Desmin Mutations Disrupt Filament Elongation and Drive Polymorphic Aggregation

In muscle cells, desmin intermediate filaments form a cytoskeletal network that maintains the structural integrity and mechanical coupling of myofibrils. Dominant missense mutations in desmin cause myopathies characterized by intracellular protein aggregation. To explore the process of aggregate formation, we investigate the assembly kinetics of wild-type desmin and four disease-associated variants (N342D, L345P, R350P, and R406W) using dual-wavelength stopped-flow spectroscopy, complemented by atomic force microscopy and molecular dynamics simulations. At low ionic strength, all proteins form uniform tetramers. Increasing the ionic strength initiates assembly byrapid lateral association of tetramers into unit-length filaments (ULFs), followed by longitudinal elongation and radial filament compaction. R406W forms ULFs with wild-type-like kinetics, whereas N342D, L345P, and R350P exhibit delayed lateral assembly. After short filaments have formed, all four mutants diverge from productive filament maturation, but through distinct pathways. Quantitative analysis of atomic force microscopy images together with kinetic modelling of the spectroscopic data shows that wild-type filaments elongate continuously, whereas R406W filaments progressively associate into fibrillar clusters and cease elongating. By contrast, N342D, L345P, and R350P rapidly collapse into globular complexes that subsequently coalesce into larger aggregates. Molecular dynamics simulations indicate that the mutations differentially destabilize coil 2, which leads to local structural perturbations and mutation-specific assembly defects. Together, these findings identify early filament maturation - when elongating ULF-derived filaments would normally undergo radial compaction to form stable, mature filaments - as a critical time point in desmin assembly. At this stage, pathogenic mutations redirect the internal reorganization of the filament from productive stabilization toward mutation-specific structural collapse and aggregation. SignificanceDominant mutations in the intermediate filament protein desmin cause myofibrillar myopathies characterized by protein aggregation and progressive muscle degeneration. Using in vitro filament-assembly experiments, we identify intrinsic assembly defects of disease-associated desmin mutants. Mutant proteins initially enter the normal assembly pathway, albeit with delayed kinetics. They subsequently fail at an early maturation step in which short filaments would normally reorganize to sustain productive elongation. Instead, mutant proteins form morphologically distinct aggregates. Similar structural abnormalities occur when mutant proteins co-assemble with wild-type desmin. Our findings identify the radial compaction phase during early longitudinal assembly as a critical checkpoint that determines whether desmin assembly yields functional intermediate filaments or is redirected into pathogenic aggregation pathways.