Local accumulation of membrane-associated calcium according to cell pattern formation inMicrasterias denticulata,visualized by chlorotetracycline fluorescence

Summary Developing cells ofMicrasterias denticulata Bréb. show a characteristic fluorescence of the plasma membrane (or cortical protoplasm) after treatment with chlorotetracycline (CTC), which is known to be an indicator for membrane-bound Ca²⁺. Depending on the stage of development the fluorescing sites of the young half cell are distributed in a specific pattern which corresponds to cell pattern formation. Therefore growth and thus cytomorphogenesis inMicrasterias seem to be mediated by a patterned accumulation of Ca²⁺ at the periphery of the differentiating cell. Participation of Ca²⁺ in a membrane-recognition process responsible for local vesicle incorporation is discussed.     

Ca2+-requirement for a blue-light-induced chloroplast translocation inEremosphaera viridis

Summary The light-mediated translocation of chloroplasts inEremosphaera viridis is dependent on blue light near 450 nm, while wavelengths longer than 500 nm are inactive. The plastid translocation results in an aggregation of the organelles close to the nucleus in the center of the cell. After cessation of irradiation, the cells begin to redistribute their plastids in the cytoplasm immediately. Treatments that alter the Ca²⁺ concentration in the cytoplasm ofEremosphaera suggest that the translocation is regulated by calcium. Ultrastructural investigation ofEremosphaera reveals a very characteristic, multilayered and highly-ordered cell wall.     

TRPV1 expression and activity during retinoic acid-induced neuronal differentiation

The transient receptor potential vanilloid subtype 1 (TRPV1) is a Ca²⁺-permeable channel primarily expressed in dorsal root ganglion neurons. Besides its function in thermogenic nociception and neurogenic inflammation, TRPV1 is involved in cell migration, cytoskeleton re-organisation and in neuronal guidance. To explore the TRPV1 level and activity during conditions for neuronal maturation, TRPV1-expressing SHSY5Y neuroblastoma cells were differentiated into a neuronal phenotype using all-trans-retinoic acid (RA). We show that RA highly up-regulated the total and cell surface TRPV1 protein expression but the TRPV1 mRNA level was unaffected. The up-regulated receptors were localised to the cell bodies and the developed neurites. Furthermore, RA increased both the basal intracellular free Ca²⁺ concentration by 30% as well as the relative capsaicin-induced Ca²⁺ influx. The results show that TRPV1 protein expression increases during RA-induced differentiation in vitro, which generates an altered intracellular Ca²⁺ homeostasis.     

Simultaneous recording of cytosolic Ca2+ levels inDidinium andParamecium during aDidinium attack onParamecium

Summary The carnivorous ciliateDidinium nasutum captures prey such asParamecium by discharging extrusomes, known as toxicysts, while the attackedParamecium defensively discharges trichocysts. Several authors have suggested that both discharges, the toxicysts ofDidinium and the trichocysts ofParamecium, are evoked by the rise in cytosolic Ca²⁺ level in each cell. However, these putative increases in cytosolic Ca²⁺ levels have not as yet been recorded simultaneously in these cells during aDidinium attack onParamecium. We injected the fluorescent Ca²⁺ indicator Ca-Green 1 dextran into bothDidinium andParamecium, and simultaneously observed the cytosolic Ca²⁺ levels in these cells asDidinium attackedParamecium. When aParamecium came into contact with theDidinium proboscis, theDidinium showed a significant rise in cytosolic Ca²⁺ in the basal portion of the proboscis. One video frame (33 ms) after the onset of the Ca²⁺ rise inDidinium, theParamecium also showed an increase in cytosolic Ca²⁺. This is the first simultaneous recording of changes in the Ca²⁺ level during a predator-prey interaction in ciliates. The possible roles of these Ca²⁺ increases are discussed in relation to the discharge of toxicysts during theDidinium attack and of trichocysts as a defensive behavior ofParamecium.Abbreviations AED aminoethyldextran - P_i inorganic phosphate - FITC fluorescein isothiocyanate     

Electrophysiological evidence suggests a defective Ca2+ control mechanism in a newParamecium mutant

Summary A new mutant ofParamecium tetraurelia, k-shyA, was characterized behaviorally and electrophysiologically. The mutant cell exhibited prolonged backward swimming episodes in response to depolarizing conditions. Electrophysiological comparison of k-shyA with wild type cells under voltage clamp revealed that the properties of three Ca²⁺-regulated currents were altered in the mutant. (i) The voltage-dependent Ca²⁺ current recovered from Ca²⁺-dependent inactivation two- to 10-fold more slowly than wild type. Ca²⁺ current amplitudes were also reduced in the mutant, but could be restored by EGTA injection. (ii) The decay of the Ca²⁺-dependent K⁺ tail current was slower in the mutant. (iii) The decay of the Ca²⁺-dependent Na⁺ tail current was also slower in the mutant. All other membrane properties studied, including the resting membrane potential and resistance and the voltage-sensitive K⁺ currents, were normal in k-shyA. Considered together, these observations are consistent with a defect in the ability of k-shyA to reduce the free intracellular Ca²⁺ concentration following stimulation. The possible targets of the genetic lesion and alternative explanations are discussed. The k-shy mutants may provide a useful tool for molecular and physiological analyses of the regulation of Ca²⁺ metabolism inParamecium.     

Ca2+ reversibly inhibits active rotation of chloroplasts in isolated cytoplasmic droplets ofChara

Summary The effects of Ca²⁺ and other cations on chloroplast rotation in isolated cytoplasmic droplets ofChara were investigated by iontophoretically injecting them. Chloroplast rotation stopped immediately after Ca²⁺ injection and recovered with time, suggesting the existence of a Ca²⁺-sequestering system in the cytoplasm. The Ca²⁺ concentration necessary for the stoppage was estimated to be >10^–4M. Sr²⁺ had the same effect as Ca²⁺. Mn²⁺ and Cd²⁺ induced a gradual decrease in the rotation rate with low reversibility. K⁺ and Mg²⁺ had no effects. Ba²⁺ had effects sometimes similar to Ca²⁺ or Sr²⁺ and sometimes similar to Mn²⁺ or Cd²⁺.Reversible inhibition by Ca²⁺, together with its specificity, strongly supports the hypothesis that a transient increase in the Ca²⁺ concentration in the cytoplasm upon membrane excitation directly stops the cytoplasmic streaming inCharaceae internodes (Hayama et al. 1979).     

Development of spatio-temporal pattern of Ca2+ on the chemotactic behaviour ofPhysarum plasmodium

Summary The development of a spatio-temporal pattern of Ca²⁺ concentration by a plasmodium ofPhysarum polycephalum during chemotaxis was studied using fura-2. Whenever the cell displayed coordinated migration in one direction as a whole body, a spatiotemporal pattern was established with a characteristic feature along the longitudinal axis. Calcium concentration oscillated with a period of a few minutes within the cell; the mean concentration at the front was higher than that at the rear. When the cell was given an attractant only at the rear end, the mean concentration rose at the site of application with an immediate increase in the frequency of oscillation. First, the change of the frequency is propagated toward the other end and then the mean level of the Ca²⁺ concentration at the non-stimulated site decreases. As a result, the Ca²⁺ gradient is reversed along the cell, which then begins to migrate in a coordinated manner in the reverse direction. This study showed that the spatiotemporal pattern of Ca²⁺ concentration is closely related to information processing for coordinated migration in chemotaxis. The role of the pattern in that process is discussed.     

Effects of microtubule-inhibitors on nuclear migration and rhizoid differentiation in germinating fern spores (Onoclea sensibilis)

Summary Germinating spores of the sensitive fern,Onoclea sensibilis L., undergo premitotic nuclear migration before a highly asymmetric cell division partitions each spore into a large protonemal cell and a small rhizoid initial. Nuclear movement and subsequent rhizoid formation were inhibited by the microtubule (MT) inhibitors, colchicine, isopropyl-N-3-chlorophenyl carbamate (CIPC) and griseofulvin. Colchicine prevented polar nuclear movement and cell division so that spores developed into enlarged, uninucleate single cells. CIPC and griseofulvin prevented nuclear migration, but not cell division, so that spores divided into daughter cells of approximately equal size. In colchicine-treated spores, MT were not observed at any time during germination. CIPC prevented MT formation at a time coincident with nuclear movement in the control and caused a disorientation of the spindle MT. Both colchicine and CIPC appeared to act at a time prior to the onset of normal nuclear movement. The effects of colchicine were reversible but those of CIPC were not. Cytochalasin b had no effect upon nuclear movement or rhizoid differentiation. These results suggests that MT mediate nuclear movement and that a highly asymmetric cell division is essential for rhizoid differentiation.     

Proteolytic activation of a hyperpolarization- and calcium-dependent potassium channel inParamecium

Summary The effects of proteolysis on a hyperpolarization- and Ca²⁺-dependent K channel from the surface membrane ofParamecium tetraurelia were examined in the inside-out excised patch mode. Treatment with trypsin, pronase or thermolysin removed the Ca²⁺-dependence of the channel activation, yielding an increase in channel activity greater than 2.5-fold at all Ca²⁺ concentrations between 10^–4 and 10^–8 m. Thermolysin addition-ally removed the voltage dependence of channel opening and gave the most activation among the three proteases tested. Proteolysis did not affect the single-channel conductance. In an analogy to the mechanism of activation of many Ca²⁺-dependent enzymes it is suggested that thisParamecium channel has a cytoplasmic inhibitory domain which can be removed by proteolysis, and that the physiological activation by Ca²⁺ is due to a temporary removal of this inhibition. Moreover, these findings indicate structural differences between depolarization-, Ca²⁺-dependent K channels (BK channels) and the hyperpolarization-, Ca²⁺-dependent K channels inParamecium.     

Rapid plant movements triggered by action potentials

Rapid bendings of the pulvinus inMimosa pudica, of the trap lobes inDionaea muscipula andAldrovanda vesiculosa, and of the tentacle in Drosera are triggered by action potentials in their motor cells. The action potential ofMimosa may be a C1⁻-spike, and that ofDionaea andAldrovanda may be a Ca²⁺-spike. Propagation of action potentials in the petiole or motor organ is thought to be electrotonie, cell-to-cell, transmission. The Ca 2+ release from unidentified organelles in the pulvinus or the Ca²⁺ influx of the cells in the trap with the action current and activation of contractile fibrillar network having ATPase activity in the cytoplasm must be involved in the rapid bending. Contractions of fibrils may open pores in the membrane of the motor cells upon activation. Outward bulk flow of the vacuolar sap through these pores, due to the pressure inside the cell, must result in turgor loss of the motor cells and then the bending of the organ.     

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.     

Localization of intracellular calcium in root meristematic cells and root cap cells of maize

The localization of Ca²⁺ in cells of the periblem and dermatogen in the root meristem and the columella and peripheral cells of the root cap of maize was examined by the precipitation method of potassium pyroantimonate and EGTA-treatment. In periblem and dermatogen cells, Ca²⁺ was found to be localized in the nucleoplasm and granular zone of the nucleolus. Ca²⁺ was also found in most cell organelles: in the matrix in mitochondria, on the thylakoid membrane in proplastids, in the vacuoles and on the plasma membranes. Ca²⁺ was also distributed throughout the cytoplasmic ground matrix. Much Ca²⁺ was present in the cell wall soon after its formation during the cell division. Ca²⁺ was also conspicuous in the vesicles of Golgi in the dermatogen cells. In columella and peripheral cells, there was less Ca²⁺ in the organelles and cytoplasmic ground matrix, but Ca²⁺ was present in Golgi vesicles in the peripheral cells. Electron microscopic and X-ray microanalysis showed that Ca²⁺ was also present in the mucilaginous layer, the outermost cell wall of the peripheral cells.     

Composition of the cell wall of the phage resistant mutantRhodopseudomonas capsulata St. Louis RC1-

A mutant ofRhodopseudomonas capsulata St. Louis (R. capsulata St. Louis RC1^-), resistant against the bacteriophage RC1, was isolated and its cytoplasmic membrane and cell wall fractions (buoyant densities on sucrose density gradient centrifugation: 1.123 and 1.222 g/cm³, respectively) were obtained. Different from the wild type strain, the cell wall fraction of the mutant lacked galactose. Galactose is a characteristic component of the capsule polysaccharide ofR. capsulata St. Louis. There were no differences in lipopolysaccharide and peptidoglycan compositions as well as in polypeptide patterns of the cell wall fractions between mutant and wild-type cells. Thus, the lack of a firmly bound capsule inR. capsulata St. Louis RC1^- was the only difference found.     

The effects of CA2+ during the elicitation of shikonin derivatives inOnosma paniculatum cells

Summary A fungal elicitor extracted fromAspergillus oryzae (Ahlb.) Cobn mycelia promoted the production of shikonin derivatives inOnosma paniculatum Bur et Franch cell suspension cultures. Elicitor treatment also increased Ca²⁺ concentration in RM₉ medium, which could be measured earlier than the elicited increase of shikonin formation. Several reagents known to induce Ca²⁺-influx and increase the intracellular-free Ca²⁺ level, such as the addition of Ca (NO₃)₂·4H₂O, the Ca²⁺ ionophore A₂₃₁₈₇, and abscisic acid (ABA), appreciably suppressed the elicitor-promoted shikonin formation inOnosma cells. In contrast, the decrease of intracellular-free Ca²⁺ level by the specific Ca²⁺-chelator ethylene glycol bis (β-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) or the Ca²⁺—channel blocker, verapamil, enhanced the biosynthesis of shikonin even in the absence of elicitor. Treatment of cells with trifluoperazine (TFP) also stimulated shikonin formation inOnosma cell cultures. A rapid and transient drop of free Ca²⁺ level in one protoplast was directly determined after the addition of elicitor toOnosma cell cultures. The inhibitory effect on shikonin formation by ABA was largely on account of its ability to restore the intracellular Ca²⁺ level lowered by the elicitor. These results suggest that Ca²⁺ play a significant role in an early stage of the elicitation process ofOnosma cells. The rapid drop of cytoplasmic Ca²⁺ carries the elicitor signal and in turn regulates the biosynthesis of shikonin derivatives.     

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.     

Calcineurin Subunit B Is Required for Normal Vegetative Growth inNeurospora crassa

A series ofNeurospora crassamutants affected in the ability to regulate entry into conidiation (an asexual developmental program) were isolated by using an insertional mutagenesis procedure followed by a screening protocol. One of the mutants isolated by this approach consisted entirely of cells with an abnormal morphology. The mutant produces chains of swollen septated cells. The developmentally regulatedccg-1gene is constitutively expressed in these cells, suggesting that they have entered the conidial developmental program. The insertionally disrupted genecnb-1was isolated by plasmid rescue and found to encode calcineurin B, the regulatory subunit of the Ca²⁺and calmodulin-dependent protein phosphatase calcineurin. The data demonstrate that calcineurin B is required for normal vegetative growth inN. crassaand suggest that thecnb-1mutant is unable to repress entry into the asexual developmental program. The results suggest that Ca²⁺may play an important role in regulating fungal morphology.     

Wall appositions induced by ionophore A 23187, CaCl2, LaCl3, and nifedipine in characean cells

Summary The formation of wall appositions (plugs) by ionophore A 23187, CaCl₂, LaCl₃, and nifedipine was studied in mature internodal cells of characeaen algae. CaCl₂ at concentrations above 10^–2M induces thick fibrillar plugs without callose inNitella flexilis. InChara corallina andNitella flexilis ionophore A 23187 (1.25×10^–5 to 5×10^–5M) and LaCl₃ (7.5×10^–5 to 2.5×10^–4M) cause flat appositions which contain callose and have a more granular structure. Plug formation by ionophore A 23187, CaCl₂, and LaCl₃ is pH-dependent and occurs beneath the alkaline regions of the cell. Nifedipine (10^–4 to 10^–5M) induces plugs inNitella flexilis after previous injury. These callose-containing wall appositions consist of a heterogeneous granular core which is covered by a fibrillar layer. The results of this work are compared with previous studies on wound wall formation and chlortetracycline (CTC)-induced plug formation which reveal that abundant coated vesicles occur only when a thick fibrillar wall layer is formed. Neither LaCl₃ nor nifedipine inhibit the formation of CaCl₂- or CTC-plugs. The unusual effects of these substances, which normally act as Ca²⁺ antagonists and therefore should prevent and not induce plug formation, are discussed. It is suggested that La³⁺ mimicks the effects of calcium and that nifedipine binding to the Ca²⁺ channels is altered in the alkaline regions of characean internodes and allows an influx of Ca²⁺.Abbreviations AFW artificial fresh water - CTC chlortetracycline - DCMU dichlorphenyldimethylurea - DMSO dimethylsulfoxide - EGTA ethyleneglycoltetraacetic acid - MES 2-(N-morpholino) ethanesulfonic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - TAPS N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid     

<Emphasis Type="Italic">Gemini pollen 2</Emphasis>, a male and female gametophytic cytokinesis defective mutation

Gametophytic cytokinesis is essential for the development and function of the male and female gametophytes. We have previously described the isolation and characterisation of gemini pollen 1 (gem1) that acts gametophytically to disturb asymmetric division and cytokinesis at pollen mitosis I (PMI) in Arabidopsis. Here we describe the genetic and cytological analysis of an independent gametophytic mutant, gem2, with similar characteristics to gem1, but which maps to a different genetic locus. gem2 shows reduced genetic transmission through both male and female gametes and leads to the production of divided or twin-celled pollen. Developmental analysis revealed that gem2 does not affect karyokinesis at PMI, but leads to repositioning of the cell plate, and partial or complete failure of cytokinesis, resulting in symmetrical divisions or binucleate pollen grains, respectively. Symmetrical divisions lead to altered pollen cell fate with both sister cells displaying vegetative cell fate. Moreover, we demonstrate that the predominant female defect in gem2 is a lack of cellularisation of the embryo sac during megagametogenesis. GEM2 therefore defines an independent genetic locus that is involved in the correct specification of both male and female gametophytic cytokinesis.     

Polar localization of plasma membrane Ca2+/Mg2+ ATPase correlates with the pattern of steady ionic currents in eggs ofLymnaea stagnalis andBithynia tentaculata (Mollusca)

Summary During extrusion of the first polar body in eggs ofLymnaea stagnalis andBithynia tentaculata a localized Ca²⁺ /Mg²⁺ ATPase activity was detected, using Ando's enzyme-cytochemical method for electron microscopy [Ando et al. (1981) Acta Histochem Cytochem 14:705–726]. The enzyme activity was distributed in a polar fashion, along the cytoplasmic face of the plasma membrane. In the eggs ofLymnaea it was found only in the vegetal hemisphere, whereas inBithynia eggs it was localized both in the vegetal hemisphere and at the animal pole. This pattern of enzyme activity corresponds to the polar pattern of transcellular ionic currents measured with the vibrating probe, which we showed to be partially carried or regulated by calcium [Zivkovic and Dohmen (1989) Biol Bull (Woods Hole) 176 (Suppl):103–109]. The characteristics of the ATPase were studied using a variety of approaches such as ion and substrate depletions and substitutions, addition of specific inhibitors of ATPase activity, treatment with EDTA/EGTA and electron energy-loss spectrometry. The results indicate that, inLymnaea, there are at least two enzymatic entities. The first one is a Ca²⁺ /Mg²⁺ ATPase localized along the membrane and in the cortex of the vegetal hemisphere. The second one is a Ca²⁺-stimulated ATPase (calcium pump of the plasma membrane) localized in a small region of the membrane at the vegetal pole. We speculate that in the eggs ofLymnaea andBithynia a functional relationship exists between the plasma-membrane-associated ATPase activity and the transcellular ionic currents measured in the same region.