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Cell-shape-dependent modulation of p52(PAI-1) gene expression involves a secondary response pathway.

Expression of the rat p52(PAI-1) gene is positively regulated by agents that influence cellular microfilament organization and/or cell-to-substrate adhesion [e.g. cytochalasin D (CD) and sodium n-butyrate (NaB)] [Higgins, Chaudhari and Ryan (1991) Biochem. J. 273, 651-658; Higgins, Ryan and Providence (1994) J. Cell. Physiol. 159, 187-195]. As shape-responsive genes may be subject to inducer-specific controls, the biochemical mechanisms underlying the shape-dependent pathway of p52(PAI-1) gene regulation were examined in v-ras-transformed rat kidney (KNRK) cells. NaB and/or CD effectively stimulated p52(PAI-1) run-off transcription and augmented de novo p52(PAI-1) mRNA and protein synthesis in KNRK cells; induction at both the mRNA and protein levels was inhibited by actinomycin D. Pretreatment with cycloheximide (CX) markedly attenuated NaB- and/or CD-stimulated p52(PAI-1) expression. CX alone, however, induced low levels of p52(PAI-1) mRNA; increased p52(PAI-1) protein synthesis was evident after release of KNRK cells from CX blockade. Such CX-mediated induction was also sensitive to actinomycin D. Full stimulation of p52(PAI-1) expression in KNRK cells in response to the shape modulators NaB and/or CD involves transcriptional activation of the p52(PAI-1) gene, requires de novo RNA synthesis and occurs through a secondary-response (i.e. protein-synthesis-dependent) pathway.

Butyrates↗

Polarized growth controls cell shape and bipolar bud site selection in Saccharomyces cerevisiae.

We examined the relationship between polarized growth and division site selection, two fundamental processes important for proper development of eukaryotes. Diploid Saccharomyces cerevisiae cells exhibit an ellipsoidal shape and a specific division pattern (a bipolar budding pattern). We found that the polarity genes SPA2, PEA2, BUD6, and BNI1 participate in a crucial step of bud morphogenesis, apical growth. Deleting these genes results in round cells and diminishes bud elongation in mutants that exhibit pronounced apical growth. Examination of distribution of the polarized secretion marker Sec4 demonstrates that spa2Delta, pea2Delta, bud6Delta, and bni1Delta mutants fail to concentrate Sec4 at the bud tip during apical growth and at the division site during repolarization just prior to cytokinesis. Moreover, cell surface expansion is not confined to the distal tip of the bud in these mutants. In addition, we found that the p21-activated kinase homologue Ste20 is also important for both apical growth and bipolar bud site selection. We further examined how the duration of polarized growth affects bipolar bud site selection by using mutations in cell cycle regulators that control the timing of growth phases. The grr1Delta mutation enhances apical growth by stabilizing G(1) cyclins and increases the distal-pole budding in diploids. Prolonging polarized growth phases by disrupting the G(2)/M cyclin gene CLB2 enhances the accuracy of bud site selection in wild-type, spa2Delta, and ste20Delta cells, whereas shortening the polarized growth phases by deleting SWE1 decreases the fidelity of bipolar budding. This study reports the identification of components required for apical growth and demonstrates the critical role of polarized growth in bipolar bud site selection. We propose that apical growth and repolarization at the site of cytokinesis are crucial for establishing spatial cues used by diploid yeast cells to position division planes.

Cell Cycle↗

Use of Bacillus subtilis minicells to demonstrate an antigenic relationship between the poles and lateral cylindrical regions of rod-shaped cells.

Purified minicells produced by Bacillus subtilis div IV-B1 mutants were used to immunize rabbits. Immune serum was obtained that agglutinated minicells and was able to form precipitin lines when reacted with minicell antigens in double-diffusion or immunoelectrophoretic procedures. Antiminicell serum agglutinated rod-shaped B. subtilis cells to long filaments produced by growth of a cell division-defective mutant at restrictive temperature. These findings indicate that minicells are immunogens capable of eliciting the production of antibodies that cross-react with the lateral, cylindrical regions of B. subtilis rods. It appears, therefore, that poles share a common antigen(s) with cylindrical regions of the cell.

Animals↗

Cell shape and motility of oligodendrocytes cultured without neurons.

Oligodendrocytes, the myelin-forming cells of the central nervous system (CNS), were cultured from newborn rat brain and optic nerve to study how they differentiate in vitro in the absence of neurons. By use of galactocerebroside (GC) as a reference marker, the development of the cell phenotype was studied with video-enhanced differential interference contrast microscopy, immunofluorescence and electron microscopy. After a few days in culture, oligodendrocytes extend 5 to 10 main processes that are very rich in microtubules, but they did not stain with a monoclonal antibody reacting with all known classes of intermediate filaments. The number of processes can vary with the substrate on which the cells are grown; fewer processes form on laminin than on polylysine coated glass. Oligodendrocytes, in a fashion similar to that of neurons appear to keep their body immobile while the long processes grow. However, while neurons display motile activities mostly at the end of the cell processes called growth cones, the oligodendrocytes display motile, actin rich filopodia and lamellipodia along the entire length of all processes. The outgrowth of motile processes from oligodendrocytes sometimes occurs preferentially towards neighboring astrocytes. Oligodendrocyte processes display intense bidirectional movement of cytoplasmic organelles. Movement of surface components also occurs since GC molecules cross-linked by antibodies move from the processes towards the cell body. Thus, oligodendrocytes cultured without neurons develop on schedule a complex phenotype similar to their in vivo counterpart. In addition, their processes are capable of specific motile activities which may function in vivo to find the target axon and to transport myelin membrane components at the site of myelin assembly.

Animals↗

Evidence for changes in cell shape from a 2-dimensional to a 3-dimensional substrate.

Chick embryo mesoderm cells were explanted to culture systems in vivo and in vitro and their subsequent movements were correlated with the external morphology as studied by SEM. In vitro cell movements are exaggerations of normal in vivo movements where a 2-dimensional substrate is encountered rather than a 3-dimensional environment.

Animals↗

Control of cell shape and locomotion by external calcium.

Dependence of locomotion of Xenopus laevis epidermal cells on calcium influx from the external medium was investigated. Inhibition of Ca2+ influx by 2 mM La3+ or 4 mM Tb3+ in the culture medium causes an immediate stop to locomotion and a loss of motion at the outer margin of the lamella; microcolliculi disappear and the entire lamella becomes flat and very thin. The cell body region enlarges by spreading into the lamella to an extent approximately coincident with the distribution of myosin. The increase in thickness of this area is the result. The cytoskeletal elements actin, alpha-actinin and myosin become homogeneously distributed throughout the cell and a great number of straight microtubules extend to the margin after 20 min in La3+-containing media. Prekeratin distribution does not change. Reduction of calcium concentration in the external medium by EGTA leads to cessation of cell locomotion. Sr2+ (1-4 mM) is also able to replace calcium for triggering locomotion. These findings point to a control of Ca2+-activated contractions of actomyosin by influx of external Ca2+. According to our model of cell locomotion [14] the contractions generate a hydrostatic pressure extending the lamella by flow of hyaloplasm towards the margin. Small swellings (microcolliculi) appearing thereby will be dislocated by a calcium-dependent sol-gel transformation in this area, which contains actin but not myosin.

Animals↗

Effects of sample temperature on red blood cell shape in septic patients.

Several recent in vitro studies have observed that alterations in red blood cell (RBC) rheology depend on sample temperature. This possibility could limit the in vivo validity of the assessment of RBC shape. We investigated the effects of sample temperature on RBC shape estimated by flow cytometry in septic patients compared with those of volunteers. 0.5 ml of blood was placed at 4 degrees C, room temperature (20 degrees C) or at 37 degrees C. RBCs were analyzed at 30 minutes (T30) by flow cytometry and results compared to baseline measures. With the flow cytometry technique, the RBC shape of healthy volunteers shows a bimodal distribution related to the biconcave form. On this histogram, we calculated the second Pearson coefficient of dissymmetry--PCD--representing the asymmetry of this histogram. At baseline, RBC PCD was increased in septic patients representing a more spherical shape compared to volunteers (-0.73+/-0.18 versus -0.95+/-0.05; p=0.007). For both groups, RBC shape estimated by the flow cytometry technique was not modified by the temperature of the sample. We conclude that estimation of RBC shape by flow cytometry is not influenced by the temperature of the sample. The present study validates the flow cytometry technique to assess RBC shape in vitro.

Adult↗

Correlation of laser-Doppler-velocity measurements and endothelial cell shape in a stenosed dog aorta.

Laser-Doppler-velocity measurements were carried out in an elastic 1:1 true-to-scale silicone rubber model of a dog aorta with stenosis. The model was constructed from a cast of a severely stenosed dog aorta (71% of its area). The stenosis in the dog aorta was prepared by wrapping a cotton band around the aorta. This band was tightened until the presence of a thrill or a bruit was felt distal to the band. Twelve weeks later the animal was sacrificed and a cast was prepared from the aorta. From this vascular cast, the cross-sectional area was calculated. Endothelial cell geometry and orientation was studied using computerized analysis to determine the cell area and shape index. An elastic silicone rubber model was prepared from the cast to measure the velocity profiles and to estimate the local wall shear stress. Velocity measurements were done at steady and pulsatile flow using a Newtonian aqueous-glycerol solution and a non-Newtonian blood-like fluid. From those velocity measurements the velocity gradients near the wall were determined and the shear stress calculated. The flow distal to the stenosis separates from the wall at physiological conditions. The endothelial cells are smaller and more elongated in the throat; distal to the stenosis they are larger and rounder. The shape index distribution along the stenosed aorta is correlated with the level of wall shear stress. It is shown that even low changes in the wall shear stress have an influence on the orientation of the endothelial cells.

Animals↗

Bioeffects of moderate-intensity static magnetic fields on cell cultures.

The interaction of static magnetic fields (SMFs) with living organisms is a rapidly growing field of investigation. However, despite the increasing number of studies on the effects of the interaction of SMFs with living organisms, many gaps in our knowledge still remain. One reason why it is extremely important to deeply understand the true mode of action of MFs on living organisms, is the need to protect human health in consideration of the probable future introduction of new technologies such as magnetically levitated trains and the therapeutical use of MFs (e.g. magnetic resonance imaging, MRI, coupling of MF exposure with chemotherapy). The lack of knowledge of the morphological modifications brought about by exposure to moderate-intensity SMFs prompted us to investigate the bioeffects of 6mT SMFs on different cell types, by means of light and electron microscopy, confocal laser scanning microscopy and immuno- or cytochemistry. In the present article we report our own and other data from the literature on the morphological studies of the bioeffects of moderate-intensity SMFs. We focus on morphological modifications related to cell shape, cell surface, cytoskeleton, and plasma membrane expression of molecules and carbohydrate residues. The effects of exposure to moderate-intensity SMF for 24 or 48 h, on apoptosis, on apoptotic related gene products, on macrophagic differentiation and on phagocytosis of apoptotic cells in primary cell cultures (transformed or stabilized cell lines) will be also discussed. Moderate-intensity (6mT) SMFs induced modifications of cell shape, cell surface and cytoskeleton, progressively achieved during the entire period of exposure. In general, at the end of the exposure period, the cells had a less flat shape due to partial detachment from the culture dishes or a more round-elongated shape (in relation to adhesion growth or in suspension growth respectively) with many irregular lamellar microvilli, while the morphology of the organelles remained unmodified. In parallel with cell shape changes, the microfilaments and microtubules, as well as the quantity and distribution of surface ConA-FITC and Ricinus Comm.-FITC labelling sites, were modified in a time-dependent manner. Apoptosis was influenced in a cell type-dependent manner: for some cells spontaneous apoptosis decreased while, for others, it increased to about 20% after 24h of continuous exposure. The induction of apoptosis was likely due to the increment of [Ca(2+)]i during exposure. Cell proliferation was only slightly affected. Indeed, in addition to the cell type, the time of exposure was also an important factor in the intensity of the effects produced. Both apoptotic rate and cell and surface shape were influenced by exposure to SMFs when simultaneously administered with apoptogenic drugs. Apoptotic cells were cleared by an efficient and fast process of phagocytosis mediated by specific epitopes, externalized during the formation of the apoptotic cells, on the dead cells and by specific receptors on the phagocytes (both "professional" and "nonprofessional"). The recognition of apoptotic lymphocytes as well as of control cells exposed for at least 24h to 6mT SMF by liver sinusoidal cells was influenced by the cell surface modifications which both apoptotic or normal exposed cells underwent during the induction of apoptosis or SMF exposure. The degree of macrophagic differentiation of human pro-monocytic U937 cells induced by phorbol ester was decreased by exposure to 6mT SMFs, with a consequent fall in cell adhesion and increased polarization of pseudopodia and cytoplasmic protrusions. Differentiation alone, or in combination with exposure to SMFs, affects distribution and quantity of cell surface carbohydrate residues, surface expression of markers of macrophage differentiation, and phagocytic capability. The increasing amount of data reporting on the bioeffects of SMFs is leading researchers to an understanding of how important it is to fully understand the mode of action of MFs on living organisms. Indeed, even if the perturbations of biological systems by SMFs are sublethal at shorter times of exposure, these perturbations could, especially at longer times of exposure, evolve into a progressive accumulation of modifications, whose ultimate effects still need to be clarified.

Apoptosis↗

Extracellular matrix regulates ovine granulosa cell survival, proliferation and steroidogenesis: relationships between cell shape and function.

The extracellular matrix (ECM), constituting the follicular basal lamina and present also between follicular cells and in the follicular fluid, is believed to regulate granulosa cell (GC) function during follicular development. Ovine GCs isolated from small (1-3 mm in diameter) or large (4-7 mm in diameter) antral follicles were cultured on various pure ECM components (type I collagen, fibronectin, laminin), synthetic substrata enhancing (RGD peptides) or impairing (poly 2-hydroxyethylmethacrylate (poly-hema)) cell adhesion, or in the presence of heparin. The effects of these factors, used alone or in combination with IGF-I and/or FSH, were evaluated in terms of GC spread, survival, proliferation and steroidogenesis. When grown on type I collagen (CI) gel, poly-hema or heparin, GCs from both large and small follicles exhibited a round shape and a low proliferation rate. Compared with non-coated plastic substratum as a control, these ECM or synthetic compounds enhanced estradiol secretion and reduced progesterone secretion by large-follicle GCs. In contrast, GCs from both large and small follicles spread extensively on CI coating, fibronectin, laminin and RGD peptides. Fibronectin and laminin dramatically increased the proliferation rate and enhanced survival of GCs from both origins. Moreover, fibronectin, laminin and RGD peptides reduced estradiol secretion by large-follicle GCs. Unexpectedly, CI coating increased estradiol secretion and reduced progesterone secretion by large-follicle GCs, suggesting that type I collagen was able to maintain estradiol secretion independently of GC shape. Finally, GC responsiveness to IGF-I and FSH, in terms of proliferation and steroidogenesis, was generally maintained when cells were grown on ECM components, RGD peptides and in the presence of heparin. However, when large-follicle GCs were grown as non-adherent clusters (as observed on poly-hema) basal and IGF-I- and/or FSH-stimulated progesterone secretions were totally abolished. Overall, this study shows that GC shape, survival, proliferation and steroidogenesis can be modulated in vitro by pure ECM components in a specific and coordinated manner. It is suggested that, in vivo, fibronectin and laminin would sustain follicular development by enhancing the survival and proliferation of GCs, whereas type I collagen might participate in the maintenance of estradiol secretion in large antral follicles.

Analysis of Variance↗

Boundary-element calculations for dielectric behavior of doublet-shaped cells.

In order to simulate dielectric relaxation spectra (DRS) of budding yeast cells (Saccharomyces cerevisiae) in suspension, the complex polarization factor (Clausius-Mossotti factor) beta for a single cell and the complex permittivity of a cell suspension epsilon(sus)* were calculated with a doublet-shaped model (model RD), in which two spheres were connected with a part of a ring torus, using the boundary element method. The beta values were represented by a diagonal tensor consisting of components beta(z) parallel to the rotation axis (z axis) and beta(h) in a plane (h plane) perpendicular to the axis. The epsilon(sus)* values were calculated from the complex permittivity of the suspending medium epsilon(a)* and the components of beta. The calculation was compared with that of a conventional prolate spheroid model (model CP). It was found that model CP could be used as a first approximation to model RD. However, differences existed in beta(z) between models RD and CP; beta(z) showed three relaxation terms in the case of model RD in contrast with two terms in model CP. Narrowing the junction between the two spheres in model RD markedly decreased the characteristic frequency of one of the relaxation terms in beta(z). This suggests that the structure of the junction can be estimated from DRS. Effects of the shape change from model RD to a two-sphere model (model RD without the junction) were also examined. The behavior of beta(z) in the two-sphere model, the relaxation intensity of which was much lower than model RD, was quite similar to that in a single-sphere model. These simulations were consistent with the experimental observations of the dielectric behavior of the yeast cells during cell cycle progression.

Cell Polarity↗

MEK/ERK pathway mediates cell-shape-dependent plasminogen activator inhibitor type 1 gene expression upon drug-induced disruption of the microfilament and microtubule networks.

Changes in cellular morphology induced as a consequence of direct perturbation of cytoskeletal structure with network-specific targeting agents (i.e. microfilament- or microtubule-disrupting drugs) results in the stimulated expression of a specific subset of genes. Transcription of c-fos, collagenase, transforming growth factor-beta, actin, urokinase plasminogen activator and its type-1 inhibitor (PAI-1) appears to be particularly responsive to shape-activated signaling pathways. Cytochalasin D (CD) or colchicine treatment of contact-inhibited and serum-deprived vascular smooth muscle (R22) cells was used, therefore, as a model system to evaluate morphology-associated controls on PAI-1 gene regulation in the absence of added growth factors. PAI-1 transcript levels in quiescent R22 cells increased rapidly and in a CD-concentration-dependent fashion, with kinetics of expression paralleling the morphological changes. Colchicine concentrations that effectively disrupted microtubule structure and reduced the cellular 'footprint' area (to approximately that of CD treatment) also stimulated PAI-1 synthesis. Shape-related increases in PAI-1 mRNA synthesis were ablated by prior exposure to actinomycin D. Unlike the mechanism of induction in growth-factor-stimulated cells, CD- and colchicine-induced PAI-1 expression required on-going protein synthesis (i.e. it was a secondary response). Although PAI-1 is a TGF-beta-regulated gene and TGF-beta expression is also shape dependent, an autocrine TGF-beta loop was not a factor in CD-initiated PAI-1 transcription. Since CD exposure resulted in actin microfilament disruption and subsequent morphological changes, with uncertain effects on interactions between signaling intermediates or 'scaffold' structures, a pharmacological approach was selected to probe the pathways involved. Signaling events leading to PAI-1 induction were compared with colchicine-treated cells. CD- as well as colchicine-stimulated PAI-1 expression was effectively and dose dependently attenuated by the MEK inhibitor PD98059 (in the 10 to 25 microM concentration range), consistent with the known MAP kinase dependency of PAI-1 synthesis in growth-factor-stimulated cells. Reduced PAI-1 mRNA levels upon exposure to genistein prior to CD addition correlated with inhibition of ERK1/2 activity, implicating a tyrosine kinase in shape-dependent MEK activation. Src-family kinases, moreover, appeared to be specific upstream elements in the CD- and colchicine-dependent pathways of PAI-1 transcription since both agents effectively activated pp60(c-src) kinase activity in quiescent R22 cells. The restrictive (src-family) kinase inhibitor PP1 completely inhibited induced, as well as basal, ERK activity in a coupled immunoprecipitation myelin-basic-protein-phosphorylation assay and ablated shape-initiated PAI-1 mRNA expression. These data suggest that PP1-sensitive tyrosine kinases are upstream intermediates in cell-shape-associated signaling pathways resulting in ERK1/2 activation and subsequent PAI-1 transcription. In contrast to the rapid and transient kinetics of ERK activity typical of serum-stimulated cells, the ERK1/2 response to CD and colchicine is both delayed and relatively sustained. Collectively, these data support a model in which MEK is a focal point for the convergence of shape-initiated signaling events leading to induced PAI-1 transcription.

Actin Cytoskeleton↗

Cell shape and total adenylate concentration as important factors for posttransfusion survival of erythrocytes.

Red cells were stored for 42 days in a CPD/SAGM plastic bag system and then rejuvenated with bicarbonate, pyruvate and slow addition of adenosine. The reversibility and the time dependence of metabolic and morphologic changes were compared with autologous, posttransfusion survival. Total adenylates correlated better (r = 0.88) than ATP with viability in vivo ATP was better maintained when the blood was stored under anaerobic than aerobic conditions. A high correlation between morphology and viability was found in rejuvenated cells (r = 0.95). It is suggested that the time needed for reversal of changes is essential for viability.

Adenine Nucleotides↗

Red blood cell aggregability is increased by aspirin and flow stress, whereas dipyridamole induces cell shape alterations: measurements by digital image analysis.

We evaluated the influence of pretreatment with aspirin in vitro, alone or combined with dipyridamole, on red cell aggregability. Samples were tested before and after being exposed to well-defined flow conditions. An aggregation rate was estimated through digital analysis of light microscopy images. Red cells of untreated blood that had been exposed to flow showed a higher aggregation rate (38.54 +/- 1.63 vs. 27.52 +/- 1.36; P < 0.05). This effect was not observed in the absence of platelets. Treatment with aspirin induced a high aggregation rate, with or without exposure to flow conditions. Dipyridamole alone or combined with aspirin provoked echinocytosis, disturbing the rouleaux arrangement (rates ranging from 11.25 +/- 1.91 - 13.62 +/- 1.62). Washing red cells after treatment restored about 90% of echinocytes to their biconcave shape, but aggregation rate did not recover in parallel. These results highlight the influence of red cell-platelet interactions in the regulation of haemostasis and show how therapeutic agents can interfere with rheological phenomena.

Aspirin↗

Alterations of red blood cell shape in patients with severe trauma.

BACKGROUND: Trauma is accompanied by a decrease in red blood cell (RBC) deformability, which may manifest itself earlier than secondary septic complications. The mechanisms of this phenomenon are not clear. The aim of this study is to determine when the alterations of RBC shape appear in trauma patients. METHODS: RBC shape was examined by scanning electron microscopy in 43 patients with multisystem trauma. Blood samples were taken at admission and every 24 hours afterward for 4 to 10 days. The patients were divided into two groups: the survivors and those who died of septic complications. The control group consisted of 10 healthy volunteers. RESULTS: A significant decrease in the percentage of discoid erythrocytes, compared with the volunteers, was observed in both groups of patients at admission (77.6 +/- 11.9 and 66.7 +/- 5.8 vs. 96.0 +/- 2.9%, p < 0.01). The percentage of irreversibly changed RBC (spherostomatocytes, spherocytes) was lower in survivors (12.9 +/- 2.0% vs. 20.3 +/- 9.4%, p < 0.01). This phenomenon remained constant until the end of the study. The survivors only showed a tendency toward the improvement of RBC shape. CONCLUSION: RBC shape alterations appear within the first hours after trauma and persist for at least 7 to 10 days. These changes are more severe in patients with secondary septic complications.

Adult↗