Structure of intermediate filaments

Recent amino acid sequence data have revealed that the microfibrils in hard α-keratin contain proteins with highly significant homologies and closely similar structural characteristics to the intermediate filament (IF) proteins known as desmin and vimentin. This result implies that microfibrils in hard α-keratin may be classified as a member of the IF and that the major features of these various filamentous structures are the same. Consequently, data obtained using X-ray diffraction, electron microscopy, amino acid sequence structural analysis and physicochemical techniques have been collated from the hitherto diverse fields of keratin and IF structure and used to formulate a more detailed model for the 7–8 nm diameter filaments than has previously been possible. Two models consisting of four-chain units arranged with the helical symmetry deduced for hard α-keratin¹ (Fraser et al. J. Mol. Biol. 1976, 108, 435–452) are in accord with the data. The structural unit comprises an oppositely directed pair of molecules each consisting of a two-stranded parallel-chain coiled-coil rope of length ～45 nm stabilized by both interchain and intermolecular ionic interactions. For a perfectly regular structure the filament may be likened either to a seven-stranded cable with a supercoil pitch length of about 345 nm (pitch angle ～2.9°), or a ten-stranded cable (Fraser, R. D. B. and MacRae, T. P. Polymer 1973, 14, 61–67) with a supercoil pitch length of about 1293 nm (pitch angle ～0.8°). The models also provide some insight into the self-assembly mechanism of the IF.     

Intermediate filament structure: 2. Molecular interactions in the filament

Molecules of intermediate filament (IF) proteins contain a central rod domain in which the two constituent chains have a predominantly α-helical conformation and are coiled around one another to form segments of two-strand rope. Possible interactions between the two long segments, termed 1B and 2 were investigated by a technique successfully employed in studies of the modes of association of collagen molecules by Miller and coworkers. Prominent maxima were found in all of the six possible modes of association between the rod domain segments in individual IF proteins and certain maxima were found to be common to all IF. The surface lattice of the IF from α-keratin has been determined and possible bonding arrangements between the rod-domain segments are catalogued. A systematic search was carried out for combinations of interaction maxima which were consistent with the dimensions of the surface lattice. By the further application of stereochemical constraints, models for the topological arrangement of the rod-domain segments on the surface lattice were derived and these are illustrated and discussed.     

Association of spectrin with desmin intermediate filaments

The association of erythrocyte spectrin with desmin filaments was investigated using two in vitro assays. The ability of spectrin to promote the interaction of desmin filaments with membranes was investigated by electron microscopy of desmin filament-erythrocyte inside-out vesicle preparations. Desmin filaments bound to erythrocyte inside-out vesicles in a spectrin-dependent manner, demonstrating that spectrin is capable of mediating the association of desmin filaments with plasma membranes. A quantitative sedimentation assay was used to demonstrate the direct association of spectrin with desmin filaments in vitro. When increasing concentrations of spectrin were incubated with desmin filaments, spectrin cosedimented with desmin filaments in a concentration-dependent manner. At near saturation the spectrin:desmin molar ratio in the sedimented complex was 1:230. Our results suggest that, in addition to its well characterized associations with actin, spectrin functions to mediate the association of intermediate filaments with plasma membranes. It might be that nonerythrocyte spectrins share erythrocyte spectrin's ability to bind to intermediate filaments and function in nonerythroid cells to promote the interaction of intermediate filaments with actin filaments and/or the plasma membrane.     

Hard α-keratin IF: A structural model lacking a head-to-tail molecular overlap but having hybrid features characteristic of both epidermal keratin and vimentin IF

In intermediate filaments (IF) both epidermal keratin and vimentin molecules have been shown to have an eight residue head to-tail overlap between the rod domains of similarly directed molecules. In the case of the epidermal keratins this region has also been shown to have particular structural/functional significance since it represents a hot-spot for mutations in the four keratinopathies characterized to date. While there is good evidence that this head-to-tail overlap is present in IF containing Type III, IV, and V chains, as well as in the epidermal keratin IF (Ib/IIb), there are no data currently available for the hard α-keratin IF (Ia/IIa). Using a variety of data derived from X-ray diffraction and crosslinking studies, as well as theoretical modeling, it is now possible to demonstrate that the overlap region is not a feature of hard α-keratin IF. Indeed, it is shown that there is a nine residue gap between consecutive parallel molecules in the IF. An explanation for this observation is presented in terms of compensating disulfide bonds that occur both within the IF, and between the IF and the matrix in which the IF are embedded. © 1995 Wiley-Liss, Inc.     

植物激素对微管和纤维素微纤丝排向的调节

回顾了微管和纤维素微纤丝在细胞骨架构成和延展中的作用;综述了植物激素在微管和纤维素微纤丝排向中的调节功能,并对细胞扩大和伸长的机制进行了探讨。     

Antiparallel orientation of the two double-stranded coiled-coils in the tetrameric protofilament unit of intermediate filaments

The chymotryptically excised middle domain of desmin slightly exceeds in length the structurally conserved alpha-helical middle region documented in all intermediate filament proteins by amino acid sequence data. This rod domain is a protofilament derivative with a tetrameric organization, thus indicating the presence of two double-stranded coiled-coil units. We now show by immunoelectron microscopy that Fab fragments of a desmin-specific monoclonal antibody mixed with the rod lead to dumb-bell-shaped structures. The tagging of both ends together with the length of the rod (48 nm) argues for an antiparallel orientation of the two coiled-coils without a major stagger. This information combined with the lateral 21 nm periodicity of the intermediate filament observed by us and others leads to a structural hypothesis similar to those entertained from X-ray data on wool alpha-keratins, although here an antiparallel tetrameric unit of some 60 to 66 nm is invoked, which has never been isolated. The structure that we discuss allows for the existence of both the particles, and the antibody experiment strongly supports the antiparallel orientation postulated in both approaches. The tube-like filament structure proposed for the intermediate filament agrees with recent mass per unit length measurements and allows for two minor classes of intermediate filaments with different values in this property as also found experimentally.     

Regulation of the association of alpha 6 beta 4 with vimentin intermediate filaments in endothelial cells

The adhesion of microvascular endothelial cells to their underlying basement membrane is important for the maintenance of vascular integrity. Most integrins function in endothelial cell adhesion by forming a transmembrane link between their basement membrane ligand and the actin microfilament cytoskeleton. The alpha 6 beta 4 laminin-binding integrin, however, associates with vimentin intermediate filaments (IFs) in microvascular endothelial cells and therefore is likely to uniquely contribute to the barrier function of the endothelium. In this study, we examined the regulation of alpha 6 beta 4-vimentin IF association. We first tested the requirement for alpha 6 beta 4-laminin interactions and actin microfilament assembly. We found that alpha 6 beta 4 associated with vimentin IFs when cells were adherent to either laminin 5 or fibronectin, indicating that this association can occur independent of alpha 6 beta 4-ligand interactions. Additionally, we found that alpha 6 beta 4 was associated with vimentin IFs prior to cell spreading, indicating that changes in the microfilament cytoskeleton associated with changes in cell shape are also not required. Thus, although the association of alpha 6 beta 4 with vimentin IFs may strengthen cell adhesion by providing endothelial cells with an additional transmembrane linkage between the basement membrane and the cytoskeleton, this association is not itself regulated by alpha 6 beta 4-mediated adhesion. Finally, we tested the role of plectin in the association of alpha 6 beta 4 with vimentin IFs. Plectin is known to bind in vitro to both IFs and the beta 4 cytoplasmic domain (beta 4 tail), suggesting that it may be important for this linkage. Therefore, we generated deletion mutants of the beta 4 tail and compared the ability of alpha 6 beta 4 containing these deletions to associate with vimentin IFs. We targeted the two regions of the beta 4 tail known to bind to plectin IN VITRO: the N-terminal and C-terminal plectin binding sites. We found that deletion of the N-terminal binding site inhibited the association of alpha 6 beta 4 with vimentin IFs. Thus, plectin-beta 4 tail interactions may play an important role in connecting alpha 6 beta 4 with vimentin IFs and may prove to be important targets in the regulation of this association in endothelial cells.     

Role of cortical microtubules in the orientation of cellulose microfibril deposition in higher-plant cells

Summary Cortical microtubules (MTs) have been implicated in the morphogenesis of plant cells by regulating the orientation of newly deposited cellulose microfibrils (CMFs). However, the role of MTs in oriented CMF deposition is still unclear. We have investigated the mechanism of CMF deposition with cultured tobacco protoplasts derived from taxol-treated BY-2 cells (taxol protoplasts). The BY-2 protoplasts regenerated patches of β-l,3-glucan (callose) and fibrils of β-l,4-glucan (cellulose). Taxol protoplasts possessed the same ordered MT arrays as material cells and regenerated CMFs with patterns almost coincidental with MTs. Electron microscopy revealed that, on the surface of cultured taxol protoplasts, each CMF bundle appeared to be deposited on each cortical MT. These results suggest that MTs may attach directly to the cellulose-synthesizing complexes, by some form of linkage, and regulate the movement of these complexes in higher-plant cells.     

Intermediate filament proteins immunologically related to desmin in astrocytes: A study of chicken spinal cord by two-dimensional gel electrophoresis and immunoblotting

Co-migration experiments by two-dimensional SDS-PAGE using chicken spinal cord extracts and desmin purified from chicken gizzard showed that desmin is not present in spinal cord. However, by the immunoblotting procedure, desmin antibodies recognized 3 spinal cord antigens with different molecular weights and isoelectric points than desmin and the glial fibrillary acidic (GFA) protein. These antigens which also reacted with GFA protein antibodies were not identified in chicken gizzard extracts. The reactivity of the antigens with a monoclonal antibody recognizing an epitope common to most intermediate filament proteins (1) suggests that immunostaining of astrocytes with desmin antibodies (2, 3) is due to the presence of new intermediate filament proteins immunologically related to desmin.Special issue dedicated to Dr. Paola S. Timiras     

Evidence for the intramolecular pleating model of fibrillin microfibril organisation from single particle image analysis

Fibrillin microfibrils endow mammalian connective tissues with elasticity and are fundamental for the deposition of elastin. The microfibrils are 57nm periodic supramolecular protein polymers with a mass of 2.4MDa per repeat. The detailed structure and organisation of most matrix assemblies is poorly understood due to their large size and complexity and it has proved a major challenge to define their structural organisation. Therefore, we have used low dose electron microscopy and single particle image analysis to study the structure of fibrillin microfibrils. Three novel features were detected: a globular feature that bridges the "arm" region, a double band of density crossing the microfibril and stain penetrating holes present in the interbead region, possibly produced by the removal of microfibril associated proteins in the purification procedure. Fine filaments of approximately 2.4nm diameter are resolved in the interbead region, which correspond to the reported diameter of the fibrillin molecule. Comparison of the stain exclusion pattern of microfibrils with the theoretical stain exclusion pattern of fibrillin packing models indicates that the intramolecular pleating model, where each fibrillin molecule is pleated within one microfibril period allowing extensibility by unpleating, has the best fit to the data.     

Growth of wool follicles in culture

Summary A procedure for the culture of isolated wool follicles from Merino sheep is described. Follicles were microdissected from midside skin samples of 2-yr-old wethers and transferred, individually, to 24-well tissue culture plates. When maintained in supplemented Williams’ E medium containing 5 to 10% fetal bovine serum (FBS), insulin, hydrocortisone, and a trace element mixture, fiber growth rates of 40 to 80 μm/day were observed. Follicles maintained their morphologic integrity for up to 7 days, incorporated [methyl-³H]thymidine into DNA and [³⁵S]methionine into intermediate-filament keratins of the growing fiber. Insulin and hydrocortisone stimulated fiber growth at concentrations of 10 μg/ml and 50 ng/ml, respectively, but higher doses were inhibitory. The growth of fibers in response to hydrocortisone and the changes in follicle morphology was similar to those induced in skin after systemic administration of cortisol in vivo. A positive interaction between hydrocortisone and trace elements for follicle survival and hydrocortisone, insulin, and FBS for fiber growth was also found. The successful culture of Merino sheep follicles provides a model with which to study the direct influence of endocrine, nutritional and local factors on wool keratin synthesis independently of systemic shifts in the animals’ metabolism.     

Motile Properties of Vimentin Intermediate Filament Networks in Living Cells

The motile properties of intermediate filament (IF) networks have been studied in living cells expressing vimentin tagged with green fluorescent protein (GFP-vimentin). In interphase and mitotic cells, GFP-vimentin is incorporated into the endogenous IF network, and accurately reports the behavior of IF. Time-lapse observations of interphase arrays of vimentin fibrils demonstrate that they are constantly changing their configurations in the absence of alterations in cell shape. Intersecting points of vimentin fibrils, or foci, frequently move towards or away from each other, indicating that the fibrils can lengthen or shorten. Fluorescence recovery after photobleaching shows that bleach zones across fibrils rapidly recover their fluorescence. During this recovery, bleached zones frequently move, indicating translocation of fibrils. Intriguingly, neighboring fibrils within a cell can exhibit different rates and directions of movement, and they often appear to extend or elongate into the peripheral regions of the cytoplasm. In these same regions, short filamentous structures are also seen actively translocating. All of these motile properties require energy, and the majority appear to be mediated by interactions of IF with microtubules and microfilaments.     

Variation of cellulose microfibril angles in softwoods and hardwoods-a possible strategy of mechanical optimization

Position-resolved small-angle X-ray scattering was used to investigate the nanostructure of the wood cell wall in two softwood species (Norwegian spruce and Scots pine) and two hardwood species (pedunculate oak and copper beech). The tilt angle of the cellulose fibrils in the wood cell wall versus the longitudinal cell axis (microfibril angle) was systematically studied over a wide range of annual rings in each tree. The measured angles were correlated with the distance from the pith and the results were compared. The microfibril angle was found to decrease from pith to bark in all four trees, but was generally higher in the softwood than in the hardwood. In Norwegian spruce, the microfibril angles were higher in late wood than in early wood; in Scots pine the opposite was observed. In pedunculate oak and copper beech, low angles were found in the major part of the stem, except for the very first annual rings in pedunculate oak. The results are interpreted in terms of mechanical optimization. An attempt was made to give a quantitative estimation for the mechanical constraints imposed on a tree of given dimensions and to establish a model that could explain the general decrease of microfibril angles from pith to bark.     

Different substrates influence the expression of intermediate filaments and the deposition of basement membrane proteins

Summary A primary culture of serous cystadenocarcinoma of the ovary was used to study the expression of intermediate filament proteins and the deposition of basal lamina proteins. It was found that cells grown on type I and IV collagens or in collagen gels failed to express vimentin, which was readily demonstrable in cultures of the same cells grown on plastic or glass. Furthermore cells grown in collagen gels formed colonies demonstrating a cystic architecture Unlike what is commonly observed on glass or plastic where laminin and fibronectin are deposited as disorganized fibrils in the extracellular space, in or on collagen these proteins appear solely at the interface between the epithelial cells and matrix. The results suggest that the extracellular matrix influences the cytoskeletal organization of the intermediate filaments and determines cell polarity. They confirm that collagen substrates permit epithelial cell cultures to progress toward a more differentiated state. Supported by grants from the Italian Assciation for Cancer Research (AIRC).     

Transient increase in vimentin phosphorylation and vimentin-HSC70 association in 9L rat brain tumor cells experiencing heat-shock

Characteristic changes in vimentin were studied in 9L rat brain tumor cells treated at 45°C. During heat-shock treatment, vimentin molecules were rapidly phosphorylated and reorganized from a filamentous form into a perinuclear higher-order structure that was less extractable by nonionic detergent. These effects were found to be highly transient, peaked at 30 min after the onset of heat-shock treatment, and subsided thereafter. Simultaneously, the solubility of the constitutively expressed heat-shock protein70 (HSC70) was also temporarily decreased and the kinetics was identical to that of vimentin. The results indicated that HSC70 and vimentin were co-insolubilized during the heat-shock treatment. We propose that the reorganization of the intermediate filaments resulted from enhanced phosphorylation of vimentin leads to the concurrent association of HSC70 to the intermediate filaments. This process may play an essential role in regulating heat-shock genes.     

Roles of microtubules and cellulose microfibril assembly in the localization of secondary-cell-wall deposition in developing tracheary elements

Summary. The roles of cellulose microfibrils and cortical microtubules in establishing and maintaining the pattern of secondary-cell-wall deposition in tracheary elements were investigated with direct dyes to inhibit cellulose microfibril assembly and amiprophosmethyl to inhibit microtubule polymerization. When direct dyes were added to xylogenic cultures of Zinnia elegans L. mesophyll cells just before the onset of differentiation, the secondary cell wall was initially secreted as bands composed of discrete masses of stained material, consistent with immobilized sites of cellulose synthesis. The masses coalesced, forming truncated, sinuous or smeared thickenings, as secondary cell wall deposition continued. The absence of ordered cellulose microfibrils was confirmed by polarization microscopy and a lack of fluorescence dichroism as determined by laser scanning microscopy. Indirect immunofluorescence showed that cortical microtubules initially subtended the masses of dye-altered secondary cell wall material but soon became disorganized and disappeared. Although most of the secondary cell wall was deposited in the absence of subtending cortical microtubules in dye-treated cells, secretion remained confined to discrete regions of the plasma membrane. Examination of non-dye-treated cultures following application of microtubule inhibitors during various stages of secondary-cell-wall deposition revealed that the pattern became fixed at an early stage such that deposition remained localized in the absence of cortical microtubules. These observations indicate that cortical microtubules are required to establish, but not to maintain, patterned secondary-cell-wall deposition. Furthermore, cellulose microfibrils play a role in maintaining microtubule arrays and the integrity of the secondary-cell-wall bands during deposition.Correspondence and reprints: Department of Biological Sciences, University of Rhode Island, Kingston, RI 02881, U.S.A.Present address: Biology Editors Co., Peacedale, Rhode Island, U.S.A.Present address: Department of Biology and Marine Biology, Roger Williams University, Bristol, Rhode Island, U.S.A.Present address: Department of Crop Science and Department of Botany, North Carolina State University, Raleigh, North Carolina, U.S.A.     

角质酶/角蛋白酶一浴法处理对羊毛性能的影响

采用T.fusca产角质酶以及Bacillus subtilis产角蛋白酶一浴法的方式处理羊毛,通过毡缩率、断裂强力、碱溶解度、上染速率、K/S值和接触角等指标考察了该处理对羊毛的改性效果,并运用XPS、氨基酸分析和SEM考察了其对羊毛结构与性质的影响。实验结果表明：经一浴法处理后,羊毛织物的毡缩率下降明显,达到机可洗要求;断裂强力下降较少,碱溶解度增加较少,上染速率提高,K/S值增加;XPS分析表明,经处理后羊毛纤维表面的元素含量变化较大;氨基酸分析表明,经处理后羊毛纤维中的胱氨酸质量分数有所降低;SEM显示,羊毛鳞片层大部分被剥除,综上可以说明角质酶/角蛋白酶的一浴法处理对羊毛具有明显的改性作用。     

The control of cellulose microfibril deposition in the cell wall of higher plants

In maize (Zea mays L.) and pine (Pinus taeda L.) seedlings, cellulose microfibril impressions are present on freeze-fractured plasma membranes. It has been proposed that impressions of newly synthesized microfibrils are a record of the movement of terminal synthesizing complexes through the plasma membrane (Mueller and Brown, 1980, J. Cell Biol. 84, 315–326). The association of terminal complexes with the ends of microfibril impressions or with the ends of microfibrils torn through the membrane indicates the orientation of microfibril tips. Unidirectionally-oriented microfibril tips (all pointing in the same direction) are associated with the organized deposition of parallel arrays of microfibrils. Multidirectionally-oriented microfibril tips were observed in a cell in which microfibril deposition was unusually disorganized. Microfibril patterns around pit fields are asymmetric and resemble flow patterns. Unidirectionally-oriented tears are associated with these microfibrils. Although microfibril orientations are deflected around pit fields, the main axis of microfibril orientation is maintained across the surface of the cell. The hypothesis is proposed that the interaction of a flowing plasma membrane with microfibril synthesizing complexes in the plane of the membrane may result in unidirectional deposition and asymmetric microfibril impressions around pit fields.Some of this work has been published in preliminary form (Brown 1979)     

Modest loss of peripheral axons, muscle atrophy and formation of brain inclusions in mice with targeted deletion of gigaxonin exon 1

Mutations in the gigaxonin gene are responsible for giant axonal neuropathy (GAN), a progressive neurodegenerative disorder associated with abnormal accumulations of Intermediate Filaments (IFs). Gigaxonin is the substrate-specific adaptor for a new Cul3-E3-ubiquitin ligase family that promotes the proteasome dependent degradation of its partners MAP1B, MAP8 and tubulin cofactor B. Here, we report the generation of a mouse model with targeted deletion of Gan exon 1 (Gan(Deltaexon1;Deltaexon1)). Analyses of the Gan(Deltaexon1;Deltaexon1) mice revealed increased levels of various IFs proteins in the nervous system and the presence of IFs inclusion bodies in the brain. Despite deficiency of full length gigaxonin, the Gan(Deltaexon1;Deltaexon1) mice do not develop overt neurological phenotypes and giant axons reminiscent of the human GAN disease. Nonetheless, at 6 months of age the Gan(Deltaexon1;Deltaexon1) mice exhibit a modest hind limb muscle atrophy, a 10% decrease of muscle innervation and a 27% axonal loss in the L5 ventral roots. This new mouse model should provide a useful tool to test potential therapeutic approaches for GAN disease.