Continuous self-repair protects vimentin intermediate filaments from fragmentation

Abstract

AbstractIntermediate filaments are key regulators of cell mechanics. Vimentin, a type of intermediate filament expressed in mesenchymal cells and involved in migration, forms a dense network in the cytoplasm that is constantly remodeled through filament transport, elongation/shortening, and subunit exchange. While it is known that filament elongation involves end-to-end annealing, it is unclear how the reverse process of filament shortening by fragmentation occurs. Here, we use a combination ofin vitroreconstitution probed by fluorescence imaging and theoretical modeling to uncover the molecular mechanism involved in filament breakage. We first show that vimentin filaments are composed of two layers of subunits, half of which are exchangeable and half of which are immobile. We also show that the exchangeable subunits are tetramers. We further reveal a mechanism of continuous filament self-repair in which a soluble pool of vimentin tetramers in equilibrium with the filaments is essential to maintain filament integrity. Filaments break as a consequence of local fluctuations in the number of subunits per cross-section induced by the constant subunit exchange of tetramers. We determine that a filament tends to break if about four tetramers are removed from the same filament cross-section. Finally, we analyze the dynamics of association/dissociation and fragmentation to estimate the binding energy of a tetramer to a complete versus a partially disassembled filament. Our results provide a comprehensive description of vimentin turnover and reveal the link between subunit exchange and fragmentation.SIGNIFICANCE STATEMENTIntermediate filaments, including vimentin, are a key component of the cytoskeleton, which is essential for cell mechanics. Inside the cell, vimentin forms a dense network that is constantly remodeled to fulfill its functions. In particular, the filaments elongate and fragment, but the molecular mechanism involved in this breakage was unknown. Here we show that fragmentation is a consequence of the constant exchange of subunits along the filament length, which could locally weaken the filament. Our results provide a physical understanding of the mechanisms involved in regulating filament length, a feature that is essential for determining the dynamic organization of the network in both healthy and diseased cells in which intermediate filament assembly is disrupted.