@article{15146, abstract = {The extracellular matrix (ECM) serves as a scaffold for cells and plays an essential role in regulating numerous cellular processes, including cell migration and proliferation. Due to limitations in specimen preparation for conventional room-temperature electron microscopy, we lack structural knowledge on how ECM components are secreted, remodeled, and interact with surrounding cells. We have developed a 3D-ECM platform compatible with sample thinning by cryo-focused ion beam milling, the lift-out extraction procedure, and cryo-electron tomography. Our workflow implements cell-derived matrices (CDMs) grown on EM grids, resulting in a versatile tool closely mimicking ECM environments. This allows us to visualize ECM for the first time in its hydrated, native context. Our data reveal an intricate network of extracellular fibers, their positioning relative to matrix-secreting cells, and previously unresolved structural entities. Our workflow and results add to the structural atlas of the ECM, providing novel insights into its secretion and assembly.}, author = {Zens, Bettina and Fäßler, Florian and Hansen, Jesse and Hauschild, Robert and Datler, Julia and Hodirnau, Victor-Valentin and Zheden, Vanessa and Alanko, Jonna H and Sixt, Michael K and Schur, Florian KM}, issn = {1540-8140}, journal = {Journal of Cell Biology}, number = {6}, publisher = {Rockefeller University Press}, title = {{Lift-out cryo-FIBSEM and cryo-ET reveal the ultrastructural landscape of extracellular matrix}}, doi = {10.1083/jcb.202309125}, volume = {223}, year = {2024}, } @phdthesis{18766, abstract = {Poxviruses are large pleomorphic double-stranded DNA viruses that include well known members such as variola virus, the causative agent of smallpox, Mpox virus, as well as Vaccinia virus (VACV), which serves as a vaccination strain for formerly mentioned viruses. VACV is a valuable model for studying large pleomorphic DNA viruses in general and poxviruses specifically, as many features, such as core morphology and structural proteins, are well conserved within this family. Despite decades of research, our understanding of the structural components and proteins that comprise the poxvirus core in mature virions remains limited. Although major core proteins were identified via indirect experimental evidence, the core's complexity, with its large size, structure and number of involved proteins, has hindered efforts to achieve high-resolution insights and to define the roles of the individual proteins. The specific protein composition of the core's individual layers, including the palisade layer and the inner core wall, has remained unclear. In this study, we have merged multiple approaches, including single particle cryo electron microscopy of purified virus cores, cryo-electron tomography and subtomogram averaging of mature virions and molecular modeling to elucidate the structural determinants of the VACV core. Due to the lack of experimentally derived structures, either in situ or reconstituted in vitro, we used Alphafold to predict models of the putative major core protein candidates, A10, 23k, A3, A4, and L4. Our results show that the VACV core is composed of several layers with varying local symmetries, forming more intricate interactions than observed previously. This allowed us to identify several molecular building blocks forming the viral core lattice. In particular, we identified trimers of protein A10 as a major core structure that forms the palisade layer of the viral core. Additionally, we revealed that six petals of a flower shaped core pore within the core wall are composed of A10 trimers. Furthermore, we obtained a cryo-EM density for the inner core wall that could potentially accommodate an A3 dimer. Integrating descriptions of protein interactions from previous studies enabled us to provide a detailed structural model of the poxvirus core wall, and our findings indicate that the interactions within A10 trimers are likely consistent across orthopox- and parapoxviruses. This combined application of cryo-SPA and cryo-ET can help overcome obstacles in studying complex virus structures in the future, including their key assembly proteins, interactions, and the formation into a core lattice. Our work provides important fundamental new insights into poxvirus core architecture, also considering the recent re-emergence of poxviruses.}, author = {Datler, Julia}, isbn = {978-3-99078-049-7}, issn = {2663-337X}, keywords = {cryo-EM, cryo-ET, cryo-SPA, Structural Virology, Poxvirus, Vaccinia Virus, Structural Biology}, pages = {106}, publisher = {Institute of Science and Technology Austria}, title = {{Elucidating the structural determinants of the poxvirus core using multi-modal cryo-EM}}, doi = {10.15479/at:ista:18766}, year = {2024}, } @article{14979, abstract = {Poxviruses are among the largest double-stranded DNA viruses, with members such as variola virus, monkeypox virus and the vaccination strain vaccinia virus (VACV). Knowledge about the structural proteins that form the viral core has remained sparse. While major core proteins have been annotated via indirect experimental evidence, their structures have remained elusive and they could not be assigned to individual core features. Hence, which proteins constitute which layers of the core, such as the palisade layer and the inner core wall, has remained enigmatic. Here we show, using a multi-modal cryo-electron microscopy (cryo-EM) approach in combination with AlphaFold molecular modeling, that trimers formed by the cleavage product of VACV protein A10 are the key component of the palisade layer. This allows us to place previously obtained descriptions of protein interactions within the core wall into perspective and to provide a detailed model of poxvirus core architecture. Importantly, we show that interactions within A10 trimers are likely generalizable over members of orthopox- and parapoxviruses.}, author = {Datler, Julia and Hansen, Jesse and Thader, Andreas and Schlögl, Alois and Bauer, Lukas W and Hodirnau, Victor-Valentin and Schur, Florian KM}, issn = {1545-9985}, journal = {Nature Structural & Molecular Biology}, keywords = {Molecular Biology, Structural Biology}, pages = {1114--1123}, publisher = {Springer Nature}, title = {{Multi-modal cryo-EM reveals trimers of protein A10 to form the palisade layer in poxvirus cores}}, doi = {10.1038/s41594-023-01201-6}, volume = {31}, year = {2024}, } @article{12334, abstract = {Regulation of the Arp2/3 complex is required for productive nucleation of branched actin networks. An emerging aspect of regulation is the incorporation of subunit isoforms into the Arp2/3 complex. Specifically, both ArpC5 subunit isoforms, ArpC5 and ArpC5L, have been reported to fine-tune nucleation activity and branch junction stability. We have combined reverse genetics and cellular structural biology to describe how ArpC5 and ArpC5L differentially affect cell migration. Both define the structural stability of ArpC1 in branch junctions and, in turn, by determining protrusion characteristics, affect protein dynamics and actin network ultrastructure. ArpC5 isoforms also affect the positioning of members of the Ena/Vasodilator-stimulated phosphoprotein (VASP) family of actin filament elongators, which mediate ArpC5 isoform–specific effects on the actin assembly level. Our results suggest that ArpC5 and Ena/VASP proteins are part of a signaling pathway enhancing cell migration.}, author = {Fäßler, Florian and Javoor, Manjunath and Datler, Julia and Döring, Hermann and Hofer, Florian and Dimchev, Georgi A and Hodirnau, Victor-Valentin and Faix, Jan and Rottner, Klemens and Schur, Florian KM}, issn = {2375-2548}, journal = {Science Advances}, keywords = {Multidisciplinary}, number = {3}, publisher = {American Association for the Advancement of Science}, title = {{ArpC5 isoforms regulate Arp2/3 complex–dependent protrusion through differential Ena/VASP positioning}}, doi = {10.1126/sciadv.add6495}, volume = {9}, year = {2023}, }