Three-dimensional architecture of macrofibrils in the human scalp hair cortex

Human scalp hairs are comprised of a central cortex enveloped by plate-like cuticle cells. The elongate cortex cells of mature fibres are composed primarily of macrofibrils-bundles of hard-keratin intermediate filaments (IFs) chemically cross-linked within a globular protein matrix. In wool, three cell types (ortho-, meso- and paracortex) contain macrofibrils with distinctly different filament arrangements and matrix fractions, but in human hair macrofibril-cell type relationships are less clear. Here we show that hair macrofibrils all have a similar matrix fraction (∼0.4) and are typically composed of a double-twist architecture in which a central IF is surrounded by concentric rings of tangentially-angled IFs. The defining parameter is the incremental angle increase (IF-increment) between IFs of successive rings. Unlike the wool orthocortex, hair double-twist macrofibrils have considerable inter-macrofibril variation in IF increment (0.05–0.35°/nm), and macrofibril size and IF increment are negatively correlated. Correspondingly, angular difference between central and outer-most IFs is up to 40° in small macrofibrils, but only 5–10° in large macrofibrils. Single cells were observed containing mixtures of macrofibrils with different diameters. These new observations advance our understanding of the nano-level and cell-level organisation of human hair, with implications for interpretation of structure with respect the potential roles of cortex cell types in defining the mechanical properties of hair.     

Immunofluorescent and immunogold replica studies of desmin distribution in cultured normal and cardiomyopathic hamster heart cells.

The architecture of desmin intermediate filament arrangements in cultured cardiomyocytes from heart of normal and cardiomyopathic hamsters was studied by immunofluorescent light microscopy and immunogold replica electron microscopy. Both polyclonal and monoclonal antidesmin antibodies were used in a biotin-streptavidin system. Immunofluorescent staining of normal and cardiomyopathic myocytes for desmin at 5 days in culture exhibited filamentous staining patterns with polyclonal antidesmin and a coarse punctate staining pattern with the monoclonal antibody. At 9 days in culture, most normal myocytes showed filamentous staining with the polyclonal antibody; many of the stained filaments were associated with Z lines. With the monoclonal antidesmin, these same cells exhibited a very fine 'spotty' staining pattern. These results suggest that the arrangements and immunoreactivities of intermediate filaments change during normal cardiac myocyte development. In cardiomyopathic cells, this pattern of rearrangement and immunoreactivity appears to be delayed or possibly nonexistent. The three-dimensional electron-microscopic observation of immunogold localization of desmin achieved by a deep-etching replica technique is made on both normal and cardiomyopathic cultured heart cells. Abnormalities of desmin filament arrangements in cardiomyopathic cells are confirmed.     

From ultra-soft slime to hard {alpha}-keratins: The many lives of intermediate filaments

Intermediate filaments are filaments 10?nm in diameter that make up an important component of the cytoskeleton in most metazoan taxa. They are most familiar for their role as the fibrous component of α-keratins such as skin, hair, nail, and horn but are also abundant within living cells. Although they are almost exclusively intracellular in their distribution, in the case of the defensive slime produced by hagfishes, they are secreted. This article surveys the impressive diversity of biomaterials that animals construct from intermediate filaments and will focus on the mechanisms by which the mechanical properties of these materials are achieved. Hagfish slime is a dilute network of hydrated mucus and compliant intermediate filament bundles with ultrasoft material properties. Within the cytoplasm of living cells, networks of intermediate filaments form soft gels whose elasticity arises via entropic mechanisms. Single intermediate filaments or bundles are also elastic, but substantially stiffer, exhibiting modulus values similar to that of rubber. Hard α-keratins like wool are stiffer still, an effect that is likely achieved via dehydration of the intermediate filaments in these epidermal appendages. The diverse mechanisms described here have been employed by animals to generate materials with stiffness values that span an impressive eleven orders of magnitude.     

Attachment of mitochondria to intermediate filaments in adrenal cells: relevance to the regulation of steroid synthesis.

The rate of steroid synthesis is regulated by the rate of transport of cholesterol from lipid droplets to mitochondria. We have previously demonstrated that lipid droplets in adrenal cells are tightly attached to intermediate filaments. Here we now show that mitochondria colocalize with intermediate filaments in modified double indirect immunofluorescence and by electron microscopy of extracted adrenal cells. Direct contact between mitochondria and intermediate filaments was established by examination of stereo pairs of electron micrographs from extracted cells. The attachment of both droplets and mitochondria to intermediate filaments suggests possible mechanisms for this form of intracellular transport of cholesterol to mitochondria and hence for the regulation of steroid synthesis.     

Intermediate (10 nm) filaments in human malignant mesothelioma.

Human malignant mesotheliomas were studied by electron microscopy. Three main types of cells were seen--submesothelial epithelioid cells, epithelial lining cells and fibroblast-like cells. In submesothelial epithelioid cells prominent arrays of intermediate (10 nm) filaments were often seen attached to plasma membrane, mitochondria, nuclei and concentric whorls of rough endoplasmic reticulum. The other types of cell found in the tumors, epithelial lining cells and fibroblast-like cells, lacked such distinct filaments. The intermediate filaments were especially abundant in cells with extensive whorling of endoplasmic reticulum. The association of intermediate filaments with such deranged cytoplasmic organization suggests that they play a role in the altered behavior of malignant cells.     

In vitro reconstitution of wool intermediate filaments

Although the hard α-keratins of wool are recognized as members of the intermediate filaments by sequence comparison thus for all attempts on reconstitution of wool α-keratin in filaments in vitro have failed. Here we show the oxidative sulphitolysis rather than the previously used S-carboxymethylation is the method of choice to prepare α-keratin derivatives suitable for assembly experiments. Once the protecting S-sulpho group is removed by 2-mercaptoethanol in vitro filaments formation can be induced. Electron micrographs show filaments with a diameter of 7–11 nm as in all other intermediate filaments. Thus, filament formation of α-keratins does not require the presence of matrix proteins.     

Macrofibril assembly in trichocyte (hard alpha-) keratins

Early electron microscope studies of developing wool and hair established that trichocyte (hard alpha-) keratin fibers have a composite structure in which filaments, subsequently shown to belong to the class of intermediate filaments (IF), were embedded in a matrix of sulfur-rich proteins. These studies also showed that the IF aggregate in a variety of ways to form what have been termed macrofibrils. Assembly into sheets appears to be an important initial factor in aggregation, and in the present contribution the structural principles governing sheet formation are formulated and specific models for the interaction between neighboring IF in a sheet are proposed, based on existing X-ray diffraction, electron microscope, and crosslinking data. All of the trichocyte keratins so far examined by electron microscopy exhibit similar filament/matrix textures and the mechanism of sheet formation proposed here is likely to have general applicability.     

Immuno-electron microscopical identification of the two types of intermediate filaments in established epithelial cells

The intermediate filament systems of the established epithelial cell lines HeLa and PtK₂ have been characterized by electron microscopy using indirect immunoferritin labelling. The results provide a direct ultrastructural confirmation of the proposal based on indirect immunofluorescence microscopy, that vimentin and cytokeratin fibrils constitute two distinct 10 nm filament systems in much of the cell body. In agreement both with classical histology and with the finding that cytokeratins are typically present in many epithelial tissues, demosome-attached 10 nm filaments (tonofilaments) were found to be of the cytokeratin type. Vimentin, but not cytokeratin filaments were translocated into juxtanuclear caps after exposure of the cells to colcemid. Regions of the cytoplasm where the two distinct systems form mixed bundles were identified and both side-by-side arrangements and the occurrence of vimentin fibers in a sheath-like structure around a cytokeratin filament core are described. Our results emphasize that the two systems interact but differ in their organization and control.     

Immunolocalization of the intermediate filament-associated protein plectin at focal contacts and actin stress fibers.

The distribution of plectin in the cytoplasm of Rat1 and glioma C6 cells was examined using a combination of double and triple immunofluorescence microscopy and interference reflection microscopy. In cells examined shortly after subcultivation (less than 48 h), filamentous networks of plectin structures, resembling and partially colocalizing with vimentin filaments, were observed as reported in previous studies. In cells kept attached to the substrate without growth for periods of 72 h to 8 days (stationary cultures), thick fibrillary plectin structures were observed. These structures were located at the end of actin filament bundles and showed co-distribution with adhesion plaques (focal contacts), vinculin, and vimentin. Only relatively large adhesion plaques (dash-like contacts) were decorated by antibodies to plectin, smaller dot-like contacts at the cell edges remained undecorated. Moreover, in stationary Rat1 cells plectin structures were found to be predominantly colocalized with actin stress fibers. However, after treatment of such cells with colcemid, plectin's distribution changed dramatically. The protein was no longer associated with actin structures, but was distributed diffusely throughout the cytoplasm. After a similar treatment with cytochalasin B, plectin's association with stress fibers again was completely abolished, although stress fibers were still present. The association of plectin with focal contact-associated intermediate filaments was demonstrated also by immunogold electron microscopy of quick-frozen, deep-etched replicas of rat embryo fibroblasts. These data confirm previous reports suggesting a relationship between intermediate filaments on the one hand, and actin stress fibers and their associated plasma membrane junctional complexes, on the other. Furthermore, the data establish plectin as a novel component of focal contact complexes and suggest that plectin plays a role as mediator between intermediate filaments and actin filaments.     

Organization of desmin-containing intermediate filaments during differentiation of mouse decidual cells

Decidualization of the mouse endometrium consists of a redifferentiation of the endometrial stromal fibroblasts. During decidualization these fibroblasts undergo growth, change of shape, multinucleation, and establishment of intercellular junctions. One feature of rodent decidual cells is the accumulation of intermediate filaments. In spite of the fact that fibroblasts normally have vimentin intermediate filaments, they acquire a large amount of desmin intermediate filaments while they undergo decidualization. The light and electron microscope immunocytochemical results of the present work show that during the initial stages of decidual transformation the desmin intermediate filaments accumulate around the nuclei, often forming caps around the nuclear envelope. As the decidual cells grow, the filaments form bundles and nets that radiate from the nuclei toward the cell surface. During the final stages of differentiation, on day 8 of pregnancy, staining of differentiated decidual cells decreases and most filaments accumulate under the cell surface. A role for intermediate filaments is suggested for decidualization of mouse endometrial cells.     

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.     

Localization of plectin and other related proteins along the sarcolemma in smooth muscle cells of rat colon

Plectin is a versatile linker protein which is associated with various types of cytoskeletal components and/or filaments including intermediate filaments. To better understand the functional roles of plectin in smooth muscle cells, we examined the distribution of plectin and other related proteins in rat colon smooth muscles by confocal laser and electron microscopy. The sarcolemma of smooth muscle cells exhibits two ultrastructurally distinct domains, domains associated with dense plaques and caveola-rich domains. Staining with anti-plectin and anti-desmin antibodies showed that plectin was localized along the sarcolemma in an intermittent manner and desmin was distributed in the sarcoplasm and intermittently at the cell periphery where it was codistributed with desmin. Plectin exhibited complementary and non-overlapping distribution to caveolin-1 and dystrophin, components of caveola domains, whereas plectin was codistributed with vinculin, talin and integrin beta1, components of dense plaques. Plectin was also codistributed with beta2-chain laminin but not with beta1-chain laminin. Electron microscopic observations on the sarcolemma revealed close association of intermediate filaments with dense plaques. Correlated confocal and electron microscopy clearly demonstrated that anti-plectin fluorescence corresponded to dense plaques but not to caveola domains in electron microscopic images. These findings indicate that plectin is confined to dense plaques to which desmin intermediate filaments may be anchored in rat colon smooth muscle cells.     

Primary cultures of epithelial cells and long-term cultures of fibroblasts from the human umbilical cord: ultrastructural and immunocytochemical characterization

Repeated trypsinization of the full term human umbilical cord epithelium allows an homogeneous and exclusively epithelial primary culture, without fibroblastic growth. Transmission electron microscopy observations of desmosomes, cytokeratin intermediate filaments as revealed by indirect immunofluorescence and cultural aspects confirm the epithelial nature of this primary culture. Fibroblasts obtained by an explants culture method exhibit neither desmosomes nor cytokeratin intermediate filaments, which are epithelial markers. They yield characteristic long term cultures of fibroblastic aspect and growth.     

The cytoskeleton of isolated murine primitive erythrocytes

Summary Cytoskeletons of primitive erythrocytes have been isolated from the embryos of day 12 pregnant C57/Bl mice and examined by transmission electron microscopy, immunofluorescence microscopy, and SDS-polyacrylamide gel electrophoresis. Microtubules are the most prominent cytoskeletal component. They are found either singly or organized into loose bundles just under the plasma membrane, but do not form classical marginal bands in most cells. Immunofluorescence with a polyclonal tubulin antiserum confirms this distribution and further reveals numerous mitotic figures among the cells. Rhodamine-conjugated phalloidin and heavy meromyosin labeling reveal that actin is localized in the cortex of the primitive erythrocyte in the form of 6 nm filaments. Antibody directed against avian erythrocyte alpha spectrin demonstrates that spectrin is also found in the cortex. Occasional 10-nm intermediate filaments, observed in the primitve erythrocytes by electron microscopy, are believed to be of the vimentin class based on positive reaction of the cells with vimentin-specific antiserum. In addition, a band in erythrocyte cytoskeletons comigrates in SDS-polyacrylamide gels with vimentin isolated from mouse kidney. Spectrin and actin were also found to be associated with the membrane of primitive erythrocytes when membrane ghost preparations were analyzed by SDS-polyacrylamide gel electrophoresis.     

On the presence of high glycogen concentration and intermediate filaments in the flat cells of the electroreceptive epidermis of mormyrid fish

The cytoplasm of the flat cells of the electroreceptive epidermis of Mormyrids was examined in the light and the electron microscope, in order to reveal the presence of glycogen and to study its distribution. In the electroreceptive epidermis, which consists of three layers, the periodic acid Schiff reaction used to stain polysaccharides is strongly positive in the superficial polyhedral cells and in the flat cells of the intermediate layer. Polysaccharides are absent in the basal polyhedral cells. Pre-incubation with alpha-amylase shows that glycogen is present only in the intermediate cell layer. In the electron microscope, after reaction with periodic acid, thiocarbohydrazide and silver proteinate, glycogen is seen in the form of rosettes of monoparticles. These rosettes occupy both the central region of the cytoplasm of these cells, and the more peripheral parts, where alignments of desmosomes are found. In the cytoplasm of certain flat cells, the rosettes are grouped to form accumulations of glycogen which cover several mu2. Observation in the electron microscope reveals that in addition to glycogen, these cells contain tonofilaments or intermediate filaments, common to epithelial cells, which may group themselves in bundles. Glycogen and the intermediate filaments are thus the principal constituents of the cytoplasm of the flat cells of the electroreceptive epidermis of Mormyrids. The possible role of the filaments, and especially of the glycogen which is a polysaccharide high in energy, in the flat cells which apparently have a low metabolic rate, is discussed.     

Adhesion of intermediate filaments and lipid droplets in adrenal cells studied by field emission scanning electron microscopy

High-resolution field emission scanning electron microscopy was used to study the organisation of intermediate filaments around lipid droplets and their binding to these droplets, in primary culture of bovine adrenal cells. Whole-mount preparations of intermediate filaments and bound lipid droplets were prepared from cells grown on Formvar-coated grids and processed by freeze-drying. Intermediate filaments were seen as an interconnected network enveloping the entire droplet. The bound filaments appear to be directly adherent to the surface of the droplet and hence take on its curved contour. The binding of the filaments to the droplets was determined by means of tilting. This study provides a new approach to investigate the cytoskeleton and its associated structures with high-resolution three-dimensional images.     

Antibodies against merokeratin from sheep wool decorate cytokeratin filaments in non-keratinizing epithelial cells

Merokeratin is an easily soluble proteolytic derivative of mature alpha-keratin. Guinea pig antibodies have been raised to merokeratin prepared from sheep wool. These antibodies decorate in immunofluorescence microscopy arrays of bundles of intermediate sized filaments present in established epithelial cell lines growing in culture. Thus, the highly helical soluble proteolytic fragments of mature alpha-keratin contains antigenic determinants shared by the cytokeratins present in non-epidermal cells, and antibodies to these keratin fragments can be used for the demonstration of at least some cytokeratin-containing structures in other cells and tissues.     

The Effect of Colchicine on the Induction of the Stellate Configuration in Dorsal Iris Epithelial Cells of the Newt Notophthalmus viridescens, in vitro

Neuroblastoma cells, grown in monolayer, transform, emit cytoplasmic processes, and acquire morphological and functional properties resembling those of mature neurons, whereas in suspension culture they remain in the undifferentiated anaplastic form. The appearance of intermediate (10 nm) filamentous structures in neuroblastoma cells is generally considered to indicate a state of cellular differentiation, one of a progressive sequence of maturing phases which lead the cell to the final differentiated state.
We have examined by electron microscope murine C 1300 neuroblastoma cloned cells, grown in suspension or in monolayer cultures in the presence or absence of BrdU as an inducing agent and have compared the expression of intermediate filaments. These filaments were present in five clones of cells grown in suspension still in undifferentiated anaplastic form. One clone in particular showed a massive expression of filaments, particularly visible in the perinuclear region. One hundred per cent of the cells observed presented filaments whose number apparently increased when cells were grown in the presence of BrdU in suspension or in monolayer. One clone never showed intermediate filaments under any circumstances. The original line from which clones were derived showed poor expression of filaments which were visible only in cells grown in monolayer. These results suggest that the expression of intermediate filaments in neuroblastoma cells should be viewed as the result of a positive genetic control of phenotype expression rather than the result of a progressive sequence of differentiating events.     

痘苗病毒的复制与中间纤维的关系

应用细胞选择性抽提与核酸点杂交技术业示,痘苗病毒的DNA 存在于中间纤维-核纤层-核基质组分(IF-L-NM)中。进而用DGD包埋-去包埋和电镜放射自显影技术相结合,结果表明新合成的病毒的DNA 优先结合在中间纤维上。在此基础上采用DNA 与蛋白质的体外吸附杂交实验,进一步说明痘苗病毒DNA序列与中间纤维及某些核基质蛋白质有较高的亲和性。