Marcel Dickmanns

Cryo-electron tomography (cryo-ET) allows to visualize the molecular architecture of pristinely preserved cells and tissues in situ. The workflow of sample preparation for cryo-ET is rather complex; it involves vitrification by rapid freezing followed by cryo-focused ion beam (FIB) milling rendering the volumes of interest thin enough for cryo-ET data acquisition. The established protocols for single cells grown on or deposited on EM-grids are not suitable for multicellular plant tissues. Plunge-freezing does not yield vitrified samples in most cases and must be replaced by high-pressure freezing. This, in turn, necessitates extensive modifications of the subsequent FIB milling procedures. In this communication we describe procedures for sample screening, targeted FIB milling guided by cryo-fluorescence light microscopy and a novel lamella trimming step that allows to obtain homogenously thin lamellae suitable for cryo-ET. We have tested all the steps along the workflow with a variety of plant tissues including the moss Physcomitrium patens, Arabidopsis thaliana seed embryos and Limonium bicolor salt glands. We could demonstrate that the workflow optimized for plant tissues allows to attain subnanometer resolution in cases where subtomogram averaging is applicable.

Plasmodesmata (PD) are conduits between plant cells that are essential to multicellularity: they allow a wide range of molecules, such as nutrients, phytohormones, RNAs and proteins including transcription factors, to be transported from cell to cell. PD are essential to intercellular communication and have been shown to be important for developmental transitions. PD were discovered using light microscopy. Since then, advanced imaging approaches have been utilised to resolve PD ultrastructure, analyse their spatial distribution, and probe the translocation of diverse cargo. In this review, we first summarise the microscopy techniques used for studying PD, with a focus on fluorescence and electron microscopy (EM). We then discuss emerging techniques and new advances, such as Correlative Light and Electron Microscopy (CLEM), single-molecule imaging, and cryo-electron tomography (cryo-ET), approaches that will likely lead to exciting discoveries in the field, such as capturing dynamics at PD and revealing previously unknown aspects of PD structure. This review discusses the strengths and weaknesses of the different techniques and how combining them could pave the way to a more detailed understanding of the diverse PD types, their distribution and function.

Plasmodesmata are nanoscopic channels that traverse plant cell walls, enabling direct intercellular exchange through membrane and cytosolic continuity. Although numerous plasmodesmal components have been identified, their molecular organization remains poorly defined. Here we used cryo-electron tomography to resolve the in situ architecture of plasmodesmata in Physcomitrium patens across tissues and physiological states. We show how callose-related cell wall remodelling shapes pore architecture to modulate permeability, including a previously undescribed fully sealed state, and resolve helical protein assemblies scaffolding the central, endoplasmic-reticulum-derived desmotubule. Candidate screening via proteomics and structure prediction indicates Multiple C2 Domain and Transmembrane Proteins (MCTPs) as key constituents of these assemblies. In this model, MCTPs tether the desmotubule to the plasma membrane, while their disordered linker regions with polyampholyte charge patterning may populate the cytosolic sleeve. These findings define core architectural features of plasmodesmata and provide a structural framework for understanding how membrane, protein and cell wall components coordinate intercellular connectivity in plants.

Plasmodesmata are nanoscopic channels that traverse plant cell walls, establishing membrane and cytoplasmic continuity between adjacent cells to enable direct intercellular exchange. Although numerous components have been identified, their molecular organization and roles in controlling passage remain unclear. Here, we used cryo-electron tomography to resolve the in situ architecture of plasmodesmata in Physcomitrium patens across tissues and physiological states. We show how callose deposition at the cell wall shapes channel architecture to modulate permeability and identify helical protein assemblies formed by Multiple C2 domain and Transmembrane Proteins (MCTPs), which scaffold a central endoplasmic reticulum tubule and tether it to the plasma membrane. These findings define core architectural features of plasmodesmata and establish a structural framework for understanding how membrane, protein and cell wall components coordinate intercellular connectivity in plants.

We study the role of the fuzzy coat of α-synuclein amyloid fibrils via proteolytic degradation. Bulk solution assays offer limited insight, while Quartz crystal microbalance can reveal detailed mechanisms of growth of the “shaven” amyloid fibrils.

Cryo-Electron Tomography (cryo-ET) allows to visualize the molecular architecture of pristinely preserved cells and tissues. The workflow of sample preparation for cryo-ET is rather complex; it involves vitrification by rapid freezing followed by cryo-Focused Ion Beam (FIB) milling rendering the volumes of interest thin enough for cryo-ET data acquisition. The established protocols for single cells grown on or deposited on EM-grids are not suitable for multicellular plant tissues. Plunge-freezing does not yield vitrified samples in most cases and must be replaced by high-pressure freezing. This, in turn, necessitates extensive modifications of the subsequent FIB milling procedures. In this communication we describe procedures for sample screening, targeted FIB milling guided by cryo-fluorescence microscopy and a novel lamella trimming step that allows to obtain homogenously thin lamellae suitable for cryo-ET. We have tested all the steps along the workflow with a variety of plant tissues including the moss Physcomitrium patens and tissues of Arabidopsis thaliana and Limonium bicolor. We could demonstrate that the workflow optimized for plant tissues allows to attain subnanometer resolution in cases where subtomogram averaging is applicable.

Cryo-Electron Tomography (cryo-ET) allows to visualize the molecular architecture of pristinely preserved cells and tissues. The workflow of sample preparation for cryo-ET is rather complex; it involves vitrification by rapid freezing followed by cryo-Focused Ion Beam (FIB) milling rendering the volumes of interest thin enough for cryo-ET data acquisition. The established protocols for single cells grown on or deposited on EM-grids are not suitable for multicellular plant tissues. Plunge-freezing does not yield vitrified samples in most cases and must be replaced by high-pressure freezing. This, in turn, necessitates extensive modifications of the subsequent FIB milling procedures. In this communication we describe procedures for sample screening, targeted FIB milling guided by cryo-fluorescence microscopy and a novel lamella trimming step that allows to obtain homogenously thin lamellae suitable for cryo-ET. We have tested all the steps along the workflow with a variety of plant tissues including the moss Physcomitrium patens and tissues of Arabidopsis thaliana and Limonium bicolor . We could demonstrate that the workflow optimized for plant tissues allows to attain subnanometer resolution in cases where subtomogram averaging is applicable.

In most bacteria, cell division depends on the tubulin-homolog FtsZ that polymerizes in a GTP-dependent manner to form the cytokinetic Z-ring at the future division site. Subsequently, the Z-ring recruits, directly or indirectly, all other proteins of the divisome complex that executes cytokinesis. A critical step in this process is the precise positioning of the Z-ring at the future division site. While the divisome proteins are generally conserved, the regulatory systems that position the Z-ring are more diverse. However, these systems have in common that they modulate FtsZ polymerization. In Myxococcus, PomX, PomY, and PomZ form precisely one MDa-sized, nonstoichiometric, nucleoid-associated assembly that spatiotemporally guides Z-ring formation. Here, using cryo-correlative light and electron microscopy together with in situ cryoelectron tomography, we determine the PomXYZ assembly’s architecture at close-to-live conditions. PomX forms a porous meshwork of randomly intertwined filaments. Templated by this meshwork, the phase-separating PomY protein forms a biomolecular condensate that compacts and bends the PomX filaments, resulting in the formation of a selective PomXYZ co-condensate that is associated to the nucleoid by PomZ. These studies reveal a hitherto undescribed supramolecular structure and provide a framework for understanding how a nonstoichiometric co-condensate forms, maintains number control, and nucleates GTP-dependent FtsZ polymerization to precisely regulate cell division.

Amyloid fibrils typically consist of a dense core made up of β-strands, with disordered flanks on either side, and sometimes interrupted by disordered loop regions (the fuzzy coat). α-synuclein found in Lewy Bodies of Parkinson’s Disease patients are mostly C-terminally truncated, meaning that a large fraction of the fuzzy coat of disease-related fibrils is enzymatically degraded in the amyloid state. We demonstrate that the proteolytic removal of the fuzzy coat leads to enhanced fibril-fibril interactions and flocculation, which renders the study of the role of the fuzzy coat in bulk solution very challenging. In order to overcome these challenges, here we use Quartz Crystal Microbalance with Dissipation (QCM-D), a surface based biosensing technique, to study the effects of proteolytic removal of the fuzzy coat of α-synuclein amyloids. We demonstrate that DissipationFrequency analysis can illuminate multiple simultaneous reactions and characterize the monomer-fibril interactions in detail. We find that removal of the fuzzy coat increases apparent fibril elongation rates permanently upon each proteinase treatment. Utilizing kinetic models, we demonstrate that our results cannot be rationalized by alterations of the elongation rate of fibrils alone, but indicates that removal of the fuzzy coat of α-synuclein fibrils enable secondary processes at neutral pH. We propose that the nature of the secondary processes investigated here may be highly relevant for understanding disease-related α-synuclein amyloid formation. Furthermore we suggest that the Quartz Crystal Microbalance with Dissipation is a particularly attractive platform for studying the effects of post-translational modifications on amyloid growth and molecular interactions.

Plasmodesmata (PD) mediate intercellular exchange of small molecules, RNAs and proteins between plant cells with an apparent exclusion limit for passive non-specific transport, and transport of specific cargo mediated by mediators. PD and nuclear pore complexes (NPC) are nanometer sized micropores with strikingly similar properties. Cargo translocation through NPC is mediated by phase separating FG-nucleoporins (FG-NUP). Here, bioinformatics, proteomics and fluorescence imaging identified FG-NUPs at PD. Transient expression of GFP fusions at low and intermediate expression levels supported dual localization of 12 NUPs to NPC and PD. Structured illumination microscopy detected the transmembrane anchor NUP CPR5 close to orifices of PD. cpr5 mutants showed reduced intercellular short-root (SHR) transport. However, transport defects cannot be excluded due to indirect effects in the mutants. Identification of FG-NUPs at PD is consistent with the recruitment of NUPs to form a PD pore gating complex consistent with phase separation domains as diffusion barriers at PD. Further analyses will be required to determine whether NUPs are bona fide PD components, or accumulate at PD in certain conditions, or may serve intermediate NPC storage.