Continuous self-repair protects vimentin intermediate filaments from fragmentation

Bioengineering Biophysics Cell Biology Biochemistry Life Sciences
intermediate filaments vimentin cytoskeleton subunit exchange self-repair fragmentation mechanism

Academic Background and Research Motivation

The cytoskeleton is the core structural support responsible for maintaining cell morphology and mechanical properties and is mainly composed of three major classes: actin filaments, microtubules, and intermediate filaments. Among these, intermediate filaments play an irreplaceable role in maintaining cell shape and withstanding stress. Although the important physiological functions of intermediate filaments have been widely recognized, and some structural features and dynamic processes have been extensively studied, there remain many unresolved mysteries regarding one of its representative members—vimentin intermediate filaments—especially concerning their assembly and disassembly mechanisms. Existing studies indicate that the assembly and disassembly mechanisms of actin filaments and microtubules are relatively well clarified, while those of intermediate filaments remain less explored in depth. Meanwhile, vimentin plays a crucial role in the development of various diseases, with its state changes closely related to many pathological processes, making elucidation and understanding of its dynamic mechanisms increasingly urgent and practically significant.

Furthermore, vimentin is widely expressed in mesenchymal-derived cells and is a classic molecular marker of epithelial-mesenchymal transition (EMT). It not only provides mechanical support but also influences various biological phenomena such as cell polarity, signal transduction, migration, and division. Within cells, vimentin forms a densely interwoven three-dimensional network, which is highly dynamic and frequently undergoes transport, elongation-shortening, and subunit exchange. Although previous research has clarified that the elongation of vimentin long chains is mainly achieved through end-to-end annealing, there has long been no clear description of the specific molecular mechanism by which it undergoes disassembly or fragmentation. In particular, the natural fragmentation mechanism in the absence of exogenous modifications (such as phosphorylation or other PTMs) remained unresolved. Moreover, it was unclear whether the protein subunits required for vimentin intermediate filament disassembly disassociate only at the two ends or are continuously exchanged along the entire filament, lacking direct quantitative physical evidence.

Therefore, revealing whether and how the subunit exchange, disassembly, and self-repair of vimentin intermediate filaments are tightly coupled has become a frontier scientific issue in the cytoskeleton field and forms an important academic foundation for advancing disease research and biomaterial development. This paper is carried out in this context, aiming to use multidisciplinary means to elucidate the molecular mechanism of vimentin filament breakage and to explore its subunit dynamic exchange and self-repair processes.

Source of the Article and Author Team

This research article, entitled “continuous self-repair protects vimentin intermediate filaments from fragmentation,” was published in June 2025 in the Proceedings of the National Academy of Sciences of the United States of America (PNAS). The author team includes experts from leading French universities and research institutions such as Brandeis University, Université Paris Cité, Université Paris-Saclay, among others, with key contributors including Quang D. Tran, Martin Lenz, Guillaume Lamour, and several other experts in cell mechanics and biophysics. Both core experiments and theoretical modeling were completed collaboratively at these institutions. The article is edited by Paul A. Janmey of the University of Pennsylvania, fully reflecting the high level of recognition for this research in the international academic community.

Research Design and Overall Workflow

1. Problem Definition and Hypothesis

Building upon previous findings that vimentin intermediate filaments can break in the absence of special modified proteins or enzymes, the authors noted that while axially overall exchange of filament subunits after assembly has been observed, the essential process, the rate of subunit loss, and the critical conditions leading to breakage remain to be clarified. Consequently, the authors hypothesize that the subunit exchange and loss occurring along the length of the vimentin filament cause local fluctuations in the number of subunits in the filament cross-section, thus triggering structural weakening and eventual breakage. The pool of soluble vimentin tetramers dynamically balanced with the filaments may be crucial for maintaining filament integrity.

2. Research Subjects and Experimental Grouping

This study mainly focuses on recombinant, fluorescently-labeled vimentin proteins assembled in vitro. The main experimental subjects include:

  • Pre-assembled vimentin filaments (some with fluorescent labels, some without)
  • Soluble tetrameric vimentin proteins (added or replaced at different proportions and concentrations)
  • Protein samples treated under different physicochemical conditions (different salt concentrations, assembly/disassembly environments, etc.)

3. Multi-faceted Experimental Techniques and Detailed Procedures

The authors employ several conventional and innovative experimental techniques, aiming to reveal the kinetic nature of vimentin filament subunit exchange, self-repair, and fragmentation from the single-molecule to ensemble level:

a) Fluorescence Imaging to Monitor Subunit Dynamic Exchange

The authors ingeniously designed mixing experiments by combining two types of vimentin filaments with different fluorescent labels (or unlabeled), and used confocal or TIRF microscopy to collect real-time and time-series images of filament length and fluorescence intensity distribution across bright and dim segments. With high-throughput image analysis algorithms and higher-order Gaussian fitting and mathematical models, they quantitatively analyzed subunit exchange along the entire filament and derived dynamic parameters.

b) SDS-PAGE Electrophoresis to Quantify the Soluble Pool

By ultracentrifugation and concentration, combined with SDS-PAGE gel quantitative analysis, the soluble vimentin concentration remaining after filament assembly was accurately measured, and experiments were conducted to test whether these tetramers can form filaments by themselves at the existing concentrations.

c) Dilution and Tetramer Supplementation Experiments

Systematic high-ratio dilution (such as 1:200, 1:500) was performed on assembled filaments, and subsequent changes in filament fluorescence intensity and length were monitored to evaluate subunit loss (thinning) and fragmentation. Different concentrations of vimentin tetramers were then supplemented to test the key role of the soluble subunit pool in self-repair and in preventing fragmentation.

d) Atomic Force Microscopy (AFM) to Measure Filament Diameter Change

AFM was used to precisely measure the cross-sectional height of filaments before and after dilution, to verify whether the thinning/loss of subunits reflected by fluorescence signals corresponds to real structural thinning.

e) Single-molecule Photobleaching to Determine Oligomeric State of Exchanged Subunits

Using a single-molecule sensitive imaging system and high-power laser photobleaching, the bleaching steps of subunit particles dissociated and bound to the substrate were analyzed. Coupled with controls of different labeling ratios, it was quantitatively determined whether these subunits exist predominantly as tetramers.

f) Theoretical Modeling and Kinetic Simulation

Based on experimental data, the authors constructed two levels of kinetic (first-passage-time theory, etc.) and statistical physics models to elucidate the probabilistic basis for subunit disassembly and filament breakage, and further deduced key energy barriers and fragmentation conditions.

Main Experimental Results and Data Logic

1. Dynamic Subunit Exchange and Distribution along Filaments

Fluorescence imaging revealed that during a dynamic process lasting up to 24 hours after mixing, labeled and unlabeled subunits are continuously exchanged along the entire length of the filament. Through Gaussian peak analysis, it was found that after mixing, the fluorescence distribution of the filaments exhibited a bimodal rather than uniform feature, suggesting that not all subunits can freely exchange. Quantitative analysis showed that about 50% of the subunits are dynamically exchangeable, while the remaining half constitute a structurally “immobile” fraction. This finding is highly consistent with filament oligomeric models and supports the theoretical prediction of vimentin filaments containing heterogeneous inner core and outer layer subunits.

By fitting experimental curves with a two-state kinetic model, the dissociation rate constant for tetrameric subunits was found to be k_off = 0.2 ± 0.1 h^-1, outlining the physical timescale of subunit disassembly.

2. Quantification and Function of the Soluble Vimentin Tetramer Pool

Through SDS-PAGE and gel comparison, the authors found that after aggregation at 0.2 mg/ml, about 2% of vimentin remained soluble, corroborating the persistent existence of a soluble tetrameric pool in the experimental system. Furthermore, single-molecule imaging verified that at such low concentrations (5 × 10^-3 mg/ml), tetramers cannot spontaneously form new filaments or precursors, confirming the delicate balance between total subunit concentration in the system and structural integrity maintenance.

3. Dilution Experiments Reveal: Subunit Loss Leads to Filament Thinning and Fragmentation

In high-dilution experiments (1:200, 1:500), filaments showed rapid declines in average fluorescence intensity and length, indicating synchronous subunit loss and filament fragmentation. AFM further confirmed that after 6 hours of dilution, the mean filament diameter decreased by 15%, corresponding to a 28% reduction in cross-sectional subunit area, closely matching changes in fluorescence signals. Upon tetramer supplementation, filament intensity and length could be restored, demonstrating that the filament structure possesses reversible self-repair capability. Only when the supplemented tetramer concentration reached ~2% of the system’s natural soluble pool was complete prevention of thinning and fragmentation achieved.

4. Single Molecule Experiments Directly Observe Subunit Disassociation and Self-Repair

With TIRF microscopy, the dissociation process of single attached and free filaments in flow cells was tracked in situ. The results showed that subunit dissociation depends on the solution volume of the system and stabilizes at a certain equilibrium. Supplementation with tetramers at certain concentrations significantly reduced subunit loss and structural decay. Single-molecule photobleaching directly proved that dissociated low-labeling subunits mainly exhibited one or two photobleaching steps, perfectly matching the tetramer model rather than higher-order oligomers.

5. Kinetic Model Fitting Reveals Physical Fragmentation Mechanism

Statistical kinetic modeling indicated that the loss of a single subunit does not result in structural breakage; instead, breakage occurs only when four movable subunits (about half) are lost in the same cross-section. Simulation results closely matched the average fragmentation time observed in previous experiments and the minimum threshold for fluorescence intensity (~75%) in the current study. The authors further deduced that as the inter-subunit binding energy drops by 4 k_BT, further dissociation is accelerated until the breakage threshold is reached.

Major Conclusions and Significance

This study systematically reveals for the first time that vimentin intermediate filaments maintain resistance to natural breakage through continuous subunit exchange and self-repair. The research not only quantifies the dynamic dissociation rates and self-assembly/disassembly kinetic parameters of subunits but also discovers that functionally and structurally distinct subunit populations exist within filaments. This finding reshapes our understanding of the structural and property transitions of intermediate filaments and provides a new molecular mechanism for how cells maintain mechanical integrity and sense/respond to microenvironmental stress fluctuations over long timescales.

Specific significance includes:

  1. Scientific Value: This work provides direct experimental validation and theoretical support for the dynamic mechanism and self-repair function of intermediate filaments, laying the theoretical foundation for a deeper understanding of the mechanical steady state of cytoskeletal networks.
  2. Applied Value: It offers innovative scientific ideas for drug development, disease mechanism research (such as genetic disorders associated with vimentin mutations, cancer cell EMT, etc.), and the design of novel biomaterials.
  3. Potential Bioengineering Beacon: The dynamic self-repair mechanism inspires the design of biomimetic polymers and self-healing materials, especially suitable for load-bearing systems under multi-stress environments.

Research Highlights and Innovations

  • First to Reveal Two Types of Subunit Heterogeneity: Clearly distinguishes exchangeable and immobile subunits, a feature previously unresolvable by structural biology.
  • Dynamically Coupled Mechanism of Self-Repair and Fragmentation: Demonstrates that self-repair relies solely on the presence of a subunit pool, and its level can directly regulate breakage probability.
  • Comprehensively Interdisciplinary Workflow: Integrates single-molecule and ensemble experiments, biophysical simulations, and theoretical modeling to realize a full-chain explanation from microscopic molecules to macroscopic structure.
  • Kinetic Model Provides Accurate Predictions: The theoretically derived breakage conditions and repair responses closely match real-world data and experimental observations.

Additional Valuable Information

The study further discusses physiological and experimental factors regulating self-repair and fragmentation—including phosphorylation modifications (which can enhance subunit dissociation), diversity of subunit populations brought about by different assembly methods, and the effect of filament surface anchoring (such as antibody binding) on structural dynamics. These findings provide several actionable new directions for further research on vimentin and other intermediate filaments. At the same time, the research team’s data, experimental materials, and analysis codes are all openly accessible on Zenodo, in line with current open science and reproducibility standards.

Summary

Through original experimentation and theoretical innovation, this paper systematically elucidates the molecular mechanism by which vimentin intermediate filaments self-repair to resist breakage and introduces new concepts of subunit heterogeneity and dynamic equilibrium, bringing significant new insights to both basic and translational research fields related to the cytoskeleton.