Alternative assembly mechanisms of desmin disease mutants: filaments in competition with super-aggregation structures

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Intermediate filaments (IFs) represent one of three filament systems that establish the functional architecture of animal cells. In muscle, the protein desmin builds a stress-bearing IF-system that encases individual myofibers and that connects to their central structural anchorage elements such as Z bands and costameres at the plasma membrane. Mutations in the human desmin gene usually translate into single amino acid changes, and they cause severe myopathies including cardiomyopathies. At the cellular level, the disease generally manifests with massive desmin aggregates and damage of the myofibrillar apparatus of myocytes. Using a panel of biochemical and biophysical techniques in an in vitro assembly approach, we have previously shown that desmin mutations can be classified into four groups with respect to the phase of assembly and network formation, where the mutation interrupts the ordered assembly process. The first three phases describe (i) the lateral association of the basic rod-like tetrameric protein complexes into unit-length filaments (ULFs), (ii) successive longitudinal annealing of ULFs to filaments, and (iii) a radial reorganization (“compaction”) occurring as elongation of IF proceeds; (iv) in the fourth phase, filaments organize into functional networks. Whereas the first phase is complete within less than one second, as determined by stopped-flow experiments, the second phase of elongation carries on for minutes and is taken over by the third phase of continuous elongation of IF with compaction occurring in a less defined time frame. Here, we propose to study four selected desmin mutants, which decay from the normal assembly pathway in one of the four phases each, in order to get insight into the molecular process leading to the formation of essentially “noxious” structures. In a dedicated in vitro assembly work schedule, we will conduct state-of-the-art biophysical investigations of recombinant proteins, including electron microscopy, stopped-flow experiments, microfluidics, x-ray scattering and fluorescence correlation spectroscopy. In a complementing approach, we will investigate cells carrying the respective mutant proteins, both in stably transfected cell lines and in cell lines established from a R350P knock-in mouse with respect to the question how newly found IF-reactive small molecules and drugs will impact desmin aggregates over time. In addition, we will follow the cellular reorganization of major cytoskeletal factors by immunofluorescence microscopy. Moreover, we will investigate the consequences of aggregate formation for the structural organization of cells by scanning small-angle X-ray scattering. With this combined approach, we expect to get deep insight into an important filament system of mammalian muscle concerning the sequence of steps in both the regular assembly pathway and the deviation from it into aggregate formation.