Deletion Mutagenesis of the Amino-Terminal Head Domain of Vimentin Reveals Dispensability of Large Internal Regions for Intermediate Filament Assembly and Stability

2002 ◽  
Vol 279 (2) ◽  
pp. 344-353 ◽  
Author(s):  
Robert L. Shoeman ◽  
Roland Hartig ◽  
Monika Berthel ◽  
Peter Traub
Keyword(s):  
Intermediate Filament ◽  
Amino Terminal ◽  
Deletion Mutagenesis ◽  
Filament Assembly ◽  
Head Domain

Structural Elements the Amino-Terminal Head Domain of Vimentin Essential for Intermediate Filament Formation in Vivo and in Vitro

1994 ◽  
Vol 213 (1) ◽  
pp. 128-142 ◽  
Author(s):  
Michael Beuttenmüller ◽  
Ming Chen ◽  
Alfred Janetzko ◽  
Siegfried Kühn ◽  
Peter Traub
Keyword(s):  
Intermediate Filament ◽  
Structural Elements ◽  
Filament Formation ◽  
Amino Terminal ◽  
Head Domain

Analysis of the roles of the head domains of type IV rat neuronal intermediate filament proteins in filament assembly using domain-swapped chimeric proteins

1999 ◽  
Vol 112 (13) ◽  
pp. 2233-2240
Author(s):  
G.Y. Ching ◽  
R.K. Liem
Keyword(s):  
Intermediate Filament ◽  
Self Assembly ◽  
Chimeric Proteins ◽  
Filament Formation ◽  
Intermediate Filament Proteins ◽  
L Protein ◽  
Type Iv ◽  
Filament Assembly ◽  
Head Domain ◽  
Neuronal Intermediate Filament Proteins

Type IV neuronal intermediate filament proteins consist of alpha-internexin, which can self-assemble into filaments and the neurofilament triplet proteins, which are obligate heteropolymers, at least in rodents. These IF proteins therefore provide good systems for elucidating the mechanism of intermediate filament assembly. To analyze the roles of the head domains of these proteins in contributing to their differential assembly properties, we generated chimeric proteins by swapping the head domains between rat alpha-internexin and either rat NF-L or NF-M and examined their assembly properties in transfected cells that lack their own cytoplasmic intermediate filament network. Lalphaalpha and Malphaalpha, the chimeric proteins generated by replacing the head domain of alpha-internexin with those of NF-L and NF-M, respectively, were unable to self-assemble into filaments. In contrast, alphaLL, a chimeric NF-L protein generated by replacing the head domain of NF-L with that of alpha-internexin, was able to self-assemble into filaments, whereas MLL, a chimeric NF-L protein containing the NF-M head domain, was unable to do so. These results demonstrate that the alpha-internexin head domain is essential for alpha-internexin's ability to self-assemble. While coassembly of Lalphaalpha with NF-M and coassembly of Malphaalpha with NF-L resulted in formation of filaments, coassembly of Lalphaalpha with NF-L and coassembly of Malphaalpha with NF-M yielded punctate patterns. These coassembly results show that heteropolymeric filament formation requires that one partner has the NF-L head domain and the other partner has the NF-M head domain. Thus, the head domains of rat NF-L and NF-M play important roles in determining the obligate heteropolymeric nature of filament formation. The data obtained from these self-assembly and coassembly studies provide some new insights into the mechanism of intermediate filament assembly.

scholarly journals Deletions in epidermal keratins leading to alterations in filament organization in vivo and in intermediate filament assembly in vitro.

1990 ◽  
Vol 111 (6) ◽  
pp. 3049-3064 ◽  
Author(s):  
P A Coulombe ◽  
Y M Chan ◽  
K Albers ◽  
E Fuchs
Keyword(s):  
Intermediate Filament ◽  
Intermediate Filament Proteins ◽  
Amino Terminal ◽  
Filament Assembly ◽  
Keratin Filaments ◽  
Keratin Filament ◽  
Filament Networks ◽  
10 Nm Filaments

To investigate the sequences important for assembly of keratins into 10-nm filaments, we used a combined approach of (a) transfection of mutant keratin cDNAs into epithelial cells in vivo, and (b) in vitro assembly of mutant and wild-type keratins. Keratin K14 mutants missing the nonhelical carboxy- and amino-terminal domains not only integrated without perturbation into endogenous keratin filament networks in vivo, but they also formed 10-nm filaments with K5 in vitro. Surprisingly, keratin mutants missing the highly conserved L L E G E sequence, common to all intermediate filament proteins and found at the carboxy end of the alpha-helical rod domain, also assembled into filaments with only a somewhat reduced efficiency. Even a carboxy K14 mutant missing approximately 10% of the rod assembled into filaments, although in this case filaments aggregated significantly. Despite the ability of these mutants to form filaments in vitro, they often perturbed keratin filament organization in vivo. In contrast, small truncations in the amino-terminal end of the rod domain more severely disrupted the filament assembly process in vitro as well as in vivo, and in particular restricted elongation. For both carboxy and amino rod deletions, the more extensive the deletion, the more severe the phenotype. Surprisingly, while elongation could be almost quantitatively blocked with large mutations, tetramer formation and higher ordered lateral interactions still occurred. Collectively, our in vitro data (a) provide a molecular basis for the dominance of our mutants in vivo, (b) offer new insights as to why different mutants may generate different phenotypes in vivo, and (c) delineate the limit sequences necessary for K14 to both incorporate properly into a preexisting keratin filament network in vivo and assemble efficiently into 10-nm keratin filaments in vitro.

Role of the Aromatic Residues in the Near-amino Terminal Motif of Vimentin in Intermediate Filament Assembly In Vitro

2008 ◽  
Vol 144 (5) ◽  
pp. 675-684 ◽  
Author(s):  
R. Gohara ◽  
S. Nishikawa ◽  
Y. Takasaki ◽  
S. Ando
Keyword(s):  
Intermediate Filament ◽  
Aromatic Residues ◽  
Amino Terminal ◽  
Filament Assembly

scholarly journals Roles of head and tail domains in alpha-internexin's self-assembly and coassembly with the neurofilament triplet proteins

1998 ◽  
Vol 111 (3) ◽  
pp. 321-333 ◽  
Author(s):  
G.Y. Ching ◽  
R.K. Liem
Keyword(s):  
Intermediate Filament ◽  
Self Assembly ◽  
Deletion Mutant ◽  
Deletion Mutants ◽  
Intermediate Filament Protein ◽  
Intermediate Filament Proteins ◽  
Amino Terminal ◽  
Head Domain ◽  
Normal Filament ◽  
Filament Network

The roles of the head and tail domains of alpha-internexin, a type IV neuronal intermediate filament protein, in its self-assembly and coassemblies with neurofilament triplet proteins, were examined by transient transfections with deletion mutants in a non-neuronal cell line lacking an endogenous cytoplasmic intermediate filament network. The results from the self-assembly studies showed that the head domain was essential for alpha-internexin's ability to self-assemble into a filament network and the tail domain was important for establishing a proper filament network. The data from the coassembly studies demonstrated that alpha-internexin interacted differentially with the neurofilament triplet protein subunits. Wild-type NF-L or NF-M, but not NF-H, was able to complement and form a normal filament network with the tailless alpha-internexin mutant, the alpha-internexin head-deletion mutant, or the alpha-internexin mutant missing the entire tail and some amino-terminal portion of the head domain. In contrast, neither the tailless NF-L mutant nor the NF-L head-deletion mutant was able to form a normal filament network with any of these alpha-internexin deletion mutants. However, coassembly of the tailless NF-M mutant with the alpha-internexin head-deletion mutant and coassembly of the NF-M head-deletion mutant with the tailless alpha-internexin mutant resulted in the formation of a normal filament network. Thus, the coassembly between alpha-internexin and NF-M exhibits some unique characteristics previously not observed with other intermediate filament proteins: only one intact tail and one intact head are required for the formation of a normal filament network, and they can be present within the same partner or separately in two partners.

Identification of a nonapeptide motif in the vimentin head domain involved in intermediate filament assembly

1992 ◽  
Vol 223 (3) ◽  
pp. 637-650 ◽  
Author(s):  
Harald Herrmann ◽  
Ilse Hofmann ◽  
Werner W. Franke
Keyword(s):  
Intermediate Filament ◽  
Filament Assembly ◽  
Head Domain

scholarly journals Modulation of desmin intermediate filament assembly by a monoclonal antibody.

1988 ◽  
Vol 106 (3) ◽  
pp. 735-745 ◽  
Author(s):  
W Ip
Keyword(s):  
Monoclonal Antibody ◽  
Electron Microscope ◽  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Building Blocks ◽  
Trypsin Digestion ◽  
Amino Terminal ◽  
The Core ◽  
Filament Assembly ◽  
Different Diameters

We have used a monoclonal antibody against desmin to examine the assembly of intermediate filaments (IF) from their building blocks, the tetrameric protofilaments. The antibody, designated D76, does not cross react with any other IF proteins (Danto, S.I., and D.A. Fischman. 1984. J. Cell Biol. 98:2179-2191). It binds to a region amino-terminal to cys-324 of avian desmin that is resistant to chymotrypsin and trypsin digestion, and in the electron microscope appears to bind to the ends of tetrameric protofilaments. In combination, these findings suggest that the epitope of the antibody resides at the amino-terminal end of the alpha-helical rod domain. Preincubation of desmin protofilaments with an excess of D76 antibodies blocks their subsequent assembly into IF. In the presence of sub-stoichiometric amounts of antibodies, IF are assembled from protofilaments but they are morphologically aberrant in that (a) they are capped by IgG molecules at one or both ends; (b) they are unraveled to varying degree, revealing a characteristic right-handed helical arrangement of sub-filamentous strands of different diameters. The antibody binds only to the ends but not along the length of desmin IF. The most straightforward explanation for this is that the epitope resides in a part of the desmin molecule that becomes buried within the core of the filament upon polymerization and is therefore inaccessible to the antibody.

scholarly journals Human keratin 1/10‐1B tetramer structures reveal a knob‐pocket mechanism in intermediate filament assembly

2019 ◽  
Vol 38 (11) ◽  
Author(s):  
Sherif A Eldirany ◽  
Minh Ho ◽  
Alexander J Hinbest ◽  
Ivan B Lomakin ◽  
Christopher G Bunick
Keyword(s):  
Intermediate Filament ◽  
Filament Assembly ◽  
Keratin 1

Effects of truncated neurofilament proteins on the endogenous intermediate filaments in transfected fibroblasts

1991 ◽  
Vol 99 (2) ◽  
pp. 335-350 ◽  
Author(s):  
S.S. Chin ◽  
P. Macioce ◽  
R.K. Liem
Keyword(s):  
Intermediate Filament ◽  
Dominant Negative ◽  
Deletion Mutants ◽  
Tail Domain ◽  
Cultured Fibroblasts ◽  
Mutant Proteins ◽  
Amino Terminal ◽  
Novel Approach ◽  
Carboxyl Terminal

The expression and assembly characteristics of carboxyl- and amino-terminal deletion mutants of rat neurofilament low Mr (NF-L) and neurofilament middle Mr (NF-M) proteins were examined by transient transfection of cultured fibroblasts. Deletion of the carboxyl-terminal tail domain of either protein indicated that this region was not absolutely essential for co-assembly into the endogenous vimentin cytoskeleton. However, deletion into the alpha-helical rod domain resulted in an inability of the mutant proteins to co-assemble with vimentin into filamentous structures. Instead, the mutant proteins appeared to be assembled into unusual tubular-vesicular structures. Additionally, these latter deletions appeared to act as dominant negative mutants which induced the collapse of the endogenous vimentin cytoskeleton as well as the constitutively expressed NF-H and NF-M cytoskeletons in stably transfected cell lines. Thus, an intact alpha-helical rod domain was essential for normal IF co-assembly whereas carboxyl-terminal deletions into this region resulted in dramatic alterations of the existing type III and IV intermediate filament cytoskeletons in vivo. Deletions from the amino-terminal end into the alpha-helical rod region gave different results. With these deletions, the transfected protein was not co-assembled into filaments and the endogenous vimentin IF network was not disrupted, indicating that these deletion mutants are recessive. The dominant negative mutants may provide a novel approach to studying intermediate filament function within living cells.

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