Desmin is an intermediate filament protein that builds a cytoskeletal network in muscle cells to maintain structural integrity and mechanical coupling of myofibrils. The authors compared the assembly properties of wild-type desmin with four disease-associated missense variants (N342D, L345P, R350P, and R406W) to define how pathogenic substitutions drive intracellular aggregation.
Under low ionic strength conditions, all proteins examined formed uniform tetramers, indicating that early oligomeric state formation is preserved across the variants. Increasing ionic strength initiated the canonical assembly pathway: tetramers associate laterally into unit-length filaments (ULFs), which then elongate longitudinally and undergo radial compaction to form mature filaments.
The transition from tetramers to ULFs marks the first assembly step sensitive to mutation. Using dual-wavelength stopped-flow spectroscopy, the investigators observed rapid lateral association of tetramers into ULFs in wild-type desmin. Among the variants, R406W produced ULFs with kinetics resembling wild-type, whereas N342D, L345P, and R350P displayed delayed lateral assembly. This indicates that while some pathogenic substitutions spare initial ULF formation, others retard this early step and thereby alter the downstream maturation trajectory.
After short filaments assembled from ULFs, the mutants diverged from the wild-type maturation pathway at an early filament maturation stage. Quantitative analysis and kinetic modelling defined distinct, mutation-specific outcomes:
Wild-type filaments progressed through continuous longitudinal elongation and radial compaction to yield stable, mature filaments.
R406W filaments initially formed ULFs at near-normal rates but then failed to elongate productively. Instead, these filaments progressively associated into fibrillar clusters, at which point elongation effectively ceased.
N342D, L345P, and R350P followed a different route: shortly after short filament formation they rapidly collapsed into globular complexes. These globular species subsequently coalesced into larger aggregates rather than undergoing the radial compaction and stabilization typical of productive filament maturation.
These distinct pathways highlight that different desmin mutations redirect the internal reorganization of nascent filaments away from stabilization and toward aggregation by separate structural mechanisms.
Atomic force microscopy (AFM) provided morphological evidence for the mutation-specific assembly outcomes observed in the spectroscopic data. Quantitative AFM image analysis showed continuous elongation and compact morphology for wild-type filaments. In contrast, R406W samples displayed clustered, fibrillar accumulations, while N342D, L345P, and R350P samples contained collapsed globular assemblies that merged into larger aggregates.
Kinetic modelling of the stopped-flow spectroscopy data supported these morphological observations and allowed the authors to time the divergence from productive assembly: the critical juncture occurs after initial elongation of ULF-derived filaments but before completion of radial compaction and stabilization.
Molecular dynamics (MD) simulations were used to probe structural consequences of the disease-associated substitutions. The simulations indicate that the mutations differentially destabilize coil 2 of desmin, producing local structural perturbations. These local instabilities correlate with the assembly defects seen experimentally and provide a plausible mechanistic link between point mutations and the distinct aggregation pathways observed for different variants.
Together, the experimental and computational data identify an early filament maturation stage—when elongating ULF-derived filaments normally undergo radial compaction to form stable, mature filaments—as a critical checkpoint vulnerable to pathogenic desmin substitutions. At this stage, mutations redirect reorganization from productive stabilization toward structural collapse and aggregation, but they do so by mutation-specific mechanisms: progressive fibrillar clustering for R406W versus rapid collapse into globular aggregates for N342D, L345P, and R350P.
These findings refine the temporal and structural understanding of how desmin mutations produce intracellular aggregates in dominant desmin myopathies. The work emphasizes that not all pathogenic substitutions act identically: some disturb lateral association kinetics, and others perturb internal filament reorganization during early maturation. This preprint has not been certified by peer review; the authors declared no competing interests and funding sources were reported in the original article.