Molecular Parameters of Type IV α-Internexin and Type IV-Type III α-Internexin-Vimentin Copolymer Intermediate Filaments

Abstract

During neuronal development, a dynamic replacement mechanism occurs in which the type VI nestin and type III vimentin intermediate filament proteins are replaced by a series of type IV proteins beginning with ␣-internexin. We have explored molecular details of how the type III to type IV replacement process may occur. First, we have demonstrated by cross-linking experiments that bacterially expressed forms of ␣-internexin and vimentin form heterodimer molecules in vitro that assemble into copolymer intermediate filaments. We show using a urea disassembly assay that ␣-internexin molecules are likely to be more stable than those of vimentin. Second, by analyses of the induced cross-links, we have determined the axial lengths of ␣-internexin homodimer and ␣-internexin-vimentin heterodimer molecules and their modes of alignments in filaments. We report that these dimensions are the same as those reported earlier for vimentin homopolymer molecules and, by implication, are also the same for the other neuronal type IV proteins. These data suggest that during neuronal development, ␣-internexin molecules are readily assimilated onto the pre-existing vimentin cytoskeletal intermediate filament network because the axial lengths and axial alignments of their molecules are the same. Furthermore, the dynamic replacement process may be driven by a positive equilibrium due to the increased stability of the ␣-internexin network.

Alexander-disease mutation ofGFAPcauses filament disorganization and decreased solubility of GFAP

“…Indeed, the L12 and 2A rod-domain regions flanking this residue are well conserved between GFAP, vimentin, desmin and peripherin (Quinlan et al, 1995). In vimentin, they are involved in intra-and intermolecular cross-links (Steinert et al, 1993;Steinert et al, 1999). They also play a role in determining subfilament architecture (Parry et al, 2001).…”

α-Internexin Is Structurally and Functionally Associated with the Neurofilament Triplet Proteins in the Mature CNS

“…Subsequently, the structural properties of ␣-internexin and the ability to assemble into filaments by itself or together with either neurofilament proteins or vimentin established ␣-internexin as an IF protein (Kaplan et al, 1990;Ching and Liem, 1993;Steinert et al, 1999). Although its specificity for neurons and abundance in axons led early investigators to suspect that ␣-internexin, like peripherin, may be a subunit of neurofilaments (Portier et al, 1983;Chiu et al, 1989;Kaplan et al, 1990), little evidence has been brought to bear on this possibility (Al-Chalabi and Miller, 2003;Lariviere and Julien, 2004;Liu et al, 2004).…”

Residues in the 1A Rod Domain Segment and the Linker L2 Are Required for Stabilizing the A11 Molecular Alignment Mode in Keratin Intermediate Filaments

“…The proteins were cross-linked with 25 mM disulfosuccinimidyl tartrate (DST) for 1 h at 23°C, and terminated with 0.1 M NH 4 HCO 3 (final concentration) (16). Although significant random cross-linking also occurs, these conditions were used because the near quantitative modification of all lysines allows for less diffuse bands on 3.75-7.5% gradient PAGE gels.…”