Propidium Iodide Competes with Ca2+ to Label Pectin in Pollen Tubes and Arabidopsis Root Hairs

We have used propidium iodide (PI) to investigate the dynamic properties of the primary cell wall at the apex of Arabidopsis (Arabidopsis thaliana) root hairs and pollen tubes and in lily (Lilium formosanum) pollen tubes. Our results show that in root hairs, as in pollen tubes, oscillatory peaks in PI fluorescence precede growth rate oscillations. Pectin forms the primary component of the cell wall at the tip of both root hairs and pollen tubes. Given the electronic structure of PI, we investigated whether PI binds to pectins in a manner analogous to Ca²⁺ binding. We first show that Ca²⁺ is able to abrogate PI growth inhibition in a dose-dependent manner. PI fluorescence itself also relies directly on the amount of Ca²⁺ in the growth solution. Exogenous pectin methyl esterase treatment of pollen tubes, which demethoxylates pectins, freeing more Ca²⁺-binding sites, leads to a dramatic increase in PI fluorescence. Treatment with pectinase leads to a corresponding decrease in fluorescence. These results are consistent with the hypothesis that PI binds to demethoxylated pectins. Unlike other pectin stains, PI at low yet useful concentration is vital and specifically does not alter the tip-focused Ca²⁺ gradient or growth oscillations. These data suggest that pectin secretion at the apex of tip-growing plant cells plays a critical role in regulating growth, and PI represents an excellent tool for examining the role of pectin and of Ca²⁺ in tip growth.The apical wall of tip-growing cells participates directly in the process of growth regulation (McKenna et al., 2009; Winship et al., 2010), yet few methods permit monitoring the wall properties of living cells. Despite this, several recent studies have enhanced our understanding of the apical cell wall. Chemical analyses of isolated pollen tube wall material have revealed a complex mixture of pectic polysaccharides with regions comprising long sequences of polygalacturonic acid. Important patterns of pectin methoxylation have been detected using immunocytochemical approaches, but these are limited to fixed cells (Dardelle et al., 2010). In a recent study, Parre and Geitmann (2005) used microindentation to show significant correlations between wall strength and growth rate. None of these techniques allow for easy investigation of the cell wall during growth.In an earlier study, we found that propidium iodide (PI) vitally stains pollen tubes of lily (Lilium formosanum) and tobacco (Nicotiana tabacum) and in particular reveals with great clarity the thickened apical cell wall (Fig. 1; McKenna et al., 2009). In addition, the apical PI fluorescence oscillates and in lily pollen tubes correlates tightly with oscillations in wall thickness measured by differential interference contrast (DIC) optics. Finally, these studies indicated that the PI fluorescence predicted cell growth rates with high confidence, suggesting that PI binding may provide useful information about the physical and chemical properties of the cell wall.Open in a separate windowFigure 1.PI fluorescence and growth rate oscillate in lily pollen tubes (A and B), Arabidopsis root hairs (C–E), and Arabidopsis pollen tubes (F and G). A, The top panel shows a DIC image of a lily pollen tube, and the bottom panel shows PI fluorescence of the same tube. The PI fluorescence is pseudocolored, with white representing high signal and blue representing low signal. Bar = 10 μm. B, Growth rate (blue) and PI fluorescence (red) are plotted on a line graph. Both oscillate with the same period but with different phases. C, DIC image (top panel) and PI fluorescence image (bottom panel) of an Arabidopsis root hair. Bar = 10 μm. D, Two PI fluorescence images of the same root hair focused on the apex representing peak (top) and trough (bottom) PI signals. Bar = 5 μm. E, A line graph showing the growth rate (blue) and peak PI fluorescence at the apex (red) for the same root hair shown in C and D. F, The top panel shows a DIC image of an Arabidopsis pollen tube, and the bottom panel shows PI fluorescence of the same tube. The PI fluorescence is pseudocolored, with white representing high signal and blue representing low signal. Bar = 5 μm. G, Growth rate (blue) and PI fluorescence (red) are plotted on a line graph. Both oscillate with the same period but with different phases. The growth rate between individual 3-s frames was smaller than the pixel size for our optics in both Arabidopsis cell types; to remove the noise this generated, a four-image (pollen) or five-image (root hair) running average is shown. A.U., Arbitrary units.PI is commonly used to visualize plant cell walls by wide-field fluorescence and confocal microscopy (Fiers et al., 2005; Tian et al., 2006; Estevez et al., 2008) and to select viable cells during cell sorting (Deitch et al., 1982; Jones and Senft, 1985). A positively charged phenanthridine derivative, the propidium ion stains cell walls but does not pass through the intact cell membranes of living cells. It readily diffuses into dead cells and forms highly fluorescent complexes by intercalation between base pairs of double-stranded nucleic acids, thus acting as an excellent indicator for cell vitality (Hudson et al., 1969). Binding to cell walls presumably occurs by a different mechanism, since it is not accompanied by the dramatic increase in fluorescence and shift in absorption and emission maxima observed when PI binds to nucleic acids. The mechanism of PI binding needs further exploration, as does the potential for broader use in other tip-growing plant cells.In this report, we test two hypotheses: first, that PI stains other tip-growing cells with pectin-containing cell walls; and second, that PI and Ca²⁺ bind to the same sites in these walls. This binding would occur through the interaction of partial positive charges caused by localized deficits in π-orbital electrons associated with three of the four nitrogen atoms of PI (Luedtke et al., 2005) coordinating with negatively charged carboxyl and hydroxyl groups on homogalacturonans (HGs), as has been suggested in Oedogonium bharuchae (Estevez et al., 2008).Our findings indicate that both hypotheses are satisfied. Notably, oscillatory changes in apical PI fluorescence occur and are observed to anticipate oscillations in growth rate in Arabidopsis (Arabidopsis thaliana) root hairs and Arabidopsis pollen tubes. In addition, competition studies indicate that PI and Ca²⁺ bind to the same sites in cell walls. Supporting these studies, we demonstrate that pectin methyl esterase (PME) creates more sites for PI binding, presumably by demethoxylating HGs as they are secreted, and that pectinase reduces PI fluorescence dramatically. However, unlike other pectin-binding dyes, PI does not block Ca²⁺ channels at the concentration used in live cell studies, nor does it alter oscillatory growth characteristics. Our findings provide evidence that PI may be employed as a quantitative measure of Ca²⁺-binding sites and thus may have use as an indicator of the degree of cross-linking of HGs and of cell wall extensibility.     

Regeneration of the cell wall of protoplasts of the fission yeastSchizosaccharomyces versatilis in liquid media and their reversion to cells

Regeneration of the cell wall and reversion of protoplasts with a completely regenerated cell wall to cells were studied by light and electron microscopy in protoplasts of the fission yeastsSchizosaccharomyces versatilis. On their surface the protoplasts regenerated a complete new wall even m liquid media The wall regeneration began with the formation of a thin irregular net of flat bundles of long microfibrils and the net was gradually filled with aggregates of short straight microfibrils and small piles of amorphous material. Osmotically resistant organisms with regenerated walls were detected after a 4–6 h cultivation Depending on the nutrient medium used 10–80 % of protoplasts with the regenerated wall were obtained that reverted subsequently to cells. The high percentage of the wall regeneration and reversion to cells was reached by combining cultivation in a poor medium with that in a rich medium Reversion to cells could only occur after the protoplasts had regenerated rigid cell walls These walled protoplasts underwent septation, and, by polar growth, produced cylindrical cells, further dividing by fission.     

Rise in chlorotetracycline fluorescence accompanies tracheary element differentiation in suspension cultures ofZinnia

Summary Developing tracheary elements in suspension cultures ofZinnia elegans fluoresce intensely relative to non-differentiating cells when stained with chlorotetracycline (CTC), a fluorescent chelate probe for membrane associated calcium. This suggests that a change in calcium uptake or subcellular distribution accompanies the onset of tracheary element differentiation. A few cells in early differentiating cultures were brightly fluorescent, but did not have visible cell wall thickenings, suggesting that a rise in sequestered calcium may precede visible differentiation. Diffuse CTC fluorescence in early differentiation most likely results from sequestration of calcium in the endoplasmic reticulum. Late in differentiation, CTC fluorescence becomes punctate in appearance, probably due to loss of plasma membrane integrity occurring at the onset of autolysis.Zinnia suspension culture cells were found to be very sensitive to CTC and low concentrations (10 M) were used to assure accurate localization of membrane-associated calcium in healthy cells.Abbreviations CTC chlorotetracycline - DIC differential interference contrast - DiOC₆ 3,3-dihexyloxacarbocyanine iodide - ER endoplasmic reticulum - EGTA ethylene glycol bis-(amino-ethyl ether) N,N,N¹N¹-tetraacetic acid - NPN n-phenylnaphthylamine - OsFeCN osmium tetroxide and potassium ferricyanide - TE tracheary element - TEM transmission electron microscopy     

Sucrose concentration in the growth medium affects the cell wall composition of tobacco pollen tubes

The cell wall of pollen tubes is organized in both spatial and temporal order to allow the pollen tube to grow according to external conditions. The deposition of methyl-esterified and acid pectins in addition to callose/cellulose occurs according to a series of temporally succeeding events. In this work, we attempted to determine how the composition of the external growth medium (in terms of osmolarity) could affect the deposition of cell wall components. Pollen tubes of tobacco were grown in a hypotonic medium and then analyzed for the distribution of pectins and callose/cellulose [as well as for the distribution of the enzyme callose synthase (CALS)]. The data indicate that pollen tubes grown in a hypotonic medium show changes of the initial growth rate followed by modification of the deposition of acid pectins and, to a lesser extent, of CALS. These observations indicate that, under the osmolarity determined by the growth medium, pollen tubes adapt their cell wall to the changing conditions of growth.     

Experimental and ultrastructural studies on cell shape formation in the defect mutant cellmicrasterias thomasiana f. uniradiata

Summary Cell development and ultrastructure are studied in the defect mutant cellMicrasterias thomasiana f. uniradiata which lacks cell pattern at one side of the cell.The ultrastructural studies reveal an uneven distribution of vesicles, preponderating at the normally growing side of the cell, as well as the presence of a special kind of dark vesicles.By means of turgor reduction and treatment with chlorotetracycline and cycloheximide some processes involved in cell shape formation are pointed out and are compared with those already described for biradiateMicrasterias cells.It is demonstrated that the asymmetric cell shape of the mutant cell is already determined at the early stage of bulb formation and is due to a unilateral growth during the later stages of development. The asymmetric arrangement of the growth areas during cell development of the mutant is expressed by an asymmetric distribution of primary wall accumulations induced by turgor reduction as well as by the presence of fluorescence zones after treatment with the Ca²⁺ -chelate probe chlorotetracycline at only one side of the cell. Inhibition of protein synthesis by cycloheximide during cell growth of the mutant leads to the formation of a characteristically reduced cell pattern (anuclear type of development) similar to that ofMicrasterias denticulata andMicrasterias thomasiana under the same conditions. Nevertheless, this cell pattern develops at only one side of the cell, indicating that the mutant does not have any information for cell pattern formation at the defective side.     

Microsporogenesis,pollen mitosis and pollen tube growth in Gagea villosa (Liliaceae)

In this study, Gagea villosa (Bieb.) Duby was investigated by using light microscopy methods in cytological and cytoembryological respects. Anthers were tetrasporangiate. Anther wall was formed with an epidermis, endothecium, middle layer and tapetum. Tapetum was glandular type and it began to degenerate when microspores released from tetrads. Tapetum cells generally have one or two nuclei. Mitosis seen in tapetum cells was generally normal but micronuclei were found in some of them. Fibrous thickenings were determined in endothecium. Microsporogenesis and pollen mitosis were generally regular. Cytokinesis was successive type. Meiosis in pollen mother cells was asynchronous in one anther locus. Mature pollen grains were 2-celled. Pollen sterility was found to be 24%. Some of the fertile pollen grains, smaller than the normal were seen at the end of the pollen mitosis. Microgametophyte development was examined in vivo and in vitro. Germination ratio of pollen grains in vitro was 4%. Generally swollen pollen tube tips and weak development of some curled pollen tubes were seen. Callose plug formation was seen only in vivo pollen tube growth.     

Altered mechano-chemical environment in hip articular cartilage: effect of obesity

The production of extracellular matrix (ECM) components of articular cartilage is regulated, among other factors, by an intercellular signaling mechanism mediated by the interaction of cell surface receptors (CSR) with insulin-like growth factor-1 (IGF-1). In ECM, the presence of binding proteins (IGFBP) hinders IGF-1 delivery to CSR. It has been reported that levels of IGF-1 and IGFBP in obese population are, respectively, lower and higher than those found in normal population. In this study, an experimental–numerical approach was adopted to quantify the effect of this metabolic alteration found in obese population on the homeostasis of femoral hip cartilage. A new computational model, based on the mechano-electrochemical mixture theory, was developed to describe competitive binding kinetics of IGF-1 with IGFBP and CSR, and associated glycosaminoglycan (GAG) biosynthesis. Moreover, a gait analysis was carried out on obese and normal subjects to experimentally characterize mechanical loads on hip cartilage during walking. This information was deployed into the model to account for effects of physiologically relevant tissue deformation on GAG production in ECM. Numerical simulations were performed to compare GAG biosynthesis in femoral hip cartilage of normal and obese subjects. Results indicated that the lower ratio of IGF-1 to IGFBP found in obese population reduces cartilage GAG concentration up to 18 % when compared to normal population. Moreover, moderate physical activity, such as walking, has a modest beneficial effect on GAG production. The findings of this study suggest that IGF-1/IGFBP metabolic unbalance should be accounted for when considering the association of obesity with hip osteoarthritis.     

Calcium gradients in tip growing plant cells visualized by chlorotetracycline fluorescence

With chlorotetracycline (CTC)-fluorescence a tip-to-base Ca²⁺ gradient is visualized in all tested, tip-growing plant cells: pollen tubes of Lilium longiflorum, root hairs of Lepidium sativum, moss caulonema of Funaria hygrometrica, fungal hyphae of Achlya and in the alga Acetabularia mediterranea. The fluorescence gradients in the different species vary in intensity and extension. Sometimes a punctate mobile CTC-fluorescence, in the size range of mitochondria, is observed. Bursting cells lose their fluorescence rapidly, indicating a cytoplasmic localization of the gradient. Only in Acetabularia is the wall also fluorescent with CTC. The results are interpreted as evidence for a general role of a calcium gradient in tip growth.Abbreviation CTC chlorotetracycline     

Interaction between the antimicrobial peptide Aurein 1.2 dimer and mannans

We have previously described the structure and the ability of a dimeric analog of the antimicrobial peptide Aurein 1.2 to aggregate Candida albicans. In this study, circular dichroism and fluorescence spectroscopy data showed that this aggregation is related to the interaction between the peptide and mannans, the main component of yeast cell wall. In this context, we propose a model in which dimers interact with the polysaccharide leading to cells aggregation.     

Influence of surface topography on the human epithelial cell response to micropatterned substrates with convex and concave architectures

Background

Understanding the fundamental mechanisms underlying the cellular response to topographical surface features will extend our knowledge regarding the regulation of cell functions. Analyzing the cellular response to different topographical features, over multiple temporal and spatial scales, is central to understanding and guiding several biological functions. We used micropatterned substrates with convex and concave architectures to evaluate the behaviors of human epithelial cells on these substrates.

Results

Pillar and pit substrates caused heterogeneous spatial growth and distribution, with differences in cell density, over 48 h. Regional densities and distribution were significantly increased at pillar sidewalls, and at pit sidewalls and bottoms compared with those on flat unpatterned areas. Time-lapse observations revealed that different mechanisms of cell migration were dependent upon pillar and pit features. Cells on pillar substrate migrated towards the sidewall, whereas cells on pit substrate tended to move towards the sidewalls and bottom. Cytoskeletal staining of F-actin and vinculin showed that this migration can be attributed to difference in spatial reorganization of actin cytoskeleton, and the formation of focal adhesions at various points on the at the convex and concave corners of pillar and pit substrates. Cells cultured on the pillar substrate had stress fibers with extended filopodia and immature focal contacts at the sidewalls and convex corners, similar to those on the flat unpatterned substrate. Cells at the sidewalls and concave corners of pit substrate had more contractile stress fibers and stable focal contacts compared with cells on the pillar substrate. We also found that the substrate structures affect cell-cell contact formation via E-cadherin, and that this was associated with reorganization of the actin cytoskeleton at the sidewall, and at the convex and concave corners of the substrate.

Conclusion

Migration is an important factor affecting spatial growth and distribution. Heterogeneity at various locations was caused by different migratory behaviors at the convex and concave corners of pillar and pit substrates. We propose that this investigation is a valuable method for understanding cell phenotypes and the heterogeneity during spatial growth and distribution of epithelial cells during culture.

     

Membrane-bound Ca2+ distribution visualized by chlorotetracycline fluorescence during morphogenesis of soredia in a lichen

Summary In the lichenParmelia caperata (L.) Ach. the distribution pattern of membrane-bound Ca²⁺ is investigated in the symbionts by chlorotetracycline (CTC)-induced fluorescence during the development of propagative structures, the soredia. The results demonstrate that Ca²⁺ accumulation in the alga and the fungus is associated with this morphogenetic process; particularly, polarized hyphal growth involves a tip-to-base Ca²⁺ gradient.CTC fluorescence distribution is coincident with that of cholinesterase (ChE) activity during morphogenesis of soredia. A comparison is suggested with embryonic ChE of animal cells, where developmental events are regulated by a cholinergic mechanism that also modulates Ca²⁺ levels.     

Adenovirus-Mediated NDRG2 Inhibits the Proliferation of Human Renal Cell Carcinoma Cell Line OS-RC-2 in Vitro

The objective of the study is to investigate the inhibitory effects of adenovirus-mediated N-Myc downstream-regulated gene 2 (NDRG2) on the proliferation of human renal cell carcinoma cell line OS-RC-2 in vitro. NDRG2 was harvested by RT-PCR, confirmed by DNA sequencing, and then cloned into the eukaryotic expression vector pIRES2-EGFP, which encodes green fluorescent protein (GFP), to construct pIRES2-EGFP-NDRG2 plasmid. OS-RC-2 cells with NDRG2 negative expression were transfected with pIRES2-EGFP-NDRG2 plasmid. The growth of transfected OS-RC-2 cells was observed under the light and fluorescence microscopes. After colony-forming cell assays, cell proliferation detection, and MTT assays, the growth curves of cells in each group were plotted to investigate the inhibitory effects of adenovirus-mediated NDRG2 on the proliferation of OS-RC-2 cells. Cell cycle was determined by flow cytometry. Confocal laser scanning microscopy was applied to determine the specific location of NDRG2 protein in subcellular level. A eukaryotic expression vector pIRES2-EGFP-NDRG2 was successfully constructed. After NDRG2 transfection, the growth of OS-RC-2 cells was inhibited. Flow cytometry showed that cells were arrested in S phase but the peak of cell apoptosis was not present, and confocal laser scanning microscopy showed that NDRG2 protein was located in mitochondrion. In conclusion, NDRG2 can significantly inhibit the proliferation of OS-RC-2 cells in vitro and its protein is specifically expressed in the mitochondrion.     

Patterned distribution of membrane-associated Ca2+ during pore formation inMicrasterias

Summary During the stage of pore formation developing cells ofMicrasterias denticulata show a patterned distribution of fluorescent dots on the plasma membrane after treatment with chlorotetracycline. The center-to-center spacing of these dots corresponds with the distances between the individual cell wall pores ofMicrasterias. Therefore it is supposed that the patterned distribution of pores and their formation which is mediated by special pore vesicles are related to local accumulations of membrane-associated Ca²⁺. Membrane-associated Ca²⁺ seems not only to be functional in tip growth but to be a general mediator for recognition and fusion processes between various vesicles and the plasma membrane.     

Not-so-tip-growth

In tip-growing plant cells such as pollen tubes and root hairs, surface expansion is confined to the cell apex. Vesicles containing pectic cell wall material are delivered to this apical region to provide the material necessarily to build the expanding cell wall. Quantification of wall expansion reveals that the surface expansion rates are not highest at the pole but instead in an annular region around the pole. These findings raise the question of the precise localization of exocytosis events in these cells. Recently, we used spatio-temporal image correlation spectroscopy (STICS) in combination with high temporal resolution confocal imaging to characterize the intracellular movement of vesicles in growing pollen tubes. These observations, together with the analysis of FRAP (fluorescence recovery after photobleaching) experiments, indicate that exocytosis is likely to occur predominantly in the same annular region where wall expansion rates are greatest. Therefore, tip growth in plant cells does not seem to happen exactly at the tip.Key words: tip growth, pollen tube, exocytosis, cell wall, expansion, root hair, plant cell growth, allometric growth, cytomechanics, cell mechanics, vesicle transport     

Identification and immunocytochemical localization of α-galactosidase in resting and germinated date palm (Phoenix dactylifera L.) seeds

α-Galactosidases (EC 3.2.1.22) from resting and germinated date (Phoenix dactylifera L.) seeds were compared and localized using immunocytochemical methods. The enzyme was present in both the endosperm and embryo of resting seeds, in the endosperm undergoing digestion where the greatest specific activity was present, and in the haustorium of seedlings. The enzyme had a molecular mass of 140000 as determined by gel filtration and a pH optimum of 4.5. At least seven forms of the enzyme with isoelectric points ranging from 3.85 to 5.2 were detected in the haustorium whereas only four of these forms were present in the endosperm. The relative activity levels of the various forms also differed between the two tissues. On Western blots all enzyme forms were recognized by antibodies raised against mung-bean (Vigna radiata) α-galactosidase. Using immunogold techniques, label was shown to be present in the protein bodies of the resting embryo cells but to decrease in this organelle as the reserve protein was mobilized and to appear diffusely in the cytoplasm in subsequent stages. In resting endosperm cells, label occurred in the protein bodies and in a thin region of inner wall. In endosperm undergoing digestion, where different stages of protoplast and wall breakdown occurred, immunogold staining was localized in the flocculent contents of vacuoles which resulted from storageprotein breakdown, then dense staining occurred in the inner wall of cell cavities formed by the complete dissolution of the cytoplasm, and finally, staining was uniformly diffuse throughout the remaining endosperm wall adjacent to the haustorium surface. These observations indicate that the α-galactosidase present in cell walls of the date palm endosperm during mannan mobilization is not secreted by the haustorium but instead is probably a pregermination product stored mainly in the protein bodies of resting endosperm and is released to the wall following loss of membrane integrity.     

Studies ofNajas pollen tubes. Fine structure and the dependence of chlorotetracycline fluorescence on external free ions

Summary The distribution of membrane-associated calcium was investigated in pollen grains and tubes of the underwater pollinated angiospermNajas marina L. using chlorotetracycline (CTC). Tubes grown in distilled water (pH 6) showed the highest fluorescence in a subapical region that tapered basally into a fluorescent strand centrally located in the tube and extending back towards the pollen grain. The apical cap had low fluorescence as did the cytoplasm surrounding the fluorescent strand, the tube base and the pollen grain. Tubes grown in different pond waters (pH 8) revealed no intracellular CTC fluorescence. Instead there was an external fluorescence forming a distinct layer around the whole tube, frequently enhanced in a subapical region to form an external collar.Modification of the patterns of fluorescence could be induced by manipulation pH of the growth media and content of specific ions. For example tubes grown in distilled water with 10^–3 M Mg²⁺ salts showed a similar CTC fluorescence as those grown in pond water. In contrast, Ca²⁺ enrichment had no visible influence on the patterns of fluorescence. The pattern of fluorescence displayed by tubes grown in distilled water, could be reproduced in pond water if the pH was artificially reduced to pH 6.Ultrastructurally, there was no detectable difference in the markedly polar distribution of organelles between pollen tubes grown in the various growth media. The secretory vesicles found in the pollen grain prior to germination become distributed throughout the pollen tube but are least concentrated in regions that show highest internal CTC fluorescence. These regions appear to have large amounts of endoplasmic reticulum and include mitochondria.These results are discussed in relation to the significance of calcium gradients for tip growth and limitations in the use of CTC.Abbreviations CTC chlorotetracycline - SV secretory vesicle - ER endoplasmic reticulum - PIXE proton induced X-ray emissions