Assembly characteristics of plant keratin intermediate filamentsin vitro

After selective extraction and purification, plant keratin intermediate filaments were reassembledin vitro. Scanning tunneling microscope (STM) and transmission electron microscope (TEM) micrographs showed that acidic keratins and basic keratins can assemble into dimers and further into 10 nm filamentsin vitro. In higher mcation images, it can be seen that fully assembled plant keratin intermediate filaments consist of several thinner filaments of 3 nm in diameter, which indicates the formation of protofilaments in the assembly processes. One of the explicit features of plant keratin intermediate filaments is a 24–25 nm periodic structural repeat alone the axis of both the 10 nm filaments and protofilarnents. The periodic repeat is one of the fundamental characteristic of all intermediate filaments, and demonstrates the half staggered arrangement of keratin molecules within the filaments.     

Assembly characteristics of plant keratin intermediate filaments in vitro

After selective extraction and purification, plant keratin intermediate filaments were reassembled in vitro. Scanning tunneling microscope (STM) and transmission electron microscope (TEM) micrographs showed that acidic keratins and basic keratins can assemble into dimers and further into 10 nm filaments in vitro. In higher magnification images, it can be seen that fully assembled plant keratin intermediate filaments consist of several thinner filaments of 3 nm in diameter, which indicates the formation of protofilaments in the assembly processes. One of the explicit features of plant keratin intermediate filaments is a 24—25 nm periodic structural repeat alone the axis of beth the 10 nm filaments and protofilaments. The periodic repeat is one of the fundamental characteristic of all intermediate filaments, and demonstrates the half staggered arrangement of keratin molecules within the filaments.     

The assembly of keratins from higher plant cells

Summary In vitro assembly and morphological characteristics of purified 58 kDa, 52 kDa, 50 kDa, and 45 kDa polypeptides in the leaves and the cotyledons of the cabbage (Brassica pekinensis Rupt.) were investigated by electron microscopy and scanning tunneling microscopy. The three or four purified intermediate filament (IF) polypeptides can spontaneously assemble into intermediate filaments in vitro with a 23–24 nm axial repeat, which indicates that keratin IFs in higher plant cells have the same molecular arrangement as in animal cells. STM images suggest that the plant keratin filaments display a pronounced structural polymorphism, which can be composed of 3 nm, 4.5 nm, or 6 nm wide keratin protofilaments.Abbreviation IF intermediate filament - STM scanning tunneling microscopy - SDS sodium dodecyl sulfate - BCIP 5-bromo-4-chloro-3-indolyl phosphate-toluidine - NBC p-nitroblue tetrazolium chloride - PMSF phenylmethyl sulfonylfluoride - HOPG high oriented pyrolytic graphite     

植物细胞中间纤维角蛋白性质的分析

角蛋白是植物细胞中间纤维的主要成分。应用选择性抽提和生物化学技术,分离纯化了豌豆根尖细胞58-、52 kD、白菜子叶52kD和胡萝卜悬浮细胞64kD角蛋白,测定了它们的氨基酸组成,结果表明上述角蛋白与动物细胞中间纤维角蛋白的氨基酸组成有较大的相似性。比较了动、植物细胞角蛋白的肽谱,结果显示它们之间存在较大的差异,但是植物细胞间角蛋白的肽谱比较一致,这提示它们属于同一蛋白家族,为植物中间纤维及其角蛋白的存在提供了新的论据。     

Microinjection of monoclonal antibodies specific for one intermediate filament protein in cells containing multiple keratins allow insight into the composition of particular 10 nm filaments

Monoclonal antibodies specific for vimentin (V9), keratin 7 (CK 7) and keratin 18 (CK5) have been microinjected into three human epithelial cell lines: HeLa, MCF-7 and RT-4. The effect of the injection on other keratin polypeptides and vimentin filaments has been observed by double label immunofluorescence and in some instances by immunoelectron microscopy using gold labels of different sizes. Microinjection of V9 into HeLa cells causes the vimentin to collapse into a perinuclear cap leaving the keratin filaments unaffected. Injection of CK5 does not affect the vimentin filaments but disrupts the keratin filaments revealing keratin aggregates similar to those seen in some epithelial cell lines during mitosis. The keratin aggregates obtained after microinjection in HeLa contain the keratins 8 and 18 and probably also other keratins, as no residual keratin filaments are observed with a keratin polyclonal antibody of broad specificity. Aggregates in mitotic HeLa cells contain at least the keratins 7, 8, and 18. In MCF-7 cells keratins 8, 18, and 19 are observed in the aggregates seen 3 h after microinjection which, however, show a different morphology from those seen in HeLa cells. In MCF-7 cells a new keratin filament is built within 6 h after the injection which is composed mainly of keratin 8 and 19. The antibody-complexed keratin 18 remains in spherical aggregates of different size. The results suggest that in HeLa cells vimentin and keratin form independent networks, and that individual 10 nm filaments in epithelial cell lines can contain more than two keratins.     

The in vitro assembly of hair follicle keratins: comparison of cortex and companion layer keratins

The hair follicle consists of a complex system of multiple tissue compartments that are clearly distinguishable by their morphology and type of differentiation. We have synthesized hair follicle-specific keratins from the companion layer (K6hf, K17) and the hair cortex (Ha1, Hb3, Hb6) in Escherichia coli. The assembly of purified keratins in mixtures of K6hf/K17 and in mixtures of hair cortex keratins was compared in urea solutions, low ionic strength and physiological strength buffers, by urea melting gels, electron microscopy and analytical ultracentrifugation. Both types of keratin mixtures, keratins from the companion layer and keratins from the hair cortex, formed heterotypic complexes at 5 M urea. In low ionic strength buffers, the keratins from the companion layer were assembled to bona fide intermediate filaments. In contrast, mixtures of hair cortex keratins stayed in an oligomeric state with a mean s value of 9 as determined in sedimentation velocity experiments. Hair cortex keratins were, however, assembled into intermediate filaments at physiological salt conditions. A point mutated hair cortex keratin [Hb6(Glu402Lys)] formed no long filaments when mixed with Ha1; instead, the assembled structures showed a length distribution of 50.8 +/- 13.4 nm, comparable to the size distribution of assembly intermediates called 'unit-length' filaments.     

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

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.     

Assemblies of psoriatic keratin and their relation to normal intermediate filament structures

Protein extracts from normal human epidermis reassemble in vitro into 8-10 nm diameter filaments characteristic of intermediate filaments, whereas extracts from psoriatic epidermal scales reassemble, under identical conditions, into a variety of paracrystalline bundles. Optical diffraction and image analysis of these paracrystalline bundles reveal an axial repeat of 16.5 nm, which subdivides into three bands of 5.5 nm, and a lateral spacing of 5.1 nm. This information, together with available sequence studies of intermediate filaments and biochemical data, suggests that the subunit of psoriatic keratin is made up essentially from the coiled-coil alpha-helical rod domain of the normal keratin subunits, whereas the random coil domains are missing or greatly reduced in size.     

The fibrillar substructure of keratin filaments unraveled

We show that intermediate-sized filaments reconstituted from human epidermal keratins appear unraveled in the presence of phosphate ions. In such unraveling filaments, up to four "4.5-nm protofibrils" can be distinguished, which are helically twisted around each other in a right-handed sense. Lowering the pH of phosphate-containing preparations causes the unraveling filaments to further dissociate into "2-nm protofilaments." In addition, we find that reconstitution of keratin extracts in the presence of small amounts of trypsin yields paracrystalline arrays of 4.5-nm protofibrils with a prominent 5.4-nm axial repeat. Limited proteolysis of intact filaments immobilized on an electron microscope grid also unveils the presence of 4.5-nm protofibrils within the filament with the same 5.4-nm axial repeat. These results, together with other published data, are consistent with a 10-nm filament model based on three distinct levels of helical organization: (a) the 2-nm protofilament, consisting of multi-chain extended alpha-helical segments coiled around each other; (b) the 4.5-nm protofibril, being a multi-stranded helix of protofilaments; and (c) the 10-nm filament, being a four-stranded helix of protofibrils.     

The role of keratin subfamilies and keratin pairs in the formation of human epidermal intermediate filaments

The four major keratins of normal human epidermis (molecular mass 50, 56.5, 58, and 65-67 kD) can be subdivided on the basis of charge into two subfamilies (acidic 50-kD and 56.5-kD keratins vs. relatively basic 58-kD and 65-67-kD keratins) or subdivided on the basis of co-expression into two "pairs" (50-kD/58-kD keratin pair synthesized by basal cells vs. 56.5-kD/65-67-kD keratin pair expressed in suprabasal cells). Acidic and basic subfamilies were separated by ion exchange chromatography in 8.5 M urea and tested for their ability to reassemble into 10-nm filaments in vitro. The two keratins in either subfamily did not reassemble into 10-nm filaments unless combined with members of the other subfamily. While electron microscopy of acidic and basic keratins equilibrated in 4.5 M urea showed that keratins within each subfamily formed distinct oligomeric structures, possibly representing precursors in filament assembly, chemical cross-linking followed by gel analysis revealed dimers and larger oligomers only when subfamilies were combined. In addition, among the four major keratins, the acidic 50-kD and basic 58-kD keratins showed preferential association even in 8.5 M urea, enabling us to isolate a 50-kD/58-kD keratin complex by gel filtration. This isolated 50-kD/58-kD keratin pair readily formed 10-nm filaments in vitro. These results demonstrate that in tissues containing multiple keratins, two keratins are sufficient for filament assembly, but one keratin from each subfamily is required. More importantly, these data provide the first evidence for the structural significance of specific co-expressed acidic/basic keratin pairs in the formation of epithelial 10-nm filaments.     

Organization and characterization of the keratin cytoskeleton in the previtellogenic ovarian follicle of the lizard Podarcis sicula raf

The cytokeratin (CK) cytoskeleton, previously described by immunofluorescence in the ovarian follicle of Podarcis sicula, at the electron microscope results constituted by bundles of 10 nm thick intermediate filaments containing keratin. These bundles are better evident in the cytoplasm of the pyriform cell apex pointed toward the oocyte surface and mostly in the intercellular bridges connecting fully differentiated pyriform cells to the oocyte. During the differentiation of pyriform cells, the intermediate filament bundles first appear inside the intercellular bridge, when the small follicle cells progressively enlarge after their fusion with the oocyte and assume a morphology of "intermediate" cells. The present study also reports a comparative analysis by immunolabeling, SDS-PAGE, and immunoblotting with anticytokeratins CK8, CK18, and CK19 antibodies of both the ovarian follicle and the intestine of Podarcis sicula. These antibodies, specific to the keratins of monolayered intestinal cells, react also against those expressed in the oocytes of Xenopus laevis. This study shows the presence in the ovarian follicle of this reptile only of keratin forms of homologues to the CK8 and CK18 of mammals and the lack of CK19. The same analysis carried out utilizing AE1 and AE3 antibodies, which recognize most of the acidic and basic keratins in mammals, has shown additional forms of keratins specifically expressed in the ovarian follicle (50 kDa) and in both the examined tissues (49 and 60 kDa). The reported results indicate that in the ovarian follicle of this reptile, keratins have peculiar characteristics that can be explained by the unique structural function of the cytoskeleton in this system.     

Synthesis and fate of keratins 8 and 18 in nonepithelial cells transfected with cDNA

To study the assembly of intermediate filaments in vivo we have transfected fibroblast cell lines with the cDNAs coding for keratins 8 and 18 under the control of the promoter of the SV40 early region and followed keratin expression by RNA hybridization, two-dimensional gel electrophoresis, and immunofluorescence analysis. When expressed individually, keratins 8 and 18 failed to polymerize into intermediate filaments but formed granular aggregates of variable size distributed throughout the cytoplasm as seen by staining with specific antibodies. The expression of one of these two keratins did not induce the synthesis of its partner or of any other keratin. Coexpression of the two keratins produced filamentous structures, frequently perinuclear, indicating that the two types of polypeptides were able to assemble into intermediate filaments but could not form the cytoskeleton characteristic of epithelial cells. These results demonstrate that assembly in heterocomplexes stabilizes keratins against cellular degradation, helping to explain why excess pools of simple keratins have never been detected.     

Integration of different keratins into the same filament system after microinjection of mRNA for epidermal keratins into kidney epithelial cells

We have isolated poly (A)+ RNA, highly enriched in keratin mRNA from bovine muzzle epidermis, and injected it into epithelial cells of a different type, i.e., cultured kidney epithelial cells of the same (MDBK) or taxonomically distant (PtK2) species. Both recipient cell lines contain keratin polypeptides that are different from those present in epidermal cells. Using keratin subtype-specific antibodies in immunofluorescence and immunoelectron microscopy, we show that foreign keratin mRNAs when injected into a different type of epithelial cell can recruit polyribosomes and are translated together with the keratin mRNAs of the host cell. Foreign epidermal keratins are excluded from vimentin filaments and other structures but readily coassemble with the endogenous keratins and appear to be integrated into the meshwork of the preexisting kidney-type keratin filaments. Our observations indicate that different sets of keratin polypeptides from the same or different species can coassemble in the living cell into a common filament system. Thus we have developed a procedure that allows experimental alteration of the intermediate filament cytoskeleton within living epithelial cells.     

Intermediate filaments reminiscent of immature cells expressed by goldfish (Carassius auratus) astrocytes and oligodendrocytes in vitro

The expression of intermediate filaments is developmentally regulated. In the mammalian embryo keratins are the first to appear, followed by vimentin, while the principal intermediate filament of the adult brain is glial fibrillary acidic protein. The intermediate filaments expressed by a cell thus reflect its state of differentiation. The differentiation state of cells, and especially of glial cells, in turn determines their ability to support axonal growth. In this study we used three new antibodies directed against three fish intermediate filaments (glial fibrillary acidic protein, keratin 8 and vimentin), in order to determine the identity and level of expression of intermediate filaments present in fish glial cells in culture. We found that fish astrocytes and oligodendrocytes are both able to express keratin 8 and vimentin. We further demonstrate that under proliferative conditions astrocytes express high keratin 8 levels and most oligodendrocytes also express keratin 8, whereas under nonproliferative conditions the astrocytes express only low keratin 8 levels and most oligodendrocytes do not express keratin 8 at all. These results suggest that the fish glial cells retain characteristics of immature cells. The findings are also discussed in relation to the fish glial lineage.     

'Hard' and 'soft' principles defining the structure, function and regulation of keratin intermediate filaments.

Keratins make up the largest subgroup of intermediate filament proteins and represent the most abundant proteins in epithelial cells. They exist as highly dynamic networks of cytoplasmic 10-12 nm filaments that are obligate heteropolymers involving type I and type II keratins. The primary function of keratins is to protect epithelial cells from mechanical and nonmechanical stresses that result in cell death. Other emerging functions include roles in cell signaling, the stress response and apoptosis, as well as unique roles that are keratin specific and tissue specific. The role of keratins in a number of human skin, hair, ocular, oral and liver diseases is now established and meshes well with the evidence gathered from transgenic mouse models. The phenotypes associated with defects in keratin proteins are subject to significant modulation by functional redundancy within the family and modifier genes as well. Keratin filaments undergo complex regulation involving post-translational modifications and interactions with self and with various classes of associated proteins.     

Fine structure and assembly in vitro of nuclear lamina in plant cells

The fine structure of the nuclear lamina (NL) in sperm cells ofGinkgo biloba was visualised using high resolution low-voltage scanning electron microscopy (LVSEM). It was shown that the nuclear lamina was composed of 10 nm filaments which formed a fine network. Lamins were purified from cultured carrot suspension cells and assembledin vitro. Long 8–12 nm diameter filaments were seen and sometimes subfilaments could be distinguished. Western blot of filament preparations showed that these contained the 66 and 84 ku lamins. These data demonstrate that plant lamins are capable of assembling into filamentsin vitro. Project supported by the National Natural Science Foundation of China (Grant No. 3500073).     

Keratin and vimentin expression in early organogenesis of the rabbit embryo

Summary The expression of vimentin and keratins is analysed in the early postimplantation embryo of the rabbit at 11 days post conceptionem (d.p.c.) using a panel of monoclonal antibodies specific for single intermediate filament polypeptides (keratins 7, 8, 18, 19 and vimentin) and a pan-epithelial monoclonal keratin antibody. Electrophoretic separation of cytoskeletal preparations obtained from embryonic tissues, in combination with immunoblotting of the resulting polypeptide bands, demonstrates the presence of the rabbit equivalents of human keratins 8, 18, and vimentin in 11-day-old rabbit embryonic tissues. Immunohistochemical staining shows that several embryonic epithelia such as notochord, surface ectoderm, primitive intestinal tube, and mesonephric duct, express keratins, while others (neural tube, dermomyotome) express vimentin, and a third group (coelomic epithelia) can express both. Similarly, of the mesenchymal tissues sclerotomal mesenchyme expresses vimentin, while somatopleuric mesenchyme (abdominal wall) expresses keratins, and splanchnopleuric mesenchyme (dorsal mesentery) expresses both keratins and vimentin. While these results are in accordance with most results of keratin and vimentin expression in embryos of other species, they stand against the common concept of keratin and vimentin specificity in adult vertebrate tissues. Furthermore, keratin and vimentin are not expressed in accordance with germ layer origin of tissues in the mammalian embryo; rather the expression of these proteins seems to be related to cellular function during embryonic development.Supported by the Deutsche Forschungsgemeinschaft and by the Netherlands Cancer Foundation     

Polymorphism of reconstituted human epidermal keratin filaments: determination of their mass-per-length and width by scanning transmission electron microscopy (STEM)

We have determined the mass-per-length (MPL) and the width of unstained freeze-dried reconstituted human epidermal keratin filaments by scanning transmission electron microscopy (STEM). Filaments were reassembled from keratins extracted from four different sources: cultured human epidermal cells (CHEC), human callus (CAL), and the living layers (LL) and stratum corneum (SC) of normal human epidermis. MPL histograms of all four keratin filament types could be fitted by a superposition of two or three Gaussians, with their respective major peaks located between 17 and 20 kDa/nm. We interpreted the multiple MPL peaks to represent different polymorphic forms of the reconstituted filaments. The number of subunits per filament cross section calculated from MPL peak positions, average subunit molecular weight, and an axial repeat of the subunits within the filament of 46.5 nm revealed an average difference between polymorphic variants of 7.5 +/- 0.9 subunits. These data suggest that reconstituted human epidermal keratin filaments are made of two to four 8-stranded "protofibrils" (i.e., made of two laterally aggregated 4-stranded protofilaments), in agreement with earlier observations. The average widths of unstained freeze-dried keratin filaments were larger than those of negatively stained filaments: 12.6 nm (9.6 nm) for CHEC, 12.3 nm (9.7 nm) for CAL, 11.6 nm (8.3 nm) for LL, and 11.3 nm (7.9 nm) for SC keratin filaments, with the values in brackets corresponding to negatively stained samples. Assuming the MPL to be proportional to the square of the filament width, there is a good correlation between the MPL and width measurements both for filaments within a given type as well as among those reconstituted from different types of keratin extracts.     

Chromosomal localization of acidic and basic keratin genes of the domestic dog

Our laboratories are interested in characterizing genes involved in the myriad of heritable diseases affecting the domestic dog, Canis lupus familiaris, and in development of detailed genetic and physical maps of the canine genome. Included in these efforts is examination of conservation of the genetic organization, structure, and function of gene families involved in diseases of the canine skin, skeleton, and eye. To that end, study of the highly conserved keratin gene family was undertaken. Keratins belong to the superfamily of intermediate filaments and are the major structural proteins of the epidermis, hair, and nail. The keratins are highly conserved throughout vertebrate evolution both at the DNA and amino acid sequence levels. Mutations in genes encoding epithelial keratins are known to cause various diseases in humans, and similar histopathological presentations have been reported in the dog. The keratins are divided into two groups, type I (acidic) and type II (basic). In the human, the genes encoding the acidic and basic keratins are clustered on Chrs 17 and 12, respectively. The same genetic arrangement is seen in the mouse with the acidic and basic keratin gene clusters found on Chrs 11 and 15, respectively. Reported here are the chromosomal localization of acidic and basic canine keratin genes as well as supportive sequence data. Fluorescence in situ hybridization (FISH) experiments with clones isolated from a canine genomic library suggest that the acidic keratin gene cluster resides on CFA9 and the basic keratin gene cluster is located on CFA27. Received: 25 September 1998 / Accepted: 1 December 1998     

Linkage of human keratin genes

Two families of keratins, type I and type II, can be distinguished within the intermediate filament family of proteins, and at least 20 genes in the human genome code for the 20 known keratin proteins. In epithelial intermediate filaments, keratins from both families appear to be coordinately expressed. We have screened a library of human genomic DNA and have identified several cases of linkage among homologous and heterologous pairs of keratin genes. Genes coding for type I keratins were found linked to those coding for type II keratins. Linkage was discovered also among homologous genes coding for type I keratins and among genes encoding type II keratins. In addition, we found genes coding for glycine-rich keratins linked to genes coding for those that do not contain glycine-rich regions. Our results raise the possibility that all keratin genes are linked in a single region of the human genome.