Cytoplasmic filaments in the endothelial cells of the sheathed capillary: an ultrastructural and immunocytochemical study in the pig spleen.

Cytoplasmic filaments of the endothelial cells of sheathed capillaries in the pig spleen were identified and their ultrastructure was studied. Two types of cytoplasmic filaments were found: intermediate filaments (diameter: 10 nm) which filled most of the interior of the cells, and thin filaments (diameter: 5 nm) which were located just beneath the cell membrane and filled the lateral cytoplasmic processes. In immunocytochemical preparations, the intermediate filaments were positive for vimentin and desmin, and were negative for keratin. Staining of the thin filaments with heavy meromyosin resulted in arrowhead formations. These observations suggest that the intermediate filaments maintain the cytoarchitecture, possibly protecting the cell from structural alterations induced by blood pressure changes. Concurrently, thin filaments may facilitate the passage of red blood cells and blood platelets through the interendothelial fenestrae of the sheathed endothelial cell to the reticular meshwork in the capillary sheath.     

上皮细胞分裂过程中中等纤维的变化

Immunofluorescence microscopy was used to follow the rearrangement of keratin filaments and vimentin filaments during mitosis in Vero and HeLa cell lines. The experiment results showed that the three dimensional organization and structure of intermediate filaments changed drastically during mitosis. The behavior of intermediate filaments was different in these two epithelial cell lines. In mitotic Vero cells the keratin filaments and vimentin filaments maintained their filamentous structure and formed a cage around the mitotic apparatus. In mitotic HeLa cells the keratin filaments and vimentin filaments reorganized extensively and formed granular cytoplasmic bodies. The ratio of granular cytoplasmic body formation changed in different mitotic phase. The interphase intermediate filament network was reconstructed after mitosis. It is proposed that the state of intermediate filament network in these cells is cell cycle-dependent and intermediate filaments may have some skeletal role in mitosis.     

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.     

上皮细胞分裂过程中中等纤维的变化

应用制备的血清抗体,采用免疫细胞化学方法观察了两株培养上皮细胞的分裂过程中IF的动态变化过程。实验结果显示,在上皮细胞分裂过程中,IF形态结构及空间分布发生了显著变化,不同细胞之间存在差异,分裂的Vero细胞中角蛋白纤维和波形纤维都维持纤维形态,围绕分裂器形成纤维网罩或纤维束环,随着细胞分裂的进行,IF网的空间组织结构和外观发生动态变化;分裂的HeLa细胞中,角蛋白纤维和波形纤维广泛重组形成颗粒状胞质小体,分裂结束后重建IF网。实验结果表明,IF变化具有细胞周期依赖性和一定的细胞特异性。本文对IF在细胞分裂过程中的功能意义作了讨论。     

Intermediate (skeletin) filaments in heart Purkinje fibers. A correlative morphological and biochemical identification with evidence of a cytoskeletal function

Cow Purkinje fibers contain a population of free cytoplasmic filaments which consistently differ in ultrastructural appearance from actin and myosin filaments, irrespective of preparation technique. The fixation and staining techniques, however, influenced the filament diameter, which was found to be 7.4--9.5 nm for filaments in plastic-embedded material, and 7.0 nm in cryo-sectioned material, thus intermediate as compared to actin and myosin filaments. Cross-sectional profiles suggested that the intermediate-sized filaments are composed of four subfilaments. To provide a basis for further biochemical investigations on the filaments, extraction procedures were carried out to remove other cell organelles. Electron microscopy showed that undulating bundles of intermediate filaments converging towards desmosomes still remained, after the extractions, together with Z-disk material. In spite of the extensive extraction, the shape of the individual cells and the assemblies of cell bundles remained intact. This confirms that the intermediate filaments of cow Purkinje fibers together with desmosomes do in fact have a cytoskeletal function. On account of (a) the cytoskeletal function of the filaments, (b) the similarities to the smooth muscle "100-A filament" protein subunit skeletin, and (c) the inadequate and confusing existing terminology, we suggest that the filaments be named "skeletin filaments."     

Characterization of alpha-actinin from Acanthamoeba

Characterization of a protein from Acanthamoeba that was originally called gelation protein [T.D. Pollard, J. Biol. Chem. 256:7666-7670, 1981] has shown that it resembles the actin filament cross-linking protein, alpha-actinin, found in other cells. It comprises about 1.5% of the total amoeba protein and can be purified by chromatography with a yield of 13%. The native protein has a molecular weight of 180,000 and consists of two polypeptides of 90,000 Da. The Stokes' radius is 8.5 nm, the intrinsic viscosity is 0.35 dl/dm, and the extinction coefficient at 280 mm is 1.8 X 10(5)M-1 X cm-1. Electron micrographs of shadowed specimens show that the molecule is a rod 48 nm long and 7 nm wide with globular domains at both ends and in the middle of the shaft. On gel electrophoresis in sodium dodecylsulfate the pure protein can run as bands with apparent molecular weights of 60,000, 90,000, 95,000, or 134,000 depending on the method of sample preparation. Rabbit antibodies to electrophoretically purified Acanthamoeba alpha-actinin polypeptides react with all of these electrophoretic variants in samples of purified protein and cell extracts. By indirect fluorescent antibody staining of fixed amoebas, alpha-actinin is distributed throughout the cytoplasmic matrix and concentrated in the hyaline cytoplasm of the cortex. The protein cross-links actin filaments in the presence and absence of Ca++. It inhibits slightly the time course of the spontaneous polymerization of actin monomers but has no effect on the critical concentration for actin polymerization even though it increases the apparent rate of elongation to a small extent. Like some other cross-linking proteins, amoeba alpha-actinin inhibits the actin-activated ATPase of muscle myosin subfragment-1. Although Acanthamoeba alpha-actinin resembles the alpha-actinin from other cells in shape and ability to cross-link actin filaments, antibodies to amoeba and smooth muscle alpha-actinins do not cross react and there are substantial differences in the amino acid compositions and molecular dimensions.     

The Supramolecular Organization of the C. elegans Nuclear Lamin Filament

Nuclear lamins are involved in most nuclear activities and are essential for retaining the mechano-elastic properties of the nucleus. They are nuclear intermediate filament (IF) proteins forming a distinct meshwork-like layer adhering to the inner nuclear membrane, called the nuclear lamina. Here, we present for the first time, the three-dimensional supramolecular organization of lamin 10 nm filaments and paracrystalline fibres. We show that Caenorhabditis elegans nuclear lamin forms 10 nm IF-like filaments, which are distinct from their cytoplasmic counterparts. The IF-like lamin filaments are composed of three and four tetrameric protofilaments, each of which contains two partially staggered anti-parallel head-to-tail polymers. The beaded appearance of the lamin filaments stems from paired globular tail domains, which are spaced regularly, alternating between 21 nm and 27 nm. A mutation in an evolutionarily conserved residue that causes Hutchison-Gilford progeria syndrome in humans alters the supramolecular structure of the lamin filaments. On the basis of our structural analysis, we propose an assembly pathway that yields the observed 10 nm IF-like lamin filaments and paracrystalline fibres. These results serve also as a platform for understanding the effect of laminopathic mutations on lamin supramolecular organization.     

Immunocytochemical demonstration of a new vimentin-associated protein in 3T3 fibroblasts

Summary Using a xanthophore cytoskeletal preparation as immunogen, we have produced a monoclonal antibody, A2, which recognized a 160 kDa protein in 3T3 fibroblasts. This protein makes up a cytoplasmic filamentous system, which colocalizes with vimentin filaments. When microtubules and actin filaments are dissolved by high salt extraction, staining with antibody A2 is unaffected. Immunoblot analysis confirms that the 160 kDa protein is co-isolated with vimentin duringin vivo high salt extraction. Following vinblastine treatment, both the 160 kDa protein and vimentin become localized to perinuclear caps, as do other intermediate filaments and their associated proteins; after vinblastine removal, the immunostaining produced by A2 becomes filamentous. Immunoelectron microscopy demonstrates that antibody A2 stains a filament system with a diameter of about 10 nm. Our observations suggest that the 160 kDa protein may be a new vimentin-associated protein which differs from the intermediate filament-associated proteins previously reported, and is widely distributed in several cell types.     

Immunocytochemical studies of intermediate filament aggregates and their relationship to microtubules in cultured skin fibroblasts from patients with giant axonal neuropathy

Giant axonal neuropathy (GAN) is a severe autosomal recessive disease affecting both the peripheral and central nervous systems. It is characterized by segmental axonal ballooning due to large neurofilamentous masses and abnormal aggregation of filaments in other cell types including glial cells. Coomassie blue staining of the detergent-resistant cytoskeleton of cultured skin fibroblasts from three patients with GAN revealed the presence of large cytoplasmic filamentous aggregates in the great majority of cells. The aggregates were birefringent when viewed under polarization microscopy and electron microscopy showed that they were composed of aggregates of 8 to 10 nm intermediate filaments. The aggregates stained with antisera specific for vimentin but did not stain with antibodies to actin, tubulin, or the high molecular weight (HMW) microtubule associated protein. Examination of the fibroblasts containing the vimentin aggregates with antibodies to tubulin and the HMW protein showed that they had a normal distribution of microtubules and that the microtubules present were normally associated with the HMW protein. The results suggest that giant axonal neuropathy is a generalized inborn error of organization of intermediate filaments and that a defect in microtubules or their association with HMW protein is not responsible for the observed aggregation of intermediate filaments in this disease. Further study of GAN may be useful in understanding the function of intermediate filaments.     

History and phylogeny of intermediate filaments: Now in insects

Intermediate filaments include the nuclear lamins, which are universal in metazoans, and the cytoplasmic intermediate filaments, which are much more varied and form cell type-specific networks in animal cells. Until now, it has been thought that insects harbor lamins only. This view is fundamentally challenged by the discovery, reported in BMC Biology, of an intermediate filament-like cytoplasmic protein, isomin, in the hexapod Isotomurus maculatus. Here we briefly review the history of research on intermediate filaments, and discuss the implications of this latest finding in the context of what is known of their structure and functions.     

THE CONTRACTILE BASIS OF AMOEBOID MOVEMENT : I. The Chemical Control of Motility in Isolated Cytoplasm

Cytoplasm has been isolated from single amoeba (Chaos carolinensis) in physiological solutions similar to rigor, contraction, and relaxation solutions designed to control the contractile state of vertebrate striated muscle. Contractions of the isolated cytoplasm are elicited by free calcium ion concentrations above ca. 7.0 x 10^-7 M. Amoeba cytoplasmic contractility has been cycled repeatedly through stabilized (rigor), contracted, and relaxed states by manipulating the exogenous free calcium and ATP concentrations. The transition from stabilized state to relaxed state was characterized by a loss of viscoelasticity which was monitored as changes in the capacity of the cytoplasm to exhibit strain birefringence when stretched. When the stabilized cytoplasm was stretched, birefringent fibrils were observed. Thin sections of those fibrils showed thick (150–250 Å) and thin (70 Å) filaments aligned parallel to the long axis of fibrils visible with the light microscope. Negatively stained cytoplasm treated with relaxation solution showed dissociated thick and thin filaments morphologically identical with myosin aggregates and purified actin, respectively, from vertebrate striated muscle. In the presence of threshold buffered free calcium, ATP, and magnesium ions, controlled localized contractions caused membrane-less pseudopodia to extend into the solution from the cytoplasmic mass. These experiments shed new light on the contractile basis of cytoplasmic streaming and pseudopod extension, the chemical control of contractility in the amoeba cytoplasm, the site of application of the motive force for amoeboid movement, and the nature of the rheological transformations associated with the circulation of cytoplasm in intact amoeba.     

Reduction of density and anisotropic distribution of intermediate filaments occur during avian skeletal myogenesis

Chicken skeletal muscle taken from embryos in ovo was examined by thin-section electron microscopy. Measurements of filament diameters reveal three nonoverlapping groups of filaments: thin (actin myofibrillar) filaments with mean diameters of 5.3 +/- 0.6 nm (S.D.), thick (myosin myofibrillar) filaments with mean diameters of 15 +/- 1.4 nm, and intermediate filaments with mean diameters of 9.3 +/- 0.9 nm. During muscle development these diameters do not change. By counting the number of filaments observed in the sarcoplasm at different stages, we find that the spatial density of intermediate filaments decreases during avian myogenesis in ovo, from 91 intermediate filaments/micron 2 at 6 days to 43 intermediate filaments/micron 2 at 17 days in ovo. Initially randomly arranged, some intermediate filaments become associated with Z discs, sarcoplasmic reticulum, nuclear membrane, and the sarcolemma between 6 and 10 days in ovo. These associated intermediate filaments course both parallel and transverse to myofibrils, forming lateral connections between myofibrillar Z discs and longitudinal connections from Z disc to Z disc within myofibrils. Intermediate filaments also appear to connect Z discs with the nuclear membrane. The intermediate filament associations persist through day 17 of development, after which the presence of cytoskeletal filaments is obscured by the densely packed myofibrils and membranes. Intermediate filament distribution becomes anisotropic during development. A greater proportion of intermediate filaments in the immediate perimyofibrillar area are oriented parallel to myofibrils than in other areas, so that the majority of the intermediate filaments nearest the myofibrils course parallel to them. The longitudinal intramyofibrillar intermediate filaments persist throughout development, as shown by their existence in KI-extracted adult myofibrils.     

Fine structural study of the abdominal muscle receptor organs of the crayfish (Procambarus clarkii). Fast and slow receptor muscles

Receptor muscles of the abdominal muscle receptor organs of the crayfish, Procambarus clarkii, were examined by electron microscopy. Both the fast and the slow receptor strand comprises a single muscle fibre which is divided by invagination of the cell membrane into numerous cytoplasmic processes in its intermediate region (the so-called intercalated tendon). Most of these myofibrillar processes insert in this region, but some of them pass through the intermediate region without interruption and join the other portion of the fibre. Thus the receptor muscles, whilst maintaining cytoplasmic continuity throughout their whole length, are modified in their intermediate regions, becoming fasciculated and providing spaces which are occupied by the connective tissue and the dendrites of the sensory neurone. Clear-cut differences in fine structure are shown between the muscle of the two types of receptor unit. The fast receptor muscle shows the typical features of arthropod fast muscles, including short sarcomere length (on average 3.3 μm), cylindrical myofibrils, well-developed sarcoplasmic reticulum, and regular hexagonal array of the myofilaments. By contrast, the slow receptor muscle fibre is characterized by long sarcomeres (average 6.5 μm) and unique organization of the myofilaments, with very thick ‘thick’ filaments having diameters in the range of 25–36 nm surrounded by about 12 thin filaments.     

Detection of dense intra- and perinuclear 10 nm filament systems by whole mount and embedment-free electron microscopy in several species of the green algal order Dasycladales

Summary In contrast to the immense body of evidence supporting the occurrence of intermediate filament (IF) proteins in the animal kingdom, there is only limited information on their distribution in plants. Nevertheless, a number of immunocytochemical and electron microscopical observations indicate that particularly in higher plant cells IFs contribute to the construction of the cyto- and karyoskeleton. Here we show by whole mount electron microscopy of the giant nuclei extruded together with adhering cytoplasm from the rhizoids of some species of the algal order Dasycladales that cytoplasmic 10 nm filament networks also occur in unicellular, mononucleated green organisms of early evolutionary origin. The filament systems were associated with the residual nuclear envelope which consisted of a dense arrangement of pore complexes suspended by a meshwork of short 5 to 6 nm filaments; structurally it was very similar to the nuclear envelopes obtained from mammalian cells. When the Dasycladales nuclei were processed side by side with mouse skin fibroblasts, the algal filament systems were physically almost indistinguishable from the mammalian vimentin filament network. Embedment-free thin sections of rhizoids have not only confirmed the existence of the perinculear 10 nm filaments and their seamless association with the nuclear envelope, but have demonstrated the existence of an extensive intranuclear meshwork of 10 nm filaments. The latter were morphologically indistinguishable from the perinuclear 10 nm filaments and seem to be connected to these via the nuclear envelope to form a continuum. Among a variety of antibodies directed against mammalian IF proteins, only polyclonal anti-mouse lamin B antibodies decorated the cytoplasmic filaments of the Dasycladales cells. Surprisingly, none of the antibodies decorated the thinner filaments of the nuclear envelope, which possibly represent the nuclear lamina. In accord with this observation, one anti-lamin B antibody recognized in Western blot analysis of a urea extract ofAcetabularia acetabulum rhizoids three polypeptides with M_rs of approximately 47,000, 64,000, and 76,000. The proteins did not react with the -IFA antibody. Since the Dasycladales have a fossil record of nearly 600 million years — an extant genus, Acicularia, also investigated here, evolved about 170 million years ago -, the molecular characterization of the subunit proteins of their cytoplasmic filament systems might throw further light on the evolution and biological role of IFs.Dedicated to Professor Sir Henry Harris on the occasion of his 70th birthday     

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 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.     

Mechanism of interaction of Acanthamoeba actophorin (ADF/Cofilin) with actin filaments.

We characterized the interaction of Acanthamoeba actophorin, a member of ADF/cofilin family, with filaments of amoeba and rabbit skeletal muscle actin. The affinity is about 10 times higher for muscle actin filaments (Kd = 0.5 microM) than amoeba actin filaments (Kd = 5 microM) even though the affinity for muscle and amoeba Mg-ADP-actin monomers (Kd = 0.1 microM) is the same (Blanchoin, L., and Pollard, T. D. (1998) J. Biol. Chem. 273, 25106-25111). Actophorin binds slowly (k+ = 0.03 microM-1 s-1) to and dissociates from amoeba actin filaments in a simple bimolecular reaction, but binding to muscle actin filaments is cooperative. Actophorin severs filaments in a concentration-dependent fashion. Phosphate or BeF3 bound to ADP-actin filaments inhibit actophorin binding. Actophorin increases the rate of phosphate release from actin filaments more than 10-fold. The time course of the interaction of actophorin with filaments measured by quenching of the fluorescence of pyrenyl-actin or fluorescence anisotropy of rhodamine-actophorin is complicated, because severing, depolymerization, and repolymerization follows binding. The 50-fold higher affinity of actophorin for Mg-ADP-actin monomers (Kd = 0.1 microM) than ADP-actin filaments provides the thermodynamic basis for driving disassembly of filaments that have hydrolyzed ATP and dissociated gamma-phosphate.     

In vitro assembly properties of mutant and chimeric intermediate filament proteins: insight into the function of sequences in the rod and end domains of IF

The factors and mechanisms regulating assembly of intermediate filament (IF) proteins to produce filaments with their characteristic 10 nm diameter are not fully understood. All IF proteins contain a central rod domain flanked by variable head and tail domains. To elucidate the role that different domains of IF proteins play in filament assembly, we used negative staining and electron microscopy (EM) to study the in vitro assembly properties of purified bacterially expressed IF proteins, in which specific domains of the proteins were either mutated or swapped between a cytoplasmic (mouse neurofilament-light (NF-L) subunit) and nuclear intermediate filament protein (human lamin A). Our results indicate that filament formation is profoundly influenced by the composition of the assembly buffer. Wild type (wt) mouse NF-L formed 10 nm filaments in assembly buffer containing 175 mM NaCl, whereas a mutant deleted of 18 NH2-terminal amino acids failed to assemble under similar conditions. Instead, the mutant assembled efficiently in buffers containing CaCl2 > or = 6 mM forming filaments that were 10 times longer than those formed by wt NF-L, although their diameter was significantly smaller (6-7 nm). These results suggest that the 18 NH2-terminal sequence of NF-L might serve two functions, to inhibit filament elongation and to promote lateral association of NF-L subunits. We also demonstrate that lengthening of the NF-L rod domain, by inserting a 42 aa sequence unique to nuclear IF proteins, does not compromise filament assembly in any noticeable way. Our results suggests that the known inability of nuclear lamin proteins to assemble into 10 nm filaments in vitro cannot derive solely from their longer rod domain. Finally, we demonstrate that the head domain of lamin A can substitute for that of NF-L in filament assembly, whereas substitution of both the head and tail domains of lamins for those of NF-L compromises assembly. Therefore, the effect of lamin A "tail" domain alone, or the synergistic effect of lamin "head" and the "tail" domains together, interferes with assembly into 10-nm filaments.     

Motility in Echinosphaerium nucleofilum. II. Cytoplasmic contractility and its molecular basis

Echinosphaerium nucleofilum exhibits at least three kinds of movement: locomotion by the bending and shortening of its many axopodia, feeding by means of food-cup pseudopodia formed from its cortical cytoplasm, and saltatory motion of cytoplasmic particles, especially in the cortex and axopodia. Since previously presented evidence indicated that the microtubular axoneme is not essential for particle motion, the cytoplasm was investigated for the possible existence of contractile behavior and for the possible presence of linear elements other than microtubules. Cytoplasm can be isolated in physiological media in which rigor, relaxation, and contraction can be induced, as in muscle, by manipulating the concentrations of calcium ions and magnesium-adenosine triphosphate. Contraction is initiated by calcium ions at concentrations above 2.4 times 10-minus 7 M. The rigor-to-relaxation transition occurs at subthreshold calcium concentrations on the addition of 10-minus 3 M ATP. Negatively stained preparations of isolated cytoplasm show two types of filaments: thin filaments identified as cytoplasmic actin by virtue of their binding heavy meromyosin from striated muscle in characteristic arrowhead arrays, and thicker filaments which do not strictly resemble myosin aggregates from muscle or amoeba but could conceivably by myosin aggregated in an unfamiliar form.     

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.