument: Methodology and application of cryo-electron tomography to vitrified plant tissue: Insights into plasmodesmata architecture, composition and gating.
| Titel: | Methodology and application of cryo-electron tomography to vitrified plant tissue: Insights into plasmodesmata architecture, composition and gating. | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=74698 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20261023-141429-5 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Dickmanns, Marcel [Autor] | |||||||
| Dateien: |
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| Beitragende: | Prof. Dr. Frommer, Wolf B. [Gutachter] Prof. Dr. Baumeister, Wolfgang [Gutachter] | |||||||
| Stichwörter: | cryo-ET, cryo-EM, plant physiology, plasmodesmata | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 570 Biowissenschaften; Biologie | |||||||
| Beschreibung: | Multicellular organisms rely on intercellular communication and exchange of resources. In plants, direct molecular exchange is mediated by plasmodesmata: nanoscopic pores that traverse the cell wall and establish continuity between the plasma membrane, cytosol, and endoplasmic reticulum of adjacent cells. Despite their essential role in plant development and physiology, the molecular composition, internal organization, and mechanisms controlling plasmodesmal permeability remain poorly defined. Proteomic studies have identified hundreds of putative plasmodesmal proteins, but such datasets are inherently noisy and likely include many false positives. Systematic mapping of the molecular inventory within plasmodesmata is further complicated by their nanometer-scale dimensions, placing them below the resolution limit of most light microscopy techniques, while preparation artifacts in classical electron microscopy prevent reliable analysis of the fine molecular detail.
Here, we applied a combination of methods to advance the characterization of plasmodesmal organization and function, focusing on the model plant Physcomitrium patens. We established a proteome of plasmodesmata-enriched cell wall fractions and analyze multiple uncharacterized candidate proteins. Among them, we found several cell wall-modifying enzymes including a clade of glycosyl hydrolase family 17 (GHL17) proteins that localize to plasmodesmata and identified a homolog modulating plasmodesmal permeability. In addition, several nucleoporins (NUPs) were identified in the proteome. Intrigued by the similarities between nuclear and plasmodesmal pores, we examined their subcellular localization and assessed plasmodesmal permeability in NUP mutants. As multiple NUPs show dual localization to nuclei and plasmodesmata and plasmodesmal permeability is altered in NUP mutants, we probed the possibility of a NUP-containing plasmodesmal pore complex. To visualize plasmodesmata components in the native context, we developed cryo-electron tomography workflows for imaging multicellular plant samples and assessed them on a set of tissues from Physcomitrium patens, Arabidopsis thaliana and Limonium bicolor. Applying these workflows, we resolved the in situ architecture of plasmodesmata in Physcomitrium patens. By comparing wild-type with overexpression of the previously identified glycosyl hydrolase GHL17, as well as abscisic acid-treated wild-type tissue, we could link architectural changes of plasmodesmal pores to permeability phenotypes. Under GHL17 overexpression associated with increased intercellular flux, plasmodesmata were shorter and wider. In contrast, abscisic acid treatment, known to reduce solute flux between cells, induced excessive deposition of cell wall material around plasmodesmal apertures. Unexpectedly, this deposition resulted in complete channel sealing in the majority of imaged plasmodesmata, abolishing membrane and cytosolic continuity between cells. Beyond this cell wall-sided gating mechanism, we resolved the structure of a protein assembly, present within all imaged plasmodesmata where it coats the central, ER-derived desmotubule membrane. Through integration of subtomogram averaging with proteomics and structure predictions, we could identify Multiple C2 domain and Transmembrane Proteins (MCTPs) as the constituents of the assembly. Bioinformatic analysis further revealed that MCTPs contain extended intrinsically disordered linker regions with electrostatic sticker-spacer characteristics. As mapping back MCTPs into the tomographic representations of plasmodesmata indicated these disordered linker domains populate the cytosolic route within plasmodesmata, we hypothesized they may constitute a charge-selective permeability barrier. In summary, our observations define molecular and architectural features of plasmodesmata and provide a structural framework for how membrane, protein, and cell wall components coordinate intercellular connectivity in plants. | |||||||
| Lizenz: |  Dieses Werk ist lizenziert unter einer Creative Commons Namensnennung 4.0 International Lizenz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät | |||||||
| Dokument erstellt am: | 23.10.2026 | |||||||
| Dateien geändert am: | 23.10.2026 | |||||||
| Promotionsantrag am: | 03.02.2026 | |||||||
| Datum der Promotion: | 07.10.2026 |
