Cytosolic factors govern vimentin network architecture and mechanics

Vimentin intermediate filaments are key cytoskeletal components forming networks with architectures distinct from other intermediate filament types, enabling specialized functions. Although assembly of individual filaments from soluble subunits is well characterized, dissecting how vimentin networks are organized has been challenging, as existing in vitro systems do not mimic the structures observed in cells. Thus, how cells establish higher-order vimentin organization remains unclear. Here, we reconstitute cell-like vimentin networks in vitro, using purified vimentin and extracts from mammalian and non-mammalian cells. Systematic variation of parameters reveals that cytosolic biomolecules, rather than intrinsic filament properties or generic ionic components, are the primary determinants of network architecture and mechanical behaviour. Importantly, network architecture is not universal but varies strongly according to the cell type from which the extract is derived, indicating that vimentin assemblies are tailored in a cell type-specific manner. Thus, this extract-based reconstitution system enables mechanistic dissection of intermediate filament regulation under near-native biochemical conditions, bridging the current gap between purified systems and the cellular environment. Our findings show that vimentin architecture and mechanics emerge primarily from cytosolic biomolecular factors that organize filaments into cell type-specific networks. These results establish cytosolic regulation as a central mechanism specifying intermediate filament network architecture and function.