Intermediate filaments: a role in epithelial polarity

Intermediate filaments have long been considered mechanical components of the cell that provide resistance to deformation stress. Practical experimental problems, including insolubility, lack of good pharmacological antagonists, and the paucity of powerful genetic models have handicapped the research of other functions. In single-layered epithelial cells, keratin intermediate filaments are cortical, either apically polarized or apico-lateral. This review analyzes phenotypes of genetic manipulations of simple epithelial cell keratins in mice and Caenorhabditis elegans that strongly suggest a role of keratins in apico-basal polarization and membrane traffic. Published evidence that intermediate filaments can act as scaffolds for proteins involved in membrane traffic and signaling is also discussed. Such a scaffolding function would generate a highly polarized compartment within the cytoplasm of simple epithelial cells. While in most cases mechanistic explanations for the keratin-null or overexpression phenotypes are still missing, it is hoped that investigators will be encouraged to study these as yet poorly understood functions of intermediate filaments.     

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.     

Synemin and vimentin are components of intermediate filaments in avian erythrocytes

Synemin, a high-molecular-weight protein associated with intermediate filaments in muscle, and vimentin, an intermediate-filament subunit found in many different cell types, have been identified by immunologic and electrophoretic criteria as components of intermediate filaments in mature avian erythrocytes. Desmin, the predominant subunit of intermediate filaments in muscle, has not been detected in these cells. Two dimensional immunoautoradiography of proteolytic fragments of synemin and vimentin demonstates that the erythrocyte proteins are highly homologous, if not identical, to their muscle counterparts. Double immunoflurorescence reaveals that erythrocyte synemin and vimentin co-localize in a cytoplasmic network of sinuous filaments that extends from the nucleus to the plasma membrane and resists aggregation by colcemid. Erythrocytes that are attached to glass cover slips can be sonicated to remove nuclei and nonadherent regions of the plasma membrane; this leaves elliptical patches of adherent membrane that retain mats of vimentin- and synemin-containing intermediate filaments, as seen by immunofluorescence and rotary shadowing. Similarly, mechanical enucleation of erythrocyte ghosts in suspension allows isolation of plasma membranes that retain a significant fraction of the synemin and vimentin, as assayed by electrophoresis, and intermediate filaments, as seen in thin sections. Both synemin and vimentin remain insoluble along with spectrin and actin, in solutions containing nonionic detergent and high salt. However, brief exposure of isolated membrane to distilled water releases the synemin and vimentin together in nearly pure form, before the release of significant amounts of spectrin and actin. These data suggest that avian erythrocyte intermeditate filaments are somehow anchored to the plasma membrane; erythrocytes may thus provide a simple system for the study of intermediate filaments and their mode of interaction with membranes. In addition, these data, in conjunction with previous data from muscle, indicate that synemin is capable of associating with either desmin or vimentin and may thus perform a special role in the structure or function of intermediate filaments in erythrocytes as well as muscle.     

Site specificity in vimentin-membrane interactions: intermediate filament subunits associate with the plasma membrane via their head domains

Fragments of vimentin, generated by chemical or enzymatic cleavages, were analyzed for their capacity to bind to human inverted erythrocyte membrane vesicles. Only peptides comprising the amino-terminal head domain of vimentin molecules were competent in associating with the membranes. In vitro studies also demonstrated that isolated ankyrin (the major vimentin acceptor site on the membrane) binds to an oligomeric species of vimentin and prevents the formation of characteristic 10-nm filaments. These data, taken together with the observation that the NH2-terminal end of vimentin is implicated in the polymerization process (Traub, P., and C. Vorgias, J. Cell Sci., 1983, 63:43-67), imply that intermediate filaments may contact the membrane in an end-on fashion, using the exposed head domains of their terminal subunits.     

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.     

辛德毕斯病毒装配及其6K蛋白与中间纤维的关系

用温和的选择性抽提方法与整装细胞电镜技术,DGD包埋-去包埋剂电镜技术相结合,对辛德毕斯病毒的装配与宿主细胞中间纤维的关系进行了探讨。电镜观察清晰地显示了病毒“装配中心”被中间纤维所网络,病毒的装配过程显然是以中间纤维为支架;正在装配的核壳体与已装配的核壳体紧密结合在中间纤维丝上。根据电镜照片分析,核壳体可能是沿中间纤维由装配中心向外扩散。应用人工合成6K蛋白所得抗体进行胶体金标记,证明辛德毕斯病毒非结构性6K蛋白也与中间纤维紧密结合。     

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.     

Intermediate filament structure.

In the past year, several new developments concerning the structure of intermediate filament proteins and their assembly into intact intermediate filaments have been made: the coiled-coil structure of a rod domain has been elucidated; the basis of the chain interaction and its role in intermediate filament assembly has been specified; the organization of nearest-neighbour molecules in keratin intermediate filaments has been determined; and the glycine loop structures of the terminal domains of epidermal keratin chains have been defined. In addition, mutations in intermediate filament chains that promote pathology have been reported for the first time.     

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.     

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.     

Assembly characteristics of plant keratin intermediate filaments in 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. Project supported by the National Natural Science Foundation of China (Grant No. 39370352) and the Doctor Foundation of Minishy of Education of China.     

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.     

Distribution of epinemin in colloidal gold-labelled, quick-frozen, deep- etched cytoskeletons

In this study I describe the ultrastructural distribution of epinemin (Lawson, D., 1983, J. Cell Biol., 97:1891-1905) in antibody-labelled, helium-cooled, quick-frozen, deep-etched cytoskeletons. This technique reveals that epinemin is expressed asymmetrically at discrete sites on the vimentin core polymer and that usually one (occasionally two or three) antiepinemin molecules are found at each of these discrete foci. Single receptor-bound antiepinemin (IgM) molecules are easily identified in deep-etched cytoskeletons by the use of colloidal gold. Epinemin does not cross-link adjacent intermediate filaments and is not associated with the many 2-3-nm filaments found associated with intermediate filaments in these preparations. The directional changes and interactions undergone by microtubules in taxol-stabilized, antibody-labelled cytoskeletons are also discussed.     

Muscle cell attachment in Caenorhabditis elegans

In the nematode Caenorhabditis elegans, the body wall muscles exert their force on the cuticle to generate locomotion. Interposed between the muscle cells and the cuticle are a basement membrane and a thin hypodermal cell. The latter contains bundles of filaments attached to dense plaques in the hypodermal cell membranes, which together we have called a fibrous organelle. In an effort to define the chain of molecules that anchor the muscle cells to the cuticle we have isolated five mAbs using preparations enriched in these components. Two antibodies define a 200-kD muscle antigen likely to be part of the basement membrane at the muscle/hypodermal interface. Three other antibodies probably identify elements of the fibrous organelles in the adjacent hypodermis. The mAb IFA, which reacts with mammalian intermediate filaments, also recognizes these structures. We suggest that the components recognized by these antibodies are likely to be involved in the transmission of tension from the muscle cell to the cuticle.     

Expression of fetal-type intermediate filaments by 17-day-old rat Sertoli cells cultured on reconstituted basement membrane

Summary The expression of cytokeratin- and vimentin-type intermediate filaments was studied by means of immunohistochemistry in Sertoli cells cultured on two types of reconstituted basement membrane in two-compartment culture chambers. In situ, the Sertoli cells of 17-day-old rats contained only vimentin intermediate filaments. During culture, a gradual reorganization of intermediate filaments accompanied by an increased cytokeratin immunoreactivity was observed. After 6 days, Sertoli cells contained both cytokeratin and vimentin, and the same cytokeratin type as in fetal and newborn testis was revealed by electrophoresis and immunoblotting. The present study shows that the isolation and culture of Sertoli cells causes, even in an improved culture system qualitative changes in the expression of intermediate filament proteins.     

Dynamic organisation of intermediate filaments and associated proteins during the cell cycle

Intermediate filaments, which form the structural framework of both the cytoskeleton and the nuclear lamina in most eukaryotic cells, have been found to be highly dynamic structures. A continuous exchange of subunit proteins at the filament surface and a stabilisation of soluble subunits by chaperone-type proteins may modulate filament structure and plasticity. Recent studies on the cell cycle-dependent interaction of intermediate filaments with associated proteins, and a detailed analysis of intermediate filament phosphorylation in defined subcellular locations at various stages of mitosis, have brought new insights into the molecular mechanisms involved in the mitotic reorganisation of intermediate filaments. Some of these studies have allowed new speculations about the possible cellular functions of cytoplasmic intermediate filaments, and increased our understanding of the specific functions of the lamins and the lamina-associated membrane proteins in the post-mitotic reassembly of the nucleus.     

The beaded intermediate filaments and their potential functions in eye lens

The elongated fiber cells of the eye lens contain a unique cytoskeletal system, the beaded chain filaments (BFs). The BFs had been morphologically identified more than two decades ago, but the precise identity of their subunit molecules remained unknown. Recently, use of recombinant DNA approaches, refined morphological and immunochemical studies and experiments with mutant mice have allowed the molecular dissection of these structures and provided clues about their potential functins. The BFs represent a highly specialized network of intermediate filaments (IFs) juxtaposed to the plasma membrane. They are obligate heteropolymers composed of two lens-specific polypeptides, filensin and phakinin. In this review we discuss the properties, molecular interactions and in situ arrangement of these two proteins, and comment on their potential roles during lens development.     

Cytoplasmic filaments in fetal and neonatal pig testis

Leydig cells in developing fetal pig testis contained during the fetal regressive phase large accumulations of intermediate filaments. Before and after this period these filaments were arranged in a criss-cross fashion. In the pig as well as in the dog testis these filaments have been characterized as vimentin. Within the vimentin aggregates occasionally a weak positive actin reaction was seen in pig but not in dog Leydig cells. Microfilaments were hardly observed. Most Sertoli cells contained a layer of actin microfilaments close to the basal cell membrane. In the lower cell compartment and around the nucleus (intermediate) vimentin filaments could be observed in a criss-cross configuration.     

Intermediate filaments and lipoprotein cholesterol

The ability of cells to utilize cholesterol derived from lipoprotein is important in plasma membrane biosynthesis, steroidogenesis and the regulation of sterol synthesis. While the endocytosis of lipoprotein-derived cholesterol has been well characterized, the subsequent events that mediate its post-lysosomal intracellular transport are not understood. Recent studies have suggested that vimentin-type intermediate filaments may have a role in cholesterol transport. The mechanism by which vimentin filaments affect this process is not known, but future studies promise to provide new insights into both the post-lysosomal transport of cholesterol and the intracellular functions of intermediate filaments.     

First ultrastructural differentiation of myoblasts of chicken embryos: appearance of the initial filaments

A study is made of the structure and ultrastructure of myoblasts located in the myotome in chicken embryos, between stages 18 and 19, for electron microscope observation of the occurrence of the first myogenetic filamentous molecules. We suggest a hypothesis for the formation of initial filaments consisting of an initial synthesis of actin globular molecules, carried out in the centre of the myoblast, near the nucleus, with the participation of RNA and the ribosomes. These molecules accumulate peripherically in areas below the plasmatic membrane where they polymerize into actin filamentous molecules which form the initial filaments. These move towards the sharp ends of the myoblast, under the plasmatic membrane, and thence to the interior of the cytoplasm, where they are evenly distributed. This genesis of initial filaments is independent of the influence of the nervous system. We postulate the existence of a single type of myoblast, of fibrillar form, with a dark central area containing the nucleus and the cell organelles, and two sharp, light end zones which contain only the initial filaments in a very light cytoplasmic matrix.