Actin filaments of guard cells are reorganized in response to light and abscisic acid.

We recently showed that treatment with actin antagonists perturbed stomatal behavior in Commelina communis L. leaf epidermis and therefore suggested that dynamic changes in actin are necessary for signal responses in guard cells (M. Kim, P.K. Hepler, S.O. Eun, K.-S. Ha, Y. Lee [1995] Plant Physiol 109: 1077-1084). Here we show that actin filaments of guard cells, visualized by immunofluorescence microscopy, change their distribution in response to physiological stimuli. When stomata were open under white-light illumination, actin filaments were localized in the cortex of guard cells, arranged in a pattern that radiates from the stomatal pore. In marked contrast, for guard cells of stomata closed by darkness or by abscisic acid, the actin organization was characterized by short fragments randomly oriented and diffusely labeled along the pore site. Upon abscisic acid treatment, the radial pattern of actin arrays in the illuminated guard cells began to disintegrate within a few minutes and was completely disintegrated in the majority of labeled guard cells by 60 min. Unlike actin filaments, microtubules of guard cells retained an unaltered organization under all conditions tested. These results further support the involvement of actin filaments in signal transduction pathways of guard cells.     

Microtubules are necessary for lamellae retraction of Vero cells

Behavior of Vero cells under the 2,3-butaneodione monoxime (BDM) treatment was examined using video-microscopy with contrast enhancement. After addition of BDM to the culture medium the area of cell contact with substratum gradually reduced--within 5 min of treatment cell lamellae became thicker, after 60 min the cell area decreased approximately 70 %, and the cells became nearly rounded. At the same time actin bundles (stress fibers) depolymerized, and microtubule network became denser. Partial depolymerization of microfilaments by treatment with latrunculin B at a concentration of 5 nM resulted in complete loss of stress fibers, yet cells slightly change their form, and microtubule system remained the same as in the control cells. However, after addition of BDM in the presence of latrunculin B cells retracted their lamellae more quickly then under BDM sole treatment. To evaluate the role of microtubules in the process of cell retraction we depolymerized them with nocodazole taken at the concentration of 5 ng/ml. Under nocodazole treatment the cell area decreased approximately 20 %, and stress fibers became more thick and abandon. The cells did not change their form, and stress fibers depolymerized very slowly under BDM treatment in the absence of microtubules. After 1 h of BDM treatment in the presence ofnocodazole stress fibers were still more numerous than in the control cells. Complete depolymerization of stress fibers happened in 90 % of cells only in 24 h after addition of BDM. When nocodazole had been washed out of the culture medium in the presence of BDM, lamellae started shrinking in 6 min. This time corresponds to the time required for the partial restoration of microtubule system. On the bases of the results obtained we conclude that retraction of the lamellae in Vero cells is guided rather mainly by microtubules, than stress-fibers.     

Microtubules are stabilized in confluent epithelial cells but not in fibroblasts

Rhodamine-tagged tubulin was microinjected into epithelial cells (MDCK) and fibroblasts (Vero) to characterize the dynamic properties of labeled microtubules in sparse and confluent cells. Fringe pattern fluorescence photobleaching revealed two components with distinct dynamic properties. About one-third of the injected tubulin diffused rapidly in the cytoplasm with a diffusion coefficient of 1.3-1.6 x 10(- 8) cm2/s. This pool of soluble cytoplasmic tubulin was increased to greater than 80% when cells were treated with nocodazole, or reduced to approximately 20% upon treatment of cells with taxol. Fluorescence recovery of the remaining two-thirds of labeled tubulin occurred with an average half-time (t1/2) of 9-11 min. This pool corresponds to labeled tubulin associated with microtubules, since it was sensitive to treatment of cells with nocodazole and since taxol increased its average t1/2 to greater than 22 min. Movement of photobleached microtubules in the cytoplasm with rates of several micrometers per minute was shown using very small interfringe distances. A significant change in the dynamic properties of microtubules occurred when MDCK cells reached confluency. On a cell average, microtubule half-life was increased about twofold to approximately 16 min. In fact, two populations of cells were detected with respect to their microtubule turnover rates, one with a t1/2 of approximately 9 min and one with a t1/2 of greater than 25 min. Correspondingly, the rate of incorporation of microinjected tubulin into interphase microtubules was reduced about twofold in confluent MDCK cells. In contrast to the MDCK cells, no difference in microtubule dynamics was observed in sparse and confluent populations of Vero fibroblasts, where the average microtubule half- life was approximately 10 min. Thus, microtubules are significantly stabilized in epithelial but not fibroblastic cells grown to confluency.     

Melanophores of Zacco temmincki (Teleostei) are light sensitive

The responses of melanophores of a cyprinid fish Zacco temmincki to changes in illumination were examined in isolated scale preparations of the adult fishes. Melanosomes in the melanophores aggregated in darkness and dispersed in light. These responses were invariably induced, even in denervated melanophores. These light responses, the dark-induced aggregation and the light-induced dispersion, were not affected by a number of alpha and beta adrenergic blocking agents. It was concluded that the melanophores of Zacco temmincki were themselves light sensitive and responded directly to light by melanosome translocations. The light responses were quantitatively assessed in relation to the intensity of illumination.     

Changes in surface area of intact guard cells are correlated with membrane internalization

Guard cells must maintain the integrity of the plasma membrane as they undergo large, rapid changes in volume. It has been assumed that changes in volume are accompanied by changes in surface area, but mechanisms for regulating plasma membrane surface area have not been identified in intact guard cells, and the extent to which surface area of the guard cells changes with volume has never been determined. The alternative hypothesis-that surface area remains approximately constant because of changes in shape-has not been investigated. To address these questions, we determined surface area for intact guard cells of Vicia faba as they underwent changes in volume in response to changes in the external osmotic potential. We also estimated membrane internalization for these cells. Epidermal peels were subjected to external solutions of varying osmotic potential to shrink and swell the guard cells. A membrane-specific fluorescent dye was used to identify the plasma membrane, and confocal microscopy was used to acquire a series of optical paradermal sections of the guard cell pair at each osmotic potential. Solid digital objects representing the guard cells were created from the membrane outlines identified in these paradermal sections, and surface area, volume, and various linear dimensions were determined for these solid objects. Surface area decreased by as much as 40% when external osmotic potential was increased from 0 to 1.5 MPa, and surface area varied linearly with volume. Membrane internalization was approximated by determining the amount of the fluorescence in the cell's interior. This value was shown to increase approximately linearly with decreases in the cell's surface area. The changes in surface area, volume, and membrane internalization were reversible when the guard cells were returned to a buffer solution with an osmotic potential of approximately zero. The data show that intact guard cells undergo changes in surface area that are too large to be accommodated by plasma membrane stretching and shrinkage and suggest that membrane is reversibly internalized to maintain cell integrity.     

Microtubules containing detyrosinated tubulin are less dynamic

Peptide antibodies specific for tyrosinated (tyr-tubulin) or detyrosinated alpha-tubulin (glu-tubulin) have been generated for studying the relative stability of microtubules enriched in either form of alpha-tubulin. Treatment of Vero cells with nocodazole has revealed that interphase microtubules rich in glu-tubulin (glu-microtubules) are resistant to higher concentrations of the microtubule-disrupting drug than the microtubules containing only tyr-tubulin (tyr-microtubules). Glu-tubulin is enriched in centrioles and mid-bodies, but absent from the first interphase microtubules that have repolymerized in late telophase. Tubulin (including both forms) has been labeled with rhodamine (rh-tubulin) and microinjected into Vero cells to study in vivo the dynamic properties and incorporation rates of tubulin into microtubules rich in either glu- or tyr-tubulin. Tyr-microtubules are significantly more rapidly labeled by the microinjected rh-tubulin than glu-microtubules. Ten minutes after injection, rh-tubulin is present in virtually all tyr-microtubules. The half-time of turnover of glu-microtubules is approximately 1 h. Even several hours after microinjection, some of the glu-microtubules have consistently not incorporated visible amounts of rh-tubulin. These results suggest that tyr- and glu-microtubules respectively represent relatively dynamic and stable subclasses of interphase microtubules.     

Epithelial cells are sensitive detectors of bacterial pore-forming toxins

Epithelial cells act as an interface between human mucosal surfaces and the surrounding environment. As a result, they are responsible for the initiation of local immune responses, which may be crucial for prevention of invasive infection. Here we show that epithelial cells detect the presence of bacterial pore-forming toxins (including pneumolysin from Streptococcus pneumoniae, alpha-hemolysin from Staphylococcus aureus, streptolysin O from Streptococcus pyogenes, and anthrolysin O from Bacillus anthracis) at nanomolar concentrations, far below those required to cause cytolysis. Phosphorylation of p38 MAPK appears to be a conserved response of epithelial cells to subcytolytic concentrations of bacterial poreforming toxins, and this activity is inhibited by the addition of high molecular weight osmolytes to the extracellular medium. By sensing osmotic stress caused by the insertion of a sublethal number of pores into their membranes, epithelial cells may act as an early warning system to commence an immune response, while the local density of toxin-producing bacteria remains low. Osmosensing may thus represent a novel innate immune response to a common bacterial virulence strategy.     

CO2 provides an intermediate link in the red light response of guard cells

Guard cells in intact leafs display light-induced membrane potential changes, which alter the direction of K+-transport across the plasma membrane (Roelfsema et al., 2001). A beam of blue light, but not red light, directed at the impaled guard cell triggers this response, while both light qualities induce opening of stomata. To gain insight into this apparent contradiction, we explored the possible interaction between red light and CO2. Guard cells in the intact plant were impaled with double-barrelled electrodes and illuminated with red light. Cells that were hyperpolarized in CO2-free air, depolarized after a switch to air with 700 micro l l(-1) CO2, in a reversible manner. As a result, K+-fluxes across the plasma membrane changed direction, to favour K+ extrusion and stomatal closure in the presence of CO2. Concurrent with the depolarization, an inward current across the plasma membrane appeared, most likely due to activation of anion channels. Guard cell responses to CO2 could be recorded in darkness as well as in red light. However, in darkness some cells spontaneously depolarized, these cells hyperpolarized again in red light. Here, red light was projected on a large area of the leaf and decreased the intracellular CO2 concentration by about 250 micro l l(-1), as measured with a miniature CO2 sensor placed in the substomatal cavity. We conclude, that in intact leaves the red light response of guard cells is mediated through a decrease of the intercellular CO2 concentration.     

Microtubules self-repair in living cells

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Microtubules are involved in transport of macromolecules by vesicles in cultured bovine aortic endothelial cells

The macromolecular transport in bovine aortic endothelial monolayers, cultured in vitro, was studied by fluorescence microscopy, confocal laser scanning microscopy, and transmission electron microscopy. A fluid-phase endocytic tracer, fluorescein isothiocyanate dextran 70 kD (FITC-dextran 70), was found to be transported into and out of endothelial cells via vesicles arranged as chains stretching between the luminal surface and the cell interior and also from cell interior to the abluminal surface. The endocytic activity was reduced by colchicine, which disrupts microtubules, and increased during treatment with cytochalasin B, which blocks microfilament polymerization. These findings indicate that microtubules are required for fluid-phase endocytosis and that microfilaments hinder this process. © 1993 Wiley-Liss, Inc.     

Mesangial cells of lupus-prone mice are sensitive to chemokine production

Infectious antigens may be triggers for the exacerbation of systemic lupus erythematosus. The underlying mechanism causing acceleration and exacerbation of lupus nephritis (LN) is largely unknown. Bacterial lipopolysaccharide (LPS) is capable of inducing an accelerated model of LN in NZB/W mice, featuring diffuse proliferation of glomerular resident cells. We hypothesized that mesangial cells (MCs) from LN subjects are more responsive to LPS than normal subjects. Cultured primary NZB/W and DBA/W (nonautoimmune disease-prone strain with MHC class II molecules identical to those of NZB/W) MCs were used. Monocyte chemoattractant protein-1 (MCP-1) and osteopontin (OPN) expressions either in the baseline (normal culture) condition or in the presence of LPS were evaluated by real-time PCR, ELISA, or western blot analysis. NF-κB was detected by ELISA, electrophoresis mobility-shift assay, and immunofluorescence. First, either in the baseline condition or in the presence of LPS, NZB/W MCs produced significantly higher levels of MCP-1 and OPN than the DBA/W MC controls. Second, NZB/W MCs expressed significantly higher levels of Toll-like receptor 4, myeloid differentiation factor 88, and NF-κB than the DBA/W MC controls, both receiving exactly the same LPS treatment. In conclusion, NZB/W MCs are significantly more sensitive than their normal control DBA/W MCs in producing both MCP-1 and OPN. With LPS treatment, the significantly elevated levels of both chemokines produced by NZB/W MCs are more likely due to a significantly greater activation of the Toll-like receptor 4-myeloid differentiation factor 88-associated NF-κB pathway. The observed abnormal molecular events provide an intrarenal pathogenic pathway involved in an accelerated type of LN, which is potentially infection triggered.     

Microtubules are not altered in the dystrophic chicken

The association of an altered cytoplasmic microtubule complex in cells of the dystrophic chicken was investigated. Dystrophic chickens of lines 304 and 413 were compared with their genetically matched control, 412 (obtained from UC, Davis). Explants and trypsin-dissociated tissues were prepared from breast and heart muscles of chickens at 1, 3, 7, 14, 20, 40, 80 and 120 days ex ovo. The cells were cultured for 7 days and then processed for antitubulin immunofluorescence. Over 90% of the cells displayed an extensive cytoplasmic microtubule complex, although there was significant elevation of creatine phosphokinase in the dystrophic chickens after 20 days ex ovo. In both dystrophic and control preparations, one to two distinct functionally intact microtubule-organizing centers per cell were observed. Dystrophic and control chicken brain extracts demonstrated essentially the same extent of microtubule assembly as assayed by turbidity increase and protein in sedimentable polymer. SDS-PAGE revealed no significant differences in the microtubule proteins polymerized from the dystrophic and control brains. These results suggest that no significant alteration occurs in the structure, assembly or distribution of cytoplasmic microtubules in the cells of the dystrophic chicken.     

Microtubules in human aortic intimal cells

Summary Microtubules were observed in aortic smooth muscle cells. These observations made it unlikely that microtubules function as a transport mechanism or a contractile system. The characteristic location of microtubules seems to favour the suggestion that they may function as a cytoskeleton.