Early response of cultured lepidopteran cells to exposure to delta-endotoxin from Bacillus thuringiensis: involvement of calcium and anionic channels.

The role of ion channels in the initial steps following exposure of SF-9 lepidopteran insect cells in culture to the delta-endotoxin CryIC from the insecticidal bacterium Bacillus thuringiensis was investigated using single ionic channel measurements and microspectrofluorescence of the calcium-sensitive probe fura-2. It was found that: (1) the toxin triggers an immediate rise in intracellular calcium; (2) the surge is due to calcium entering the cells via calcium channels; (3) the toxin recruits or introduces anionic channels in the cell's plasma membrane in a time-dependent manner. These channels, not seen in the absence of the toxin, are induced by toxin exposure to either side of the cell membrane. They have a conductance of 26 picosiemens (pS) and are mainly permeable to chloride. This study provides the first evidence of the primary role of calcium and chloride ions in the action of delta-endotoxin on cultured insect cells.     

Reduction in rat mesentery mast cell staining after degranulation by protamine. A competitive antagonism.

Degranulation of rat mesentery mast cells by increasing concentrations of protamine causes a parallel decrease in the numbers of mast cells stained with toluidine blue or with berberine sulfate. No decrease in mast cell numbers occurs when degranulation is inhibited. Since protamine does not enter into non stimulated mast cells, these results suggest that this reduction in mast cell numbers is caused by the binding of protamine to the anionic sites of heparin of exocytosed granules thereby preventing their staining. There seems to be a competitive antagonism between protamine and toluidine blue at the anionic sites of heparin for increasing concentrations of toluidine blue progressively reverse the reduction in mast cell numbers.     

Intracellular calcium signaling by jurkat T-lymphocytes exposed to a 60 hz magnetic field

To explore possible biochemical mechanisms whereby electromagnetic fields of around 0.1 mT might affect immune cells or developing cancer cells, we studied intracellular calcium signaling in the model system Jurkat E6-1 human T-leukemia cells during and following exposure to a 60 Hz magnetic field. Cells were labeled with the intracellular calcium-sensitive fluorescent dye Fluo-3, stimulated with a monoclonal antibody against the cell surface structure CD3 (associated with ligand-stimulated T-cell activation), and analyzed on a FACScan flow-cytometer for increases in intensity of emissions in the range of 515–545 nm. Cells were exposed during or before calcium signal-stimulation to 0.15 mT_rms 60 Hz magnetic field. The total DC magnetic field of 78.2 μT was aligned 17.5° off the vertical axis. Experiments used both cells cultured at optimal conditions at 37 °C and cells grown under suboptimal conditions of 24 °C, lowered external calcium, or lowered anti-CD3 concentration. These experiments demonstrate that intracellular signaling in Jurkat E6-1 was not affected by a 60 Hz magnetic field when culture and calcium signal-stimulation were optimal or suboptimal. These results do not exclude field-induced calcium-related effects further down the calcium signaling pathway, such as on calmodulin or other calcium-sensitive enzymes. Bioelectromagnetics 18:439–445, 1997. © 1997 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

CHANGES IN CHARGED GROUPS ON THE CELL SURFACE DURING DEVELOPMENT OF THE CELLULAR SLIME MOLD DICTYOSTELIUM DISCOIDEUM: AN ELECTRON MICROSCOPIC STUDY

The distribution of charged groups on the surface of Dictyostelium cells and their change during development were examined by electronmicroscopy using cationic and anionic ferritins. The number of anionic sites on the cell surface decreased greatly during the course of development. The whole surface of vegetative cells stained strongly with cationic ferritin (CF). On the other hand, the surface of aggregation-competent cells had fewer negative charges and these were unequally distributed, the surface of the advancing area (lobopodial region) being devoid of anionic sites. The number of anionic sites on the cell surface decreased progressively during further development, and the suface of slug cells did not stain at all with CF. The cell surface did not stain with anionic ferritin at any developmental stage, indicating the absence of detectable cationic sites. The biological significance of these findings is discussed in connection with cell adhesiveness and movement.     

Ultrastructural localization of anionic sites on the surface of yeast, hyphal and germ-tube forming cells of Candida albicans

The cell wall of Candida albicans contains chitin, beta-glucans and phosphorylated mannoproteins, and possesses a fuzzy coat which is thought to play a role in pathogenicity, phagocytosis, and adherence of this dimorphic yeast. Using scanning electron microscopy and the gold method, mannoproteins were detected on the whole surface of blastoconidia including the bud scars, but chitin was absent even after alpha-mannosidase treatment of the cells. The presence of surface beta-(1----6)glucan (but not beta(1----3)glucan) was observed only after extensive alpha-mannosidase and alkaline phosphatase treatments of blastoconidia. Using transmission and scanning electron microscopy, the locations of anionic sites were revealed by polycationic colloidal gold-chitosan complexes on the surface of blastoconidia, germ tubes and hyphae. Anionic sites were dispersed evenly over the surface of blastoconidia bearing bud scars. Depending upon the growth conditions, anionic sites could be detected on emerging buds and young cells. However, bud scars were always free of marking. When germ-tube formation was induced, anionic sites were present at different densities on all cell surfaces, the highest density being observed on cells with bud scars. Anionic sites were detected at a remarkably high density on all hyphal surfaces. An apical concentration of anionic sites was observed on germ tubes and hyphae. The distribution of anionic sites was not modified by endoglucosaminidase treatment of blastoconidia, germ tubes and hyphae. The anionic sites were associated with the fuzzy coat. As the hyphal form is regarded as possessing the greatest invasiveness, it is suggested that anionic sites play an important role in establishing tissue colonization by this human pathogen.     

Distribution of the anionic sites in the cell wall of apple fruit after calcium treatment

Summary The ripening and softening of fleshy fruits involves biochemical changes in the cell wall. These changes reduce cell wall strength and lead to cell separation and the formation of intercellular spaces. Calcium, a constituent of the cell wall, plays an important role in interacting with pectic acid polymers to form cross-bridges that influence cell wall strength. In the present study, cationic colloidal gold was used for light and electron microscopic examinations to determine whether the frequency and distribution of anionic binding sites in the walls of parenchyma cells in the apple were influenced by calcium, which was pressure infiltrated into mature fruits. Controls were designed to determine the specificity of this method for in muro labelling of the anionic sites on the pectin polymers. The results indicate that two areas of the cell wall were transformed by the calcium treatment: the primary cell walls on either side of the middle lamella and the middle lamella intersects that delineate the intercellular spaces. The data suggest that calcium ions reduce fruit softening by strengthening the cell walls, thereby preventing cell separation that results in formation of intercellular spaces.Abbreviations EDTA ethylenediaminotetraacetic acid - PATAg periodic acid-thiocarbohydrazide-silver proteinate     

The contribution of the calcium-dependent interaction of aggregation factor molecules to recognition: A system providing additional specificity forces?

Cells from the sponge Microciona prolifera display on their surfaces large but defined proteoglycan complexes (Microciona aggregation factor = MAF) that mediate species-specific cell aggregation by a process requiring high calcium ion concentrations. An analysis of MAF-MAF interactions based on binding studies of MAF to glutaraldehyde-fixed sponge cells and MAF-derivatized beads demonstrates that the requirement for high calcium concentrations can be overcome by extremely small amounts of certain polycations such as polybrene, polylysine, or histones. For measurements of the affinity of these substances to MAF, a method was adopted that partitions ¹²⁵I-labeled MAF between dextran and polyethyleneglycol in an aqueous two-phase polymer system depending on the net charge of the complex formed. Since only polymers of positive charges affect binding and partitioning at low concentrations, large areas of interaction similar to those found in glycosaminoglycans are proposed for MAF. Through a multitude of appropriately spaced interaction sites, the rather weak selectivity of single charged sites could in such a system still provide strong enough specificities to explain species-specific cell sorting. The biological significance of naturally occurring polycations as well as extracellular calcium includes their role in cell recognition, sorting out as well as the ordered and continual streaming movements of groups of cells seen in the mesohyl of live sponges.     

A model for the stimulation of taste receptor cells by salt.

A taste cell mucosal surface is regarded as a planar region containing bound anionic sites and openings to ionic channels. It is assumed that the bulk aqueous properties of the exterior phase are not continuous with the surface but terminate at a plane near the surface. The region between the (Stern) plane and the membrane is regarded as having a lower dielectric constant than bulk water. This fact admits the possibility of ion pair formation between fixed sites and mobile cations. Mobile ion pairs entering the region may also bind to a fixed anionic site. Thus, it is assumed that mobile cations and ion pairs are potential determining species at the surface. Binding cations neutralizes surface charges, whereas binding mobile ion pairs does not. This competition accounts for the observed anion effect on stimulation of tast receptors by sodium salts. The potential profile is constructed by superimposing the phase boundary potentials with an ionic diffusion potential across the membrane. The model accounts for the anion effect on receptor potential, pH effects, the reversal of polarity when cells are treated with FeCl3, and the so-called "water reponse," depolarization of the taste cell upon dilution of the stimulant solution below a critical lower limit. The proposed model does not require both bound cationic and anionic receptors, and further suggests that limited access to a Stern-like region continuous with membrane channels may generally serve to control transport of ions.     

The distribution and mobility of anionic sites on the surfaces of baby hamster kidney cells

The distribution and mobility of anionic sites on the surfaces of baby hamster kidney cells were studied by utilizing the multivalent ligand, polycationic ferritin, as a visual probe. Our observations revealed that anionic sites are distributed over the entire cell surface, with the highest density of sites being located on cell surface microextensions. Following the initial binding of polycationic ferritin to the surface of unfixed cells, the ligand-bound anionic sites redistributed by migrating from the surface of microextensions to the surface of the cell body. In 20 min, this migration resulted in a total clearing of anionic sites from the surface of microextensions concomitant with the formation of patches of anionic sites on the surface of the cell body. Polycationic ferritin-induced migration and patch formation of anionic sites was not prevented by 2,4- dinitrophenol, N-ethylmaleimide, colchicine, or cytochalasin B. However, the ligand-induced redistribution of cell surface anionic sites was prevented by prefixation of cells with glutaraldehyde.     

Calcium signaling during convergent extension in Xenopus

BACKGROUND: During Xenopus gastrulation, cell intercalation drives convergent extension of dorsal tissues. This process requires the coordination of motility throughout a large population of cells. The signaling mechanisms that regulate these movements in space and time remain poorly understood. RESULTS: To investigate the potential contribution of calcium signaling to the control of morphogenetic movements, we visualized calcium dynamics during convergent extension using a calcium-sensitive fluorescent dye and a novel confocal microscopy system. We found that dramatic intercellular waves of calcium mobilization occurred in cells undergoing convergent extension in explants of gastrulating Xenopus embryos. These waves arose stochastically with respect to timing and position within the dorsal tissues. Waves propagated quickly and were often accompanied by a wave of contraction within the tissue. Calcium waves were not observed in explants of the ventral marginal zone or prospective epidermis. Pharmacological depletion of intracellular calcium stores abolished the calcium dynamics and also inhibited convergent extension without affecting cell fate. These data indicate that calcium signaling plays a direct role in the coordination of convergent extension cell movements. CONCLUSIONS: The data presented here indicate that intercellular calcium signaling plays an important role in vertebrate convergent extension. We suggest that calcium waves may represent a widely used mechanism by which large groups of cells can coordinate complex cell movements.     

PldB, a putative phospholipase D homologue in Dictyostelium discoideum mediates quorum sensing during development

Quorum sensing, also known as cell-density sensing in the unicellular eukaryote Dictyostelium discoideum, is required for efficient entry into the differentiation and development segment of its life cycle. Quorum sensing is accomplished by simultaneously secreting and sensing the glycoprotein Conditioned Medium Factor, or CMF. When the density of starving cells is high, CMF levels are high, which leads to aggregation followed by development. Here, we describe the role of pldB, a gene coding for a putative phospholipase D (PLD) homologue, in quorum sensing. We find that in submerged culture, adding butanol, an inhibitor of PLD-catalyzed phosphatidic acid production, allows cells to bypass the requirement for CMF mediated quorum sensing and aggregate at low cell density. Deletion of pldB mimics the presence of butanol, allowing cells to aggregate at low cell density. pldB- cells also initiate and finish aggregation rapidly. Analysis of early developmental gene expression in pldB- cells reveals that the cyclic AMP receptor cAR1 is expressed at higher levels earlier than in wild-type cells, which could explain the rapid aggregation phenotype. As would be predicted, cells overexpressing pldB are unable to aggregate even at high cell density. Adding CMF to these pldB- overexpressing cells does not rescue aggregation. Both of these phenotypes are cell autonomous, as mixing a small number of pldB- cells with wild-type cells does not cause the wild-type cells to behave like pldB- cells.     

Adrenal medullary cyclic nucleotide phosphodiesterase: lack of activation by the calcium-dependent regulator.

Calcium-dependent regulator, a calcium-binding protein isolated from brain and adrenal medulla, has been shown to activate a brain calcium-sensitive cyclic nucleotide phosphodiesterase. To determine if this protein has the same role in the adrenal medulla, the cyclic nucleotide phosphodiesterase of adrenal medulla was characterized. Neither crude nor partially purified adrenal medullary phosphodiesterase was inhibited by EGTA or stimulated by calcium and the calcium-dependent regulator, whereas similar brain preparations displayed sensitivity to these agents. As the calcium-dependent regulator does not appear to stimulate adrenal medullary cyclic nucleotide phosphodiesterase activity, alternate roles of this protein in adrenal medulla are suggested.     

Role of calcium in growth inhibition induced by a novel cell surface sialoglycopeptide

Our laboratory has purified an 18 kDa cell surface sialoglycopeptide growth inhibitor (CeReS-18) from intact bovine cerebral cortex cells. Evidence presented here demonstrates that sensitivity to CeReS-18-induced growth inhibition in BALB-c 3T3 cells is influenced by calcium, such that a decrease in the calcium concentration in the growth medium results in an increase in sensitivity to CeReS-18. Calcium did not alter CeReS-18 binding to its cell surface receptor and CeReS-18 does not bind calcium directly. Addition of calcium, but not magnesium, to CeReS-18-inhibited 3T3 cells resuts in reentry into the cell cycle. A greater than 3-hour exposure to increased calcium is required for escape from CeReS-18-induced growth inhibition. The calcium ionophore ionomycin could partially mimic the effect of increasing extracellular calcium, but thapsigargin was ineffective in inducing escape from growth inhibition. Increasing extracellular calcium 10-fold resulted in an approximately 7-fold increase in total cell-associated ⁴⁵Ca⁺², while free intracellular calcium only increased approximately 30%. However, addition of CeReS-18 did not affect total cell-associated calcium or the increase in total cell-associated calcium observed with an increase in extracellular calcium. Serum addition induced mobilization of intracellular calcium and influx across the plasma membrane in 3T3 cells, and pretreatment of 3T3 cells with CeReS-18 appeared to inhibit these calcium mobilization events. These results suggest that a calcium-sensitive step exists in the recovery from CeReS-18-induced growth inhibition. CeReS-18 may inhibit cell proliferation through a novel mechanism involving altering the intracellular calcium mobilization/regulation necessary for cell cycle progression. © 1995 Wiley-Liss, Inc.     

Role of cadherins 5 and 13 in the aortic endothelial barrier

We investigated the role of the cadherins 5 and 13 in the solute barrier formed by aortic endothelial cells in vitro. In confluent monolayers of bovine aortic endothelial cells, immunofluorescence with antibodies to the external domain of cadherin 5 (Mab 9H7) or to cadherin 13 (Mab Ec6C10) found staining for both cadherins at endothelial cell borders. Western blotting with an antibody to the characteristic cadherin cytoplasmic tail or with an antibody to the extracellular domain of cadherin 5 revealed a single 125 kD protein band. A second larger band was found at 130 kD with the anti-cadherin 13 Mab which was not recognized by an antibody to the cadherin cytoplasmic tail. A calcium switch strategy was used to investigate the involvement of these cadherins in the endothelial barrier. Changes in the permeability of small solutes in an endothelial cell column produced by a decrease in calcium concentration followed by a return to normal calcium, with or without antibody, were recorded. We found that anti-cadherin 5 IgG (10 μg/ml) interfered with the reforming of interendothelial junctions after restoration of calcium at every time point tested for a total of 45 min after restoration of calcium. The anti-cadherin 13 IgG (10 μg/ml) did not block reforming of the endothelial barrier in a similar manner. The presence of this antibody delayed only by 15 min the restoration of the normal barrier. Without calcium switch, addition of either monoclonal antibody (10 μg/ml) to the endothelial cell column had no effect on solute permeability. These results suggest that cadherin 5 in bovine aortic endothelial cells has a major functional role in forming the calcium-sensitive endothelial junction in vitro and may play an important role in the normal structure and function of the in vivo barrier. J. Cell. Physiol. 171:243–251, 1997. © 1997 Wiley-Liss, Inc.     

Calcium-sensitive cells of the pars intermedia and osmotic balance in the eel

Summary The structure of the PAS-positive calcium-sensitive (Ca-s) cells of the pars intermedia was investigated in eels kept in deionized water (DW) or fresh water (FW) supplemented with Ca²⁺ or Mg²⁺. Ca²⁺ (2mM) reduces considerably the response to DW; plasma osmolarity, Na⁺ and Ca²⁺ levels are not significantly affected. In eels adapted to DW for 21 or 28 days, showing highly stimulated Ca-s cells, an addition of CaCl₂ for 2 days inhibits the release of granules, but does not immediately block their synthesis and the mitotic activity. The nuclear area is reduced, osmolarity and plasma sodium increase, but the rise in calcium is not always significant. Magnesium, at a 10-fold greater concentration than in FW (2 mM), slightly inhibits the release of secretory granules without reducing other indicators of stimulation. In Ca-enriched FW, the Ca-s cells appear inactive. These data show that the PAS-positive cells in the pars intermedia of the eel are calcium-sensitive, similar to those of the goldfish; their role in calcium regulation is briefly discussed.     

Genetically encoded calcium indicators for studying long-term calcium dynamics during apoptosis

Intracellular calcium release is essential for regulating almost all cellular functions. Specific spatio-temporal patterns of cytosolic calcium elevations are critical determinants of cell fate in response to pro-apoptotic cellular stressors. As the apoptotic program can take hours or days, measurement of long-term calcium dynamics are essential for understanding the mechanistic role of calcium in apoptotic cell death. Due to the technical limitations of using calcium-sensitive dyes to measure cytosolic calcium little is known about long-term calcium dynamics in living cells after treatment with apoptosis-inducing drugs. Genetically encoded calcium indicators could potentially overcome some of the limitations of calcium-sensitive dyes. Here, we compared the performance of the genetically encoded calcium indicators GCaMP6s and GCaMP6f with the ratiometric dye Fura-2. GCaMP6s performed as well or better than Fura-2 in detecting agonist-induced calcium transients. We then examined the utility of GCaMP6s for continuously measuring apoptotic calcium release over the course of ten hours after treatment with staurosporine. We found that GCaMP6s was suitable for measuring apoptotic calcium release over long time courses and revealed significant heterogeneity in calcium release dynamics in individual cells challenged with staurosporine. Our results suggest GCaMP6s is an excellent indicator for monitoring long-term changes cytosolic calcium during apoptosis.     

Cholesterol influences voltage-gated calcium channels and BK-type potassium channels in auditory hair cells

The influence of membrane cholesterol content on a variety of ion channel conductances in numerous cell models has been shown, but studies exploring its role in auditory hair cell physiology are scarce. Recent evidence shows that cholesterol depletion affects outer hair cell electromotility and the voltage-gated potassium currents underlying tall hair cell development, but the effects of cholesterol on the major ionic currents governing auditory hair cell excitability are unknown. We investigated the effects of a cholesterol-depleting agent (methyl beta cyclodextrin, MβCD) on ion channels necessary for the early stages of sound processing. Large-conductance BK-type potassium channels underlie temporal processing and open in a voltage- and calcium-dependent manner. Voltage-gated calcium channels (VGCCs) are responsible for calcium-dependent exocytosis and synaptic transmission to the auditory nerve. Our results demonstrate that cholesterol depletion reduced peak steady-state calcium-sensitive (BK-type) potassium current by 50% in chick cochlear hair cells. In contrast, MβCD treatment increased peak inward calcium current (~30%), ruling out loss of calcium channel expression or function as a cause of reduced calcium-sensitive outward current. Changes in maximal conductance indicated a direct impact of cholesterol on channel number or unitary conductance. Immunoblotting following sucrose-gradient ultracentrifugation revealed BK expression in cholesterol-enriched microdomains. Both direct impacts of cholesterol on channel biophysics, as well as channel localization in the membrane, may contribute to the influence of cholesterol on hair cell physiology. Our results reveal a new role for cholesterol in the regulation of auditory calcium and calcium-activated potassium channels and add to the growing evidence that cholesterol is a key determinant in auditory physiology.     

Plasma membrane calcium ATPase proteins as novel regulators of signal transduction pathways

Emerging evidence suggests that plasma membrane calcium ATPases (PMCAs) play a key role as regulators of calcium-triggered signal transduction pathways via interaction with partner proteins. PMCAs regulate these pathways by targeting specific proteins to cellular sub-domains where the levels of intracellular free calcium are kept low by the calcium ejection properties of PMCAs. According to this model, PMCAs have been shown to interact functionally with the calcium-sensitive proteins neuronal nitric oxide synthase, calmodulin-dependent serine protein kinase, calcineurin and endothelial nitric oxidase synthase. Transgenic animals with altered expression of PMCAs are being used to evaluate the physiological significance of these interactions. To date, PMCA interactions with calcium-dependent partner proteins have been demonstrated to play a crucial role in the pathophysiology of the cardiovascular system via regulation of the nitric oxide and calcineurin/nuclear factor of activated T cells pathways. This new evidence suggests that PMCAs play a more sophisticated role than the mere ejection of calcium from the cells, by acting as modulators of signaling transduction pathways.     

Binding of calcium ions to an avian flight muscle troponin T

Numerous troponin T (TnT) isoforms are generated by alternative RNA splicing primarily in its N-terminal hypervariable region, but the functions of these isoforms are not completely understood. Here for the first time, we discovered that a chicken fast TnT isoform with a unique Tx motif (HEEAH)(n) binds calcium. The metal binding behavior of this TnT isoform was first investigated using terbium as a calcium analogue due to its more readily detectable fluorescence variation upon TnT binding. Both intact TnT and TnT N-terminal fragment (TnT N47) bound terbium with high affinity indicating that the N-terminal sequence was the site of binding. Since terbium often substitutes at calcium-binding sites, radioactive calcium was tested and found to bind both intact TnT and TnT N47. Fluorescence measurements using the calcium-sensitive fluorescent dye, calcium green 5N, confirmed that calcium bound to the tertiary complex of TnT and the tropomyosin dimer with a fast on-rate (10(6)-10(7) M(-1) s(-1)) as detected in stopped-flow analysis. Consistent with these observations, computational predictions suggest that TnT N47 might fold into an elongated structure with at least one high-affinity metal ion binding pocket comprised primarily of the Tx motif sequence and several lower affinity binding sites. These results suggest that TnT may play a role in modulating the calcium-mediated regulatory process of striated muscle contraction.