Biology subjects

Datler, J.

Publications and source records attributed to Datler, J..

3 recordsLinked to original sources

bioRxiv2023

Unveiling the ultrastructural landscape of native extracellular matrix via lift-out cryo-FIBSEM and cryo-ET

The extracellular matrix (ECM) is a highly hydrated, three-dimensional network composed of various macromolecules and signaling factors. It serves as a structural scaffold for cells and plays an essential role in the regulation of numerous cellular processes, including cell migration, adhesion, and proliferation. Despite its importance in metazoans, structural knowledge is rudimentary on how the components of the matrisome are secreted, remodeled, and interact with each other and with surrounding cells. Specifically, the exact molecular assembly of important ECM fibers, such as fibronectin fibrils, fibrillin microfibrils, or Collagen-VI filaments has remained enigmatic. This is largely due to methodological limitations in specimen preparation for conventional room-temperature electron microscopy (EM). To overcome these limitations, we have developed a cell culture-based 3D-ECM platform compatible with sample thinning by cryo-lift out focused ion beam (FIB) milling and cryo-electron tomography (cryo-ET). Our workflow involves the implementation of cell-derived matrices (CDMs) grown on EM grids, resulting in a highly adaptable and versatile tool to closely mimic ECM environments. This allows us to visualize native ECM and its components for the first time in their fully hydrated, cellular context. Our data reveals an intricate network of ECM fibers and their positioning with respect to matrix-secreting cells. In addition to D-spaced collagen fibers, we visualize previously unresolved fibrous structures, and an amorphous matrix co-assembling in proximity to ECM fibers and delineating the boundary between ECM and empty extra-cellular space. Intra- and extracellular granules presumably represent assembly intermediates of the ECM. Our results add to the structural atlas of the ECM and provide novel insights into ECM secretion, assembly and maintenance.

cell biology↗

bioRxiv2023

Multi-modal cryo-EM reveals trimers of protein A10 to form the palisade layer in poxvirus cores

Poxviruses are among the largest double-stranded DNA viruses with members such as Variola virus, Monkeypox virus and the famous vaccination strain Vaccinia virus (VACV). Knowledge about the structural proteins that form the viral core, found in all infectious poxvirus forms, 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 the individual architectural features of the core. Hence, which proteins constitute which layers of the core, such as the so-called palisade layer and the inner core wall has remained enigmatic. Here, we have performed a multi-modal cryo-electron microscopy (cryo-EM) approach to elucidate the structural determinants of the VACV core. In combination with molecular modeling using AlphaFold, we unambiguously identify trimers formed by the cleavage product of A10 as 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 substantially revised model of poxvirus core architecture. Importantly, we show that interactions within A10 trimers are likely identical among Poxviridae, implying that our structural observations should be generalizable over most, if not all members of this important virus family. One sentence summarySingle-particle cryo-EM, cryo-electron tomography, and AlphaFold modeling reveal the structural architecture of the poxvirus core and identify trimers of protein A10 as the key component of the palisade layer.

molecular biology↗

bioRxiv2022

ArpC5 isoforms regulate Arp2/3 complex-dependent protrusion through differential Ena/VASP positioning

Tight regulation of Arp2/3 complex is required to allow productive nucleation of force-generating, branched actin networks. An emerging aspect of regulation is the incorporation of subunit isoforms into Arp2/3 complex. Specifically, both isoforms of the ArpC5 subunit, ArpC5 and ArpC5L, have been reported to fine-tune nucleation activity and branch junction stability. Elevated levels of ArpC5 have also been linked to increased cancer progression and metastasis. Here, we have combined genetic engineering of cells and cellular structural biology to describe how ArpC5 and ArpC5L differentially regulate cell migration. They do so by defining the structural stability of ArpC1 in branch junctions and, in turn, by determining protrusion characteristics, protein dynamics, and actin network ultrastructure. ArpC5 isoforms also have an impact on the positioning of actin assembly factors from the Ena/VASP family, which act downstream of Arp2/3 complex-mediated nucleation. This suggests that ArpC5 and Ena/VASP proteins, both predictors for poor outcome in cancer, are part of a signaling pathway enhancing cell migration and, by inference, metastasis.

cell biology↗