A general mathematical model for the in vitro assembly dynamics of intermediate filament proteins.

Intermediate filament (IF) proteins assemble into highly flexible filaments that organize into complex cytoplasmic networks: keratins in all types of epithelia, vimentin in endothelia, and desmin in muscle. Since IF elongation proceeds via end-to-end annealing of unit-length filaments and successively of progressively growing filaments, it is important to know how their remarkable flexibility, i.e., their persistence length lp, influences the assembly kinetics. In fact, their lp ranges between 0.3 µm (keratin K8/K18) and 1.0 µm (vimentin and desmin), and thus is orders of magnitude lower than that of microtubules and F-actin. Here, we present a unique mathematical model, which implements the semiflexible nature of the three IF types based on published semiflexible polymers theories and depends on a single free parameter k0. Calibrating this model to filament mean length dynamics of the three proteins, we demonstrate that the persistence length is indeed essential to accurately describe their assembly kinetics. Furthermore, we reveal that the difference in flexibility alone does not explain the significantly faster assembly rate of keratin filaments compared with that of vimentin. Likewise, desmin assembles approximately six times faster than vimentin, even though both their filaments exhibit the same lp value. These data strongly indicate that differences in their individual amino acid sequences significantly impact the assembly rates. Nevertheless, using a single k0 value for each of these three key representatives of the IF protein family, our advanced model does accurately describe the length distribution and mean length dynamics and provides effective filament assembly rates. It thus provides a tool for future investigations on the impact of posttranslational modifications or amino acid changes of IF proteins on assembly kinetics. This is an important issue, as the discovery of mutations in IF genes causing severe human disease, particularly for desmin and keratins, is steadily increasing.

Dynamics of a fluorophore attached to superhelical DNA: FCS experiments simulated by Brownian dynamics.

We investigated the dynamics of a single-fluorophore-labeled pUC18 plasmid through a Brownian dynamics algorithm, followed by a simulation of the fluorescence correlation spectroscopy (FCS) process. Recent experimental FCS measurements indicated a sensitivity of the monomer mean square displacements in DNA circles towards superhelicity. Simulations with homogeneous DNA elasticity and local straight equilibrium are not sufficient to reproduce this observed behavior. But inserting permanently bent sequences into the DNA, which favor end loop formation, caused a dependence of the calculated FCS correlation curves on superhelical density. Furthermore, our simulations allow us to take into account the orientation of the fluorophore in polarized excitation, which might explain the observed appearance of a Rouse-like regime at intermediate time scales.