Mitochondria DNA synthesis in oocytes of Xenopus laevis

The process of attachment was studied in primary mouse kidney epithelial cell cultures by means of reflexion contrast microscopy, a method developed for studying the cell membrane-substrate relationship. The first in a series of events is simple adherence to the substrate, called close contact. This phenomenon is associated with the greatest extension of lamellar cytoplasm and the fewest number of cell nuclei/unit area. The nuclei of such cells are in close contact with the bottom portion of the cell membrane. Approx. 24 h after planting, as the cultures become more crowded, cells develop a different kind of attachment to the substrate—focal contacts—that are correlated with a decrease in lamellar cytoplasm. Cells detached from the substrate after close contact formation readily reattach, while cells detached after formation of focal contacts do not reattach. After incubation for periods greater than 5 days, the dense cultures degenerate and cells lose their attachment to the glass surface.     

Spreading of normal and transformed fibroblasts in dense cultures

Cell spreading in dense cultures of normal mouse embryo fibroblasts and of the two lines of mouse transformed fibroblasts was examined by electron microscopy. The mean number of cell layers in culture and cell population density per unit area of the substrate were detetmined; the mean area of the cell projection on the substratum was found from these data.Normal fibroblasts formed multilayefed sheet in dense culture. The cells in this sheet were well-spread. These cells formed thin lamellae (lamellar cytoplasm) over the surface of other cells and over the intercellular substance. The mean cell area in dense culture was not smaller than that of the cell spread on the substratum in sparse culture.Dense cultures of two transformed lines (M 22 and L) had differing morphologies: cultures of one line (M 22) were multilayered, those of the other line (L) were monolayered. Decreased spreading and almost complete (M 22) or complete (L) absence of lamellar cytoplasm were characteristic of both transformed lines. The mean area of the cell in dense cultures of both lines was several times smaller than that of their normal progenitors.It is concluded that similar reactions leading to the spreading accompanied by the formation of lamellar cytoplasm can be induced by the contact of fibroblast with various surfaces: that of the substratum in sparse culture, that of other cells and of intercellular structures in dense culture. Deficiency of these reactions characteristic for transformed fibroblasts may be responsible for abnormal morphology of their cultures.     

Structural organization of interphase 3T3 fibroblasts studied by total internal reflection fluorescence microscopy

We studied the laminar organization of 3T3 fibroblast cells growing on glass slides by use of total internal reflection illumination to excite fluorescence emission (TIRF) from labeled molecules and stained cellular compartments that are very close to the cell-substrate contact region. Mitochondria, distant from the contact regions and stained with the water-soluble cationic dye, dil-C3-(3), fluoresced only as the glass/cytoplasm critical angle was approached. A similar result was obtained when the nuclei were stained with Hoechst dye 33342. From this measured angle a cytoplasmic refractive index in the range 1.358-1.374 was computed. The plasma membrane of 3T3 cells was stained with dil-C18-(3), and the cytoplasmic compartment was stained with fluoresceinyl-dextran (FTC-dextran) or with carboxyfluorescein. We have demonstrated a high degree of correspondence between the low-reflectance zones in the reflection interference image of a live cell and the TIRF images of both the plasma membrane and cytoplasmic compartment. TIRF photometry of selected contact regions of cells provided data from which the absolute separation of cell and substrate was computed. From a population of 3T3 cells microinjected with fluorescein-labeled actin, motile and adherent interphase cells were selected for study. For adherent cells, which displayed fluorescent stress fibers, the TIRF image was composed of intense patches and less intense regions that corresponded, respectively, to the focal contact and close-contact zones of the reflection-interference image. The intense patches corresponded to the endpoints of the stress fibers. Cells of motile morphology, which formed some focal contacts and extensive close-contact zones, gave AF-actin TIRF images of relatively even intensity. Thin lamellar regions of the cytoplasm were found to contain concentrations of actin not significantly different from other close-contact regions of the cell. The major analytical problem of TIRF microscopy is separation of the effects of proximity to substrate, refractive index, and fluorescent probe concentration on the local brightness of the TIRF image. From our results, it appears possible to use TIRF microscopy to measure the proximity of different components of substrate contact regions of cells.     

Serial propagation of mammalian cells on microcarriers

For the large-scale operation of microcarrier culture to be successful, a technically feasible method for sequential inoculation is essential. Using human foreskin fibroblasts, FS-4, we have achieved this by detaching cells viably from microcarriers employing a selection pH trypsinization technique. Cells thus detached are able to reattach to microcarriers and grow normally after subsequent reinoculation into new cultures. However, after reinoculation cells attach to new microcarriers at a higher rate than to used microcarriers on which cells have previously grown. The effect of this differential cell attachment was analyzed and overcome by employing a low inoculum concentration. FS-4 cells could thus be serially propagated on microcarriers and subsequently used for beta-interferon production. This technique has also been applied to the cultivation of a monkey kidney cell line, Vero. We have also shown that Vero cells directly inoculated from a seed microcarrier culture could be used for virus production.     

Changes in the interior surface of newly mesodermalized ectoderm and its contact activity with competent ectoderm in the newtCynops (Amphibia)

Summary The presumptive ectoderm (pE) ofCynops gastrulae was artificially mesodermalized by contact with teleost swimbladder. The newly mesodermalized ectoderm (mE) acquired the capacity for neural induction (Suzuki et al. 1986a). SEM observations revealed that the mE cells altered their cellular profiles immediately after mesodermalization. The characteristics of the cell surface and the cell architecture became similar to those of invaginated mesoderm cells. There were distinct differences in the cellular contact between mE—pE and pE—pE combinations. The mE-pE combinations kept close contact at their interior surfaces, while the pE—pE combinations did not keep contact. Both TEM and SEM observations also indicated that there were tight contacts between mE and pE cells. These findings suggest that neural-inducing activity of the newly mesodermalized ectoderm cells is coupled with acquisition of cellular affinity toward the interior surface of competent ectoderm cells, and probably requires close cell contacts.     

Detection of Tobacco Mosaic Virus Movement Protein in Association with Tobacco Nuclei Isolated from Intact and Detached Leaves

The movement protein (MP) of tobacco mosaic virus was observed to cofractionate with nuclei from intact and detached tobacco (Nicotiana tahacum cv. Xanthi-nn) leaves. When purified nuclei were treated with proteinase K, MP disappeared indicating that MP is localized close to but not inside nuclei. Moreover, the amount of MP was showti to increase greatly in nuclei isolated from detached leaves, thus facilitating the detection of MP. Accumulation close to nuclei was strongest early in infection, results indicating that it may be an early step in the pathway of MP from cell cytoplasm to plasmodesmata. Other TMV-specific proteins were not detected in the nuclei fraction.     

Ultrastructure of the cell surface cellulosome of Clostridium thermocellum and its interaction with cellulose.

The ultrastructural distribution of the cellulosome (a cellulose-binding, multicellulase-containing protein complex) on the cell surface of Clostridium thermocellum YS was examined by cytochemical techniques and immunoelectron microscopy. When cells of the bacterium were grown on cellobiose, cellulosome complexes were compacted into quiescent exocellular protuberant structures. However, when the same cells were grown on cellulose, these polycellulosomal organelles underwent extensive structural transformation; after attachment to the insoluble substrate, the protuberances protracted rapidly to form fibrous "contact corridors." The contact zones mediated physically between the cellulosome (which was intimately attached to the cellulose matrix) and the bacterial cell surface (which was otherwise detached from its substrate). In addition, cell-free cellulosome clusters coated the surface of the cellulose substrate. The cellulose-bound cellulosome clusters appear to be the site of active cellulolysis, the products of which are conveyed subsequently to the cell surface via the exocellular contact zones.     

The distribution of fibronectin in attachment sites of chick fibroblasts

Primary chick heart fibroblasts were cultured on glass coverslips and examined by reflection contrast microscopy and then by indirect immunofluorescence microscopy using affinity purified antifibronectin antibody. Fibronectin was found to be primarily localized in areas of ‘close’ contact and in intensely staining fibrous webs that were usually external to the cytoplasmic matrix. Focal contacts, in which there is intimate contact between the cell and the substratum by localized attachment, failed to react with the anti-fibronectin antibody despite a variety of extraction procedures designed to increase the accessibility of antibody to this type of attachment site. It is concluded that in chick fibroblasts, fibronectin participates in cell attachment at areas of close contact and fibrillar attachment sites, but is often excluded from the attachment pads of focal contacts.     

Human osteoblast cell spreading and vinculin expression upon biomaterial surfaces

Any biomaterial implanted within the human body is influenced by the interactions that take place between its surface and the surrounding biological milieu. These interactions are known to influence the tissue interface dynamic, and thus act to emphasize the need to study cell-surface interactions as part of any biomaterial design process. The work described here investigates the relationship between human osteoblast attachment, spreading and focal contact formation on selected surfaces using immunostaining and digital image processing for vinculin, a key focal adhesion component. Our observations show that a relationship exists between levels of cell attachment, the degree of vinculin-associated plaque formation and biocompatibility. It also suggests that cell adhesion is not indicative of how supportive a substrate is to cell spreading, and that cell spreading does not correlate with focal contact formation.     

Quantitative reflection contrast microscopy of living cells

Mammalian cells in culture (BHK-21, PtK2, Friend, human flia, and glioma cells) have been observed by reflection contrast microscopy. Images of cells photographed at two different wavelengths (546 and 436 nm) or at two different angles of incidence allowed discrimination between reflected light and light that was both reflected and modulated by interference. Interference is involved when a change in reflected intensity (relative to glass/medium background reflected intensity) occurs on changing either the illumination wavelength or the reflection incidence angle. In cases where interference occurs, refractive indices can be determined at points where the optical path difference is known, by solving the given interference equation. Where cells are at least 50 nm distant from the glass substrate, intensities are also influenced by that distance as well as by the light's angle of incidence and wavelength. The reflected intensity at the glass/medium interface is used as a standard in calculating the refractive index of the cortical cytoplasm. Refractive indices were found to be higher (1.38--1.40) at points of focal contact, where stress fibers terminate, than in areas of close contact (1.354--1.368). In areas of the cortical cytoplasm, between focal contacts, not adherent to the glass substrate, refractive indices between 1.353 and 1.368 were found. This was thought to result from a microfilamentous network within the cortical cytoplasm. Intimate attachment of cells to their substrate is assumed to be characterized by a lack of an intermediate layer of culture medium.     

Disassembly of F-actin filaments in human endothelial cells cultured on type V collagen.

We examined the inhibitory activity of type V collagen on cell attachment and cell growth and the role of stress fibers and beta 1 integrin in cultured human endothelial cells. Human endothelial cells cultured on type V collagen attached temporarily to the substrate and formed stress fibers. However, the cells failed to proliferate and gradually detached from the substrate. After 24 h, the cells on type V collagen lacked discernible stress fibers (F-actin filaments) and exhibited dots in small aggregates of F-actin. In addition, the cells expressed little or no proteins as focal adhesions, including vinculin and beta 1 integrin. In contrast, the cells on fibronectin and type I collagen formed complete F-actin filaments, exhibited sufficient vinculin and beta 1 integrin, and grew logarithmically from 2 days. On the other hand, human smooth muscle cells formed complete F-actin filaments, revealed typical focal adhesions, and started to proliferate rapidly after 24 h on type V collagen as well as on fibronectin and type I collagen. Thus, the disassembly of F-actin filaments was observed as a specific phenomenon in human endothelial cells cultured on type V collagen. Moreover, the F-actin filaments disappeared from endothelial cells treated with cytochalasin D after 24 h and the cells detached from fibronectin and type I collagen with time, a result consistent with the observations on type V collagen. Accordingly, the disassembly of F-actin filaments in focal adhesions may result in the detachment of endothelial cells from type V collagen.     

A theoretical analysis for the effect of focal contact formation on cell-substrate attachment strength.

For many cell types, growth, differentiation, and motility are dependent on receptor-mediated adhesion to ligand-coated surfaces. Focal contacts are strong, specialized, adhesive connections between cell and substrate in which receptors aggregate and connect extracellular ligand to intracellular cytoskeletal molecules. In this paper, we present a mathematical model to examine how focal contact formation affects cellular adhesive strength. To calculate adhesive strength with and without focal contacts, we use a one-dimensional tape peeling analysis to determine the critical tension necessary to peel the membrane. Receptor-ligand bonds are modeled as adhesive springs. In the absence of focal contacts, we derive analytic expressions for the critical tension at low and high ligand densities and show how membrane morphology affects adhesion. Then, focal contacts are modeled as cytoplasmic nucleation centers which bind adhesion receptors. The extent of adhesive strengthening upon focal contact formation depends on the elastic rigidity of the cytoskeletal connections, which determines the structural integrity of the focal contact itself. We consider two limits to this elasticity, very weak and rigid. Rigid cytoskeletal connections give much greater attachment strengths. The dependence of attachment strength on measurable model parameters is quite different in these two limits, which suggests focal contact structure might be deduced from properly performed adhesion experiments. Finally, we compare our model to the adhesive strengthening response reported for glioma cell adhesion to fibronectin (Lotz et al., 1989. J. Cell Biol. 109:1795-1805). Our model successfully predicts the observed detachment forces at 4 degrees C and yields values for the number of fibronectin receptors per glioma cell and the density of cytoskeletal connection molecules (talin) involved in receptor clusters which are consistent with measurements for other cell types. Comparison of the model with data at 37 degrees C suggests that while cytoskeletal cross-linking and clustering of fibronectin receptors significantly increases adhesion strength, specific glioma cell-substratum attachment sites possess little mechanical rigidity and detach through a peeling mechanism, consistent with the view that these sites of < or = 15 nm cell-substrate separation are precursors to fully formed, elastically rigid focal contacts.     

Self-contact inhibition of movement in cultured chick heart fibroblasts

Collisions between two lamellar processes extended from a single locomoting cultured cell were examined by time-lapse cinemicrography and transmission electron microscopy. In most cases after contact the forward movement of either one or both of the lamellae ceased and was followed by a phase of retraction of the lamellae resulting in the breaking of the contact. The events correspond well to the contact inhibition of movement expressed when two cells collide. The similarity is also shown in the ultrastructure of the cell contacts which exhibit a close parallel arrangement of the apposed cell membranes and an alignment of microfilaments in the regions of the cytoplasm at the contacts.     

The Cytoskeleton Regulates Cell Attachment Strength

Quantitative information about adhesion strength is a fundamental part of our understanding of cell-extracellular matrix (ECM) interactions. Adhesion assays should measure integrin-ECM bond strength, but reports now suggest that cell components remain behind after exposure to acute force for radial shear assays in the presence of divalent cations that increase integrin-ECM affinity. Here, we show that focal adhesion proteins FAK, paxillin, and vinculin but not the cytoskeletal protein actin remain behind after shear-induced detachment of HT1080 fibrosarcoma cells. Cytoskeletal stabilization increased attachment strength by eightfold, whereas cross-linking integrins to the substrate only caused a 1.5-fold increase. Reducing temperature—only during shear application—also increased attachment strength eightfold, with detachment again occurring between focal adhesion proteins and actin. Detachment at the focal adhesion-cytoskeleton interface was also observed in mouse and human fibroblasts and was ligand-independent, highlighting the ubiquity of this mode of detachment in the presence of divalent cations. These data show that the cytoskeleton and its dynamic coupling to focal adhesions are critically important for cell adhesion in niche with divalent cations.     

Substratum attachment of embryonic mesoderm cells in culture

Summary The cell-substratum adhesive characteristics of cultured chick embryo primary mesoderm cells have been examined by inteference reflection microscopy and transmission electron microscoy under various conditions. Correlations were drawn between the type of adhesion and the degree of motility shown by the cells. During the rapid spreading and motility of cells cultured on fibronectin-containing substrate, focal contacts (10 to 15-nm gap) were rare and close contacts (about 30-nm gap) were pedominant. By contrast, when the cells were immobile, after 5 d in cultue, extensive focal contacts were present, together with stress fibers. The results indicate that tight cell-substratum contact is incompatible with rapid cell motility and that fibronectin acts by inducing the formation of close contacts rather than focal contacts. This work was supported by grants from the Medical Research Council of Canada and the Alberta Heritage Foundation for Medical Research.     

Characteristics of the upper cell surface in cultures of normal and SV-40 virus-transformed epithelium of mouse kidney. Study with the aid of scanning electron microscopy]

Central cells of the normal epithelial sheet are sparsely covered by microvilli. Numerous microvilli were seen in the regions of intercellular contacts. Marginal cells of sheets had a finely developed lamellar cytoplasm (lameloplasm) with smooth upper surface at their free margins. A transformed cell line (MPTR) resembled normal parent cells by its ability to form monolayered sheets in cultures. More microvilli of increased length appeared on the upper surface of central MPTR cells. The normal structure of lamelloplasm was changed at the free edge of the MPTR sheets. It is suggested that abnormal cell attachment to the substratum may be responsible for the altered cell surface morphology (increased length of microvilli, defective, structure of lamelloplasm) in the MPTR cultures.     

The Physical Interaction of Myoblasts with the Microenvironment during Remodeling of the Cytoarchitecture

Integrins, focal adhesions, the cytoskeleton and the extracellular matrix, form a structural continuum between the external and internal environment of the cell and mediate the pathways associated with cellular mechanosensitivity and mechanotransduction. This continuum is important for the onset of muscle tissue generation, as muscle precursor cells (myoblasts) require a mechanical stimulus to initiate myogenesis. The ability to sense a mechanical cue requires an intact cytoskeleton and strong physical contact and adhesion to the microenvironment. Importantly, myoblasts also undergo reorientation, alignment and large scale remodeling of the cytoskeleton when they experience mechanical stretch and compression in muscle tissue. It remains unclear if such dramatic changes in cell architecture also inhibit physical contact and adhesion with the tissue microenvironment that are clearly important to myoblast physiology. In this study, we employed interference reflection microscopy to examine changes in the close physical contact of myoblasts with a substrate during induced remodeling of the cytoarchitecture (de-stabilization of the actin and microtubule cytoskeleton and inhibition of acto-myosin contractility). Our results demonstrate that while each remodeling pathway caused distinct effects on myoblast morphology and sub-cellular structure, we only observed a ∼13% decrease in close physical contact with the substrate, regardless of the pathway inhibited. However, this decrease did not correlate well with changes in cell adhesion strength. On the other hand, there was a close correlation between cell adhesion and β1-integrin expression and the presence of cell-secreted fibronectin, but not with the presence of intact focal adhesions. In this study, we have shown that myoblasts are able to maintain a large degree of physical contact and adhesion to the microenvironment, even during shot periods (<60 min) of large scale remodeling and physiological stress, which is essential to their in-vivo functionality.     

Licht- und elektronenmikroskopische Untersuchungen von Entwicklungsstadien der FlechteEndocarpon pusillum unter Kulturbedingungen

The lichenEndocarpon pusillum Hedw. was cultivated under laboratory conditions on agar, silica gel and soil substrate. Selected developmental stages of the life cycle (germination, contact between the symbionts, cortex, squamule and perithecia development) were studied by light and scanning electron microscope.—It could be shown that the spores had no rigid spore walls with germination colpies and the spore cells which are in contact with the substrate were formed directly into germination tubes.— Further studies showed that the initial contact between the components was thigmotropic and both the form and the gelatinous matrix around the algal cells play an important role in this process. — The development of the cortex occurs under reduced moisture conditions resulting in a reduced algal reproduction. The thickness of the cortex was dependent on light intensity during cultivation. The cortex originated from hyphae, which developed beyond the algal layer and were combined to a tight network.—Fruiting bodies with spores and hymenial algae were only formed in cultures on soil substrate.

Frau Prof. Dr.Elisabeth Tschermak-Woess zu ihrem 70. Geburtstag gewidmet.     

Anatomy and ultrastructure of epidermal cells in the haustorium of a parasitic flowering plant,Cuscuta japonica, during attachment to the host

Changes of epidermal cells in the haustorium of the parasiticCuscuta japonica during its attachment to the host plantimpatiens balsamina were studied with light and electron microscopy. In the transverse sections of dodder stems not in contact with the host, epidermal cells had rounded outlines. However, when haustorial initials developed in the cortex of the parasite stem at the contact site, the epidermal cells had more dense cytoplasm and conspicuous nuclei than before, and their outline was flat in the longitudinal section. As meristem cells developed from those initials, the epidermal cells became more elongated. When the haustorium was fully matured, the apical tips of the elongated epidermal cells at the contact site branched like toes, producing numerous projections via cell wall invaginations. This event caused spaces to form between the projections; coincidently, the surface area of the apical ends of the epidermal cells increased. The dense cytoplasm at those projections contained prominent nuclei and abundant other organelles, suggesting a active metabolism. Osmiophilic particles, releasing into the cell walls from the cytoplasm, were though to be associated with the loosening and elongating of the epidermal cell walls. Dense and homogeneous materials were secreted within the spaces between the projections. These materials could play an important role in cementing the haustorium onto the surface of the host organ.