Diversity of intermediate filament structure. Evidence that the alignment of coiled-coil molecules in vimentin is different from that in keratin intermediate filaments.

P M Steinert,L N Marekov,D A Parry

doi:10.1016/s0021-9258(19)74552-9

Abstract

Although vimentin intermediate filaments (IF) are morphologically similar to all other IF types, cells have evolved different ways of manipulating vimentin and keratin IF. The structural basis for such differences is unknown. We have explored this by use of cross-linking experiments on vimentin oligomers, polymers, and intact IF to determine the axial length of vimentin molecules and the degrees to which neighboring molecules are aligned in IF. Our data reveal that the homodimer vimentin molecule (43.9 nm) is clearly shorter than a keratin heterodimer molecule (46.2 nm). Vimentin assemblies contain three modes of antiparallel molecular alignments: A11 and A22 in two-molecule or larger oligomeric assemblies, in which the two molecules are staggered so as to bring their 1B and 2B rod domain segments, respectively, into register; and A12 in higher order molecular assemblies in which the two neighboring molecules are largely overlapped. Since the repeat axial length of the vimentin assemblies (42.6 nm) is less than the molecular length, this means there is an overlap (designated as alignment ACN) of about 1 nm (5-10 residues) between the end of the 2B and beginning of the 1A rod domain segments of similarly directed molecules in the IF. Interestingly, these four modes of nearest neighbor molecular alignments also occur in keratin IF. However, the degree of stagger of alignments in the A11 and A22 modes is different (staggers of -19.5 for vimentin versus -16.6 nm for keratin, and 23.3 and 28.6 nm, respectively). Two-dimensional surface lattice maps of the two IF types are very similar, except for differences in molecule alignments and different axial repeats of 21.4 nm in vimentin and 22.6 nm in keratin IF. Although vimentin-keratin hybrid molecules can be induced to form in vitro, they do not assemble into higher order structures. The data suggest that vimentin and keratin are incapable of assembly into IF in vitro or in vivo simply because their molecules are of different axial lengths and because the exact axial alignments of neighboring molecules are different.

Highlights

From the SkinBiology Branch, National Institute ofdrthritis,Musculoskeletal, and Skin Diseases, National Institutesof Health, Bethesda, Maryland20892 and the §Departmentof Physics and Biophysics, Massey University, Palmerston North, NewZealand
The data suggest that vimentin and keratin are incapable of assembly into intermediate filaments (IF) in vitro or in vivo because of the five types of vertebrate cytoplasmic IF, each segment is of identical size and likely secondary structure [4, 5]
Cross-linking of Vimentin Oligomers, Polymers, and IF with DST-Cross-linking of vimentin protein (0.5 mg/ml) in the a low ionic strength buffer of 10 mM triethanolamine-HC1 with a range of concentrations of the bifunctional cleavable reagent DSTshows that thereaction appears tobe near complete in 30 min at 1-2 mM (Fig. 1).the appearance of a series of odd-numbered products indicates the occurrence of an unacceptable degree of cross-linking of random collisioncomplexesabove about 0.6 mM

Summary

RESULTS

Cross-linking of Vimentin Oligomers, Polymers, and IF with DST-Cross-linking of vimentin protein (0.5 mg/ml) in the a low ionic strength buffer of 10 mM triethanolamine-HC1 (pH 8.0) with a range of concentrations of the bifunctional cleavable reagent DSTshows that thereaction appears tobe near complete in 30 min at 1-2 mM (Fig. 1).the appearance of a series of odd-numbered products indicates the occurrence of an unacceptable degree of cross-linking of random collisioncomplexesabove about 0.6 mM. Expected,since the fundamental coiled-coil molecule of vimentin IF is a homodimer [12] In this way, 38 cross-linked peptides were positively identified and consisted of 16 unique species (Table I). The two-molecule oligomer obtained by cross-linking with 1mM reagent was not assembly-competent (Fig. 2F), indicating that the high degree of chemicalmodification interferedwithnormal assembly in tions, suggesting that vimentin anddesmin IF areassembled from similar molecular alignments. Following equilibration intobuffer Calculation of Rod Axial Alignments and Linker Segments containing 150 mM KC1, short IF of 10-nm diameter were in' Vimentin ZF-Using the 11 unique intermolecular crossassembled with the former, but no IF structurews ere formed links, separate equationws ere derived which relate directly to in the latter (Fig. 2, G and If) Together, these data provide the positions of the lysines in adjacent molecules (Table 11). The coiled-coil rod domain segments of the two chain types may have become associated in an in-register alignment of parallel chains to

18 EL vz Vimentin Intermediate Filament Structure

L12-15

DISCUSSION