Self-assembly of bovine epidermal keratin filaments in vitro

Steinert, Peter M.; Idler, William W.; Zimmerman, Steven B.

doi:10.1016/s0022-2836(76)80136-2

Cited by 383 publications

(210 citation statements)

References 34 publications

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“…We conclude that the tonofilamentous structures, which are indistinguishable in different epithelial cells, can be formed by completely different sets of proteins of the cytokeratin family . These compositional differences apparently do not result in profound structural and, probably also, functional differences, in agreement with observations that the different combinations of polypeptides of epidermal prekeratin and cytokeratins of other epithelial cells can reconstitute, after denaturation, tonofilament-like structures in vitro (e.g., 1, 13,17,39,55,56) . It is conceivable that only a portion common to all the different polypeptides of the cytokeratin family, probably the core region that is enriched in a-helical conformation and relatively resistant to tyrpsin treatment, is critical for the establishment and maintenance of the tonofilament structure (13,(70)(71)(72) .…”

Section: Franke Et Alsupporting

confidence: 88%

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Isolation and characterization of desmosome-associated tonofilaments from rat intestinal brush border.

Franke¹,

Winter

Gründ

et al. 1981

The Journal of Cell Biology

117

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Epithelial cells of the small intestine, like those of other internal organs, contain intermediate-sized filaments immunologically related to epidermal prekeratin which are especially concentrated in the cell apex. Brush-border fractions were isolated from rat small intestine, and apical tonofilaments attached to desmosomal plaques and terminal web residues were prepared therefrom by extraction in high salt (1 .5 M KCI) buffer and Triton X-100. The structure of these filaments was indistinguishable from that of epidermal tonofilaments and, as with epidermal prekeratin, filaments could be reconstituted from solubilized, denatured intestinal tonofilament protein. On SIDS polyacrylamide gel electrophoresis of proteins of the extracted desmosome-tonofilament fractions, a number of typical brush-border proteins were absent or reduced, and enrichment of three major polypeptides of M r 55,000, 48,000, and 40,000 was noted. On two-dimensional gel electrophoresis, the three enriched major polypeptides usually appeared as pairs of isoelectric variants, and the two smaller components (M r 48,000 and 40,000) were relatively acidic (isoelectric pH values of 5.40 and below), compared to the M r 55,000 protein which focused at pH values higher than 6.4 . The tonofilament proteins were shown to be immunologically related to epidermal prekeratin by immunoreplica and blotting techniques using antibodies to bovine epidermal prekeratins . Similar major polypeptides were found in desmosome-attached tonofilaments from small intestine of mouse and cow. However, comparisons with epidermal tissues of cow and rat showed that all major polypeptides of intestinal tonofilaments were different from the major prekeratin polypeptides of epidermal tonofilaments. The results present the first analysis of a defined fraction of tonofilaments from a nonepidermal cell . The data indicate that structurally identical tonofilaments can be formed, in different types of cells, by different polypeptides of the cytokeratin family of proteins and that tonofilaments of various epithelia display tissue-specific patterns of their protein subunits .

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Section: Franke Et Alsupporting

confidence: 88%

“…As described for epidermal prekeratin dissolved in denaturing concentrations of urea (13,17,39,55,56), filamentous structures were formed from such solutions upon removal of the urea by dialysis (Fig. 6).…”

Section: Reconstitution Of Filament Structures From Denatured Proteinmentioning

confidence: 76%

Isolation and characterization of desmosome-associated tonofilaments from rat intestinal brush border.

Franke¹,

Winter

Gründ

et al. 1981

The Journal of Cell Biology

117

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“…In vitro data have supported the notion that single keratins are unable to form IFs (Steinert et al, 1976;Hatzfeld and Franke, 1985) and quickly become proteolyzed in the absence of their partner (Domenjoud et al, 1988;Kulesh and Oshima, 1988;Kulesh et al, 1989;Lersch et al, 1989; Magin et al., 1990;Bader et al, 1991). In addition to a consistent suprabasal expression of type I keratins, a punctuate basal staining was observed in epidermal blisters of K5 Ϫ/Ϫ mice.…”

Section: Expression and Accumulation Of Type I Keratinsmentioning

confidence: 77%

Complete Cytolysis and Neonatal Lethality in Keratin 5 Knockout Mice Reveal Its Fundamental Role in Skin Integrity and in Epidermolysis Bullosa Simplex

Peters

Kirfel

Büssow³

et al. 2001

MBoC

110

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In human patients, a wide range of mutations in keratin (K) 5 or K14 lead to the blistering skin disorder epidermolysis bullosa simplex. Given that K14 deficiency does not lead to the ablation of a basal cell cytoskeleton because of a compensatory role of K15, we have investigated the requirement for the keratin cytoskeleton in basal cells by inactivating the K5 gene in mice. We report that the K5 Ϫ/Ϫ mice die shortly after birth, lack keratin filaments in the basal epidermis, and are more severely affected than K14 Ϫ/Ϫ mice. In contrast to the K14 Ϫ/Ϫ mice, we detected a strong induction of the wound-healing keratin K6 in the suprabasal epidermis of cytolyzed areas of postnatal K5 Ϫ/Ϫ mice. In addition, K5 and K14 mice differed with respect to tongue lesions. Moreover, we show that in the absence of K5 and other type II keratins, residual K14 and K15 aggregated along hemidesmosomes, demonstrating that individual keratins without a partner are stable in vivo. Our data indicate that K5 may be the natural partner of K15 and K17. We suggest that K5 null mutations may be lethal in human epidermolysis bullosa simplex patients. INTRODUCTIONKeratin (K) intermediate filaments (IFs) belong to a gene family that is organized into two subfamilies, coding for type I (K9 -20) and type II keratins (K1-8). At least one member of each family is necessary to form heterodimeric IFs. In epidermis, keratins display a complex expression pattern that is widely assumed to reflect the structural requirements of distinct epidermal compartments (Fuchs and Cleveland, 1998).The major keratins in the basal layer of stratified epithelia are K5, K14, and K15 (Fuchs and Green, 1978, 1980;Moll et al., 1982;Leube et al., 1988;Lloyd et al., 1995). In wound healing or in hyperproliferative disorders, the keratin pair K6 and K16 is transiently expressed in the suprabasal layers instead of K1, K2e, and K10 (for review, see McGowan and Coulombe, 1998). The functional significance of the highly patterned expression profile of keratins is still poorly understood, but evidence is accumulating that they have distinct functions in epidermis (Paladini and Coulombe, 1999).The structural function of keratins was elucidated by the discovery of point mutations in human epidermal keratin genes (Corden and McLean, 1996), preceded by studies on transgenic mice expressing mutant keratin subunits . These mutations cause a number of inherited human skin disorders such as epidermolysis bullosa simplex (EBS) (Bonifas et al., 1991;Coulombe et al., 1991;Lane et al., 1992;Rothnagel et al., 1995;Corden and McLean, 1996). The characteristic feature of EBS is epidermal blistering resulting from cytolysis of basal keratinocytes. On the basis of these observations, it was suggested that the overall function of epidermal keratins was to provide the reinforcement of the epidermis and the maintenance of cellular integrity under mechanical and thermal stress. Several mice carrying null or dominant keratin mutations that affect epidermal integrity have added further support to t...

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“…Subsequent removal of the urea or guanidine by dialysis allows for the spontaneous reassembly of denatured proteins into native-looking IFs (11)(12)(13). Although this feature is beneficial to the study of IF assembly in vitro, it has created great difficulties in efforts to generate IF protein crystals suitable for high-resolution structural studies.…”

mentioning

confidence: 99%

Structural Characterization of Human Vimentin Rod 1 and the Sequencing of Assembly Steps in Intermediate Filament Formation in Vitro Using Site-directed Spin Labeling and Electron Paramagnetic Resonance

Hess¹,

Budamagunta

Voss

et al. 2004

Journal of Biological Chemistry

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We have previously established the utility of site-directed spin labeling and electron paramagnetic resonance to determine structural relationships among proteins in intact intermediate filaments. Using this same approach we have introduced spin labels at 21 residues between amino acids 169 and 193 in rod domain 1 of human vimentin. The electron paramagnetic resonance spectra provide direct evidence for the coiled coil nature of the vimentin dimer in this region. This finding is consistent with predictions but has never been demonstrated previously. In a previous study we identified residue 348 in the rod domain 2 as one point of overlap between adjacent dimers in intact filaments. In the present study we defined residue 191 in the rod domain 1 as a second point of overlap and established that the dimers are arranged in an anti-parallel and staggered orientation at this site. Finally, by isolating spin-labeled samples at successive stages during the dialysis that lead to filament assembly in vitro, we have been able to establish a sequence of interactions that occurs during in vitro assembly, starting with the ␣ helix and loose coiled coil dimer formation, then the formation of tetrameric species centered on residue 191, followed by interactions centered on residue 348 suggestive of octamer or higher order multimer formation. A continuation of this strategy revealed that both 191-191 and 348 -348 interactions are present in low ionic strength Tris buffers when vimentin is maintained at the "protofilament" stage of assembly.

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Self-assembly of bovine epidermal keratin filaments in vitro

Cited by 383 publications

References 34 publications

Isolation and characterization of desmosome-associated tonofilaments from rat intestinal brush border.

Isolation and characterization of desmosome-associated tonofilaments from rat intestinal brush border.

Complete Cytolysis and Neonatal Lethality in Keratin 5 Knockout Mice Reveal Its Fundamental Role in Skin Integrity and in Epidermolysis Bullosa Simplex

Structural Characterization of Human Vimentin Rod 1 and the Sequencing of Assembly Steps in Intermediate Filament Formation in Vitro Using Site-directed Spin Labeling and Electron Paramagnetic Resonance

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