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PubMed1999

Acto-myosin drug effects and aqueous outflow function.

PURPOSE: Previous studies have identified the cytoskeletal proteins actin and tubulin as potential cellular targets in the trabecular meshwork for novel glaucoma therapy. The authors and others have hypothesized that acto-myosin interactions may be important for outflow function. The current study was conducted to evaluate 2,3-butanedione 2-monoxime (BDM), a compound that interferes with acto-myosin function through the myosin adenosine triphosphatase (ATPase) reaction; 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7), a proposed myosin light-chain kinase inhibitor; and the direct actin disrupter, latrunculin B, in an outflow pathway cell culture and perfused excised eye model system. METHODS: Freshly enucleated porcine eyes were perfused using the constant-pressure method at 15 mm Hg and 25 degrees C. Human trabecular meshwork (HTM) cells and Schlemm's canal (SC) cells were grown in culture, treated with BDM, H-7, and latrunculin B, and then fixed, stained for beta-tubulin and filamentous actin, and observed by epifluorescence. RESULTS: Twenty millimolar BDM, 100 microM H-7, and 1 microM latrunculin B increased outflow facility 36%, 63%, and 72%, respectively, compared to sham-treated controls, 13%, 15%, and 4% (n=7, 8, and 8; P=0.01, 0.0001, and 0.0002), respectively. In cultured HTM and SC cells, 100 microM H-7 caused a rapid loss of filamentous actin staining but did not produce a change in cell shape or cell- cell attachment. In contrast, 20 mM BDM induced a loss of cell- cell attachment and a change in cell shape that was associated with a 50% to 60% loss of filamentous actin staining, often in a distinct stick-and-ball pattern. Latrunculin B caused a severe loss of actin staining and cell shape changes. No drug altered beta-tubulin staining. CONCLUSIONS: Interference with myosin function can cause a secondary loss of actin organizational structure. Our study indicates that myosin, perhaps through its various phosphorylation reactions, may have a potential regulatory role in outflow function.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1985

Cell density and cell shape-related regulation of vimentin and cytokeratin synthesis. Inhibition of vimentin synthesis and appearance of a new 45 kD cytokeratin in dense epithelial cell cultures.

The pattern of the intermediate type filament protein synthesis was examined in cultured bovine mammary gland epithelial (BMGE) cells under conditions of varied cell shape and cell-cell contact. In dense monolayer and suspension cultures BMGE cells expressed a new cytokeratin of 45 kD identified as a member of the acidic subfamily of cytokeratins. This polypeptide has a phosphorylated component and is dissociated from the cytokeratins complex in the presence of 6.5 M urea. The mRNA of the new cytokeratin accumulated in dense cell cultures, as revealed by in vitro translation in a cell-free system. In BMGE-H cells that express also vimentin, the synthesis of vimentin decreased dramatically in dense cell cultures, while the synthesis of the 45 kD cytokeratin was maximal under these conditions. The results suggest that the expression of certain cytokeratins and that of vimentin can be coordinately regulated by factors in the cellular environment that effect cell shape and cell surface contacts.

Animals↗

PubMed1995

Shape changes of osteoblastic cells under gravitational variations during parabolic flight--relationship with PGE2 synthesis.

The relationship existing between cell morphology and cell metabolism, and the role of mechanical load in bone remodelling are well-known. In osteoblasts, PGE2 mediates part of the response to mechanical stress and induce cell shape changes. We studied the influence of gravity variations on osteoblast morphology and its relationship with PGE2 synthesis during a parabolic flight. ROS 17/2.8 osteosarcoma cells flew 15 or 30 parabolae. We measured cell area and shape factor after fluorescein staining with a semi-automatic image analyser and PGE2 levels by RIA. Significant flight-induced shape changes consisted in a decrease in cell area and an increase in shape factor (cell irregularity), in some cells, as compared to ground controls. This heterogeneity in cell response might be explained by a cell-cycle sensitivity to mechanical stress. A 45 min pretreatment with indomethacin inhibited the flight-induced increase in cell irregularity whereas cell area remained decreased. PGE2 levels were higher in flight than in ground controls. Linear regression analysis showed a significant negative relationship between cell area and PGE2 synthesis. We concluded that ROS 17/2.8 are highly sensitive to gravitational variations and that PGE2 is partly implicated in cell shape changes observed during parabolic flight. However, other mechanisms than PGE2 synthesis condition ROS 17/2.8 morphology in response to mechanical changes.

Animals↗

PubMed2005

Reduction of Pnn by RNAi induces loss of cell-cell adhesion between human corneal epithelial cells.

PURPOSE: Pinin (Pnn/DRS/memA) plays an important role in regulating cell-cell adhesion of corneal epithelial cells. In the nucleus, Pnn interacts with both transcriptional repressor and pre-mRNA processing machinery. Here we investigated the consequences of "knocking down" Pnn expression with short hairpin RNAi (shRNAi) on the corneal epithelial cell phenotype. METHODS: Cultured human corneal epithelial (HCE-T) cells were cotransfected with a shRNAi-expressing construct containing an inverted repeat of a Pnn specific 21 nucleotide sequence (Pnn shRNAi) and a GFP vector as a marker of transfected cells. After 24-48 h, cells were fixed and immunostained with antibodies against Pnn, keratin, desmoplakin, desmoglein, E-cadherin, ZO-1, SR-proteins, and SRm300. To demonstrate specificity of the Pnn knock down, a rescue vector was designed by incorporating three conservative nucleotide substitutions within the Pnn-shRNAi targeting sequences of the full length Pnn-GFP construct, thus generating a Pnn construct to produce mRNA that Pnn shRNAi could not target (Pnn-CS3-GFP). RESULTS: HCE-T cells were cotransfected with Pnn shRNAi and GFP vectors and after 24 and 48 h exhibited significantly reduced immunostaining for Pnn. Western blot analyses of Pnn and E-cadherin protein expression in cells transfected with Pnn-shRNAi and GFP vectors revealed marked reduction in levels of both proteins compared to those observed in cells transfected with GFP alone. The cells receiving Pnn-shRNAi appeared to be less adherent to neighboring nontransfected cells, often exhibited altered cell shape, downregulated cell adhesion and cell junction molecules, and escaped from the epithelium. The Pnn shRNAi transfected cells exhibited fewer keratin filaments anchored to desmosomes and a concurrent increase in the perinuclear bundling of filaments. SR proteins and SRm300 showed an altered distribution in the Pnn knock down cells. Cotransfection of Pnn-CS3-GFP with Pnn shRNAi demonstrated that the conservatively mutated Pnn could maintain cell-cell adhesion. CONCLUSIONS: Our results indicate that knocking down Pnn expression leads to a loss of epithelial cell-cell adhesion, changes in cell shape, and movement of Pnn shRNAi transfected cells out of the epithelium. We suggest that Pnn plays an integral role in the establishment and maintenance of epithelial cell-cell adhesion via its activity within nuclear multi-protein complexes.

Blotting, Western↗

PubMed1991

[The cellular polymorphism of the myointimal thickening in the rabbit aorta].

Partly endothelialized myointimal thickening formed in rabbit aorta after balloon catheter injury. It was populated by cells of different shape. There were spindle-shaped cells. Y-shaped cells, cells of irregular shape with processes and stellate-shaped cells. As a rule stellate cells situated just near the endothelium. It is supposed, that the endothelium can influence the shape of myointimal cells.

Animals↗

PubMed1999

Embryonic mesenchymal cells share the potential for smooth muscle differentiation: myogenesis is controlled by the cell's shape.

Undifferentiated embryonic mesenchymal cells are round/cuboidal in shape. During development, visceral myogenesis is shortly preceded by mesenchymal cell elongation. To determine the role of the cell's shape on smooth muscle development, undifferentiated embryonic mesenchymal cells from intestine (abundant visceral muscle), lung (some visceral muscle) or kidney (no visceral muscle) were plated under conditions that maintained cell rounding or promoted elongation. Regardless of their fate in vivo, all the cells differentiated into smooth muscle upon elongation as indicated by the expression of smooth muscle-specific proteins and the development of membrane potentials of -60 mV and voltage-dependent Ca2+ currents, characteristic of excitable cells. Smooth muscle differentiation occurred within 24 hours and was independent of cell proliferation. Regardless of their fate in vivo, all the round cells remained negative for smooth muscle markers, had membrane potentials of -30 mV and showed no voltage-activated current. These cells, however, differentiated into smooth muscle upon elongation. The role of the cell's shape in controlling smooth muscle differentiation was not overcome by treatment with retinoic acid, TGF-beta1, PDGF BB or epithelial-conditioned medium (all modulators of smooth muscle differentiation). These studies suggest that the mesenchymal cell shape plays a main role in visceral myogenesis.

Animals↗

PubMed1994

Pattern formation in plant development: four vignettes.

In their development, plants precisely control the patterns of cell size, cell shape and cell division, the position of differentiation of cell types, and the position and number of organs. In the past year, progress has been made in understanding four of these five levels of control of plant pattern formation, including specification of cell fate by cell size in the alga Volvox carteri, genetic control of cell shape in the leaf hairs of the flowering plant Arabidopsis thaliana, precise control of cell division and cellular enlargement in Arabidopsis root meristems, and control of cell number in meristems of Arabidopsis, tobacco, and maize. These examples indicate that both the fate and shape of differentiated plant cells can be determined by the size of their undifferentiated precursors, and that the primary effect of mutations that alter cell fate and cellular shape can be on cell size. Furthermore, specific genes have been found that are necessary for normal patterns and numbers of cell divisions. Molecular cloning of these genes is revealing the molecular basis of plant cell division control.

Arabidopsis↗

PubMed2005

Synergistic interaction of topographic features in the production of bone-like nodules on Ti surfaces by rat osteoblasts.

The objective of this study was to study the responses of osteoblast-like cells to rough Titanium (Ti)-coated epoxy surfaces of differing topographic complexity. Four topographies were studied: polished (PO), coarse-blasted (CB), acid-etched (AE) and coarse-blasted+acid-etched (SLA). Rat osteoblasts were cultured on these surfaces and their morphology, thickness as well as the number and size of bone-like nodules measured. To determine cell shape and cell thickness, fluorescein-5-thiosemicarbazide was used to stain the cell components including the cell membrane, the stained cells were optically sectioned using epifluorescent microscopy and the optical sections were computationally reconstructed to obtain three-dimensional images in which cell volume and cell thickness could be determined. Similarly optical sections of bone-like nodules labeled with tetracycline were also reconstructed to determine their size. The different surface topographies were found to alter the thickness and morphology of osteoblasts cultured on these surfaces. Osteoblasts produced significantly more and larger nodules on SLA compared to other surfaces. Nevertheless and perhaps surprisingly, given the evidence in various cell populations that cell shape can affect cell differentiation, cell thickness was not directly correlated with an increase in bone-like nodule formation. Data were analyzed by factorial analysis of variance. In this way the primary effect of each surface treatment ( i.e. blasting and acid etching) could be assessed as well as their interaction. Both the acid etching and blasting processes significantly affected the number and size of bone-like nodules cultured on Ti surfaces. Moreover there were significant interaction effects indicating that surface topographic features can act synergistically to enhance bone formation. This result suggests that a useful approach to the optimization of surfaces for bone production could involve systematic investigation of combinations of processes each of which produces distinct surface topographical features.

Animals↗

PubMed1993

Quantitative ultrastructural analysis of the relationship between cell growth, shape change, and mucosecretory differentiation in cultured hamster tracheal epithelial cells exposed to retinoic acid.

Hamster tracheal epithelial cells in extended (32 degrees C) primary culture with and without supplemental retinoic acid (RA) were studied during the proliferative (5 days) and differentiation phases (11 days) by correlative transmission electron microscopy (EM) and light microscopic (LM) autoradiography to quantify the relationship between cell proliferation, shape change, and mucin granule expression. In retinyl acetate-containing control medium, cell numerical density was higher and [3H]thymidine labeling index (LI) lower at day 11 compared with day 5. The addition of 10(-7) M RA to the medium caused an increase in cell numerical density at both times. LI was increased by RA at 5 days and decreased at 11 days. Measurements of cell shape in ultrathin sections adjacent to LM autoradiographs made in the vertical plane demonstrated an RA-induced change from flat to cuboidal at 5 days and a more columnar phenotype at 11 days. Cells containing mucin granules were of two main types based on their ultrastructure. One type, seen at 5 and 11 days, contained diminutive mucin granules and had an LI of 50% at 11 days. Its LI and frequency (26%) were unaltered by RA. The other type, less frequent (15%) and present only at 11 days, was more columnar and contained mucous granules similar to those found in vivo. RA doubled the frequency of this cell type but did not affect its LI (11%). Cells of this type with more than five mucin granules in EM profile did not incorporate thymidine. The data indicate that RA accelerates and enhances cell shape change toward a more cuboidal phenotype.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2005

In vitro study of the interaction of polyalkilimide and polyvinyl alcohol hydrogels with cells.

Hydrogels are a class of polymers that in the last decade have had a great development and application for soft tissue augmentation, due to their similarity to this tissue for their high water content. The in vitro effects of polyalkylmide hydrogel (pAI) and polyvinyl alcohol hydrogel (pVOH) on human lymphocytes and U937 cells viability, apoptosis and cell shape were investigated. Cell viability was always higher than 70%, thus showing the hydrogels were not cytotoxic for both cell lines. Some differences were, however, found. At short time, lymphocytes were very sensitive to the hydrogels incubation, while at long time, U937 cells were the most sensitive cells. Other differences on cell viability were related to the time of incubation, to the type of hydrogel and to the polymers concentration. Cell viability decreased only at the longest time of incubation and with the highest hydrogel concentration. Accordingly, cell death by apoptosis increased; necrosis was never observed in the cultures. Concentration- and hydrogel-dependent modifications of cell shape (bigger cell volume, elongations of cells) were observed in a few percentage of viable cells. In conclusion, the very high in vitro degree of biocompatibility shown by both hydrogels encourages their use as dermal fillers.

Acrylic Resins↗

PubMed1995

Culturing of acoustic neuroma--methodological aspects.

We have been able to culture acoustic neuroma (AN) tissues by taking small tumor pieces during surgery from the cisternal or, preferably, metal part of the tumor where macroscopic tissue degeneration is not prominent. The AN pieces were transported in culture tubes to our cell laboratory where they were sliced and transferred to culture medium. The AN tissue demands a crude culture suspension containing laminin. We have been successful in culturing 15 successive ANs. The cells have different morphological features with characteristics of type Anthony A and B. Four main cell types were recognized: ameboid microglia-like cells, spindle-shaped cells, racket-shaped cells, and kite-shaped cells. However, no immortal cell line could be established. Some of the cultures were viable for up to 4 months.

Culture Media↗

PubMed1986

Cell-substratum and cell-cell interactions promote testicular peritubular myoid cell histotypic expression in vitro.

Peritubular cells, prepared from seminiferous tubules from testes of 20-day-old-rats, were seeded onto different substrata and cultured under varying conditions. When plated onto polystyrene or glass surfaces, peritubular cells assumed a typical fibroblast-like cell shape and cell association pattern, together with a fibroblast-like migration behavior. They maintained high rates of proliferation even after achieving confluency. In contrast, when peritubular cells were plated onto a seminiferous tubule biomatrix (ST-biomatrix) surface, they spread to form a continuous cell layer having a myoepithelioid histotype similar to that of peritubular myoid cells in the intact seminiferous tubule. The characteristics of the myoepithelioid histotype described include a squamous, polyhedral cell shape; a cobblestone-like cell association pattern, with closely apposing or slightly overlapping cell borders, and a very low mitotic index. When peritubular cells were plated onto laminin, collagen, fibronectin, heparin, or a liver biomatrix, a fibroblast-like pattern resulted, indicating that ECM components listed and liver biomatrix are unable to substitute for ST-biomatrix in maintaining normal myoepithelioid characteristics in vitro. In cocultures of Sertoli cells plated on top of peritubular cells, the peritubular cells directly in contact with Sertoli cell aggregates developed a myoepithelioid histotype, whereas peritubular cells in regions not in direct contact had a fibroblast-like histotype. The data are discussed in relation to the possible role of cell-cell interactions, and cell-substratum interactions, in the acquisition and stabilization of the histotype of peritubular cells in the seminiferous tubule during development.

Animals↗

PubMed2000

The relation between the rod-like inner structures and the shapes of the cells.

The shapes of the cells with simple rod-like inner structures are studied theoretically. Since the cell with inner structure can be bent, the possibility of non-axisymmetric shapes is considered. The equilibrium shape of the cell, obtained by minimizing the sum of the membrane bending energy and the bending energy of the rod, depends on the ratio between the bending constant of the membrane and the bending rigidity of the polymer rod. The dependence of the cell shape on the length of the rod and on the difference between inner and outer membrane layer areas is presented.

Cell Size↗

PubMed1982

The effect of ophthalmic preservatives on the shape of corneal endothelial cells.

The mean area (293.6 +/- 53.6 micrometers 2) and perimeter (65.5 +/- 5.8 micrometers) of individual cells were determined from the photographed endothelium of a total of 54 guinea pig corneas, after staining with silver nitrate and removal of the epithelium and stroma. In addition a quantitative assessment of cell shape S (= P2/A) was calculated. The mean cell density (3406 +/- 619 cells per mm2) was similar to that for young humans, and the S value (14.76 +/- 0.73) was as expected for slightly irregular hexagonal and pentagonal shapes. Incubation of the corneas in glutathione-bicarbonate-Ringer solution for periods up to 4 h resulted in only slight changes in the endothelial cell shape (S = 15.21 +/- 0.89). However, incubation with thimerosal (0.01%) for 2 h (S = 16.78 +/- 1.79) or with disodium ethylenediaminetetraacetate (Na2EDTA) (0.2%) for 1 h (S = 17.20 +/- 2.36) gave a marked increase in the tortuosity of the cell 'outlines'. Longer periods of contact with Na2EDTA resulted in a rounding up and separation of endothelial cells.

Animals↗

PubMed2005

Adhesion remodeling underlying tissue morphogenesis.

Cell-adhesion molecules localized at adherens junctions (AJs) maintain the polarized architecture of epithelial cells but limit their movements. The morphogenesis of a developing epithelium is associated with the control of both cell shape and cell contacts. Epithelial cells remodel their contacts, and intercellular adhesion controlled by cadherin molecules is spatially and temporally regulated. Cell shape depends, in part, on the regulation of cell adhesion between different groups of cells. Patterned epithelial cell movements such as those that occur during cell intercalation--a universal process whereby cells exchange neighbors--rely on the polarized remodeling of AJs. Recent studies show that the understanding of adhesion will benefit from studies of developing organisms in which adhesion is regulated.

Animals↗

PubMed1987

Analysis of polarization and orientation of human polymorphonuclear leukocytes by computer-interfaced video microscopy.

Chemotactic behavior is a complex cellular response to chemical environmental stimuli. For polymorphonuclear leukocytes (PMNs), such behavior involves net migration as well as changes in cell shape and cell orientation. Accordingly, we have applied computer-interfaced video microscopy to analyze cell shape and orientation in control and patient PMNs migrating under agarose. From a digitized tracing of the PMNs at the leading front of migration, cells were characterized in terms of area, circumference, and longest dimension. A shape factor and angle of orientation were computed. Numerical shape factors discriminated three PMN morphologies: polar, apolar, and hyperpolar. Only polar cells could be oriented. Orientation of polar cells was defined as toward, away, or disoriented with respect to the chemotactic gradient. Apolar cells were considered to be nonoriented. Of PMNs from healthy controls, 30 +/- 5% of the cells were oriented toward and 11 +/- 4% of the cells were oriented away from the gradient. For PMNs from patients with localized juvenile periodontitis, a 40% deficit in net migration was associated with reduced orientation toward (5 +/- 2%) and elevated orientation away from the gradient (33 +/- 9%). PMNs from a panel of patients with thermal injury showed reduced migration and orientation toward the gradient associated with elevated percentages of apolar cells. Such analysis of PMN polarization and orientation of the leading front permitted calculation of a chemotactic behavior index. Application of this multiparameter index to the analysis of the chemotactic response may identify PMNs that are defective, but not by evaluation of any single variable.

Burns↗

PubMed2001

Theoretical analysis of shape transformation of V-79 cells after treatment with cytochalasin B.

We observed that after treatment of V-79 fibroblasts with cytochalasin B the area of cell contact with the substrate is essentially reduced, the microtubules are organized into rodlike structures and the actin filaments are disintegrated. Remnants of the actin cortex become concentrated in the form of discrete patches under the plasma membrane. The described changes in the organization of the cytoskeleton and of the cortical shell are accompanied by the formation of a cell shape resembling the Greek letter phi. We calculated that the phi shape corresponds to the minimum of the stretching energy of the cortical shell at relevant geometrical constraints. In line with this result, if cytochalasin B treatment was followed by colchicine application which disrupted the microtubular rod, the characteristic phi shape completely disappeared. This study suggests that the effect of the microtubular rod on the cell shape can be theoretically well described by taking into account some basic conditions for the mechanical equilibrium of the cell cortical shell and the appropriate geometrical constraints.

Actins↗

PubMed2004

The effect of resting transmembrane voltage on cell electropermeabilization: a numerical analysis.

The transmembrane voltage induced due to applied electric field superimposes to the resting transmembrane voltage of the cell. On the part of the cell membrane, where the transmembrane voltage exceeds the threshold transmembrane voltage, changes in the membrane occur, leading to increase in membrane permeability known as electropermeabilization. This part of the cell membrane represents the permeabilized area through which the transport of molecules occurs. In this paper we calculated numerically the permeabilized area for different electric field strength, resting transmembrane voltage, cell shape and cell orientation with respect to the applied electric field. Results show that when the transmembrane voltage is near the threshold transmembrane voltage, the permeabilized area of the cell is increased on the anodic side and decreased on the cathodic side due to the resting transmembrane voltage. In some cases, only anodic side of the cell is permeabilized. Therefore, by using bipolar pulses, the permeabilized area can be significantly increased and consequentially also the efficiency of electropermeabilization. However, when the induced transmembrane voltage is far above the threshold, the effect of the resting transmembrane voltage is negligible. These observations are valid for different cell shapes and orientations.

Animals↗