Vimentin intermediate filaments: the origin of stretchability and their direct interaction with actin filaments

Información del proyecto

VimAc

Identificador del acuerdo de subvención: 101148781

DOI

10.3030/101148781

Proyecto cerrado

Fecha de la firma de la CE 12 Marzo 2024

Fecha de inicio 1 Mayo 2024

Fecha de finalización 30 Abril 2026

Financiado con arreglo a

• Marie Skłodowska-Curie Actions (MSCA)

Coste total

Sin datos

Aportación de la UE

€ 189 687,36

Coordinado por

GEORG-AUGUST-UNIVERSITAT GOTTINGEN STIFTUNG OFFENTLICHEN RECHTS
Germany

Periodic Reporting for period 1 - VimAc (Vimentin intermediate filaments: the origin of stretchability and their direct interaction with actin filaments)

Período documentado: 2024-05-01 hasta 2026-04-30

Resumen del contexto y de los objetivos generales del proyecto

Living cells constantly experience mechanical forces during movement, division, and tissue remodeling. Their ability to withstand and respond to these forces depends on the cytoskeleton, a dynamic network of protein filaments that provides structural support and coordinates cellular functions. The cytoskeleton consists of three major filament types: actin filaments, microtubules, and intermediate filaments (IFs). The organization of and interactions between these filaments are essential for processes such as force transmission, intracellular transport, cell migration, and cell division. While actin filaments and microtubules have been studied extensively, IFs, particularly vimentin IFs, remain less well understood despite their importance in cellular resilience and their involvement in processes such as wound healing and cancer progression.

Two key questions motivated this project. First, although actin and vimentin networks frequently cooperate in cells, it remained unclear whether their coupling arises from direct filament–filament interactions or is mediated by protein crosslinkers. Previous studies of reconstituted actin–vimentin networks yielded inconclusive results, leaving the physical basis of actin–vimentin crosstalk unresolved. Second, vimentin IFs are among the most extensible protein filaments known in biology. Their remarkable stretchability has been attributed to structural transitions involving the unfolding of alpha-helical domains into beta-sheet structures during stretching. However, direct structural evidence for these transitions at the single-filament level is still lacking.

To address these questions, the project combined reconstituted systems of purified protein filaments with advanced biophysical techniques, including optical tweezers, microfluidics, confocal fluorescence microscopy, atomic force microscopy (AFM), tip-enhanced Raman spectroscopy (TERS), and computer simulation. This reductionist approach enabled direct and quantitative investigation of filament interactions, mechanics, and structure under well-controlled conditions.

Trabajo realizado desde el comienzo del proyecto hasta el final del período abarcado por el informe y los principales resultados hasta la fecha

WP1 – Unifying buffer conditions.
We developed common buffer conditions and experimental workflows that enabled actin filaments and vimentin IFs to be studied simultaneously at the single-filament level. Using optical tweezers combined with confocal fluorescence microscopy and microfluidics, we established robust and reproducible protocols for filament preparation, handling, and force measurements, providing the experimental foundation for the subsequent interaction studies.

WP2 – Vimentin–actin interactions on a single filament level.
Using quadruple optical tweezers combined with confocal microscopy and microfluidics, we precisely controlled the interaction conditions, visualized actin–vimentin interactions in real time, and measured the forces involved. We provided direct evidence that actin and vimentin filaments interact directly and quantified the interaction forces between them. The measured interaction strengths ranged from a few to several tens of piconewtons and remained largely unaffected by changes in ionic conditions. The interaction strengths also depended on the mechanical loading geometry. We further quantified the interaction kinetics, including binding and unbinding rates, through a combination of experiments, theoretical modeling, and Bayesian inference.

WP3 – Unfolding of alpha helices within vimentin IFs upon filament stretching.
We developed AFM-based protocols for stretching single filaments and established workflows combining AFM with TERS for nanoscale structural characterization. We successfully stretched single filaments to approximately twice their original length, systematically evaluated different substrates, and identified conditions suitable for controlled stretching experiments. We also acquired reference TERS spectra of unstretched filaments. Although complete structural characterization of stretched filaments was not achieved within the project period, we generated important methodological advances, reference datasets, and optimized protocols for future studies.

The main findings on actin–vimentin interactions have been published as a bioRxiv preprint (https://doi.org/10.64898/2026.06.11.731581) and submitted to a peer-reviewed journal.

Avances que van más allá del estado de la técnica e impacto potencial esperado (incluida la repercusión socioeconómica y las implicaciones sociales más amplias del proyecto hasta la fecha)

Prior to this project, it remained unclear whether the coupling between actin and vimentin originated from direct filament–filament interactions or required additional crosslinking proteins. We provide direct experimental evidence that single actin and vimentin filaments form force-bearing interactions in the absence of crosslinking proteins. We further provide a quantitative characterization of these interactions at the single-filament level, including interaction strengths, binding rates, and unbinding kinetics under various experimental conditions. We also demonstrated that the measured interaction strengths depend on the mechanical loading geometry, revealing the importance of filament mechanics in determining interaction behavior.

The project also advances the state of the art by combining single-filaments force and binding-rate measurements with theoretical modeling and Bayesian inference to extract unbinding kinetics. These approaches provide new tools for linking molecular-scale interactions to the mechanics of cytoskeletal networks.

In addition, we established experimental workflows combining AFM-based stretching of single vimentin filaments with TERS to investigate force-induced structural changes. Although the structural characterization objective was only partly achieved, the developed methodologies, reference datasets, and identification of critical technical bottlenecks provide a foundation for future studies of force-induced structural changes in IFs.

The project generates knowledge, datasets, and methodologies that provide a foundation for future studies linking molecular-scale filament interactions with network- and cellular-scale mechanics. In the longer term, these advances may support research on diseases associated with altered cell mechanics, including cancer, and the development of bio-inspired materials.