Biology subjects

Faessler, F.

Publications and source records attributed to Faessler, F..

6 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↗

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↗

bioRxiv2022

Bimodally oriented cellulose fibers and reticulated homogalacturonan networks - A direct visualization of Allium cepa primary cell walls

One hallmark of plant cells is their pecto-cellulosic cell walls. They protect cells against the environment and high turgor and mediate morphogenesis through the dynamics of their mechanical and chemical properties. The walls are a complex polysaccharidic structure. Although their biochemical composition is well known, how the different components organize in the volume of the cell wall and interact with each other is not well understood and yet is key to the walls mechanical properties. To investigate the ultrastructure of the plant cell wall, we imaged the walls of onion (Allium cepa) bulbs in a near-native state via cryo-Focused Ion Beam milling (cryo-FIB-milling) and cryo-Electron Tomography (cryo-ET). This allowed the high-resolution visualization of cellulose fibers in situ (in muro). We reveal the coexistence of dense fiber fields bathed in a reticulated matrix we termed "meshing," which is more abundant at the inner surface of the cell wall. The fibers adopted a regular bimodal angular distribution at all depths in the cell wall and bundled according to their orientation, creating layers within the cell wall. Concomitantly, employing homogalacturonan (HG)-specific enzymatic digestion, we observed changes in the meshing, suggesting that it is at least in part composed of HG pectins. We propose the following model for the construction of the abaxial epidermal primary cell wall: The cell deposits successive layers of cellulose fibers at -45{degrees} and +45{degrees} relative to the cells long axis and secretes the surrounding HG-rich meshing proximal to the plasma membrane, which then migrates to more distal regions of the cell wall.

plant biology↗

bioRxiv2021

Computational toolbox for ultrastructural quantitative analysis of filament networks in cryo-ET data

A precise quantitative description of the ultrastructural characteristics underlying biological mechanisms is often key to their understanding. This is particularly true for dynamic extra- and intracellular filamentous assemblies, playing a role in cell motility, cell integrity, cytokinesis, tissue formation and maintenance. For example, genetic manipulation or modulation of actin regulatory proteins frequently manifests in changes of the morphology, dynamics, and ultrastructural architecture of actin filament-rich cell peripheral structures, such as lamellipodia or filopodia. However, the observed ultrastructural effects often remain subtle and require sufficiently large datasets for appropriate quantitative analysis. The acquisition of such large datasets has been enabled by recent advances in high-throughput cryo-electron tomography (cryo-ET) methods. However, this also necessitates the development of complementary approaches to maximize the extraction of relevant biological information. We have developed a computational toolbox for the semi-automatic quantification of filamentous networks from cryo-ET datasets to facilitate the analysis and cross-comparison of multiple experimental conditions. GUI-based components simplify the manipulation of data and allow users to obtain a large number of ultrastructural parameters describing filamentous assemblies. We demonstrate the feasibility of this workflow by analyzing cryo-ET data of untreated and chemically perturbed branched actin filament networks and that of parallel actin filament arrays. In principle, the computational toolbox presented here is applicable for data analysis comprising any type of filaments in regular (i.e. parallel) or random arrangement. We show that it can ease the identification of key differences between experimental groups and facilitate the in-depth analysis of ultrastructural data in a time-efficient manner.

cell biology↗

bioRxiv2020

Novel cryo-electron tomography structure of Arp2/3 complex in cells reveals mechanisms of branch formation

The actin-related protein (Arp)2/3 complex nucleates branched actin filament networks pivotal for cell migration, endocytosis and pathogen infection. Its activation is tightly regulated and involves complex structural rearrangements and actin filament binding, which are yet to be understood. Here, we report a 9.0[A] resolution structure of the actin filament Arp2/3 complex branch junction in cells using cryo-electron tomography and subtomogram averaging. This allows us to generate an accurate model of the active Arp2/3 complex in the branch junction and its interaction with actin filaments. Our structure indicates a central role for the ArpC3 subunit in stabilizing the active conformation and suggests that in the branch junction relocation of the ArpC5 N-terminus and the C-terminal tail of Arp3 is important to fix Arp2 and Arp3 in an actin dimer-like conformation. Notably, our model of the branch junction in cells significantly differs from the previous in vitro branch junction model.

cell biology↗

bioRxiv2020

3D printed cell culture grid holders for improved cellular specimen preparation in cryo-electron microscopy

Cryo-electron microscopy (cryo-EM) of cellular specimens provides insights into biological processes and structures within a native context. However, a major challenge still lies in the efficient and reproducible preparation of adherent cells for subsequent cryo-EM analysis. This is due to the sensitivity of many cellular specimens to the varying seeding and culturing conditions required for EM experiments, the often limited amount of cellular material and also the fragility of EM grids and their substrate. Here, we present low-cost and reusable 3D printed grid holders, designed to improve specimen preparation when culturing challenging cellular samples directly on grids. The described grid holders increase cell culture reproducibility and throughput, and reduce the resources required for cell culturing. We show that grid holders can be integrated into various cryo-EM workflows, including micro-patterning approaches to control cell seeding on grids, and for generating samples for cryo-focused ion beam milling and cryo-electron tomography experiments. Their adaptable design allows for the generation of specialized grid holders customized to a large variety of applications.

cell biology↗