Mitochondrial morphology is dynamic and varied

The morphology of mitochondria is dynamic, often changing within a cell and from one cell type to the next. In the past few years, significant advances have been made in the study of mechanisms that help determine the morphologies of mitochondria and their intracellular distributions. It has become apparent that the distribution of mitochondria is determined by movement along the cytoskeleton, driven by molecular motors, and attachment to the cytoskeleton, using specific connector proteins. However, not all cells use the same cytoskeletal elements and motor proteins for mitochondrial movement and attachment. The shapes of mitochondria are also influenced by the extent of mitochondrial division and fusion. A number of proteins that affect mitochondrial division and fusion were recently discovered. Here, we review the proteins involved in the distribution and morphology of mitochondria and discuss how they may be physiologically regulated.     

Eph-modulated cell morphology, adhesion and motility in carcinogenesis

Eph receptor tyrosine kinases (Ephs) and their membrane anchored ephrin ligands (ephrins) form an essential cell-cell communication system that directs the positioning, adhesion and migration of cells and cell layers during development. While less prominent in normal adult tissues, there is evidence that up-regulated expression and de-regulated function of Ephs and ephrins in a large variety of human cancers may promote a more aggressive and metastatic tumour phenotype. However, in contrast to other RTKs, Ephs do not act as classical proto-oncogenes and do not effect cell proliferation or differentiation. Mounting evidence suggests that Eph receptors, through de-regulated re-emergence of their mode of action in the embryo may direct cell movements and positioning during metastasis, invasion and tumour angiogenesis. This review discusses these and other emerging roles of Eph receptors during oncogenesis.     

Human gaze is systematically offset from the center of cone topography

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The relative importance of topography and RGD ligand density for endothelial cell adhesion

The morphology and function of endothelial cells depends on the physical and chemical characteristics of the extracellular environment. Here, we designed silicon surfaces on which topographical features and surface densities of the integrin binding peptide arginine-glycine-aspartic acid (RGD) could be independently controlled. We used these surfaces to investigate the relative importance of the surface chemistry of ligand presentation versus surface topography in endothelial cell adhesion. We compared cell adhesion, spreading and migration on surfaces with nano- to micro-scaled pyramids and average densities of 6×10(2)-6×10(11) RGD/mm(2). We found that fewer cells adhered onto rough than flat surfaces and that the optimal average RGD density for cell adhesion was 6×10(5) RGD/mm(2) on flat surfaces and substrata with nano-scaled roughness. Only on surfaces with micro-scaled pyramids did the topography hinder cell migration and a lower average RGD density was optimal for adhesion. In contrast, cell spreading was greatest on surfaces with 6×10(8) RGD/mm(2) irrespectively of presence of feature and their size. In summary, our data suggest that the size of pyramids predominately control the number of endothelial cells that adhere to the substratum but the average RGD density governs the degree of cell spreading and length of focal adhesion within adherent cells. The data points towards a two-step model of cell adhesion: the initial contact of cells with a substratum may be guided by the topography while the engagement of cell surface receptors is predominately controlled by the surface chemistry.     

Biosynthesis and membrane topography of the neural cell adhesion molecule L1.

The biosynthesis and membrane topography of the neural cell adhesion molecule L1 have been studied in cerebellar cell cultures by metabolic labeling and immunoprecipitation. Pulse and pulse-chase experiments with [35S]methionine show that L1 is synthesized in its high mol. wt. form, the 200 kd component. The lower mol. wt. components with 40, 80 and 140 K apparent mol. wts. can be generated by proteolysis in intact cellular membranes. Peptide maps generated by protease treatment of L1 isolated from adult mouse brain show that the 80 and 140 kd components are related to the 200 kd component, but not to each other. The 200, 80 and 40 kd components can be biosynthetically phosphorylated. The 140 kd component is not phosphorylated and not released from the surface membrane during tryspinization. The phosphorylated amino acid is serine. In the presence of tunicamycin the 200 kd component is synthesized as a 150 kd protein. Pulse-chase experiments in the presence of tunicamycin indicate that the carbohydrate moieties are predominantly N-glycosidically linked and that the contribution of O-glycosylation is minimal. The carbohydrate moieties are of the complex type as shown by treatment with endoglycosidase H. Since monensin inhibits processing of the carbohydrate moieties, the 200 kd component appears to be transported to the surface membrane via the Golgi apparatus.     

Rapid and quantitative measurement of cell adhesion and migration activity by time-series analysis on biomimetic topography

Glioblastoma Multiforme (GBM) is the most malignant brain tumor in adults, highly infiltrative and difficult to cure. According to the histopathological evidence, the glioma cells are found to infiltrate into the surround normal brain tissue, along the Scherer’s structure (e.g. white matter tract and microvasculature). As a major invasion route of microenvironments, these pre-existing anatomic structures should be considered in studying infiltrative movement of glioblastoma. In our previous work, we introduced in vitro biomimetic platform as alternative model of brain-anatomical structures to study about migratory phenotypes of glioblastoma. By applying this proper biomimetic platform, we further investigated the influence of integrin, which is one of mechanoreceptors to sense mechanical cues, on phenotype of glioblastoma cells in this study. On in vitro biomimetic platform, glioblastoma cells show elongated morphology with highly aligned along the patterned direction, which is similar to that on in vivo condition. These morphological changes were gradually progressed in time-dependent manner, which might be mediated by a representative mechanoreceptor, integrin. Treatment of cell adhesive motif for integrin inhibition hinders the morphological dynamics on in vitro biomimetic platform in early time-point compared with cell proliferation cycle. Since cell adhesion mediated by mechanoreceptors is one of essential steps in migration/invasion, our results imply that effect of integrin on glioblastoma invasion is mediated by the mechanosensing process on topography and indicated by morphological changes. For further application, this quantitative analysis of glioblastoma morphology on biomimetic platform can be contributed to simple and ease investigation and effective anti-cancer drug screening.     

Dynamics of adhesion molecule domains on neutrophil membranes: surfing the dynamic cell topography

Lateral organization and mobility of adhesion molecules play a significant role in determining the avidity with which cells can bind to target cells or surfaces. Recently, we have shown that the lateral mobility of the principal adhesion molecules on neutrophils is lower for rolling associated adhesion molecules (RAAMs: L-selectin and PSGL-1) than for β₂ integrins (LFA-1 and Mac-1). Here we report that all four adhesion molecules exhibit distinct punctate distributions that are mobile on the cell surface. Using uniform illumination image correlation microscopy, we measure the lateral mobility of these topologically distinct domains. For all four molecules, we find that diffusion coefficients calculated from domain mobility agree with measurements we made previously using fluorescence recovery after photobleaching. This agreement indicates that the transport of receptors on the surface of the resting neutrophil is dominated by the lateral movement of domains rather than individual molecules. The diffusion of pre-assembled integrin domains to zones of neutrophil/endothelial contact may provide a mechanism to facilitate high avidity adhesion during the earliest stages of firm arrest.     

Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion

The morphology and cytoskeletal structure of fibroblasts, endothelial cells, and neutrophils are documented for cells cultured on surfaces with stiffness ranging from 2 to 55,000 Pa that have been laminated with fibronectin or collagen as adhesive ligand. When grown in sparse culture with no cell-cell contacts, fibroblasts and endothelial cells show an abrupt change in spread area that occurs at a stiffness range around 3,000 Pa. No actin stress fibers are seen in fibroblasts on soft surfaces, and the appearance of stress fibers is abrupt and complete at a stiffness range coincident with that at which they spread. Upregulation of alpha5 integrin also occurs in the same stiffness range, but exogenous expression of alpha5 integrin is not sufficient to cause cell spreading on soft surfaces. Neutrophils, in contrast, show no dependence of either resting shape or ability to spread after activation when cultured on surfaces as soft as 2 Pa compared to glass. The shape and cytoskeletal differences evident in single cells on soft compared to hard substrates are eliminated when fibroblasts or endothelial cells make cell-cell contact. These results support the hypothesis that mechanical factors impact different cell types in fundamentally different ways, and can trigger specific changes similar to those stimulated by soluble ligands.     

Influence of proteolytic enzymes and calcium-binding agents on nuclear and cell surface topography

Summary Transmission electron microscopy was used to study the effects of proteolytic enzymes (collagenase, trypsin, clostripain), the calcium chelator ethyleneglycol-bis-(-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA), and the calcium ionophore A 23187 on substrate adhesion and fine structure of chondrocytes and fibroblasts. Monolayer cultured cells responded to treatment with the proteolytic enzymes followed by EGTA or A 23187 by rounding and detaching from the substrate. This was accompanied by the formation of a microvillous surface, deep nuclear folds, and numerous cytoplasmic vacuoles. Labeling experiments with colloidal thorium dioxide indicated that the vacuoles were formed by endocytosis and fusion of endocytic vesicles with preexisting lysosomes. To a variable extent, similar changes were produced by trypsin or EGTA alone. The cells regained their normal fine structure after withdrawal of the reagents and when seeded onto a substrate. In suspension culture, recovery was incomplete; the cells retained a rounded shape and an increased number of cytoplasmic vacuoles.The results suggest that changes in plasma membrane composition and its permeability to calcium represent the primary signal for cell rounding and detachment. The cellular mechanisms responsible for the associated folding of the nuclear envelope and the cell surface remain unidentified. Nevertheless, this is believed to represent a means of handling of excess membrane during sudden transition from a flattened to a rounded shape. Membrane stored in folds and vacuoles is reutilized when the cells reattach and spread out on a substrate.Expert technical assistance was provided by Karin Blomgren and Anne-Marie Motakefi. Financial support was obtained from the Swedish Medical Research Council (06537), the King Gustaf V 80th Birthday Fund and from the Funds of Leiden University     

Influence of sodium butyrate on HeLa cell morphology and proliferation

The effect of one-week exposure to sodium butyrate on HeLa S3 cell cultures was studied with special regard to influence on prekeratin synthesis, by comparison to cultures similarly treated with the known proliferation inhibitor hydroxyurea, and not treated. Like hydroxyurea, sodium butyrate inhibited cell proliferation to a considerable degree, but accounted additionally for an increase in membrane-bound alkaline phosphatase activity, cellular prekeratin synthesis, tonofilament number, and filament bundle formation. These phenomena unequivocally indicate that sodium butyrate acted as a specific stimulator of Hela (epithelial) cell differentiation. Similar differentiation phenomena can be observed during early spontaneous keratinization of the stratified horny epithelium.     

Ephrin-A5 modulates cell adhesion and morphology in an integrin-dependent manner

The ephrins are membrane-tethered ligands for the Eph receptor tyrosine kinases, which play important roles in patterning of the nervous and vascular systems. It is now clear that ephrins are more than just ligands and can also act as signalling-competent receptors, participating in bidirectional signalling. We have recently shown that ephrin-A5 signals within caveola-like domains of the plasma membrane upon engagement with its cognate Eph receptor, leading to increased adhesion of the cells to fibronectin. Here we show that ephrin-A5 controls sequential biological events that are consistent with its role in neuronal guidance. Activation of ephrin-A5 induces an initial change in cell adhesion followed by changes in cell morphology. Both effects are dependent on the activation of beta1 integrin involving members of the Src family of protein tyrosine kinases. The prolonged activation of ERK-1 and ERK-2 is required for the change in cell morphology. Our work suggests a new role for class A ephrins in specifying the affinity of the cells towards various extracellular substrates by regulating integrin function.     

Influence of cell surface properties on adhesion ability of bifidobacteria

Adhesion ability of bifidobacteria to the intestinal mucosa is considered to be the prerequisite for colonization of bifidobacteria and can protect against gastrointestinal pathogens infection. The aim of this study was to investigate bifidobacterial surface traits related to adhesion ability in vitro and characterize the cell surface substances that may be involved in the adhesion process of bifidobacteria. Twelve strains of Bifidobacterium spp. were studied for the correlation among their adhesion ability, autoaggregation ability and surface hydrophobicity. The strain that exhibited good adhesion ability also showed high degree of hydrophobicity and strong autoaggregation ability. Pepsin treatment had negative effect on the surface traits and adhesion ability of B. bifidum KLDS2.0603 (P < 0.01), it revealed that hydrophobicity, autoaggregation and adhesion process maybe mediated by proteinaceous components on the surface of cell. Moreover, the adhesion and autoaggregation ability decreased after extraction of B. bifidum KLDS2.0603 with 5 mol l⁻¹ LiCl, and an unreported 50-kDa surface protein which can bind to Caco-2 cell was observed by western blotting. Our results indicated that surface hydrophobicity and autoaggregation ability can be used together for preliminary screening the strains with high adhesion ability, and the present of the surface proteinaceous components would contribute to understand the interactions between bifidobacteria and human intestinal mucosa.     

Influence of cis-dichlorodiamineplatinum on glioma cell morphology and cell cycle kinetics in tissue culture

Summary C6 glioma cells (CCL 107) were cultured for three days and then treated withcis-dichlorodiamineplatinum (cis-DDP) at doses of 0.2–10 µg/ml medium. Changes in DNA synthesis and DNA content, as well as morphology of cells and chromatin distribution, were examined from the first post-treatment day onwards. The number of cells labelled with [³H]thymidine, detected autoradiographically, decreased after treatment with 0.2–10 µg/ml by approximately one half on post-treatment day 1 and diminished further by the third day after treatment. The labelled cells were entirely absent only after treatment with 10µg/ml, 7 days post-treatment. Mitoses decreased from 1.4–0.6% by post-treatment day 1 and completely disappeared by day 3 (1 µg/ml). Feulgen cytophotometry and propidium iodide cytofluorimetry revealed accumulation of cells in the S-phase, especially the latter part (0.5 and 1.0 µg/ml, post-treatment day 1) and subsequently also in G2 phase (post-treatment day 3). Incomplete cyto- and karyokinesis in some cycling cells was indicated by an increased number of binucleate cells and nuclei of higher ploidy classes. Labelled cells with intermediate DNA values were, on average, labelled less intensively, as was revealed by simultaneous measurements of DNA content and [³H]thymidine incorporation. Some cells displayed reduction in grain density over heterochromatin clumps. This would be in agreement with the late S-phase block of DNA replication. After post-treatment day 3 the density of cells in cultures was substantially lower. This was due to slowed transversing through the cell cycle and cell death occurring after post-treatment day 1 with higher doses or after day 2 with lower doses (up to 1 µg/ml). The size of the nuclei of surviving cells enlarged initially (post-treatment day 1) and later (day 7) giant cells with long, branched fibres similar to those of reactive astrocytes occurred. Texture analysis of Feulgen-stained nuclei revealed that the chromatin of cells treated withcis-DDP became less evenly distributed. This might be due either to the direct influence ofcis-DDP on the DNA molecule, or mediated by changes in cytoskeleton and cAMP levels described earlier.     

Influence of cell adhesion and spreading on impedance characteristics of cell-based sensors

Impedance measurements of cell-based sensors are a primary characterization route for detection and analysis of cellular responses to chemical and biological agents in real time. The detection sensitivity and limitation depend on sensor impedance characteristics and thus on cell patterning techniques. This study introduces a cell patterning approach to bind cells on microarrays of gold electrodes and demonstrates that single-cell patterning can substantially improve impedance characteristics of cell-based sensors. Mouse fibroblast cells (NIH3T3) are immobilized on electrodes through a lysine-arginine-glycine-aspartic acid (KRGD) peptide-mediated natural cell adhesion process. Electrodes are made of three sizes and immobilized with either covalently bound or physically adsorbed KRGD (c-electrodes or p-electrodes). Cells attached to c-electrodes increase the measurable electrical signal strength by 48.4%, 24.2%, and 19.0% for three electrode sizes, respectively, as compared to cells attached to p-electrodes, demonstrating that both the electrode size and surface chemistry play a key role in cell adhesion and spreading and thus the impedance characteristics of cell-based sensors. Single cells patterned on c-electrodes with dimensions comparable to cell size exhibit well-spread cell morphology and substantially outperform cells patterned on electrodes of other configurations.     

Influence of bacteria on cell size development and morphology of cultivated diatoms

Vegetative cell division in diatoms often results in a decreased cell size of one of the daughter cells, which during long‐term cultivation may lead to a gradual decrease of the mean cell size of the culture. To restore the initial cell size, sexual reproduction is required, however, in many diatom cultures sexual reproduction does not occur. Such diatom cultures may lose their viability once the average size of the cells falls below a critical size. Cell size reduction therefore seriously restrains the long‐term stability of many diatom cultures. In order to study the bacterial influence on the size diminution process, we observed cell morphology and size distribution of the diatoms Achnanthidium minutissimum, Cymbella affiniformis and Nitzschia palea for more than two years in bacteria‐free conditions (axenic cultures) and in cultures that contain bacteria (xenic cultures). We found considerable morphological aberrations of frustule microstructures in A. minutissimum and C. affiniformis when cultivated under axenic conditions compared to the xenic cultures. These variations comprise significant cell length reduction, simplification and rounding of the frustule contour and deformation of the siliceous cell walls, features that are normally found in older cultures shortly before they die off. In contrast, the xenic cultures were well preserved and showed less cell length diminution. Our results show that bacteria may have a fundamental influence on the stability of long‐term cultures of diatoms.     

Effect of Husk morphology on grain development and topography in rice

Kernels grown within loosened glumes in three varieties of paddy were darker in color and had a smoother surface than those grown under normal conditions. The thickness of the pericarp plus seed coat layers was 33.6 ±2.8 µm, and the thickness of the aleurone layers was 21.7 ± 2.5 µm in grains of the first type, while in the normal grains, these dimensions were 13.0 ± 1.4 and 26.9 ± 2.9 µm respectively. The kernels which developed within loosened glumes tended to taper towards the distal end. They were lighter in weight than normal grains by 32 to 67 percent, the weight loss being less in the bolder variety. The lemma-palea interlocking depth was positively correlated with the groove depth on the kernel and with the clearance between husk and kernel. All three parameters showed a positive correlation with grain breadth. A low lemma-palea interlocking depth and a smaller clearance between husk and kernel are technologically desirable characteristics in rice. The reclasping of the two glume components after pollination was essential for the normal development of the rice grain.     

Morphological behavior of acidic and neutral liposomes induced by basic amphiphilic alpha-helical peptides with systematically varied hydrophobic-hydrophilic balance

Lipid-peptide interaction has been investigated using cationic amphiphilic alpha-helical peptides and systematically varying their hydrophobic-hydrophilic balance (HHB). The influence of the peptides on neutral and acidic liposomes was examined by 1) Trp fluorescence quenched by brominated phospholipid, 2) membrane-clearing ability, 3) size determination of liposomes by dynamic light scattering, 4) morphological observation by electron microscopy, and 5) ability to form planar lipid bilayers from channels. The peptides examined consist of hydrophobic Leu and hydrophilic Lys residues with ratios 13:5, 11:7, 9:9, 7:11, and 5:13 (abbreviated as Hels 13-5, 11-7, 9-9, 7-11, and 5-13, respectively; Kiyota, T., S. Lee, and G. Sugihara. 1996. Biochemistry. 35:13196-13204). The most hydrophobic peptide (Hel 13-5) induced a twisted ribbon-like fibril structure for egg PC liposomes. In a 3/1 (egg PC/egg PG) lipid mixture, Hel 13-5 addition caused fusion of the liposomes. Hel 13-5 formed ion channels in neutral lipid bilayer (egg PE/egg PC = 7/3) at low peptide concentrations, but not in an acidic bilayer (egg PE/brain PS = 7/3). The peptides with hydrophobicity less than Hel 13-5 (Hels 11-7 and Hel 9-9) were able to partially immerse their hydrophobic part of the amphiphilic helix in lipid bilayers and fragment liposome to small bicelles or micelles, and then the bicelles aggregated to form a larger assembly. Peptides Hel 11-7 and Hel 9-9 each formed strong ion channels. Peptides (Hel 7-11 and Hel 5-13) with a more hydrophilic HHB interacted with an acidic lipid bilayer by charge interaction, in which the former immerses the hydrophobic part in lipid bilayer, and the latter did not immerse, and formed large assemblies by aggregation of original liposomes. The present study clearly showed that hydrophobic-hydrophilic balance of a peptide is a crucial factor in understanding lipid-peptide interactions.