Characterization of the collagen in the hexagonal lattice of Descemet's membrane: its relation to type VIII collagen

To investigate the nature of the hexagonal lattice structure in Descemet's membrane, monoclonal antibodies were raised against a homogenate of bovine Descemet's membranes. They were screened by immunofluorescence microscopy to obtain antibodies that label Descement's membrane. Some monoclonal antibodies labeled both Descemet's membrane and fine filaments within the stroma. In electron microscopy, with immunogold labeling on a critical point dried specimen, the antibodies labeled the hexagonal lattices and long-spacing structures produced by the bovine corneal endothelial cells in culture; 6A2 antibodies labeled the nodes of the lattice and 9H3 antibodies labeled the sides of the lattice. These antibodies also labeled the hexagonal lattice of Descemet's membrane in situ in ultrathin frozen sectioning. In immunofluorescence, these antibodies stained the sclera, choroid, and optic nerve sheath and its septum. They also labeled the dura mater of the spinal cord, and the perichondrium of the tracheal cartilage. In immunoblotting, the antibodies recognized 64-kD collagenous peptides both in tissue culture and in Descemet's membrane in vivo. They also recognized 50-kD pepsin-resistant fragments from Descemet's membranes that are related to type VIII collagen. However, they did not react either in immunoblotting or in immunoprecipitation with medium of subconfluent cultures from which type VIII collagen had been obtained. The results are discussed with reference to the nature of type VIII collagen, which is currently under dispute. This lattice collagen may be a member of a novel class of long-spacing fibrils.     

Immunoelectronmicroscopic localization of extracellular matrix components produced by bovine corneal endothelial cells in vitro

Bovine corneal endothelial cells deposit an extracellular matrix in short-term cultures, which contains various morphologically distinct structures when analysed by electron microscopy after negative staining. Amongst these were long-spacing fibers with a 150 nm periodicity, which appeared also to be assembled into more complex hexagonal lattices. Another structure was fine filaments, 10-40 nm in diameter, which occasionally exhibited 67 nm periodic cross-striation. Non-striated 10-20 nm filaments sometimes formed radially oriented bundles arranged in networks and fuzzy granular material was associated with the filaments in the bundles. Often, these bundles extended into solitary filaments, 10-20 nm in diameter, with a smooth surface. In addition, amorphous patches were seen, which contained dense aggregates of fibrillar and granular material. In longer-term cultures, some of the structures coalesced to form large fibrillar bundles. By using specific antibodies to various extracellular matrix components and immunolabeling with gold some of these structures could be identified as to their protein composition. Whereas fibronectin antibodies labeled a variety of structures--fine filaments with granular materials, radially oriented bundles, patchy amorphous aggregates and small granular material scattered throughout the background--type III collagen antibody predominantly labeled filaments with periodic banding (10-40 nm in diameter). A small amount of type III specific labeling was also observed over the networks of radially oriented fibrils and fine filaments associated with granular material. Type IV collagen and laminin antibodies localized in areas of the patchy amorphous aggregates. Type VI collagen antibodies, on the other hand, labeled fine filaments and the gold particles showed a pattern of 100 nm periodicity. Many of the fine 10-20 nm filaments exhibited a tubular appearance on cross-section, but they were not reactive with any of the antibodies used. Also negative were the long-spacing fibers and assemblies--including hexagonal lattices--containing this structural element.     

Improved preservation and staining of HeLa cell actin filaments, clathrin-coated membranes, and other cytoplasmic structures by tannic acid-glutaraldehyde-saponin fixation

Fixation of HeLa cells with a mixture of 100 mM glutaraldehyde, 2 mg/ml tannic acid and 0.5 mg/ml saponin allows the tannic acid to penetrate intact cells without disruption of membranes or extraction of the cytoplasmic matrix. After subsequent treatment with OsO4 cytoplasmic structures are stained so densely that fine details are visible even in very thin (dark gray) sections. Actin filaments are protected from disruption by OsO4 so that straight, densely stained filaments are seen in the cell cortex, filopodia, ruffling membranes, and stress fibers. Stress fibers also have 15-18-nm densities similar in appearance to myosin filaments. Tannic acid staining reveals that the coats of coated vesicles, pits, and plaques have a 12-nm layer of amorphous material between the membrane and the clathrin basketwork. HeLa cells have very large clathrin-coated membrane plaques on the basal surface. These coated membrane plaques appear to be a previously unrecognized site of cell-substrate adhesion.     

Replica immunoelectron microscopic study of the upper surface layer in rat mandibular condylar cartilage by a quick-freezing method

A quick-freezing and deep-etching method in combination with replica immunoelectron microscopy was applied for examining localization of hyaluronic acid and fibronectin on the upper surface layer of rat mandibular condylar cartilage. Rat temporomandibular joints were dissected with articular disks in order to leave the articular cartilage surface intact. The disks were slightly cut with razor blades for exposing the condylar articular cartilage surface. They were quickly frozen with the isopentane-propane cryogen (–193°C) and prepared for freeze-fracturing and deep-etching replica membranes. They were additionally treated with 5% SDS and 0.5% collagenase to keep some antigens attached on the replica membranes. After such a treatment, a routine immunogold method was applied for clarifying the localization of hyaluronic acid and fibronectin in the upper surface layer. Small immunogold particles for hyaluronic acid were mainly localized around upper filamentous networks covered with amorphous materials, but large immunogold ones for fibronectin were localized on deep thicker fibrils. We have revealed the native architecture of the upper surface layer of mandibular condylar cartilage on the replica membranes and also three-dimensional localization of hyaluronic acid and fibronectin by the immunogold method.     

Paracrystalline structures in the epithelial principal cells of the epididymis of water buffalo (Bubalus bubalis).

The ultrastructure of many principal cells in the cauda epididymis of water buffaloes with ages varying between 4, 18, 24, 30 and 36 months, revealed, in many cells, the presence of long, curved paracrystalline structures that are quite large and frequently encountered in the cytoplasm, usually near the nucleus. Concomitantly or not, smaller rod-like, hexagonal or curled structures can be found in the nucleus. Both structures, cytoplasmic and intranuclear, are made up of a sheath of parallel filaments. These paracrystals may appear as thin, regularly spaced filaments that are associated with fine, evenly spaced subunits. Occasionally, the association of paracrystalline structures with membranes similar to the endoplasmic reticulum was observed, but no membranes were consistently found in close contact with the nuclear crystalloids. It is postulated that both structures are proteinaceous and may represent stored enzymes or substances present in the intraluminal fluid, which are absorbed and initially stored in numerous intraepithelial vacuoles of the corpus and cauda of the buffalo epididymis.     

Lumenal plasma membrane of the urinary bladder. II. Isolation and structure of membrane components

A technique has been devised for isolation of lumenal plasma membranes from transitional epithelial cells lining the urinary bladder in rabbits and for subsequent separation of particle-bearing plaque regions from particle-free areas of the membranes. The success of the procedures employed and their effects on the isolates were assessed by electron microscopy of conventional plastic sections, negatively stained preparations, and freeze-etch replicas. When bladders are distended with a solution of 0.01 M thioglycolic acid, which reduces sulfhydryl bridges, cytoplasmic filaments are disrupted, and large segments of the lumenal membranes rupture and float free into the lumen. A centrifugation procedure was developed for isolating a fraction enriched with the large fragments. A comparison of membranes isolated in the presence of thioglycolate with those isolated from epithelial cells homogenized in sucrose medium indicates that thioglycolate has little effect on their fine structure except for the removal of filaments which are normally associated with their cytoplasmic surface. The curved plaques of hexagonally arrayed particles and the particle-free interplaque regions, both characteristic of membranes before exposure to thioglycolate, are well preserved. Subsequent treatment of thioglycolate-isolated lumenal membranes with 1% sodium desoxycholate (DOC) severs many of the interplaque regions, releasing individual plaques in which the particles are more clearly visible than before exposure to desoxycholate. Presumably, DOC acts by disrupting the hydrophobic bonds within the membrane; therefore, this type of cohesive force probably is a major factor maintaining the structural integrity of interplaque regions. This conclusion is consistent with the observation that interplaque regions undergo freeze-cleaving like simple bilayers with a plane of hydrophobic bonding.     

Virus-Induced Cytoplasmic Membrane Structures Associated with Semliki Forest Virus Infection Studied by the Freeze-Etching Method

Intracellular membrane structures associated with the Semliki Forest virus replication process were studied from freeze-etch replicas. Cleaved membrane structures inside the CPV I type vacuoles lacked the typical membrane particles present on most other fractured membranes. CPV II type vacuoles present in thin sections were obscured in the freeze-etch replicas by the cytoplasmic ground substance.     

Cytoplasmic organization in cerebellar dendritic spines

Three sets of filamentous structures were found to be associated with synaptic junctions in slices of cerebellar tissue prepared by rapid- freezing and freeze-etch techniques. The electron-dense fuzz subjacent to postsynaptic membranes corresponds to a web of 4-6-nm-diam filaments that were clearly visualized in rapid-frozen, freeze-etched preparations. Purkinje cell dendritic spines are filled with a meshwork of 5-7-nm filaments that were found to contact the spine membrane everywhere except at the synaptic junction, and extend through the neck of the spine into the parent dendrite. In addition, 8-10-nm microfilaments, possibly actin, were seen to be associated with the postsynaptic web and to extend into the body and neck of the spine. The arrangements and attachments of the filamentous elements in the Purkinje cell dendritic spine may account for its shape.     

Ultrastructural study of upper surface layer in rat mandibular condylar cartilage by quick-freezing method

The purpose of the present study is to clarify native ultrastructures of upper surface layers of the rat mandibular condylar cartilage in vivo by a quick-freezing method. The mandibular cartilaginous tissues were removed with their articular discs attached without opening the lower joint cavity. The specimens were processed for light microscopy, transmission or scanning electron microscopy. Deep-etching replica membranes were also prepared after the routine quick-freezing method. The upper surface layer was well preserved by the quick-freezing method. The cartilaginous tissues, which were fixed without opening their articular discs, appeared to keep better morphology than those after opening them. The upper surface layer was thicker than the corresponding layer as reported before. It consisted of atypical extracellular matrices with lots of apparently amorphous components, which were distributed over typical collagen fibrils, by conventional electron microscopy. As revealed with the replica membranes, it also consisted of variously sized filaments and tiny granular components localized on the typical collagen fibrils. A pair of stereo-replica electron micrographs three-dimensionally showed compact filaments within the upper surface layer. The quick-freezing method was useful for keeping native ultrastructures of the fragile upper surface layer in the mandibular condylar cartilage, which may be functionally important to facilitate smooth movement of the temporomandibular joint.     

Immunogold quantitation of laminin, type IV collagen, and heparan sulfate proteoglycan in a variety of basement membranes

A series of basement membranes was immunolabeled for laminin, type IV collagen, and heparan sulfate proteoglycan in the hope of comparing the content of these substances. The basement membranes, including thin ones (less than 0.3 micron) from kidney, colon, enamel organ, and vas deferens, and thick ones (greater than 2 micron), i.e., Reichert's membrane, Descemet's membrane, and EHS tumor matrix, were fixed in formaldehyde, embedded in Lowicryl, and treated with specific antisera or antibodies followed by anti-rabbit immunoglobulin bound to gold. The density of gold particles, expressed per micron2, was negligible in controls (less than or equal to 1.1), but averaged 307, 146, and 23, respectively, for laminin, collagen IV, and proteoglycan over the thick basement membranes (except for Descemet's membranes, over which the density was 16, 5, and 34, respectively) and 117, 72, and 64, respectively, over the lamina densa of the thin basement membranes. Lower but significant reactions were observed over the lamina lucida. Interpretation of the gold particle densities was based on (a) the similarity between the ultrastructure of most thick basement membranes and of the lamina densa of most thin basement membranes, and (b) the biochemical content of the three substances under study in the EHS tumor matrix (Eur J Biochem 143:145, 1984). It was proposed that thick basement membranes (except Descemet's) contained more laminin and collagen IV but less heparan sulfate proteoglycan than the lamina densa of thin basement membranes. In the latter, there was a fair variation from tissue to tissue, but a tendency towards a similar molar content of the three substances.     

Fluorescence-mediated visualization of actin and myosin filaments in the contractile membrane-cytoskeleton complex of Dictyostelium discoideum

An intact complex that consisted of the cell membrane and cytoskeleton was prepared from Dictyostelium amoebae by an improved version of the method previously used by CLARKE et al. (1975). Proc. Natl. Acad. Sci. USA., 72: 1758-1762. After cells had attached tightly to a polylysine-coated coverslip in the presence of a divalent cation, the upper portions of the cells were removed with a jet of microfilament-stabilizing solution squirted from a syringe. The cell membranes left on the coverslip were immediately stained with tetramethylrhodamine-conjugated phalloidin for staining of actin filaments, and with antibody against myosin from Dictyostelium and a fluorescein-conjugated second antibody for staining of myosin. Networks of actin filaments and numerous rod-like structures of myosin (myosin filaments) aligned along them were observed on the exposed cytoplasmic surfaces of the cell membranes. These networks were similar to those observed in the cortex of fixed whole cells. Addition of ATP to these intact complexes of cell membrane and cytoskeleton caused the aggregation of both actin and myosin into several dot-like structures of actin on the cell membrane. Similar dot-like structures were also seen in the cortex of fixed whole cells, and their changes in distribution correlated with the motile activity of the cells. Transmission electron microscopy showed that these dot-like structures were composed of an electron-dense structure at the center, from which numerous actin filaments radiated outwards. These observations suggest that these novel dot-like structures are organizing centers for cortical actin filaments and may possibly be related to the adhesion of cells to the substratum.     

The association of amorphous mineral deposits with the plasma membrane of pre- and young odontoblasts and their relationship to the origin of dentinal matrix vesicles in rat incisor teeth.

Young and preodontoblasts and matrix vesicles which occur in the presecretory region of incisor teeth of growing rats were examined in stained and unstained ultrathin sections in order to characterize sites involved in the initial mineralization of dentin. Common to pre- and young odontoblasts in the presecretory region were hemispherical membrane-associated amorphous densities, measuring 5-35 nm in diameter after fixation in glutaraldehyde-osmium tetroxide or glutaraldehyde only. Amorphous densities were associated also with the limiting membranes of some vesicles in the extracellular matrix. Other vesicles in the extracellular matrix contained needle-like crystalline deposits typical of dentinal matrix vesicles. Fully differentiated odontoblasts in more incisal regions of the tooth lacked plasma membrane-associated amorphous densities. Neither amorphous nor crystalline densities were associated with any other cellular or subcellular structures in cells of the presecretory region. Flotation of ultrathin sections on solutions of EDTA or EGTA removed the amorphous densities from the plasma membranes, suggesting that the amorphous densities are calcium-containing mineral deposits. Amorphous deposits were associated with the membrane of vesicular structures protruding from the surfaces of pre- and young odontoblasts, suggesting that vesicles found in the extracellular matrix arise by budding from the plasma membranes of pre- and young odontoblasts. The occurrence of amorphous mineral deposits in association with the limiting membrane of some vesicles in the extracellular matrix, and the occurrence of needle-like mineral crystals within other matrix vesicles, suggest that an amorphous-to-crystalline phase transformation of mineral takes place within the matrix vesicle. The results of this study suggest that calcium-binding sites associated with plasma membranes of pre- and young odontoblasts act as nucleating centers for primary mineral deposition in tooth dentin.     

Deposition and ultrastructural organization of collagen and proteoglycans in the extracellular matrix of gel-cultured fibroblasts

Human skin fibroblasts were cultivated within the three-dimensional space of polymerized alginate and collagen, respectively. The in vitro synthesis of collagens and proteoglycans was measured during the first 3 days of culture, and the deposition as well as the ultrastructural organization of newly synthesized extracellular matrix components were examined by electron microscopy. The amount of collagens and proteoglycans synthesized by fibroblasts, embedded in calcium alginate gels as well as in collagen lattices, was lowered as compared to monolayer cultures. Furthermore, it was found that collagen synthesis was reduced to a greater extent in alginate gels than in collagen lattices. On the contrary, total proteoglycan biosynthesis was similarly reduced either in alginate gels or in collagen lattices. At the end of a 3-day-culture period, filamentous material as well as cross-striated banded structures were found extracellularly in the alginate gel. According to their periodicity, their banding pattern, their association with polyanionic matrix components and their sensitivity towards glycosaminoglycan-degrading enzymes we could distinguish (1) sheets of amorphous non-banded material consisting of irregularly arranged filaments and containing dermatan sulfate-rich proteoglycans (type I structures), (2) sheets of long-spacing fibrils consisting of parallel orientated filaments and containing chondroitin sulfate-rich proteoglycans (= zebra bodies; type II structures), and (3) fibrillar structures with a complex banding pattern different from that of native collagen fibrils (type III structures). In fibroblasts cultured in collagen lattices, we only sporadically found depositions which are identified as type I structures. Using indirect immunoelectron microscopy and monospecific polyclonal antibodies, we localized type VI collagen in type I structures and type II structures. Type III structures can be identified as type I collagen derived as becomes obvious by comparison with segment long spacing crystallites of type I collagen.     

MEMBRANE LESIONS IN IMMUNE LYSIS : Surface Rings, Globule Aggregates, and Transient Openings

It is known that there are 100 Å-wide circular structures associated with the erythrocyte membrane in immune lysis. To determine whether these structures were functional holes extending through the membrane, freeze-etch electron microscopy was carried out. Sheep erythrocytes incubated with either rabbit complement or rabbit antibody (anti-sheep erythrocyte antibody) did not hemolyze and did not reveal any abnormalities in freeze-etch or negative-stain electron microscopy. Erythrocytes incubated with both complement and antibody revealed rings on the extracellular surface (etch face) of the cell membrane. Allowing for the 30 Å-thick Pt/C replica, the dimensions of the surface rings were similar to those seen by negative staining. The ring's central depression was level with the plane of the membrane; some rings were closed circles, others were crescent shaped. The cleavage face of the extracellular leaflet revealed globule aggregates, each aggregate appearing to be composed of about four fused globules. The cleavage face of the cytoplasmic leaflet was normal. When immune lysis was carried out in the presence of ferritin, ferritin was subsequently detected in all lysed erythrocytes. If ferritin was added after immune lysis was complete, only 15% of the cells were permeated by ferritin, indicating that transient openings exist in the cell membrane during immune lysis. No abnormal structures were detected when C6-deficient rabbit serum was used as a source of complement. It is concluded that antibody and complement produce surface rings, prelytic leakage of K⁺, colloid osmotic swelling, membrane disruption, and membrane resealing; the surface rings persist after these events.     

Topographical difference of cytoskeletal organization in smooth muscle cells of rat duodenum revealed by quick-freezing and deep-etching method

The sarcolemmal domain of rat duodenal smooth muscle cells includes caveolae and associated cytoskeletal or filamentous elements. We have used the quick-freezing, deep-etching method to examine the three dimensional relationships between these components. Replica membranes for separated strips of rat duodenal muscle layers were routinely prepared after extraction soluble proteins from cytoplasm and extracellular matrix. As results, 1) cytoskeletal elements in smooth muscle cells consisted mainly of striated thin filaments; 2) thin filaments were connected with some plasma membranes through filaments associated with the sarcolemma, which formed fine network structures beneath the sarcolemma; 3) many bridging structures between the filaments associated with the sarcolemma and the extracellular matrix were frequently detected in the plasma membrane; and 4) compact filaments associated with the sarcolemma almost disappeared near the caveolae, and only thin filaments were anchored to their neck parts. The special arrangement of the cytoskeletal components, which is probably necessary for the intestinal motility, characterizes the topographical difference of the smooth muscle sarcolemma.     

Ultrastructural observations of astrocyte end-feet in the rat central nervous system

Astrocytic end-feet in the rat CNS were studied by thin-section electron microscopy. Astrocyte processes that enclose neuronal elements extended to blood vessels and the pia mater, where the processes expanded to form end-feet or glial limiting membranes. At the end-feet, cell junctions such as gap junctions and desmosome-like junctions were formed between the astrocyte processes. The end-foot plasma membrane facing the basal lamina was undercoated with electron-dense, layered materials, with an internal substructure of filamentous networks, with which bundles of glial filaments (GFs) appeared to be closely associated via fine filamentous structures, often showing a hemi-desmosome-like appearance. In specimens treated with Triton X-100, the internal substructure of the undercoat was better visualized and the association with GFs was well preserved. At the end-feet, some unique tubular structures were found in spatial relationship to the plasmalemmal undercoat. Plectin visualized by immunofluorescence was localized to astrocytes and their processes, especially at the end-feet facing the pia mater. Immunoelectron microscopy located plectin on fine filamentous structures lying between GFs and the plasmalemmal undercoat. These observations suggest that plasmalemmal undercoats at the astrocyte end-feet may serve as attachment sites of GFs to the plasma membrane and that plectin may be involved in such attachment.     

Ultrastructural study of human glomerular capillary loops with IgA nephropathy using quick-freezing and deep-etching method

Immunoglobulin A (IgA) nephropathy shows great variability regarding the histological features of the lesions of human renal glomeruli. In the present study, the quick-freezing and deep-etching (QF-DE) method was used to analyze the glomerular ultrastructure of biopsied kidney tissues from children with IgA nephropathy. Biopsied renal tissues were routinely prepared for light microscopy, immunofluorescence microscopy, conventional electron microscopy, and replica electron microscopy. The three-dimensional ultrastructure of glomeruli of the kidney was clearly observed by using the QF-DE method. Three layers of glomerular basement membranes, i.e., middle, inner and outer layers, were clearly detected in the replica electron micrographs. The middle layer was 343.0+/-24.2 nm (n=20) in width and formed polygonal meshwork structures. We also observed slit diaphragms, electron-dense mesangial deposits, and increased amounts of mesangial matrix and foot process effacement. Many delicate filaments were found to be distributed from the apical to the bottom portions between neighboring foot processes. The ultrastructural difference between the replica electron micrographs and conventional electron micrographs was found to be especially marked in the appearance of foot processes and connecting filaments between the neighboring foot processes. The examination of extracellular matrix changes, as revealed at high resolution by the QF-DE method, gave us some morphofunctional information relevant to the mechanism of proteinuria with IgA nephropathy.     

Immunoelectron microscopic studies of the ultrastructure of myosin filaments in Dictyostelium discoideum

We have examined the characteristics of myosin in situ in Dictyostelium amoebae. By an improved immunofluorescence method, we previously found rod-like structures that contain myosin, which we call "myosin rods", in amoebae (Yumura. S., and Fukui, Y. (1985) Nature, 314: 194-196). Although we prepared samples for electron microscopy using conventional chemical fixation to clarify the ultrastructure of the myosin rods, we could not find any filamentous structures similar to myosin thick filaments. Therefore, we examined the effects of chemical fixatives on the myosin rods in situ by immunofluorescence staining. When cells were fixed in more than 0.05% glutaraldehyde or more than 1% osmium tetroxide at 4 degrees C, the myosin rods disappeared. These effects did not result from loss of the antigenicity, because a monoclonal myosin-specific antibody was able to react with synthetic myosin filaments treated with 0.5% glutaraldehyde or 2% osmium tetroxide. Cells fixed by the procedure used for immunofluorescence staining were post-fixed with permissible concentrations of chemical fixatives and prepared for examination by transmission electron microscopy. We found discrete filaments of about 12 nm thickness between the microfilaments. These filaments were shown to contain myosin by immunoelectron microscopy with an immunogold probe. These filaments were thinner than synthetic myosin thick filaments formed in vitro in the presence of 10 mM MgCl2, but they were similar to those formed in the presence of 2 mM MgCl2, or under nearly physiological ionic conditions. The images after immunofluorescence and immunogold labeling both suggested that these 12-nm-thick filaments in Dictyostelium amoebae were myosin filaments in situ.     

Alteration of human erythrocyte plasma membranes by perfringolysin O as revealed by freeze-fracture electron microscopy. Studies on Clostridium perfringens exotoxins V.

When human erythrocyte membranes were treated with perfringolysin O (Clostridium perfringens theta-toxin) and examined by electron microscopy after freeze-fracture, two ultrastructural alterations were observed in fracture faces of membrane. (1) A random aggregation of intramembranous particles was seen in the fracture face of the protoplasmic half (PF face) of all membranes treated with the toxin, even if at a low concentration (40 hemolytic units/ml). On the other hand, the aggregation in the fracture face of the exoplasmic half (EF face) was observed only in membranes treated with a high concentration (3300 hemolytic units/ml) for 2 h. (2) Round protrusions and "cavities" with 30 nm in diameter were visible in EF and PF faces of membranes treated with a high concentration, respectively. These structures were always protruded toward cytoplasmic side, but did not appear to form holes through the membrane. Ring and arc shaped structures with a dark center of 26 nm and a distinct border of 5 nm in width were observed when the toxin alone was negatively stained at a very high concentration (170,000 hemolytic units/ml). These structures were also produced in the presence of cholesterol even if the toxin concentration was low.     

The electron microscopy of the human hair follicle. I. Introduction and the hair cortex

1. The presumptive cortical cells of hair in the undifferentiated matrix of the bulb contain mitochondria, agranular vesicles, and many small dense R.N.P. particles, but no keratin, pigment granules, or endoplasmic reticulum. 2. In the mid-bulb region intercellular adhesion is limited to small localised areas. Intercellular gaps are common and the cell surfaces are irregularly convoluted. The melanocyte processes penetrate the cell gaps. The relation between their pigment-bearing tips and the involutions of the cell membranes suggests an active phagocytosis of the tips. 3. Fibrous keratin first appears in loose parallel strands of fine filaments (ca. 60 A diameter) in the mid-bulb. The filaments, the long mitochondria, and elongated nucleus are all parallel to the long axis of the cell and the axis of the follicle. 4. At the level of the constriction of the bulb and above, a dense amorphous substance appears between the fine filaments and apparently acts as adhesive cement. The bundles of filaments now form well defined fibrils. The packing of the filaments within the fibrils is in places hexagonal and elsewhere in the form of "whorls." 5. At higher levels further filaments and interfilamentous cement are added together and the whole cytoplasmic space becomes packed with fibrils which finally condense to massive blocks of keratin. The residual cellular material occupies the interstices. 6. The addition of the interfilamentous substance is regarded as an essential factor in keratinisation. Keratin is considered to be a complex made of fine filaments (alpha-filaments) embedded in an amorphous substance (gamma-keratin) which has the higher cystine content. 7. The wide-angle fibre-type x-ray pattern is thought to be due to scattering by the fine alpha-filaments and some low angle lateral spacings to the filament-plus-cement structure.