Freeze-fracture organization of hair cell synapses in the sensory epithelium of guinea pig organ of Corti

The fine structure of both the afferent and efferent hair cell synapses in the sensory epithelium of guinea pig organ of Corti was examined by freeze-fracture electron microscopy. In the afferent synapse, barlike aggregates of intramembrane particles (IMPs) of about 10 nm in diameter were seen on the P-face of the afferent presynaptic membrane directly beneath the presynaptic dense projection which is located in the active zone of the presynaptic membrane. Small and large depressions have been seen on the presynaptic membrane. The former were observed in the proximity of the barlike aggregates, while the latter were observed some distance from the aggregate. In outer hair cells, IMPs of about 10 nm in diameter were seen on the P-face of the afferent postsynaptic membrane at a density of 3,000/μm². In the efferent synapse, many aggregates composed of from several to tens of large IMPs of 13 nm in diameter were observed on the presynaptic membrane. These aggregates were localized to small membrane depressions, which tended to be deeper as particle number per aggregate increased. Dense populations of IMPs of about 9 nm in diameter were observed on the P-face of the efferent postsynaptic membrane at a density of 4,000/μm². A fenestrated subsynaptic cistern completely covers the efferent postsynaptic membrane. Moreover, the subsynaptic cistern spans several efferent postsynaptic membranes when efferent synapses are gathered in a group. In the afferent and efferent synapses of hair cells, specializations of the synaptic membranes were represented by marked aggregates characteristic of IMPs. In the efferent synapse, IMP movement inside the synaptic membrane was proposed in relationship to retrival of synaptic vesicle membrane. Structural relationship between the subsynaptic cistern and efferent postsynaptic membrane was revealed.     

Autoradiographic studies of selective amino acid uptake by neural and nonneural elements in the gerbil cochlea

The cochlea is well suited for studies of the uptake properties of auditory neurons and nonneuronal supporting cells. Probe concentrations of radioisotopically labeled amino acids, including putative neurotransmitters and their precursors, breakdown products, and blockers, can be introduced via the natural, fluid-filled channels of the inner ear. Uptake patterns can be mapped at cellular and intracellular levels using light and electron microscopic autoradiographic methods. The procedures for introduction of label, fixation, plastic embedment, and light and electron microscopic autoradiography are described with special reference to the cochlea. Labeling patterns observed with over 20 amino acids are summarized for hair cells, spiral ganglion neurons, efferents, and nonneural elements of the stria vascularis, limbus, and modiolus. Limitations on the interpretation of results and their implications for the general usefulness of the methods are discussed.     

Ultrastructure of the intercalated body,a novel organelle associated with fluid forming cells in the organ of Corti

The intercalated body is a newly discovered organelle in the inner and outer spiral sulcus cells of the mouse organ of Corti. The organelle was found in the cochleas of 14-day and older intact mice and in organs in culture of corresponding ages. The organelle consists of a stack of interconnected cisternae of endoplasmic reticulum and of membrane bound rodlets that are intercalated between, and run parallel to, the cisternae. The cisternal membranes are predominantly smooth, but some may display ribosomes. Most rodlets are from 1 to 2 μm long, about 0.1 μm wide, and contain electron dense material. Mitochondria are commonly associated with or incorporated into the organelle. Some electron micrographs suggest that the rodlets may originate from modified mitochondria. It is our impression that the formation of the organelle begins with the apposition of cisternae and mitochondria. Cisternal-associated mitochondria appear to constrict, elongate, and lose their inner membranes. In both the intact animal and in culture, the cells of the inner and outer spiral sulci display microvilli, apical junctional complexes, lateral intercellular spaces containing interdigitating cell processes, and appear to be involved in fluid formation. Moreover, in culture, the cells of inner and outer spiral sulci as well as some cells proliferating in the outgrowth zone participate in fluid formation, producing large fluid pockets. All these cells commonly contain intercalated bodies. It is possible that in the intact animal, as in culture, intercalated bodies may play a role in fluid regulation in the immediate vicinity of the hair cells.     

Intracellular localization of the antitumour drug adriamycin in living cultured cells: A confocal microscopy study

The intracellular distribution of the anthracyclinic antibiotic adriamycin in living cultured cells has been investigated by confocal microscopy. In human melanoma cells (M14), adriamycin was localized inside the nuclei. When adriamycin-treated M14 cells were allowed to recover in drug-free medium, a complete efflux of the drug from the nucleus was revealed. In recovered cells, a weakly fluorescent signal was observed in the perinuclear region. When M14 cells were recovered in a medium containing colcemid, a microtubule depolymerizing agent, the drug transport from the nucleus to the cell periphery appeared to be inhibited, suggesting that the microtubule network is strongly involved in drug transport mechanisms. In multidrug-resistant (MDR) cells the intracellular location of adriamycin was shown to be noticeably different from that of the parental wild-type cells. In particular, in resistant human breast carcinoma cells (MCF-7), adriamycin appeared to be exclusively located within the cytoplasm whereas the nuclei were shown to be completely negative. When adriamycin treatment was performed in association with MDR revertants, such as Lonidamine (inhibitor of the energy metabolism) or verapamil (inhibitor of the P-glycoprotein efflux pump), a marked enhancement of the cytoplasmic signal was observed in resistant cells. Under these conditions, adriamycin appeared concentrated in the perinuclear region, but the nuclei were still negative. Confocal microscopy proved to be a very useful method for the study of the intracellular transport of fluorescent substances, such as anthracyclinic antibiotics, and for the investigation of the multidrug resistance phenomenon in tumour cells.     

Auditory hair cells in human fetuses: Synaptogenesis and ciliogenesis

This paper brings together the most recent findings concerning the development of human fetus cochlear hair cells, as observed using transmission and scanning electron microscopy (TEM and SEM). Specific attention is drawn to the formation of synapses and the growth of stereocilia. In both types of hair cells, synaptogenesis begins before ciliogenesis (week 10 of gestation versus week 12). In the inner hair cell (IHC), an adultlike stage is seen around week 15 for synapses, but not before week 22 for stereocilia. In the outer hair cell (OHC), both synapses and stereocilia are not yet fully mature at week 22. Classic gradients of maturation are found: a base-to-apex gradient, and an IHC-to-OHC gradient. By comparing these results with the anatomical and functional data on cochlear development in laboratory mammals, the onset of cochlear function in the human fetus can be estimated to occur around week 18. The completion of cochlear maturation based upon the same anatomical criteria should occur about 10 weeks later.     

Dissociated cells of the calcareous sponge clathrina: a model for investigating cell adhesion and cell motility in vitro

The study of cell-cell and cell-substratum adhesion in vitro is useful for understanding cell behavior in a three-dimensional pattern. We have used dissociated cells (choanocytes represent the main fraction) from the calcareous sponge Clathrina, namely C. cerebrum and C. clathrus, to illustrate our present understanding on three main aspects of cell-cell and cell-substratum adhesion in vitro: (1) cytoskeletal protrusions; (2) cell behaviours on organic substrata; and (3) paths of locomotory sponge cell. Cell locomotion occurs by the extensions of scleropodial and lamellipodial protrusions, by way of actin polymerization. The extent to which cells produce these cytoplasmic processes varies according to the substratum (e.g., collagen, fibronectin, laminin, polylysine). It was found that more cell extensions were produced on collagen substrata, and this led to greater cell movement. Advancing choanocytes are not polarized. Their paths are particularly complicated, showing linear segments, which produce a more efficent cellular translocation, and winding tracts with frequent turns or loops. Small amoeboid cells describe more linear paths with a wide range of speed variation than larger cells. The presence of cell-derived substratum reduces the progressive dispersion of cells and allows cells to encounter one another in such a way that the initial random walking later turns into non-random displacement. Even though cAMP-treated cells exhibit different aggregative tactics, cAMP 10(-8) M remarkably enhances cell encounters and supports the existing information that this cyclic nucleotide represents a signal that affects cell morphology and locomotion. The bulk of data on sponge cell-cell and cell-substratum adhesion has been evaluated by mentioning the significant advances and references concerning studies of other cell systems.     

Gene expression of 49 kDa apyrase,cytoskeletal proteins,ATPase, ADPase and amino acid contents of <i>Pisum sativum</i> (L.) cells germinated in E<i>uryops arabicus</i> (Steud. ex Jaub. & Spach) water extract

The present research reports of quick and marked changes induced by plant extract of Euryops arabicus in thegene expression of 49-kDa apyrases, cytoskeletal proteins, ATPases, ADPase and amount of amino acid of pea (Pisumsativum L. var. Alaska). Pellets of cytoskeletals proteins (27000 xg) were probed with anti-apyrase antibody, biotinylatedanti-rat, actin and alpha and beta-tubulin for Western blotting. ATPase and ADPase activities were determined basedon the hydrolytic efficacy of adenine triphosphate and adenine diphosphate. By 72 hours, the abundance of apyrases,cytoskeletal proteins and amount of amino acid in pellets of 27000 xg of germinated pea seeds in E. arabicus extractswere sharply increased than those sown in distilled water. All the samples exhibited that the stems had more amountfrom apyrases, cytoskeletal proteins, amino acids and ATPase and ADPase activities than primary leaves and primaryroots that were germinated either on E. arabicus water extract or in distilled water. Based on the enzyme’s capability tohydrolyse nucleotide triphosphate and nucleotide diphosphate as well as the direct association between expression of49-kDa apyrase and cytoskeletal proteins, E. arabicus water extract had an important effect on plant germinations.     

Segmentation of clustered cells in negative phase contrast images with integrated light intensity and cell shape information

Automated cell segmentation plays a key role in characterisations of cell behaviours for both biology research and clinical practices. Currently, the segmentation of clustered cells still remains as a challenge and is the main reason for false segmentation. In this study, the emphasis was put on the segmentation of clustered cells in negative phase contrast images. A new method was proposed to combine both light intensity and cell shape information through the construction of grey‐weighted distance transform (GWDT) within preliminarily segmented areas. With the constructed GWDT, the clustered cells can be detected and then separated with a modified region skeleton‐based method. Moreover, a contour expansion operation was applied to get optimised detection of cell boundaries. In this paper, the working principle and detailed procedure of the proposed method are described, followed by the evaluation of the method on clustered cell segmentation. Results show that the proposed method achieves an improved performance in clustered cell segmentation compared with other methods, with 85.8% and 97.16% accuracy rate for clustered cells and all cells, respectively.     

Form and distribution of actin and myosin in non-muscle cells: a study using cultured chick embryo fibroblasts.

Attempting to throw light on the mechanical basis of movement of non-muscle (cf. muscle) cells, the present work aims to determine the form and distribution of actin and myosin in chick embryo fibroblasts. These cells were cultured on formvar, fixed in glutaraldehyde then osmium tetroxide vapours, dehydrated, critical-point dried and examined, in toto, in the electron microscope (EM). Stereoscopic pairs of micrographs were studied to define more exactly the form and distribution of cytoplasmic filaments topographically associated with deformations of the cell surface and with organelle movements through the cytoplasm. Permeating the cytoplasm, interconnecting long and short filaments closely surrounded all organelles, linked with microtubules and polyribosomes and joined to the plasma membrane. These filaments, which varied greatly in width (2-13 nm) were closely associated with large numbers of 'comma-shaped' globoid bodies of approximately 15 nm diameter. Attempting to establish the identity, form and distribution of cytoplasmic myosin, cultured cells were extracted with a cold (4 degrees C) glycerol/pyrophosphate solution for 24 h before being fixed and critical-point dried. EM examination of these cells revealed a residual three-dimensional network of branching and anastomosing 4-13 nm diameter smooth filaments, devoid of fine (2 nm) filaments and globoid bodies. Examination of fixed, critical-point dried, skeletal muscle heavy meromyosin showed globoid structures similar in form and size to the globoid bodies found in cultures fibroblasts. Similarly fixed and critical-point dried paracrystals of actin, polymerized in the presence of Mg2+, appeared as branching interconnecting filaments which, in form and dimensions, resembled the network filaments observed in pyrophosphate-extracted cells. It is concluded that the pyrophosphate-extractable globoid bodies found in cultured fibroblasts represent monomers of myosin, that the broader filaments to which these attach represent actin in Mg2+ paracrystalline form and that the various subcellular movements are brought about by interactions between the two, analogous to those occurring in muscle cells.     

Streak time-lapse video microscopy: analysis of protoplasmic motility and cell division in Tradescantia stamen hair cells

We have developed a video time-lapse analogue of a streak camera using a digital image processing technique, and have used it to analyse dynamic processes in plant cells. The same image area is selected from a succession of frames in a video tape recording. The successive images are stored in an image memory, rearranged, and displayed in a single frame according to the time course. Each image area is rectangular, with selectable length and width: the closest analogy to a streak camera is obtained when one of its sides is a single pixel. The composite image displays sequential changes in the distribution of subcellular components in the selected area. The method was shown to be useful for monitoring details of cytoplasmic streaming and organelle movement, observing the time course of chemical fixation, and also for studying kinetochore movement, shortening of chromatids and cell plate formation during mitosis and cytokinesis. Cessation and resumption of cytoplasmic movement during the cell division cycle were also clearly localized outside the nuclear region in apical cells of Tradescantia stamen hairs.     

Video-rate confocal reflection microscopy of neoplastic cells: Rate of intracellular movement and peripheral motility characteristic of neoplastic cell line (RSK4) with high degree of growth independence in vitro

Video-rate laser confocal interference reflection microscopy was used to demonstrate rapid motion of intracellular organelles and features at the cell periphery in a fully transformed neoplastic cell line, RSK4, and in four other neoplastic cell populations. In the RSK4 cells, vibrational and trafficking movements of intracellular particles at a rate greater than 25 Hz and ranging down to 5 Hz were recorded. Rapidly moving processes changed to ruffles, then microspikes, and previously undetectable ephemeral intercellular contacts were seen. Dynamic cyclical changes were revealed in the sizes of the podosomal close contacts of the transformed cells. The visibility of such features and the temporal and spatial resolution are improved over earlier methods. The fact that fast cellular and intracellular movements can be detected with this microscopic technique offers new possibilities in attempting to recognise differences between unimpaired living cells, and it may prove useful in the identification of malignant cells.     

Transcytosis of plasma macromolecules in endothelial cells: a cell biological survey

The modern exploration of endothelial cell biology is a largely interdisciplinary exercise. Cell biological, physiological, and more recently molecular biology approaches were used to study the pathways and the organelles involved in transcytosis of macromolecules in endothelial cell (EC). Here we discuss mainly the cell biological findings that revealed that EC have the attributes to fulfill the transport function. They are polarized cells, heterogeneous, and, thus, structurally and functionally adapted to the vascular bed in which they reside. The structural heterogeneity involves the number and distribution of plasmalemmal vesicles (caveolae), their generated channels, and the organization of intercellular junctions. The closely related functional heterogeneity comprises the degree of permeability for plasma molecules that vary as a function of organ. The EC are endowed with the cellular machinery to perform (1) endocytosis, that is to take up plasma proteins and the molecules they carry to be used for themselves (cholesterol-carrying low density lipoproteins, fatty acid carrying albumin, iron carrying transferrin, etc.), and (2) transcytosis, which implies to transport plasma proteins to the subjacent cells and tissues. The possible pathways for transport of molecules are transcellular, via caveolae and channels, and paracellular via intercellular junctions. Most of the results obtained, so far, indicate that transcytosis of albumin, low-density lipoproteins, metaloproteases, and insulin, is performed by cargo-vesicles and their generated channels. The paracellular pathway can be used for water and ions; in postcapillary venules, at the level of which approximately 30% of junctions are open to a space of 6 nm, small molecules may take this route. Recent data obtained by molecular biology techniques revealed that caveolae are endowed with the molecular machinery for fusion/fission, docking, and movement across cells. Moreover, the various and numerous molecules that have been detected in the caveolae membrane and the different functions assumed by this differentiated microdomain strengthen the postulate that there are at least two or more types of vesicles molecularly tailored for the local physiological requirements.     

Localization of Ca2+ -stores and tissue compartments with a Ca2+ -binding capacity in the organ of Corti of the guinea-pig by electron energy-loss spectroscopy

The addition of 10 mM CaCl₂ to glutaraldehyde fixative leads to the formation of small electron-dense deposits in the organ of Corti of the guinea-pig. These precipitates are mainly attached to cell membranes in contact with different extracellular lymphatic fluids. A higher number of precipitates is localized in the acellular parts of tectorial and basilar membrane. Electron energy-loss spectroscopy (EELS) was used to determine the elemental composition of the deposits formed. The spectra showed a prominent signal at the Ca²⁺ L_2,3 ionization edge. Oxygen could also be detected in all the precipitates analysed. EELS analysis of mitochondria of the inner and outer hair cells after conventional fixation (glutaraldehyde followed by post-fixation in OsO₄) revealed a small but significant calcium signal.     

The intracellular distribution of vinculin and alpha2 integrin in epithelial cells and chondrocytes

The purpose of this study was to demonstrate the presence of vinculin and alpha₂ integrin in chondrocytes in situ and epithelial cells. We also determined that the appropriate fixation and extraction protocols for immunohistochemistry and laser scanning confocal microscopy for an integral membrane protein and an actin-associated protein in cultured cells and whole tissue was different. Cultured epithelial cells, whole mount human cornea and avian cartilage were fixed and prepared using a number of standard procedures used for indirect fluorescence immunohistochemistry. The distribution of vinculin was cell-type and fixation-specific. Chondrocytes and cultured epithelial cells demonstrated vinculin in areas that appear to be associated with filamentous actin. Vinculin was associated with cell membranes in human cornea. The expression of alpha₂ integrin observed in chondrocytes fixed with methanol, paraformaldehyde, or formaldehyde is consistent with its role in cell–substrate interaction, but may also suggest a role in dividing and differentiating cells. The localization of alpha₂ integrin in human corneal epithelia supports its role as a cell-cell adhesion molecule. The cytoplasmic distribution of vinculin and alpha₂ integrin in tissues fixed without detergent extraction suggests that the fixation step may be sufficient for antibody penetration and antigen extraction. These studies are the first report of vinculin and alpha₂ integrin in embryonic chondrocytes. In addition we have shown that confocal laser scanning microscopy combined with proper fixation and extraction protocols may optimize the localization of antigens in cultured and whole mount cells.     

The intracellular distribution of vinculin and alpha2 integrin in epithelial cells and chondrocytes

The purpose of this study was to demonstrate the presence of vinculin and alpha₂ integrin in chondrocytes in situ and epithelial cells. We also determined that the appropriate fixation and extraction protocols for immunohistochemistry and laser scanning confocal microscopy for an integral membrane protein and an actin-associated protein in cultured cells and whole tissue was different. Cultured epithelial cells, whole mount human cornea and avian cartilage were fixed and prepared using a number of standard procedures used for indirect fluorescence immunohistochemistry. The distribution of vinculin was cell-type and fixation-specific. Chondrocytes and cultured epithelial cells demonstrated vinculin in areas that appear to be associated with filamentous actin. Vinculin was associated with cell membranes in human cornea. The expression of alpha₂ integrin observed in chondrocytes fixed with methanol, paraformaldehyde, or formaldehyde is consistent with its role in cell-substrate interaction, but may also suggest a role in dividing and differentiating cells. The localization of alpha₂ integrin in human corneal epithelia supports its role as a cell-cell adhesion molecule. The cytoplasmic distribution of vinculin and alpha₂ integrin in tissues fixed without detergent extraction suggests that the fixation step may be sufficient for antibody penetration and antigen extraction. These studies are the first report of vinculin and alpha₂ integrin in embryonic chondrocytes. In addition we have shown that confocal laser scanning microscopy combined with proper fixation and extraction protocols may optimize the localization of antigens in cultured and whole mount cells.     

Intracellular localization of samarium in the lactating mammary gland cells: ultrastructural and microanalytical study

The frequent use of some rare earths in the medical and industrial domains make us worry about their intracellular behavior into the body. Reason for which we have investigated the subcellular localization of one of these elements, the samarium, in the mammary gland of lactating female wistar rats using two very sensitive methods of observation and microanalysis, the transmission electron microscopy and the secondary ion mass spectrometry. The ultrastructural study showed the presence of electron dense deposits in the lactating mammary glandular epithelial cell lysosomes of the samarium-treated rats, but no loaded lysosomes were observed in those of control rats. The microanalytical study allowed both the identification of the chemical species present in those deposits as samarium isotopes ((152) Sm(+)) and the cartography of its distribution. Our results confirm the previous ones showing that lysosomes of the glandular epithelial cells are the site of the intracellular concentration of foreign elements such as gallium. The intralysosomal deposits observed in the mammary glandular cells of the samarium-treated rats are similar in their form and density to those observed with the same element in other varieties of cells, such as liver, bone marrow, and spleen cells. Our ultrastructural and microanalytical results and those obtained in previous studies allow deducing that the intralysosomal deposits are very probably composed of an insoluble samarium phosphate salt.     

Progress in electron microscopic diagnostics: semi-quantitative determination of precipitable calcium in different cell types of the organ of Corti in the guinea-pig

Potassium antimonate was used to precipitate calcium in the cochlea of the guinea-pig. The distribution of the calcium antimonate precipitates was analysed by electron microscopy. The precipitate density was determined in different cell types in the organ of Corti by counting the number of calcium binding sites in a 10-μm² area. The size of the precipitates varied considerably, and thus the relative amount of the precipitable calcium was estimated only semi-quantitatively. As the prominent carbon signal is superimposed over the nearby small Ca²⁺ -edge signals, the combined signal of the antimony M_4,5-edge and the oxygen K-edge of the calcium antimonate salt formed was chosen for the semi-quantitative estimation. Images of the inelastically scattered electrons of the precipitates at ΔE = 570 eV were recorded by electron spectroscopic imaging. The area covered by the calcium precipitates within a given cell type was determined in different ultrathin sections of the same organ of Corti by an image processing system.