Biology subjects

Weber, M. S.

Publications and source records attributed to Weber, M. S..

5 recordsLinked to original sources

bioRxiv2026

A simplified High-Pressure Freezing Workflow in Autogrids

Cryo-electron tomography (cryo-ET) is a powerful method for studying biological structures in near-native states, yet vitrification of thick and complex specimens remains a major limitation. High-pressure freezing (HPF) allows vitrification of up to [~]200 {micro}m thick samples, but its seamless integration into cryo-electron microscopy (cryo-EM) workflows is hindered by planchettes that are not optimized for EM grids, resulting in extensive handling, reliance on user experience, and frequent grid damage. Here, we present a streamlined HPF workflow based on newly designed planchettes that directly accommodate clipped EM Autogrids. Combined with interchangeable lids for controlling sample thickness, this system enables robust vitrification of diverse specimens, including single-celled eukaryotes and tissue biopsies, while minimizing post-freezing handling. Integration with plasma-focused ion beam milling supports both on-grid lamella preparation and lift-out approaches. We demonstrate reliable preservation of cellular ultrastructure and macromolecular integrity by cryo-ET and subtomogram averaging. This resource provides a practical and scalable solution for integrating HPF into modern cryo-ET pipelines, expanding access to structurally complex biological systems and supporting future clinical applications.

cell biology↗

bioRxiv2023

Vimentin filaments integrate low complexity domains in a highly complex helical structure

Intermediate filaments (IFs) are integral components of the cytoskeleton. They provide cells with tissue-specific mechanical properties and are involved in numerous cellular processes. Due to their intricate architecture, a 3D structure of IFs has remained elusive. Here we use cryo-focused ion beam milling, cryo-electron microscopy and tomography, to obtain a 3D structure of vimentin IFs (VIFs). VIFs assemble into a modular, densely-packed and highly-ordered helical symmetric structure of 40 -helices in cross-section, organized into 5 protofibrils. Surprisingly, the intrinsically disordered head domains form an amyloid-like fiber in the center of VIFs, while the intrinsically disordered tails form lateral connections between the protofibrils. Our findings demonstrate how protein domains of low sequence complexity can complement well-folded protein domains to construct a biopolymer with striking strength and stretchability.

biochemistry↗

bioRxiv2021

Vimentin Intermediate Filaments and Filamentous Actin Form Unexpected Interpenetrating Networks That Redefine the Cell Cortex

Vimentin intermediate filaments (VIFs) and F-actin are filamentous cytoskeletal proteins generally thought to form completely independent networks that have vastly different properties and functions. Here, we show that, unexpectedly, there exist both extensive structural and functional interactions between VIFs and F-actin. We show that VIFs and F-actin form an interpenetrating network (IPN) within the cell cortex and interact synergistically at multiple length scales. This IPN structure has important functional consequences in cells: The IPN results in enhanced contractile forces in the cell. In addition, VIFs influence the diffusive behavior of actin monomers, suggesting specific associations between actin and vimentin proteins in the cytoplasm; this facilitates formation of the IPN and has downstream effects on other actin-driven processes. The results suggest that contributions of VIFs and F-actin are strongly correlated. Such interactions counter generally accepted behavior and are broadly significant given the wide range of processes currently attributed to F-actin alone.

cell biology↗

bioRxiv2021

The molecular architecture of vimentin filaments

Intermediate filaments are integral components of the cytoskeleton in metazoan cells. Due to their specific viscoelastic properties they are principal contributors to flexibility and tear strength of cells and tissues. Vimentin, an intermediate filament protein expressed in fibroblasts and endothelial cells, assembles into ~11 nm thick biopolymers, that are involved in a wide variety of cellular functions in health and disease. Here, we reveal the structure of in-situ polymerized vimentin filaments to a subnanometer resolution by applying cryo-electron tomography to mouse embryonic fibroblasts grown on electron microscopy grids. We show that vimentin filaments are tube-like assemblies with a well-defined helical symmetry. Their structure is comprised of five octameric, spring-like protofibrils harboring 40 vimentin polypeptide chains in cross-section. The protofibrils are connected by the intrinsically disordered head and helix 1A domains of vimentin. Individual filaments display two polymerization states characterized by either the presence or absence of a luminal density along the helical axis. The structure of vimentin filaments unveils the generic building plan of the intermediate filament superfamily in molecular details.

cell biology↗

bioRxiv2018

Longitudinally persistent cerebrospinal fluid B-cells resist treatment in multiple sclerosis

B-cells are key contributors to chronic autoimmune pathology in multiple sclerosis (MS). Clonally related B-cells exist in the cerebrospinal fluid (CSF), meninges, and central nervous system (CNS) parenchyma of MS patients. Longitudinally stable CSF oligoclonal band (OCB) antibody patterns suggest some local CNS B-cell persistence; however, the longitudinal B-cell dynamics within and between the CSF and blood remain unknown. We sought to address this by performing immunoglobulin heavy chain variable region repertoire sequencing on B-cells from longitudinally collected blood and CSF samples of MS patients (n=10). All patients were untreated at the time of the initial sampling; the majority (n=7) were treated with immune modulating therapies 1.2 (+/-0.3 SD) years later during the second sampling. We found clonal persistence of B-cells in the CSF of five patients; these B-cells were frequently immunoglobulin (Ig) class-switched and CD27+. We identified specific blood B-cell subsets that appear to provide input into CNS repertoires over time. We demonstrate complex patterns of clonal B-cell persistence in CSF and blood, even in patients on high-efficacy immune modulating therapy. Our findings support the concept that peripheral B-cell activation and CNS-compartmentalized immune mechanisms are in part therapy-resistant.

immunology↗