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Role of cytoskeleton in cell shaping of developing mesophyll of wheat (Triticum aestivum L.).

The effects of oryzalin and cytochalasin B (CB) on microtubule and actin microfilament arrays and on cell shaping were investigated in developing wheat mesophyll. Excised immature leaf sections capable of differentiating were incubated with the drugs. The behavior of the cytoskeleton was monitored by fluorescence microscopy after labeling with fluorescent dyes. Brief incubation with oryzalin (40 min, 10 microM) caused disassembly of microtubules. Recovery of microtubule arrays was comparatively slow after removal of the drug. Cells failed to establish transverse cortical bands of microtubules and transverse hoops of wall reinforcement. They expanded isodiametrically rather than longitudinally without forming lobes typical of wheat mesophyll cells. Brief treatment with CB (60 min, 20 micrograms ml-1) appeared to disrupt the microfilament arrays. Filaments recovered rapidly after removal of CB, and cells were able to shape in an apparently normal fashion. Continuous incubation at comparatively low concentration of CB (4 micrograms ml-1) appeared to cause selective loss of the fine transverse cortical microfilament arrays. Cortical transverse microtubule arrays persisted, but failed to form distinct bands in the majority of the cells. Cells were able to elongate in an almost normal fashion, but no lobes were formed.

Actin Cytoskeleton↗

Regulation of cell shape in Euglena gracilis. II. The effects of altered extra- and intracellular Ca2+ concentrations and the effect of calmodulin antagonists.

When cultures of Euglena gracilis Z., normally grown in medium containing 180 microM-Ca2+, are resuspended in Ca2+-free medium cells assume round shapes within 10 min, from which they recover slowly when Ca2+ is returned to the cultures. Cultures grown in 10 microM-Ca2+ do not display the typical circadian rhythm in cell shape even though the photosynthesis and cell division circadian rhythms are unaffected. Elevating intracellular Ca2+ levels by the addition of the Ca2+ ionophore A23187 prevents cells from undergoing the two daily shape changes characteristic of growth-synchronized cultures, but does not alter the ability to maintain the cell shapes found at the time of ionophore addition. When the calmodulin inhibitors trifluoperazine or chlorpromazine are added to cultures, the cells always respond by rounding. Cells are not able to maintain any cell shape other than spherical in the presence of these inhibitors and therefore cannot change shape throughout the daily cycle as is found in the control populations.

Animals↗

Axonal patterns of disc-shaped cells in the central nucleus of the cat inferior colliculus.

The axonal patterns of disc-shaped cells (Dsc) and their distribution within the central nucleus (Cn) of the inferior colliculus was studied in young cats with the rapid Golgi method. Dsc were subdivided in three main cell varieties according to their axonal branching pattern. The first type possesses local axonal collaterals inside the lamina of origin but also gives collaterals and probably terminals to adjacent lamina. The second variety is characterized by a dense axonal plexus with a restricted zone of arborization. The third axonal pattern is distributed in a radiate fashion. These results demonstrate that Dsc contribute to the intrinsic axonal system of the Cn to a larger degree than previously supposed. Axon terminals of Dsc probably establish axo-dendritic contacts with medium sized Stc which probably establish contacts with neighbouring Dsc. This suggests that reciprocal connections between Dsc and Stc could exist in the Cn.

Animals↗

Cell walls, cell shape, and bacterial actin homologs.

The synthesis of the peptidoglycan layer, one of the key determinants of cell shape in B. subtilis, has been shown by Daniel and Errington to occur in a helical pattern. This pattern is generated by the actin homolog Mbl.

Actins↗

Prostaglandin E2 effects on corneal endothelial cyclic adenosine monophosphate synthesis and cell shape are mediated by a receptor of the EP2 subtype.

Corneal endothelial cells synthesize prostaglandin E2 (PGE2), and this synthesis is necessary for the maintenance of the normal polygonal shape of these cells. A series of experiments was done to examine the receptor-effector mechanism responsible for PGE2-mediated effects on cultured rabbit corneal endothelium. When challenged with exogenous PGE2, endothelial cells synthesized cyclic adenosine monophosphate (AMP) in a dose-dependent manner, and this synthesis was not antagonized by AH6809. The synthetic agonist 11-deoxy-PGE1, but not sulprostone, stimulated increased cyclic AMP synthesis. The pharmacologic profile of the endothelial PGE2 receptor is therefore consistent with that of an EP2 receptor linked to activation of adenylate cyclase. The prostaglandin agonists were also tested for their ability to prevent cellular elongation in response to indomethacin. The PGE2, 11-deoxy-PGE1, and 16,16-dimethyl PGE1 prevented elongation, but sulprostone and PGF2 alpha did not. The authors conclude that rabbit corneal endothelium in culture expresses a specific PG receptor of the EP2 subtype which is coupled to cyclic AMP synthesis and is involved in the regulation of cell shape.

Animals↗

Changes in brain cell shape create residual extracellular space volume and explain tortuosity behavior during osmotic challenge.

Diffusion of molecules in brain extracellular space is constrained by two macroscopic parameters, tortuosity factor lambda and volume fraction alpha. Recent studies in brain slices show that when osmolarity is reduced, lambda increases while alpha decreases. In contrast, with increased osmolarity, alpha increases, but lambda attains a plateau. Using homogenization theory and a variety of lattice models, we found that the plateau behavior of lambda can be explained if the shape of brain cells changes nonuniformly during the shrinking or swelling induced by osmotic challenge. The nonuniform cellular shrinkage creates residual extracellular space that temporarily traps diffusing molecules, thus impeding the macroscopic diffusion. The paper also discusses the definition of tortuosity and its independence of the measurement frame of reference.

Animals↗

Control of cell shape in bacteria: helical, actin-like filaments in Bacillus subtilis.

In the absence of an overt cytoskeleton, the external cell wall of bacteria has traditionally been assumed to be the primary determinant of cell shape. In the Gram-positive bacterium Bacillus subtilis, two related genes, mreB and mbl, were shown to be required for different aspects of cell morphogenesis. Subcellular localization of the MreB and Mbl proteins revealed that each forms a distinct kind of filamentous helical structure lying close to the cell surface. The distribution of the proteins in different species of bacteria, and the similarity of their sequence to eukaryotic actins, suggest that the MreB-like proteins have a cytoskeletal, actin-like role in bacterial cell morphogenesis.

Actins↗

Conditional lethality of cell shape mutations of Salmonella typhimurium: rodA and mre mutants are lethal on solid but not in liquid medium.

Round-cell (rodA, mre, divD) derivatives of a conditional alaS mutant of Salmonella typhimurium were studied under conditions allowing expression of tolerance to lethal cell shape mutations (41 degrees C), and under nontolerant conditions (30 degrees C). The rodA22::Tn10d(Kan) derivative grew normally (OD650 nm) in LB-broth at 30 degrees C; however, doubling of total cell count took much longer (130 min) than at 41 degrees C (57 min). Although the cells were able to divide in LB-broth at 30 degrees C, viable count on LB-agar at 30 degrees C was 10(3)-fold lower than on LB-agar at 41 degrees C. Phase-contrast microscopy of rodA cells incubated under different conditions showed that their size increased on LB-soft agar at 30 degrees C, but they failed to divide and finally lysed. In contrast, division occurred in LB-broth at 30 degrees C and also in LB-broth and LB-soft agar at 41 degrees C. The mre-17::Tn10d(Kan) derivative acted like the rodA strain whereas the divD135::Tn10d(Kan) mutant behaved normally both at 30 degrees C and 41 degrees C. It is concluded that rodA and mre mutations delay cell division, but are lethal only on solid medium. Mutations conferring tolerance to "lethal" rodA and mre mutations improve division performance both in liquid and solid media.

Amdinocillin↗

Laminin alters cell shape and stimulates motility and proliferation of murine skeletal myoblasts.

Proliferating skeletal myoblasts show multiple specific responses to laminin, one of the major glycoprotein components of basement membranes. Using MM14Dy myoblasts, a myogenic cell strain derived from a normal adult mouse skeletal muscle, we show in this study that substrate-bound laminin but not other matrix proteins such as collagens or fibronectin specifically and rapidly induces the outgrowth of cell processes, resulting in bipolar, spindle-shaped cells. This effect is independent from the presence of collagens or serum, and was also observed in primary cultures of fetal mouse skeletal myoblasts. The outgrowth of cell processes on laminin is associated with a dramatic stimulation of cell motility: MM14 myoblasts migrate about five times faster on laminin than on fibronectin. In another series of experiments the effect of laminin and fibronectin on thymidine uptake and proliferation of myoblasts was tested. On top of a type I collagen substrate which was provided to ensure complete adhesion even at low doses of laminin or fibronectin, laminin stimulated myoblast proliferation and incorporation of [3H]thymidine in a dose-dependent manner. The stimulation is two- to threefold higher than on dishes coated with equivalent amounts of fibronectin and is observed both in the presence and in the absence of serum. These results suggest that laminin, a major component of the muscle basal lamina, may be actively involved in the development and regeneration of skeletal muscle.

Animals↗

Alteration of cell shape, adhesion, and lipid accumulation in human breast cancer cells (T-47D) by human prolactin and growth hormone.

We have demonstrated previously that many human cancer cell lines maintained in tissue culture possess specific cell surface receptors for human prolactin (HPRL) and human growth hormone (HGH). In the present studies, the biological response in vitro of one human breast cancer cell line, T-47D, to the two pituitary hormones was examined. T-47D cells, when grown on tissue culture dishes, display typical epithelioid characteristics; cells are flat and polygonal in shape and are very adhesive to the plastic substratum. Upon the addition of HPRL or HGH (10 to 1000 ng/ml), in the presence of hydrocortisone, insulin, and triiodothyronine, each at 1 microgram/ml, the T-47D cells became round and refractile. In addition, there was a dramatic reduction in the adhesiveness of the cells to the substratum; 80% of the hormone-treated cells were detached by trypsin (25 micrograms/ml) in 30 min at 37 degrees, as compared with 5% for cells not treated with hormones. These prolactin-induced changes could be abolished upon the addition of antiserum to prolactin. Neither HPRL nor the combination of hydrocortisone, insulin, and triiodothyronine alone was active, indicating a synergism between HPRL and hydrocortisone, insulin, and triiodothyronine. It was subsequently found that only hydrocortisone was required for the action of HPRL, and that human luteininzing hormone and ovine growth hormone were inactive, whereas ovine prolactin exerted a very weak effect. In addition, in the presence of hydrocortisone (or hydrocortisone, insulin, and triiodothyronine), HPRL (or HGH) retarded cell proliferation by 30%, whereas HPRL or hydrocortisone by itself had no effect on cell growth. Ultrastructural studies revealed that, accompanying cell rounding and reduced adhesion, HPRL and HGH increased the formation of intracytoplasmic lipid droplets in the T-47D cells. The increase in lipid synthesis was confirmed by the staining of cells with Oil Red O, and by monitoring the incorporation of [14C]acetate into lipid; HPRL stimulated lipid synthesis and accumulation by approximately 2-fold. Thus, receptor-positive human breast cancer cells are biologically responsive in vitro to HPRL and HGH.

Acetates↗

A novel short-root gene encodes a glucosamine-6-phosphate acetyltransferase required for maintaining normal root cell shape in rice.

Glycosylation is a posttranslational modification occurring in many secreted and membrane-associated proteins in eukaryotes. It plays important roles in both physiological and pathological processes. Most of these protein modifications depend on UDP-N-acetylglucosamine. In this study, a T-DNA insertional rice (Oryza sativa) mutant exhibiting a temperature-sensitive defect in root elongation was isolated. Genetic and molecular analysis indicated that the mutated phenotype was caused by loss of function of a gene encoding a glucosamine-6-P acetyltransferase (designated OsGNA1), which is involved in de novo UDP-N-acetylglucosamine biosynthesis. The aberrant root morphology of the gna1 mutant includes shortening of roots, disruption of microtubules, and shrinkage of cells in the root elongation zone. Our observations support the idea that protein glycosylation plays a key role in cell metabolism, microtubule stabilization, and cell shape in rice roots.

Acetyltransferases↗

Effect of incubation on red cell shape and water content as measured by gas-liquid chromatography.

A new method for measuring water content of red cells by using gas chromatography is described. Water content of healthy adult red cells was estimated to be 71.73 +/- 0.72%. Changes in water content and red cell shape following incubation are described. Intracellular water content increased in relation to the incubation time, reaching a maximum of 109% at 24 h, and then decreased to 82% at 48 h. Intercellular water content (trapped water) also increased for 24 h, reaching 209%, and then showed no remarkable change until 48 h. Observations with the scanning electron microscope revealed that these changes were associated with the process of transformation to echinocytes, both in numbers and stages.

Adult↗

Influence of cell shape and aggregate formation on the optical properties of flowing whole blood.

We studied the influence of shape and secondary, or intercellular, organization on the absorption and scattering properties of red blood cells to determine whether these properties are of any practical significance for optical evaluation of whole blood and its constituents. A series of measurements of transmittance and reflectance of light from bovine blood in a flow cuvette was conducted with a 650-900-nm integrating sphere at shear rates of 0-1600 s(-1), from which the influence of cell orientation, elongation, and aggregate formation on the absorption (mu(a)) and the reduced scattering (mu(s)') coefficients could be quantified. Aggregation was accompanied by a decrease of 4% in mu(s)' compared with the value in randomly oriented single cells. Increasing the degree of cell alignment and elongation as a result of increasing shear rate reduced mu(s)' by 6% and mu(a) by 3%, evaluated at a shear rate of 1600 s(-1). Comparison with T-matrix computations for oblate- and prolate-shaped cells with corresponding elongation and orientation indicates that the optical properties of whole blood are determined by those of its individual cells, though influenced by a collective scattering factor that depends on the cell-to-cell organization. We demonstrate that cell morphological changes must be taken into consideration when one is conducting whole blood spectroscopy.

Animals↗

Functional mapping of SPARC: peptides from two distinct Ca+(+)-binding sites modulate cell shape.

Using synthetic peptides, we have identified two distinct regions of the glycoprotein SPARC (Secreted Protein Acidic and Rich in Cysteine) (osteonectin/BM-40) that inhibit cell spreading. One of these sites also contributes to the affinity of SPARC for extracellular matrix components. Peptides representing subregions of SPARC were synthesized and antipeptide antibodies were produced. Immunoglobulin fractions of sera recognizing an NH2-terminal peptide (designated 1.1) blocked SPARC-mediated anti-spreading activity. Furthermore, when peptides were added to newly plated endothelial cells or fibroblasts, peptide 1.1 and a peptide corresponding to the COOH terminal EF-hand domain (designated 4.2) inhibited cell spreading in a dose-dependent manner. These peptides exhibited anti-spreading activity at concentrations from 0.1 to 1 mM. The ability of peptides 1.1 and 4.2 to modulate cell shape was augmented by an inhibitor of protein synthesis and was blocked by specific antipeptide immunoglobulins. In addition to blocking cell spreading, peptide 4.2 competed for binding of [125I]SPARC and exhibited differential affinity for extracellular matrix molecules in solid-phase binding assays. The binding of peptide 4.2 to matrix components was Ca+(+)-dependent and displayed specificities similar to those of native SPARC. These studies demonstrate that both anti-spreading activity and affinity for collagens are functions of unique regions within the SPARC amino acid sequence. The finding that two separate regions of the SPARC protein contribute to its anti-spreading activity lead us to propose that multiple regions of the protein act in concert to regulate the interactions of cells with their extracellular matrix.

Amino Acid Sequence↗

Class I major histocompatibility complex proteins diffuse isotropically on immune interferon-activated endothelial cells despite anisotropic cell shape and cytoskeletal organization: application of fluorescence photobleaching recovery with an elliptical beam.

Interferon gamma induces striking phenotypic alterations in confluent cultures of human vascular endothelial cells (HEC), including cell shape change from polygonal to elongated and cytoskeletal actin rearrangement from dense peripheral bands to longitudinal bundles of stress fibers. Since many transmembrane proteins, including class I major histocompatibility complex (MHC) proteins, interact with cytoskeletal actin, an interferon-gamma-induced anisotropic arrangement of stress fibers might cause anisotropic lateral diffusion of HEC class I MHC proteins. To test this hypothesis, we adapted the fluorescence photobleaching recovery technique to allow measurement of anisotropic diffusion of fluorescently labeled molecules on two-dimensional surfaces. A highly eccentric elliptical Gaussian laser beam was used to photobleach the sample and to monitor fluorescence recovery. In this technique, named "line fluorescence photobleaching recovery," lateral diffusion is measured along that axis of the sample that is perpendicular to the major axis of the elliptical beam. The lateral diffusion coefficient and fractional mobility are obtained by fitting the experimental data to a theoretical recovery curve, the form of which is determined by the solution to a modified version of the diffusion equation in which a tensor is used to describe diffusion in two orthogonal directions. Fluorescein-conjugated murine monoclonal antibodies were used to label class I MHC proteins on interferon-gamma-treated HEC and human dermal fibroblasts. These two cultured human cell types were found to be similar in their elongated shape and anisotropic stress fiber organization. Class I MHC protein lateral mobility was compared to that of fluorescein-labeled phosphatidyl-ethanolamine, a membrane phospholipid probe. Class I MHC proteins diffused anisotropically on human dermal fibroblasts, whereas fluorescein-labeled phosphatidylethanolamine diffused isotropically on this cell type. In contrast, both class I MHC proteins and fluorescein-labeled phosphatidylethanolamine diffused isotropically on interferon-gamma-treated HEC. These data suggest that neither elongated shape nor anisotropic stress fiber arrangement is sufficient to induce anisotropic diffusion of proteins on the HEC plasma membrane.

Algorithms↗

Responses to nutrient starvation in Pseudomonas putida KT2442: analysis of general cross-protection, cell shape, and macromolecular content.

The physiology of Pseudomonas putida KT2442 with respect to growth and carbon starvation was studied. During the transition from growth to nongrowth, the cell shape changes from cylindrical to spheric, a change which is accompanied by reductions in cell size, DNA and ribosome content, and the rate of total protein synthesis. In addition, a pattern of general cross-protection develops, which enables the cells to survive environmental stresses such as high and low temperatures, elevated osmolarity, solvents, and oxidative agents. Cultures are almost fully viable during 1 month of carbon, nitrogen, and multiple-nutrient starvation and are considered to be in an active nondormant state. In contrast, strain KT2442 does not survive well under conditions of sulfate and phosphate starvation.

Adaptation, Physiological↗

Ectodermal inhibition of cartilage differentiation in micromass culture of chick limb bud mesenchyme in relation to gene expression and cell shape.

Ectoderm inhibits the formation of cartilage by chick wing bud mesenchyme in micromass culture. This suggests that the pattern of cartilage formation in the limb bud may result from a restriction of cartilage cell differentiation to the limb bud core as cells leave the progress zone. We have used in situ hybridization to investigate whether ectodermal inhibition in micromass culture occurs at the level of gene transcription. We found that ectoderm completely inhibited the accumulation of cartilage-specific type II collagen transcripts in the mesenchyme cells, whilst the level of type I collagen transcripts was unaffected. Morphometric analysis of electron micrographs revealed that inhibition of chondrogenesis in micromass culture was not preceded by cell flattening. In fact, a rounded cell shape was found not to be a prerequisite for cartilage cell differentiation in micromass.

Animals↗