{"user_id":"8b945eb4-e2f2-11eb-945a-df72226e66a9","date_created":"2023-02-02T14:50:20Z","oa_version":"Published Version","oa":1,"publisher":"Institute of Science and Technology Austria","status":"public","related_material":{"record":[{"status":"public","id":"8586","relation":"part_of_dissertation"}]},"title":"Ultrastructural characterization of natively preserved extracellular matrix by cryo-electron tomography","alternative_title":["ISTA Thesis"],"degree_awarded":"PhD","day":"02","type":"dissertation","file":[{"relation":"main_file","checksum":"069d87f025e0799bf9e3c375664264f2","file_name":"PhDThesis_BettinaZens_2023_final.pdf","embargo":"2024-02-07","creator":"bzens","access_level":"open_access","content_type":"application/pdf","date_updated":"2024-02-08T23:30:04Z","date_created":"2023-02-07T13:07:38Z","file_size":23082464,"file_id":"12527"},{"relation":"source_file","checksum":"8c66ed203495d6e078ed1002a866520c","content_type":"application/vnd.openxmlformats-officedocument.wordprocessingml.document","file_name":"PhDThesis_BettinaZens_2023_final.docx","creator":"bzens","access_level":"closed","embargo_to":"open_access","date_updated":"2024-02-08T23:30:04Z","file_id":"12528","file_size":106169509,"date_created":"2023-02-07T13:09:05Z"}],"date_updated":"2024-02-08T23:30:05Z","month":"02","supervisor":[{"last_name":"Schur","first_name":"Florian KM","full_name":"Schur, Florian KM","id":"48AD8942-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0003-4790-8078"}],"article_processing_charge":"No","date_published":"2023-02-02T00:00:00Z","publication_status":"published","abstract":[{"text":"The extracellular matrix (ECM) is a hydrated and complex three-dimensional network consisting of proteins, polysaccharides, and water. It provides structural scaffolding for the cells embedded within it and is essential in regulating numerous physiological processes, including cell migration and proliferation, wound healing, and stem cell fate. \r\nDespite extensive study, detailed structural knowledge of ECM components in physiologically relevant conditions is still rudimentary. This is due to methodological limitations in specimen preparation protocols which are incompatible with keeping large samples, such as the ECM, in their native state for subsequent imaging. Conventional electron microscopy (EM) techniques rely on fixation, dehydration, contrasting, and sectioning. This results in the alteration of a highly hydrated environment and the potential introduction of artifacts. Other structural biology techniques, such as nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography, allow high-resolution analysis of protein structures but only work on homogenous and purified samples, hence lacking contextual information. Currently, no approach exists for the ultrastructural and structural study of extracellular components under native conditions in a physiological, 3D environment. \r\nIn this thesis, I have developed a workflow that allows for the ultrastructural analysis of the ECM in near-native conditions at molecular resolution. The developments I introduced include implementing a novel specimen preparation workflow for cell-derived matrices (CDMs) to render them compatible with ion-beam milling and subsequent high-resolution cryo-electron tomography (ET). \r\nTo this end, I have established protocols to generate CDMs grown over several weeks on EM grids that are compatible with downstream cryo-EM sample preparation and imaging techniques. Characterization of these ECMs confirmed that they contain essential ECM components such as collagen I, collagen VI, and fibronectin I in high abundance and hence represent a bona fide biologically-relevant sample. I successfully optimized vitrification of these specimens by testing various vitrification techniques and cryoprotectants. \r\nIn order to obtain high-resolution molecular insights into the ultrastructure and organization of CDMs, I established cryo-focused ion beam scanning electron microscopy (FIBSEM) on these challenging and complex specimens. I explored different approaches for the creation of thin cryo-lamellae by FIB milling and succeeded in optimizing the cryo-lift-out technique, resulting in high-quality lamellae of approximately 200 nm thickness. \r\nHigh-resolution Cryo-ET of these lamellae revealed for the first time the architecture of native CDM in the context of matrix-secreting cells. This allowed for the in situ visualization of fibrillar matrix proteins such as collagen, laying the foundation for future structural and ultrastructural characterization of these proteins in their near-native environment. \r\nIn summary, in this thesis, I present a novel workflow that combines state-of-the-art cryo-EM specimen preparation and imaging technologies to permit characterization of the ECM, an important tissue component in higher organisms. This innovative and highly versatile workflow will enable addressing far-reaching questions on ECM architecture, composition, and reciprocal ECM-cell interactions.","lang":"eng"}],"department":[{"_id":"GradSch"},{"_id":"FlSc"}],"acknowledged_ssus":[{"_id":"EM-Fac"},{"_id":"LifeSc"},{"_id":"Bio"}],"year":"2023","keyword":["cryo-EM","cryo-ET","FIB milling","method development","FIBSEM","extracellular matrix","ECM","cell-derived matrices","CDMs","cell culture","high pressure freezing","HPF","structural biology","tomography","collagen"],"file_date_updated":"2024-02-08T23:30:04Z","project":[{"name":"Integrated visual proteomics of reciprocal cell-extracellular matrix interactions","_id":"eba3b5f6-77a9-11ec-83b8-cf0905748aa3"},{"_id":"059B463C-7A3F-11EA-A408-12923DDC885E","name":"NÖ-Fonds Preis für die Jungforscherin des Jahres am IST Austria"}],"ddc":["570"],"author":[{"first_name":"Bettina","last_name":"Zens","full_name":"Zens, Bettina","id":"45FD126C-F248-11E8-B48F-1D18A9856A87"}],"has_accepted_license":"1","doi":"10.15479/at:ista:12491","page":"187","publication_identifier":{"isbn":["978-3-99078-027-5"],"issn":["2663-337X"]},"citation":{"ista":"Zens B. 2023. Ultrastructural characterization of natively preserved extracellular matrix by cryo-electron tomography. Institute of Science and Technology Austria.","chicago":"Zens, Bettina. “Ultrastructural Characterization of Natively Preserved Extracellular Matrix by Cryo-Electron Tomography.” Institute of Science and Technology Austria, 2023. <a href=\"https://doi.org/10.15479/at:ista:12491\">https://doi.org/10.15479/at:ista:12491</a>.","apa":"Zens, B. (2023). <i>Ultrastructural characterization of natively preserved extracellular matrix by cryo-electron tomography</i>. Institute of Science and Technology Austria. <a href=\"https://doi.org/10.15479/at:ista:12491\">https://doi.org/10.15479/at:ista:12491</a>","ama":"Zens B. Ultrastructural characterization of natively preserved extracellular matrix by cryo-electron tomography. 2023. doi:<a href=\"https://doi.org/10.15479/at:ista:12491\">10.15479/at:ista:12491</a>","short":"B. Zens, Ultrastructural Characterization of Natively Preserved Extracellular Matrix by Cryo-Electron Tomography, Institute of Science and Technology Austria, 2023.","mla":"Zens, Bettina. <i>Ultrastructural Characterization of Natively Preserved Extracellular Matrix by Cryo-Electron Tomography</i>. Institute of Science and Technology Austria, 2023, doi:<a href=\"https://doi.org/10.15479/at:ista:12491\">10.15479/at:ista:12491</a>.","ieee":"B. Zens, “Ultrastructural characterization of natively preserved extracellular matrix by cryo-electron tomography,” Institute of Science and Technology Austria, 2023."},"_id":"12491","language":[{"iso":"eng"}]}