Disordered Motifs and Domains in Cell Control - October 11-15, 2014

Disordered Motifs and Domains in Cell Control

Poster Session I

15-POS Board 15 The Non-structured Amino-terminal Domain of Intermediate Filaments Powers Assembly Harald Herrmann 1 , Tatjana Wedig 1 , Norbert Mücke 1 , Ueli Aebi 2 . 1 German Cancer Research Center, Heidelberg, Germany, 2 University of Basel, Basel, Switzerland. Intermediate filament (IF) proteins are principal structures of the metazoan cytoskeleton. The IF multi-gene family represents fibrous proteins consisting of a central alpha-helical "rod" domain flanked by non-alpha-helical amino- (“head”) and carboxy-terminal (“tail) domains. IF-proteins form robust but highly flexible fibers and networks, both in the cytoplasm and the nucleus, that are not soluble under physiological conditions. Their role for the mechanical properties of cells is being elucidated by recent work quite convincingly. However, the assembly mechanism is still not completely understood, in particular the role of the non-structured “head” is by no means clear. At the molecular level, IF-proteins form coiled coils that laterally associate in an anti- parallel fashion to yield tetrameric complexes, which are stable under low ionic strength conditions. Hence, the assembly module for IFs is non-polar. The principal reaction for the elongation of IF proteins is a “head-to-tail” association of the “rods” with a 2 to 4 nm overlap. The “head” domain is essential for assembly, both laterally and longitudinally, as headless IF- proteins form only dimers under tetramer conditions, and tetramers under filament forming conditions. In order to investigate the contribution of individual parts of the “head” domain in these two assembly reactions, we have subjected the fibroblast-specific IF-protein vimentin to systematic truncation by recombinant techniqiues within the “head” domain. The assembly properties were followed by analytical ultracentrifugation, electron and atomic force microscopy in vitro and by transfection of the corresponding cDNA clones into vimentin-free fibroblasts generated from vimentin-(-/-) embryos. We demonstrate that the first 20 amino acids of the 85 amino acid-long "head" are dispensable for both tetramer and IF formation. Further truncation abolishes filament-forming capacity completely. Truncation of more than 40 amino acids impairs tetramer formation indicating the “head” is needed for orderly coiled-coil alignment.

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