Tectorins crosslink type II collagen fibrils and connect the tectorial membrane to the spiral limbus.

All inner ear organs possess extracellular matrix appendices over the sensory epithelia that are crucial for their proper function. The tectorial membrane (TM) is a gelatinous acellular membrane located above the hearing sensory epithelium and is composed mostly of type II collagen, and α and ß tectorins. TM molecules self-assemble in the endolymph fluid environment, interacting medially with the spiral limbus and distally with the outer hair cell stereocilia. Here, we used immunogold labeling in freeze-substituted mouse cochleae to assess the fine localization of both tectorins in distinct TM regions. We observed that the TM adheres to the spiral limbus through a dense thin matrix enriched in α- and ß-tectorin, both likely bound to the membranes of interdental cells. Freeze-etching images revealed that type II collagen fibrils were crosslinked by short thin filaments (4±1.5nm, width), resembling another collagen type protein, or chains of globular elements (15±3.2nm, diameter). Gold-particles for both tectorins also localized adjacent to the type II collagen fibrils, suggesting that these globules might be composed essentially of α- and ß-tectorins. Finally, the presence of gold-particles at the TM lower side suggests that the outer hair cell stereocilia membrane has a molecular partner to tectorins, probably stereocilin, allowing the physical connection between the TM and the organ of Corti.

Isolation, purification and some ultrastructural details of discharged ejectisomes of cryptophytes.

The first successful isolation of discharged ejectisomes from pigmented cryptophytes is reported. Discharged ejectisomes from a Chroomonas and two Cryptomonas species were characterized by transmission electron microscopy using negative staining and freeze-etching. Tubular-shaped fragments of variable lengths and diameters were obtained which showed a paracrystalline lattice. Particle periodicities of 4.1 nm along the longitudinal axis and 3.1 nm in the transverse direction were measured in negative-stained fragments. The dimensions measured from freeze-etched ejectisome fragments were about 0.5-1 nm larger. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a protein banding pattern, dominated by polypeptides of 40-44, 23-25 and 16-18 kDa. The results are discussed in the context of what is currently known about extrusomes of protists.

Extraskeletal myxoid chondrosarcoma: a study using a quick-freezing and deep-etching method.

A case of extraskeletal myxoid chondrosarcoma (ESMC), which developed in the right thigh of a middle-aged Japanese woman, was studied using immunohistochemistry, conventional electron microscopy, and the quick-freezing and deep-etching (QF-DE) method. In addition to typical light microscopic findings of ESMC, conventional electron microscopy indicated that the tumor cells had features of chondrocytes. Immunohistochemically, the tumor cells showed a positive immunoreaction for S100 protein. A diagnosis of ESMC was made. An interesting observation was the ultrastructural features of collagen fibrils in the myxoid matrix highlighted by the QF-DE method. These collagen fibrils consisted of relatively thin collagen (20-35 nm) with pleated surface structures. The surface striation at 65 nm was obscure. We consider that such a finding of collagen fibrils identified by the QF-DE method is one of the characteristics of the myxoid matrix of ESMC, and this is useful for the differential diagnosis of myxoid soft tissue tumors.

Ultrastructural study of Rac1 and its effectors beneath the substratum-facing membrane.

The cytoskeletal architecture and adhesion apparatus are tightly controlled during embryogenesis, tissue development, and carcinogenesis. The Rho family GTPases play central roles in regulation of the cytoskeleton and adhesions. Rac1, one of the Rho family GTPases, appears to be activated at the plasma membrane and exert its functions through its effectors. However, where Rac1 and its effectors function at the molecular level remains to be determined. In this study, we examined the molecular organization on the cytoplasmic surface of the substratum-facing plasma membrane, focusing on Rac1 and its effectors, IQGAP1 and Sra-1, by electron microscopy. We employed deep-etch immunoreplica methods to observe the membrane cytoskeletal architecture while determining molecular locations. Beneath the plasma membrane, Rac1 and its effectors showed similar, but distinct, destinations. Rac1 localized on the membrane and associated with the membrane cytoskeleton. IQGAP1 predominantly localized beside actin filaments and occasionally near microtubules together with Rac1. On the other hand, Sra-1 localized at actin filaments, microtubules, and the plasma membrane. Sra-1 colabeled with Rac1 was mainly found at the membrane and actin filaments. These results suggest that IQGAP1 and Sra-1 colocalize with Rac1 at distinct places, including the plasma membrane and cytoskeletal architecture, for their specific functions.

Detailed glomerular ultrastructure in Japanese type 2 diabetic patients by the quick-freezing and deep-etching method.

Mesangial expansion and glomerular basement membrane (GBM) thickening did not correlate with urinary albumin excretion (UAE) in type 2 diabetic patients in our previous studies; therefore, it was necessary to elucidate more detailed ultrastructural changes in the early stages of diabetic nephropathy (DN) in type 2 diabetic patients. The quick-freezing and deep-etching (QF-DE) method allows us to examine three-dimensional ultrastructures of human renal glomeruli in vivo at high resolution. The QF-DE method was applied to six type 2 diabetic patients without definable renal diseases other than DN. Four patients were normoalbuminuric (NA) and the other two were microalbuminuria (MA). Three control specimens were the normal parts from nephrectomies due to renal cell carcinomas. Electron microscopic morphometric analyses provided quantitative glomerular structural changes. Replica membranes were prepared by the QF-DE method, and diameters of mesh structures at the GBM and mesangial matrix (MM) were measured on electron micrographs as previously described. By the QF-DE method, both the GBM middle layer and MM were composed of polygonal meshwork structures. The mesh pores of the GBM and MM were more enlarged and irregular in shape in NA diabetic patients than those of the controls, and these ultrastructural changes became more obvious in MA patients. The mesh diameters of the GBM and MM in the diabetic patients were also larger than those of the controls. Such a mesh diameter of the GBM was well correlated with the amount of UAE, while the mesh diameter of MM showed a slight correlation with UAE. Although there were small number of subjects in the present study, the detailed ultrastructural changes in NA and MA type 2 diabetic patients, which had not been disclosed by conventional electron microscopy, were revealed by the QF-DE method. Increased mesh diameters of GBM might be related with the increase of UAE.

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.

Chondrosarcoma with myxoid change: a study using a quick-freezing and deep-etching method.

A middle-aged Japanese woman visited the Orthopedics Department of Nihon University Nerima Hikarigaoka Hospital complaining of pain in the left hip joint that had started approximately 8 months earlier. Following several examinations, including imaging diagnoses, an incisional biopsy demonstrated a malignant acetabular bone tumor, which was removed and examined by a quick-freezing and deep-etching (QF-DE) method, conventional electron microscopy, and light microscopy. Histologically, the tumor was a chondrosarcoma with marked myxoid changes. An interesting extracellular matrix was observed by the QF-DE method. The myxoid area consisted of a fine meshwork of proteoglycans (PG) without obvious aggrecans, which resembled that of PG usually present in the pericellular matrix of normal cartilage. Thin collagen fibrils with pleated surface structures of regular periodicity were also seen, which were sparsely distributed in wide areas except for the pericellular matrix. These collagen fibrils were of the type that are mainly located in the pericellular side of the territorial matrix in normal cartilage. A myxoid matrix consisting of thin collagen fibrils on the background of pericellular type PG suggested that the myxoid matrix in the chondrosarcoma resembled those of the pericellular and pericellular sides of the territorial matrices in normal cartilage.

Clinical applications of the quick-freezing and deep-etching method to human diabetic nephropathy.

Mesangial expansion and glomerular basement membrane (GBM) thickening were not different between normoalbuminuric (NA) and microalbuminuric (MA) type 2 diabetic patients. The quick-freezing and deep-etching (QF-DE) method allows us to examine three-dimensional ultrastructures of human renal glomeruli in vivo at high resolution. In the present study, the QF-DE method was applied to the renal biopsy from 6 type 2 diabetic patients without definable renal diseases other than diabetic nephropathy. Four patients were NA and the other two were MA. Three control specimens were normal parts in surgically resected kidneys of renal cell carcinoma. Replica membranes were prepared by the QF-DE method as previously described. By the QF-DE method, both GBM middle layer and mesangial matrix (MM) were composed of polygonal meshwork structures. The mesh pores of GBM and MM were more enlarged in size and irregular in shape in NA diabetic patients than those of the controls, and these ultrastructural changes became more obvious in MA patients. The diameters of mesh pores in the diabetic patients were significantly larger than those in the control subjects. In conclusion, the QF-DE method could be applied to needle renal biopsy and the present study has firstly clarified the difference of ultrastructural changes between NA and MA type 2 diabetic patients, which had not been disclosed by the conventional electron microscopy, were revealed by the QF-DE method.

Application of a quick-freezing and deep-etching method to pathological diagnosis: a case of elastofibroma.

A case of elastofibroma in a middle-aged Japanese woman was examined by the quick-freezing and deep-etching (QF-DE) method, as well as by immunohistochemistry and conventional electron microscopy. The slowly growing tumor developed at the right scapular region and was composed of fibrous connective tissue with unique elastic materials called elastofibroma fibers. A normal elastic fiber consists of a central core and peripheral zone, in which the latter has small aggregates of 10 nm microfibrils. By the QF-DE method, globular structures consisting of numerous fibrils (5-20 nm in width) were observed between the collagen bundles. We could confirm that they were microfibril-rich peripheral zones of elastofibroma fibers by comparing the replica membrane and conventional electron microscopy. One of the characteristics of elastofibroma fibers is that they are assumed to contain numerous microfibrils. Immunohistochemically, spindle tumor cells showed positive immunoreaction for vimentin, whereas alpha-smooth muscle actin, desmin, S-100 protein and CD34 showed negative immunoreaction. By conventional electron microscopy, the tumor cell had thin cytoplasmic processes, pinocytotic vesicles and prominent rough endoplasmic reticulum. Abundant intracytoplasmic filaments were observed in some tumor cells. Thick lamina-like structures along with their inner nuclear membrane were often observed in the tumor cell nuclei. The whole image of the tumor cell was considered to be a periosteal-derived cell, which would produce numerous microfibrils in the peripheral zone of elastofibroma fibers. This study indicated that the QF-DE method could be applied to the pathological diagnosis and analysis of pathomechanism, even for surgical specimens obtained from a patient.

Decreased gap junctional communication in neurobiotin microinjected lens epithelial cells after taxol treatment.

The aim of the study was to examine gap-junction-mediated intercellular communication after experimentally induced aggregations of microtubules in cultured bovine lens epithelial cells. Intercellular communication between lens cells appears to be crucial for normal lens homeostasis. However, investigations on the maintenance of direct ion and metabolite exchange via gap junctions and its quantified dependency of cytoskeletal microtubules have not been available under conditions leading to bundling of microtubules. Thus, metabolic coupling of neighboring lens epithelial cells was quantified following microinjections of neurobiotin into single cells under various conditions. In controls, intensive gap-junction-mediated intercellular communication could be documented by dye-spreading of microinjected neurobiotin. In contrast, taxol treatment for 1-3 days impaired, but did not completely block gap-junction-mediated intercellular communication. After depletion of taxol, a complete recovery of intercellular communication was achieved. In addition, confocal laser scanning microscopy and rapid-freeze deep-etch electron microscopy revealed a displacement of actin-filaments from the perinuclear cytoplasm, accompanied by an abnormal aggregation of microtubules after taxol treatment, including impeded translocation of connexin 43 from the cytoplasm into the plasma membrane. Incubation of cells with nocodazole destroyed the microtubule network, accompanied by a clear reduction of plasma-membrane-integrated connexin 43 and significant impairment of dye spreading. Thus, in lens epithelial cells intercellular communication at gap junctions made by connexin 43 depends on the integrity of the microtubule network through the translocation of connexins to the plasma membrane.

Further studies on knockout mice lacking a functional dynein heavy chain (MDHC7). 1. Evidence for a structural deficit in the axoneme.

Male mice had previously been generated in which the inner dynein arm heavy chain 7 gene (MDHC7) was inactivated by the substitution of four exons encoding the ATP-binding site (P1-loop) with the neomycin resistance gene, giving a putative non-functional gene product. We have used additional techniques of electron microscopy to determine what effect the truncated, non-functional heavy chain has on the assembly of the inner dynein arm complex. From a comparison of MDHC7-/- with the wild-type morphology, we have found that the expected loss of a C-terminal (globular) domain is associated with inner dynein arm 3, a change from two visible "heads" to one. This deficit was seen in replicas of rapidly-frozen, deeply-etched spermatozoa, and was confirmed in filtered images of 20-nm-thin sections, cut in longitudinal planes. Assembly of the other IDAs appeared unaffected. This study is the first to reveal the location of a specific dynein heavy chain within the 96-nm repeat pattern of the inner dynein arms of the mammalian axoneme.

Further studies on knockout mice lacking a functional dynein heavy chain (MDHC7). 2. A developmental explanation for the asthenozoospermia.

Male mice had been previously generated in which the inner dynein arm heavy chain 7 gene (MDHC7) was disrupted. MDHC7-/- animals show asthenozoospermia and are sterile. Very few of their spermatozoa can achieve forward progression, but for those that can, we add here the information (1) that the three-dimensional aspects of their movement are normal; (2) that their maximum velocity is less than that of wild-type controls; and (3) that they are entirely unable to penetrate media of raised viscosity (25-4,000 cP). However, the large majority of the spermatozoa can achieve only a low amplitude vibration. In these sperm we find, using electron microscopy, that the outer dense fibres retain attachments to the inner surface of the mitochondria. Such attachments are present in normal epididymal mouse spermatozoa but are broken down as soon as the sperm become motile on release from the epididymis. The attachments are presumed to be essential during midpiece development and, afterwards, to require a threshold level of force to loosen them and so permit the sliding displacements necessary for normal bending. We presume that the disruption of the inner dynein arm heavy chain gene, MDHC7, means that there is insufficient force to overcome the attachments, for all but a few spermatozoa.

Interaction of myosin.ADP.fluorometal complexes with fluorescent probes and direct observation using quick-freeze deep-etch electron microscopy.

Myosin forms stable ternary complexes with ADP and phosphate analogues of fluorometals that mimic different ATPase reaction intermediates corresponding to each step of the cross-bridge cycle. In the present study, we monitored the formation of ternary complexes of myosin.ADP.fluorometal using the fluorescence probe prodan. It has been reported that the fluorescence changes of the probe reflect the formation of intermediates in the ATPase reaction [Hiratsuka (1998) Biochemistry 37, 7167-7176]. Prodan bound to skeletal muscle heavy-mero-myosin (HMM).ADP.fluorometal, with each complex showing different fluorescence spectra. Prodan bound to the HMM.ADP.BeFn complex showed a slightly smaller red-shift than other complexes in the presence of ATP, suggesting a difference in the localized conformation or a difference in the population of BeFn species of global shape. We also examined directly the global structure of the HMM.ADP.fluorometal complexes using quick-freeze deep-etch replica electron microscopy. The HMM heads in the absence of nucleotides were mostly straight and elongated. In contrast, the HMM heads of ternary complexes showed sharply kinked or rounded configurations as seen in the presence of ATP. This is the first report of the direct observation of myosin-ADP-fluorometal ternary complexes, and the results suggest that these complexes indeed mimic the shape of the myosin head during ATP hydrolysis.

Squamous cell carcinoma with sarcomatous feature (so-called carcinosarcoma) of stomach probably metastasized from esophageal tumor: a case report with quick-freezing and deep-etching method.

A squamous cell carcinoma (SCC) with sarcomatous features (so-called carcinosarcoma) of stomach is reported in a 72-year-old man. The gastric submucosal tumor (12 x 11 x 6 cm) consisted of carcinoma cells and sarcomatous spindle cells, which were immunohistochemically recognized to contain high molecular weight cytokeratin. These histological and immunohistochemical results indicated that carcinoma cells and spindle tumor cells had cytokeratin similar to that of stratified squamous epithelium. These features were consistent with so-called carcinosarcoma of esophagus. A combined type of tumor consisting of polypoid and shallow ulcerative lesions (5.5 cm in diameter) was demonstrated by the biopsy to have SCC on the polypoid surface area. Therefore, the gastric tumor was thought to have metastasized from the esophageal tumor. The quick-freezing and deep-etching (QF-DE) method demonstrated that many spindle tumor cells in the gastric tumor had abundant intermediate filaments, which were evenly distributed in more peripheral cytoplasm along the cell membrane. This feature was similar to that of the control SCC. Intramembranous protein particles in the cell membrane of the tumor cells were markedly decreased as compared with those of control SCC. These ultrastructures by QF-DE method could be used for the pathological diagnosis of so-called carcinosarcomas of esophagus.

Direct observations of freeze-etching processes of ice-embedded biomembranes by atomic force microscopy.

We have fabricated a cryogenic atomic force microscope that is designed for structural investigation of freeze-fractured biological specimens. The apparatus is operated in liquid nitrogen gas at atmospheric pressure. Freeze-fracturing, freeze-etching and subsequent imaging are carried out in the same chamber, so that the surface topography of a fractured plane is easily visualized without ice contamination. A controlled superficial sublimation of volatile molecules allows us to obtain three-dimensional views of ultrastructures of biological membranes.

Further studies on the structural analysis of the cuticle of Litomosoides chagasfilhoi (Nematoda: Filarioidea).

In order to obtain further information on the structural organization of the cuticle of nematodes, this structure was isolated from adult forms of the filariid Litomosoides chagasfilhoi. The purity of the fraction was determined by light and transmission electron microscopy, deep-etching, high resolution scanning electron microscopy, atomic force microscopy, immunocytochemistry, gel electrophoresis (SDS-PAGE) and Western blot. The epicuticle presented a rugous surface with parallel rows and several globular particles that could be involved in the absorption of nutrients and secretion of products. Analysis by SDS-PAGE of purified cuticles revealed five major polypeptides corresponding to 151, 41, 28, 13 and 11 kDa. A polyclonal antibody against a synthetic 18 amino-acid peptide that corresponds to the sequence of domain E of the Haemonchus contortus3A3 collagen gene recognized several protein bands on the Western blot of purified cuticle, and labeled all cuticular layers, as shown by immunocytochemistry.

Confocal laser scanning, conventional scanning and transmission electron microscopy of vertebrate cerebellar granule cells

Confocal laser scanning microscopy of hamster cerebellar granular layer showed in montages of z-series the presence of small, medium and large granule cells. A granule cell Golgi cell ratio of 50/4 was observed surrounding glomerular regions. Field emission high resolution scanning electron microscopy of mouse cerebellar granular and molecular layers showed SE-I images of the outer and inner surfaces of nuclear and cytoplasmic compartments of chromium coated granule cells and the axo-spinodendritic synapses of parallel fibers with Purkinje cell dendrites. Conventional scanning electron microscopy of teleost fish cerebellar cortex showed three dimensional morphology of granule cell soma and processes and the synaptic relationship with mossy and climbing fibers, Golgi cell axonal ramifications and dendrites of stellate neurons, by means of SE-II and SE-III signal image mode, in sagittally and transversally cryofractured cerebellar cortex. SE-II images of the non-synaptic segments and synaptic varicosities of parallel fiber outer surface were characterized in the molecular layer. Ultrathin sections of transmission electron microscopy (TEM) revealed somato-somatic, dendro-somatic and dendro-dendritic like-desmosomal and like-hemidesmosomal junctions in human cerebellar granule cells. Freeze-etching replicas of mouse cerebellar cortex displayed granule cell intramembrane morphology, cytoplasmic fractured face and the Bergman glial cell cytoplasm completely surrounding the parallel fibers in the molecular layer. The mossy fiber-granule cell dendrite synaptic relationship was observed in sagittally and transversally cryofractured cerebellar cortex and correlated with TEM images. SE-II images of the climbing fiber synaptic connections with granule cell dendrites were obtained in teleost fish cerebellar cortex. One to one axo-dendritic synaptic contacts between Golgi cell axonal ramifications and granule cell dendrites were also seen. The above findings provide new vistas for future studies dealing with intracortical circuits and information processing in the cerebellar cortex.

Relationship between skeinoid fibers and stromal matrix in gastrointestinal stromal tumors: morphometric analysis with quick-freezing and deep-etching method.

Our previous study of a gastrointestinal autonomic nerve tumor with skeinoid fibers (SF) using the quick-freezing and deep-etching method, suggested that the distance between one radix and a neighboring radix (DRNR) in pre-existing meshwork structures around the tumor cells is consistent with the periodicity of the SF. Therefore, measurement of the DRNR in the meshwork could clarify the significance of the pericellular matrix for SF development. In the present study, we analyzed the meshwork in three cases of gastrointestinal stromal tumor (GIST), which showed different immunohistochemical stainings, but confirmed to have smooth muscle differentiation (SMD) by immunohistochemistry and/or electron microscopy. The DRNR from the three cases of GIST showed similar histogram patterns (a peak of 20-30 nm, mean values of 28.02, 25.74 and 26.45 nm), which were significantly shorter than the periodicity of SF (a peak of 40-45 nm, mean value of 42.14). Although we need further studies with additional GIST cases, we speculate that the pericellular matrix of GIST with SMD is not suitable for SF development.