Characterization of distinct early assembly units of different intermediate filament proteins

Harald Herrmann, Markus Häner, Monika Brettel, Nam On Ku, Ueli Aebi

Research output: Contribution to journalArticle

190 Citations (Scopus)

Abstract

We have determined the mass-per-length (MPL) composition of distinct early assembly products of recombinant intermediate filament (IF) proteins from the four cytoplasmic sequence homology classes, and compared these values with those of the corresponding mature filaments. After two seconds under standard assembly conditions (i.e. 25 mM Tris-HCl (pH 7.5), 50 mM NaCl, 37°C), vimentin, desmin and the neurofilament triplet protein NF-L aggregated into similar types of 'unit-length filaments' (ULFs), whereas cytokeratins (CKs) 8/18 already yielded long IFs at this time point, so the ionic strength had to be reduced. The number of molecules per filament cross-section, as deduced from the MPL values, was lowest for CK8/18, i.e. 16 and 25 at two seconds compared to 16 and 21 at one hour. NF-L exhibited corresponding values of 26 and 30. Vimentin ULFs yielded a pronounced heterogeneity, with Major peak values of 32 and 45 at two seconds and 30, 37 and 44 after one hour. Desmin formed filaments of distinctly higher mass with 47 molecules per cross-section, at two seconds and after one hour of assembly. This indicates that individual types of IF proteins generate filaments with distinctly different numbers of molecules per cross-section. Also, the observed significant reduction of apparent filament diameter of ULFs compared to the corresponding mature IFs is the result of a 'conservative' radial compaction-type reorganization within the filament, as concluded from the fact that both the immature and mature filaments contain very similar numbers of subunits per cross-section. Moreover, the MPL composition of filaments is strikingly dependent on the assembly conditions employed. For example, vimentin fibers formed in 0.7 mM phosphate (pH 7.5), 2.5 mM MgCl2, yield a significantly increased number of molecules per cross-section (56 and 84) compared to assembly under standard conditions. Temperature also strongly influences assembly: above a certain threshold temperature 'pathological' ULFs form that are arrested in this state, indicating that the system is forced Into strong but unproductive interactions between subunits. Similar 'dead-end' structures were obtained with vimentins mutated to introduce principal alterations in subdomains presumed to be of general structural importance, indicating that these sequence changes led to new modes of intermolecular interactions.

Original languageEnglish
Pages (from-to)1403-1420
Number of pages18
JournalJournal of Molecular Biology
Volume286
Issue number5
DOIs
Publication statusPublished - 1999 Mar 12

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Intermediate Filament Proteins
Vimentin
Desmin
Keratin-8
Keratin-18
Neurofilament Proteins
Magnesium Chloride
Temperature
Sequence Homology
Recombinant Proteins
Osmolar Concentration
Phosphates

All Science Journal Classification (ASJC) codes

  • Structural Biology
  • Molecular Biology

Cite this

Herrmann, Harald ; Häner, Markus ; Brettel, Monika ; Ku, Nam On ; Aebi, Ueli. / Characterization of distinct early assembly units of different intermediate filament proteins. In: Journal of Molecular Biology. 1999 ; Vol. 286, No. 5. pp. 1403-1420.
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Characterization of distinct early assembly units of different intermediate filament proteins. / Herrmann, Harald; Häner, Markus; Brettel, Monika; Ku, Nam On; Aebi, Ueli.

In: Journal of Molecular Biology, Vol. 286, No. 5, 12.03.1999, p. 1403-1420.

Research output: Contribution to journalArticle

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AU - Herrmann, Harald

AU - Häner, Markus

AU - Brettel, Monika

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N2 - We have determined the mass-per-length (MPL) composition of distinct early assembly products of recombinant intermediate filament (IF) proteins from the four cytoplasmic sequence homology classes, and compared these values with those of the corresponding mature filaments. After two seconds under standard assembly conditions (i.e. 25 mM Tris-HCl (pH 7.5), 50 mM NaCl, 37°C), vimentin, desmin and the neurofilament triplet protein NF-L aggregated into similar types of 'unit-length filaments' (ULFs), whereas cytokeratins (CKs) 8/18 already yielded long IFs at this time point, so the ionic strength had to be reduced. The number of molecules per filament cross-section, as deduced from the MPL values, was lowest for CK8/18, i.e. 16 and 25 at two seconds compared to 16 and 21 at one hour. NF-L exhibited corresponding values of 26 and 30. Vimentin ULFs yielded a pronounced heterogeneity, with Major peak values of 32 and 45 at two seconds and 30, 37 and 44 after one hour. Desmin formed filaments of distinctly higher mass with 47 molecules per cross-section, at two seconds and after one hour of assembly. This indicates that individual types of IF proteins generate filaments with distinctly different numbers of molecules per cross-section. Also, the observed significant reduction of apparent filament diameter of ULFs compared to the corresponding mature IFs is the result of a 'conservative' radial compaction-type reorganization within the filament, as concluded from the fact that both the immature and mature filaments contain very similar numbers of subunits per cross-section. Moreover, the MPL composition of filaments is strikingly dependent on the assembly conditions employed. For example, vimentin fibers formed in 0.7 mM phosphate (pH 7.5), 2.5 mM MgCl2, yield a significantly increased number of molecules per cross-section (56 and 84) compared to assembly under standard conditions. Temperature also strongly influences assembly: above a certain threshold temperature 'pathological' ULFs form that are arrested in this state, indicating that the system is forced Into strong but unproductive interactions between subunits. Similar 'dead-end' structures were obtained with vimentins mutated to introduce principal alterations in subdomains presumed to be of general structural importance, indicating that these sequence changes led to new modes of intermolecular interactions.

AB - We have determined the mass-per-length (MPL) composition of distinct early assembly products of recombinant intermediate filament (IF) proteins from the four cytoplasmic sequence homology classes, and compared these values with those of the corresponding mature filaments. After two seconds under standard assembly conditions (i.e. 25 mM Tris-HCl (pH 7.5), 50 mM NaCl, 37°C), vimentin, desmin and the neurofilament triplet protein NF-L aggregated into similar types of 'unit-length filaments' (ULFs), whereas cytokeratins (CKs) 8/18 already yielded long IFs at this time point, so the ionic strength had to be reduced. The number of molecules per filament cross-section, as deduced from the MPL values, was lowest for CK8/18, i.e. 16 and 25 at two seconds compared to 16 and 21 at one hour. NF-L exhibited corresponding values of 26 and 30. Vimentin ULFs yielded a pronounced heterogeneity, with Major peak values of 32 and 45 at two seconds and 30, 37 and 44 after one hour. Desmin formed filaments of distinctly higher mass with 47 molecules per cross-section, at two seconds and after one hour of assembly. This indicates that individual types of IF proteins generate filaments with distinctly different numbers of molecules per cross-section. Also, the observed significant reduction of apparent filament diameter of ULFs compared to the corresponding mature IFs is the result of a 'conservative' radial compaction-type reorganization within the filament, as concluded from the fact that both the immature and mature filaments contain very similar numbers of subunits per cross-section. Moreover, the MPL composition of filaments is strikingly dependent on the assembly conditions employed. For example, vimentin fibers formed in 0.7 mM phosphate (pH 7.5), 2.5 mM MgCl2, yield a significantly increased number of molecules per cross-section (56 and 84) compared to assembly under standard conditions. Temperature also strongly influences assembly: above a certain threshold temperature 'pathological' ULFs form that are arrested in this state, indicating that the system is forced Into strong but unproductive interactions between subunits. Similar 'dead-end' structures were obtained with vimentins mutated to introduce principal alterations in subdomains presumed to be of general structural importance, indicating that these sequence changes led to new modes of intermolecular interactions.

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