Goldmann, W. H. | Search bioRxiv

bioRxiv2022

Dual-wavelength stopped-flow analysis of the lateral and longitudinal assembly kinetics of vimentin

Vimentin is a highly charged intermediate filament protein that inherently forms extended dimeric coiled-coils, which serve as the basic building blocks of intermediate filaments. Under low ionic strength conditions, vimentin filaments dissociate into uniform tetrameric complexes of two anti-parallel oriented, half-staggered coiled-coil dimers. By addition of salt, vimentin tetramers spontaneously reassemble into filaments in a time-dependent process: i) lateral assembly of tetramers into unit-length filaments (ULFs); ii) longitudinal annealing of ULFs; iii) longitudinal assembly of filaments coupled with subsequent radial compaction. To independently determine the lateral and longitudinal assembly kinetics, we measure with a stopped-flow instrument the static light scattering signal at two different wavelengths (405 and 594 nm) with a temporal resolution of 3 ms, and analyze the signals based on Rayleigh-Gans theory. This theory considers that the intensity of the scattered light depends not only on the molecular weight of the scattering object but also on its shape. This shape-dependence is more pronounced at shorter wavelengths, allowing us to decompose the scattered light signal into its components arising from lateral and longitudinal filament assembly. We demonstrate that both the lateral and longitudinal filament assembly kinetics increase with salt concentration. Significance statementThe proper formation of intermediate filament (IF) networks in the cytoplasm is important for numerous cell functions. Here, we present a stopped-flow method for measuring the in-vitro assembly kinetics of intermediate filaments with a temporal resolution of 3 ms using static light scattering at two different wavelengths. This allows us to compute the shape factor of the assembly products based on Rayleigh-Gans light scattering theory. From the shape factor, we can separately measure the lateral assembly of tetramers into unit-length filaments (ULFs), and the longitudinal annealing of ULFs and longer filaments. For the IF protein vimentin, we find that with increasing salt concentrations, both the lateral and longitudinal assembly rates increase, and unstable, hyper-aggregated assembly complexes emerge.

biophysics↗

bioRxiv2021

The desmin mutation R349P increases contractility and fragility of stem cell-generated muscle micro-tissues

Desminopathies comprise hereditary myopathies and cardiomyopathies caused by mutations in the intermediate filament protein desmin that lead to severe and often lethal degeneration of striated muscle tissue. Animal and single cell studies hinted that this degeneration process is associated with massive ultrastructural defects correlating with increased susceptibility of the muscle to acute mechanical stress. The underlying mechanism of mechanical susceptibility, and how muscle degeneration develops over time, however, has remained elusive. Here, we investigated the effect of a desmin mutation on the formation, differentiation, and contractile function of in vitro-engineered three-dimensional micro-tissues grown from muscle stem cells (satellite cells) isolated from heterozygous R349P desmin knock-in mice. Micro-tissues grown from desmin-mutated cells exhibited spontaneous unsynchronized contractions, higher contractile forces in response to electrical stimulation, and faster force recovery compared to tissues grown from wild-type cells. Within one week of culture, the majority of R349P desmin-mutated tissues disintegrated, whereas wild-type tissues remained intact over at least three weeks. Moreover, under tetanic stimulation lasting less than five seconds, desmin-mutated tissues partially or completely ruptured, whereas wild-type tissues did not display signs of damage. Our results demonstrate that the progressive degeneration of desmin-mutated micro-tissues is closely linked to extracellular matrix fiber breakage associated with increased contractile forces and unevenly distributed tensile stress. This suggests that the age-related degeneration of skeletal and cardiac muscle in patients suffering from desminopathies may be similarly exacerbated by mechanical damage from high-intensity muscle contractions. We conclude that micro-tissues may provide a valuable tool for studying the organization of myocytes and the pathogenic mechanisms of myopathies.

biophysics↗