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PubMed2002

The thrombocyte aggregation process in the turtle Phrynopys hilarii (Chelonia). An ultrastructural study.

This study investigates the thrombocyte aggregation process in the South American fresh water turtle (Phrynopys hilarii) using electron microscopy. Blood was taken from surgically exposed lateral neck vessels of ten turtles Phrynopys hilarii during the spring and summer seasons, when the mean temperature is 37 degrees C. Blood samples were fixed with Karnovsky solution for processing by transmission electron microscopy. The turtle thrombocytes were spindle-shaped with lobulated nuclei. Prominent vesicles and canaliculi were found throughout the cytoplasm. The cytoplasm organelles showed an agranular endoplasmatic reticulum, Golgi complex near the centrioles and scattered free ribosomes. These cells are similar to bird thrombocytes but distinct from fish and frog thrombocytes. Blood clotting time was 5 min +/- 30 sec measured by the Lee and White method. Structural alterations resulting from the aggregation process occurred after activation. Thrombocytes developed numerous filopodial projections, an increased number of vacuoles and changed from spindle to spherical shape. P. hilarii thrombocytes have different morphologic characteristics compared to other non-mammalian vertebrate cells. These cells can participate in the aggregation process, as observed in birds.

Animals↗

PubMed2002

Morphological analysis of active microglia--rod and ramified microglia in human brains affected by some neurological diseases (SSPE, Alzheimer's disease and Wilson's disease).

The activation of microglial cells in pathological conditions is manifested primarily by their proliferation, as well as by the occurrence of a new morphological form--rod microglia. In the present study immunohistochemical identification of rod microglial phenotype against ramified microglia was performed on segments of 17 brains derived from 7 cases of encephalitis of viral aetiology (including 5 SSPE cases), 6 cases of Wilson's disease and 4 cases of Alzheimer's disease. Segments from frontal, temporal and occipital lobes, cerebellum and brainstem were subjected to histological, histochemical and immunohistochemical reactions. The presence of activated rod and ramified microglia was observed in sections derived from all structures of the brains under study. Both morphological forms of activated microglia reacted to antibodies: HLA II, CD68, HAM56 and lectin RCA-1. Expression of HLA II molecules was less intensive on the surface of microglial rod cells. A positive reaction to PCNA antibody was mainly observed in rod/elongated/cylinder-shaped nuclei, which is a characteristic feature of rod microglia. In the study material, the localisation of microglial processes seemed to depend rather on the structural topography of the cell in the brain than on the nuclear shape of the activated microglial cell. Our observations revealed a strong similarity between immunohistochemical phenotypes of both morphological forms of microglia with the indication that rod microglia is a first developmental form of activated microglia.

Adolescent↗

PubMed2002

The neuronal structure of the inferior colliculus in the bank vole (Clethrionomys glareolus)--Golgi and Nissl studies.

The inferior colliculus (IC) of the bank vole is made up of 3 nuclei: the external and pericentral nucleus, which are located on the outer border of the IC, and the central nucleus, which is the largest part of IC and shows a laminated structure. On the basis of various morphological criteria 5 types of neurons have been distinguished in the bank vole IC: 1. The rounded cells (perikarya 10-15 microm) with 2-4 primary dendritic trunks. The dendritic tree has a spindle-like shape. The axon emerges from the soma or from the proximal portion of a dendrite. 2. The fusiform neurons (17-20 microm) with 2 primary dendrites arising from both poles of the perikaryon. The dendritic tree has the same shape as the previous type. The axon originates from the proximal dendritic trunk. The rounded and fusiform cells constitute the main neuronal type. 3. The pear-shaped neurons (10-13 microm) with 2 main stems or rarely 1. The axon emerges from the perikaryon or seldom from the dendritic trunk. 4. The multipolar cells (18-23 microm), which have from 4 to 6 primary dendrites radiating in all directions. The dendritic tree has a spherical shape. The axon emerges either from the proximal stem or directly from the soma. 5. The triangular neurons (15-18 microm) with 3 primary dendritic trunks. The axon originates from the perikaryon. The triangular cells are the least numerous. All types of neurons in the bank vole IC bear spines and protrusions.

Animals↗

PubMed2003

A role of Rnd1 GTPase in dendritic spine formation in hippocampal neurons.

Rho family of small GTPases are key regulators for morphological changes of neurons on the basis of reorganization of the actin cytoskeleton. Rnd1, a novel member of this family, is predominantly expressed in neurons in brain; however, the neuronal functions of Rnd1 are not known. Here we investigated the effect of Rnd1 on neuronal morphology. Northern blot analysis of Rnd1 expression in rat brain showed that Rnd1 mRNA was highly expressed during early postnatal period, the synaptogenic stage. In situ hybridization analysis at this period revealed that Rnd1 mRNA was strongly expressed in neurons, including the hippocampal pyramidal neurons. Furthermore, immunoblot analysis showed that Rnd1 protein was localized in synaptosomal membrane fraction. Ectopical overexpression of Rnd1 in cultured rat hippocampal neurons promoted the elongation of dendritic spines. On the other hand, suppression of endogenous Rnd1 level by antisense oligonucleotide of Rnd1 caused the increase in the percentage of headless protrusions accompanied by the reduction in the spine number and spine width and shortened the length of the headless protrusions. These results indicate that Rnd1 plays a role in spine formation in the developmental synaptogenic stage.

Animals↗

PubMedDate not supplied

Qualitative and quantitative analysis of the effects of acetaminophen and N-acetylcysteine on the surface morphology of Hep3B hepatoma cells in vitro.

Acetaminophen (AAP) is harmful to the liver if consumed in excessive doses. Its toxicity can be counteracted by N-acetylcysteine (NAC). The authors studied cultures of Hep3B cells exposed to AAP or NAC or both, at 24 and 48 h, using the scanning electron microscope. Using morphometric software, they found that cultures exposed for 24 h to AAP or AAP + NAC suffered reduction in cell confluence. Exposure increased the incidence of rounding cells and of apoptotic and autoschizic appearances. Differences between control cultures cultivated without serum versus those exposed to xenobiotics were merely quantitative, not essential.

Acetaminophen↗

PubMed2004

Surface properties and in vitro cytopathic effect of various strains of Trichomonas vaginalis.

The in vitro cytopathic effect of four strains of Trichomonas vaginalis on cultured epithelial monolayers was analyzed through electrophysiology and electron microscopy. Interaction of trichomonads of two virulent strains (GT-10 and GT-13) with cultured MDCK cell monolayers mounted in Ussing chambers produced a rapid decrease in transepithelial resistance to less than 30% of control values after only 15 min. By 30 min the electrical resistance was practically abolished by the virulent parasites. In contrast, of two attenuated strains of trichomonads (GT-3 and GT-7) analyzed under similar conditions, GT-3 trophozoites required 180 min to reduce transepithelial resistance to 9% of control values, while monolayers in contact with GT-7 parasites still showed 28% of control values at this time of incubation. Sequential scanning electron microscopy confirmed the much faster and widespread cytopathic effect of virulent parasites. In contrast, the slow lytic process produced by attenuated trophozoites was reduced to focal areas of direct contact with epithelial cells. Another difference was found by measurement of the surface charge of the four strains of T. vaginalis by means of cell microelectrophoresis. While the two virulent strains showed a negative surface charge, the two attenuated strains had no detectable surface charge at neutral pH. When parasites were incubated with cationized ferritin and studied with transmission electron microscopy the surface of virulent trichomonads appeared heavily labeled, whereas the surface of attenuated parasites had only sparse and irregular ferritin binding.

Animals↗

PubMed2004

SRC regulates actin dynamics and invasion of malignant glial cells in three dimensions.

Malignant glioma is the major brain tumor in adults and has a poor prognosis. The failure to control invasive cell subpopulations may be the key reason for local glioma recurrence after radical tumor resection and may contribute substantially to the failure of the other treatment modalities such as radiation therapy and chemotherapy. As a model for this invasion, we have implanted spheroids from a human glioma cell line (U251) in three-dimensional collagen type I matrices, which these cells readily invade. We first observed that the Src family kinase-specific pharmacologic inhibitors PP2 and SU6656 significantly inhibited the invasion of the cells in this assay. We confirmed this result by showing that expression of two inhibitors of Src family function, dominant-negative-Src and CSK, also suppressed glioma cell invasion. To characterize this effect at the level of the cytoskeleton, we used fluorescent time-lapse microscopy on U251 cells stably expressing a YFP-actin construct and observed a rapid change in actin dynamics following addition of PP2 in both two-dimensional and three-dimensional cultures. In monolayer cultures, PP2 caused the disappearance of peripheral membrane ruffles within minutes. In three-dimensional cultures, PP2 induced the loss of actin bursting at the leading tip of the invadopodium. The inhibition of Src family activity is thus a potential therapeutic approach to treat highly invasive malignant glioma.

Actin Cytoskeleton↗

PubMedDate not supplied

Arising podosomal structures are associated with neoplastic cell morphological phenotype induced by the microenvironment.

Increased numbers of rosettes of podosomes were observed in overgrown rat Rous sarcoma RsK4 cells. A possible role of these structures in nutrient uptake in tumour cell survival was investigated by exposure to acute starvation. A single cell suspension of RsK4 cells in Hanks balanced salt solution was allowed to interact with either clean uncoated or serum-coated for bait coverglasses. Confocal microscopy revealed contrasting 3D cell morphologies that were associated with conspicuous patterns of podosomal structures, which on the coated coverglasses resembled the sealing zones of osteoclasts, while on the uncoated coverglasses they resembled the marginal podosomes of migrating monocyte-derived cells. Thus, the arising podosomal structures, the involvement of which in an uptake of nutrients appeared feasible morphologically, were associated with the emerging 3D cell shapes guided by the microenvironment. Such phenotypic plasticity of neoplastic RsK4 cells in response to microenvironmental challenge suggested that uniqueness in cellular attributes within the neoplastic cell population could be crucial for the malignant potential.

Actins↗

PubMed2004

Forces required to initiate membrane tether extrusion from cell surface depend on cell type but not on the surface molecule.

When a cell adhered to another cell or substratum via surface proteins is forced to detach, lipid membrane tethers are often extruded from the cell surface before the protein bond dissociates. For example, during the inflammatory reaction leukocytes roll on the surface of activated endothelial cells. The rolling adhesion is mediated by interactions of selectins with their ligands, e.g., P-selectin glycoprotein ligand (PSGL)-1, which extrudes membrane tethers from the surfaces of both leukocytes and endothelial cells. Membrane tether extrusion has been suggested to regulate leukocyte rolling. Here we examine several factors that may affect forces required to initiate membrane tethers, or initial tether force. It was found that initial tether forces were similar regardless of the presence or absence of the cytoplasmic tail of P-selectin and regardless of whether the tethers were extruded via binding to PSGL-1 or Fcy receptors. Initial tether forces were found to depend on the cell types tested and were greatly reduced by treatment of latrunculin A, which inhibits actin polymerization. These data provide additional insights to the control of membrane tether extrusion, which should be taken into account when cellular functions such as rolling where tether extrusion plays a regulatory role are compared using different cell types expressing the same molecule.

Animals↗

PubMed1981

Mechanisms of nutritive endocytosis. I. Phagocytic versatility and cellular recognition in Chlorohydra digestive cells, a scanning electron microscope study.

The quantity of surface membrane internalized during phagocytosis by Chlorohydra digestive cells was estimated for a range of particle types. Challenge with 2 of these particles, freshly isolated symbiotic algae (FIS) and latex spheres (LS), resulted in a greater (2.5 X) quantity of surface membrane interiorized than with heat-treated symbiotic algae (HTS) and free living algae (FA), Chlorella vulgaris. This discriminatory process was investigated further by a scanning electron microscope (SEM) and transmission electron microscope (TEM) comparison of the surface events associated with phagocytosis of each of these 4 particles. Those particles that were avidly phagocytized, FIS and LS, were both enveloped by a tightly fitting extension of digestive-cell surface, and obtained a prominent surface coating after their injection into the gut of Chlorohydra. Phagocytic challenge with FIS resulted, furthermore, in the rapid formation of a dense microvillar cover on digestive-cell surfaces. HTS and FA, on the other hand, were enveloped by a less closely fitting extension of digestive-cell surface, did not obtain a prominent surface coating, and did not induce the formation of microvilli. In addition, SEM revealed that at least 3 morphologically distinct phagocytic modes were utilized by the versatile nutritive phagocyte of Chlorohydra: (I) envelopment by the progressive movement of numerous, overlapping tubular protrusions (microvilli) over the particle (FIS) surface, forming first a network of tubular interlocking members, and finally a continuous but rough enclosing surface; (2) envelopment by a single, smooth-surfaced, funnel-like extension of digestive-cell surface (FIS, LS, HTS, FA); and (3) envelopment by multiple, broad folds, often of unequal size, and with overlapping margins (Artemia particles).

Cell Membrane↗

PubMed2000

Reaction-diffusion waves of actin filament polymerization/depolymerization in Dictyostelium pseudopodium extension and cell locomotion.

Cell surface movements and the intracellular spatial patterns and dynamics of actin filament (F-actin) were investigated in living and formalin-fixed cells of Dictyostelium discoideum by confocal microscopy. Excitation waves of F-actin assembly developed and propagated several micrometers at up to 26 microm/min in cells which had been intracellularly loaded with fluorescently labeled actin monomer. Wave propagation and extinction corresponded with the initiation and attenuation of pseudopodium extension and cell advance, respectively. The identification of chemical waves was supported by the ring, sphere, spiral and scroll wave patterns, which were observed in the extensions of fixed cells stained with phalloidin-rhodamine, and by the similar, asymmetrical [F-actin] distribution in wavefronts in living and fixed cells. These F-actin patterns and dynamics in Dictyostelium provide evidence for a new supramolecular state of actin, which propagates as a self-organized, reaction-diffusion wave of reversible F-actin assembly and affects pseudopodium extension. Actin's properties of oscillation and self-organization might also fundamentally determine the nature of the eukaryotic cell's reactions of adaptation, timing and signal response.

Actins↗

PubMed1999

Identification of PA2.26 antigen as a novel cell-surface mucin-type glycoprotein that induces plasma membrane extensions and increased motility in keratinocytes.

PA2.26 antigen was identified as a cell-surface protein induced in epidermal carcinogenesis and skin remodeling processes. PA2.26 is expressed in carcinoma cell lines and cultured fibroblasts but absent in nontumorigenic keratinocytes. In tissues, PA2.26 is present in epithelial cells of the choroid plexus, ependyma, glomerulus and alveolus, in mesothelial cells, and in endothelia of lymphatic vessels. Biochemical characterization of PA2.26 protein and sequence analysis of the isolated cDNA demonstrate that PA2.26 antigen is a mucin-like transmembrane glycoprotein. Confocal and immunoelectron microscopy analysis in cultured cells reveal that PA2. 26 is concentrated in actin-rich microvilli and plasma membrane projections, such as filopodia, lamellipodia and ruffles, where it colocalizes with members of the ERM (ezrin, radixin, moesin) family protein. Ezrin and moesin, but not radixin, can be coimmunoprecipitated together with PA2.26 from cell lysates. Ectopic expression of PA2.26 in immortalized, nontumorigenic, keratinocytes induces an epithelial-fibroblastoid morphological conversion with increased plasma membrane extensions, concomitantly to a major reorganization of the actin cytoskeleton, redistribution of ezrin to cell-surface projections, and enhanced motility. These findings suggest an involvement of PA2.26 in cell migration.

Actins↗

PubMed2006

Calcium ionophore-induced de-encryption of tissue factor in monocytes is associated with extensive cell death.

INTRODUCTION: Cell surface tissue factor (TF) is normally encrypted, but can be activated by various cellular perturbations. Exposure of TF bearing cells to calcium ionophore has been reported to increase TF activity, de-encrypt TF, by phosphatidylserine (PS)-dependent and -independent mechanisms. Our aim has been to examine at the single cell level, if increased cell surface PS coincided with increased cell surface TF antigen, and cell death (necrosis, 7-AAD-intercalation), and relate this to monocyte- and microparticle (MP)-associated procoagulant activity. MATERIALS AND METHODS: We exposed lipopolysaccharide-stimulated, human, elutriation-purified, cryopreserved TF bearing monocytes to increasing concentrations of calcium ionophore (A23187) and measured procoagulant activity in cells and supernatants. These measurements were compared with quantification of cell surface TF and PS (Annexin V) and of cell necrosis (7-AAD) by flow cytometry, and complemented by confocal microscopy. RESULTS: We observed that calcium ionophore increased cellular and MP-associated TF activity, but not cell surface TF antigen. The discrepancy between TF activity and TF antigen coincided with a dose-dependent increase in the number of cells expressing PS. These cells were to a large extent necrotic and many of them also expressed TF. CONCLUSIONS: We suggest such TF positive dying cells to contribute to the discordance between TF activity and TF expression. Calcium ionophore also increased MP-associated TF activity and release of MPs may be a way to disseminate procoagulant activity. Our findings emphasize the importance of adequately assessing cell death and taking into consideration its possible role in experiments with calcium ionophore.

Blood Coagulation↗

PubMed1989

Micropipette aspiration of guinea pig megakaryocytes: absence of fragmentation and dependence on maturation stage.

Platelet release has been alternatively viewed as a fragmentation of platelet territories demarcated within the cytoplasm of mature megakaryocytes or as a later event involving segmentation of proplatelet pseudopodia extended from the cell. The mechanical constraints on platelet release were evaluated by measuring the resistance of guinea pig megakaryocytes to aspiration into micropipettes of similar diameter to the width of naturally forming proplatelet projections. Application of increasing negative pressure to the surface of the cells resulted in progressively longer extensions being drawn into the pipette until maximal extension lengths were reached. None of the passively aspirated cytoplasmic extensions fragmented off the cells even at the highest aspiration pressure under physiologic study conditions. The longest extensions were aspirated from megakaryocytes of the most advanced maturation stage, and a proportion of the mature cells yielded very long extensions over 50 mu and up to 150 mu in length. Surprisingly, the ease of aspiration did not correlate to cell size during any stage of maturation. The mechanical behavior of guinea pig megakaryocytes indicates a large availability of surface for extension in mature cells ideal for active proplatelet projection. The lack of mechanical fragility suggests that platelet release is a very late maturational event not yet initiated in the "mature" megakaryocytes available for study from marrow harvests.

Animals↗

PubMed1976

A consideration of the role of cell surface macromolecules in the process of viral transformation.

There is extensive physiological evidence implicating the cell surface as the key organelle which mediates the cell:cell interactions which underlie both normal and neoplastic growth. This information has now been supplemented with biochemical and biophysical data which indicates that surface macromolecules, in particular the heteroglycans of transformed cells, differ from those which lie at the periphery of normal cells. In the case of cells neoplastically transformed by most tumour viruses it is clear that the small virus genome (2-5 x 10(6) daltons) cannot carry the total genetic information to accomodate these various biochemical modifications, if indeed they are encoded in separate genes (1). To examine the part played in transformation by cellular genes coding for surface heteroglycan formation, we have turned to a study of SV-3T3 cells (ts H6-15) which are temperature-sensitive for expression of the transformed cell phenotype (2). The data show that cells grown under conditions permissive and non-permissive for such expression exhibit the same pattern of formation of glycolipids, and the majority of the polypeptides of the plasma membrane. There are, however, significant differences in the synthesis of some glycopeptides. A large molecular weight, trypsin-labile glycopeptide, present at the surface of untransformed fibroblasts but barely measurable in some of their virus-transformed derivatives (3), was detected, essentially at the same level, at the surface of ts H6-15 cells grown at the permissive and non-permissive temperatures. The signficance of these observations is discussed.

Cell Adhesion↗

PubMed1974

Replication of Sindbis virus. IV. Electron microscope study of the insertion of viral glycoproteins into the surface of infected chick cells.

The appearance of Sindbis virus-envelope glycoproteins in the surfaces of chicken embryo fibroblasts was studied by an indirect labeling technique. This technique involved treating infected cells sequentially with rabbit immunoglobulin G (IgG) specific for Sindbis virus followed by hemocyanin-conjugated goat (anti-rabbit IgG) IgG; surface replicas of these cells were then prepared and examined in the electron microscope. As early as 2 h after infection (and at least 1 h before mature virions were released), newly synthesized virus-envelope glycoproteins were detected at the cell surface. By 3 h after infection, cell surface membranes were extensively modified by the insertion of the Sindbis glycoproteins. When infected cells were prefixed with glutaraldehyde before labeling, the glycoproteins were distributed fairly evenly over the cell surface, although a slight clustering was observed on cells labeled early in infection. However, no evidence for large-scale clustering of virus glycoproteins corresponding to patches of budding virus was observed. Similar results were found with unfixed cells labeled at 4 C. However, when unfixed cells were labeled at 37 C, the glycoproteins were shown to be in discrete clusters, demonstrating that these glycoprotein antigens can diffuse laterally through the cell membrane at this temperature.

Animals↗

PubMed1986

Phagocytosis of Staphylococcus aureus by cultured bovine aortic endothelial cells: model for postadherence events in endovascular infections.

We examined the interaction of Staphylococcus aureus with cultured bovine aortic endothelial cells as a model for the initial events in the pathogenesis of endovascular infections. Confluent monolayers of cultured endothelial cells were incubated with S. aureus. Cell-associated bacteria were measured by washing away nonadherent organisms, disrupting the monolayers, and performing quantitative cultures. Phagocytosis was differentiated from adherence by treating the cells with lysostaphin; approximately 60% of cell-associated bacteria was found to be intracellular. Phagocytosis could be blocked by using cytochalasin B, which interferes with microfilament function. Addition of fibronectin resulted in a 63% increase in adherence of S. aureus to the endothelial cells but did not increase ingestion. Transmission electron microscopy demonstrated a sequence of events similar to that which occurs during ingestion by professional phagocytes, including: adherence of bacteria to the endothelial cell; formation and elongation of surface extensions of the endothelial cell to surround the adherent bacteria; and complete enclosure within apparent phagosomes. Phagocytosis of bacteria by endothelial cells, followed by intracellular persistence, may be an important postadherence event in the pathogenesis and pathophysiology of endovascular infections.

Animals↗

PubMed1983

Changes in cell-surface fucose-containing glycopeptides and adhesion of cultured intestinal epithelial cells as a function of cell density.

Confluent cultured intestinal epithelial cells displayed greater adhesion to the substratum than did subconfluent cells. Subconfluent and confluent cells were labelled with [3H]fucose for 24h and the cell-surface components were released by mild Pronase treatment. After extensive Pronase digestion, cell-surface and cell-residue glycopeptides were fractionated on Bio-Gel P-6. The cell surface contained a higher proportion of lower-molecular-weight glycopeptides than the residue. No significant difference in elution pattern was found between total cell-surface glycopeptides of subconfluent and confluent cells. However, confluent cells contained almost twice as much [3H]-fucose-labelled glycopeptides that were bound to concanavalin A-Sepharose and were subsequently eluted with 20mM-methyl alpha-D-glucopyranoside as subconfluent cells. When the bound glycopeptides were chromatographed on Bio-Gel P-6, it was found that confluent cells contained a larger proportion of lower-molecular-weight glycopeptides than subconfluent cells. This difference in size was eliminated after treatment of glycopeptides with sialidase. When growth of subconfluent cells was inhibited with a non-toxic concentration of retinoic acid, no significant effect on the elution pattern of [3H]fucose-labelled glycopeptides was observed on either Bio-Gel P-6 or concanavalin A-Sepharose. No significant difference was found in the total [3H]fucose-labelled glycoproteins from subconfluent and confluent cells by two-dimensional gel electrophoresis. It is suggested that the differences in [3H]fucose-labelled glycopeptides between subconfluent and confluent cells are cell-density-dependent rather than growth-dependent, and that these differences are likely to result from some changes in glycosylation mechanism(s). Furthermore, the differences in cell-surface glycopeptides may be related to the changes in the adhesion of the cells to the substratum.

Animals↗