Peptide chain elongation: A possible role of montmorillonite in prebiotic synthesis of protein precursors

Several studies have proven the ability of montmorillonite to catalyse amino acid condensation under assumed prebiotic conditions, simulating wetting-drying cycles. In this work, the oligomerization of short peptides gly₂, gly₃, gly₄ and ala₂ on Ca-and Cu-montmorillonite in drying-wetting cycles at 80 °C was studied. The catalytic effect of montmorillonite was found to be much higher than in the case of glycine oligomerization. From gly₂ after 3 weeks, 10% oligomers (up to gly6, with gly₃ as main products) are formed. Gly₃ and gly₄ give higher oligomers even after 1 cycle. Ala₂ produces both ala₃ and ala₄, whereas ala does not produce any oligomers under these conditions. Heteroologomerization was observed: ala-gly-gly is formed from ala and gly₂. Much higher yields are obtained using Ca-montmorillonite, because copper (II) oxidizes organic molecules. The influence of the reaction mechanism on the preferential oligomerization of oligopeptides is discussed.     

The effect of smectite composition on the catalysis of peptide bond formation

Clay-catalyzed glycine and diglycine oligomerizations were performed as drying/wetting cycles at 80°C. Two trioctahedral smectites (hectorite and saponite), three pure montmorillonites, a ferruginous smectite, an Fe(II)-rich smectite, and three smectites containing goethite admixture were used as catalysts. Highest peptide bond formation was found with trioctahedral smectites. About 7% of glycine was converted to diglycine and diketopiperazine on hectorite after 7 days. In the case of dioctahedral smectites, highest yields were achieved using clays with a negative-layer charge localized in the octahedral sheets (up to 2% of converted glycine after 7 days). The presence of Fe(II) in clay is reflected in a higher efficiency in catalyzing amino acid dimerization (about 3.5% of converted glycine after 7 days). The possible significance of the results for prebiotic chemistry is discussed. Received: 20 February 1996 / Accepted: 26 April 1996     

Relationships Between Genomic G+C Content,RNA Secondary Structures,and Optimal Growth Temperature in Prokaryotes

G:C pairs are more stable than A:T pairs because they have an additional hydrogen bond. This has led to many studies on the correlation between the guanine+cytosine (G+C) content of nucleic acids and temperature over the last 20 years. We collected the optimal growth temperatures (T_opt) and the G+C contents of genomic DNA; 23S, 16S, and 5S ribosomal RNAs; and transfer RNAs for 764 prokaryotic species. No correlation was found between genomic G+C content and T_opt, but there were striking correlations between the G+C content of ribosomal and transfer RNA stems and T_opt. Two explanations have been proposed—neutral evolution and selection pressure—for the approximate equalities of G and C (respectively, A and T) contents within each strand of DNA molecules. Our results do not support the notion that selection pressure induces complementary oligonucleotides in close proximity and therefore numerous secondary structures in prokaryotic DNA, as the genomic G+C content does not behave in the same way as that of folded RNA with respect to optimal growth temperature. Received: 25 September 1996 / Accepted: 21 January 1997     

Photofrin II Sensitized Modifications of Ion Transport Across the Plasma Membrane of an Epithelial Cell Line: II. Analysis at the Level of Membrane Patches

In the first part of this study, photofrin II sensitized membrane modifications of OK-cells were investigated at the level of macroscopic membrane currents. In this second part, the inside-out configuration of the patch-clamp technique is applied to analyze the phenomena at the microscopic level. It is shown that the characteristic single channel fluctuations of the electric current disappear after the start of illumination of membrane patches in the presence of photofrin II. This holds for all three types of ion channels investigated: the large-conductance Ca²⁺-dependent K⁺ channel (maxi-K_Ca), a K⁺ channel of small conductance (sK), and a stretch-activated nonselective cation channel (SA-cat). Part of the experiments show a transient activation of the channels (indicated by an increase of the probability in the open-channel state) before the channels are converted into a closed nonconductive state. Inactivation of all three channel types proceeds by a continuous reduction of their open probability, while the single channel conductance values are not affected. The process of photodynamically induced channel inactivation is followed by a pronounced increase of the leak conductance of the plasma membrane. The latter process — after light-induced initiation — is found to continue in the dark. The ionic pathways underlying the leak conductance also allow permeation of Ca²⁺ ions. The resulting Ca²⁺-flux may contribute to the photodynamically induced increase of the intracellular Ca²⁺ concentration observed in various cell lines. Received: 26 May 1998/Revised: 8 September 1998     

Evidence for Negative Charge in the Conduction Pathway of the Cardiac Ryanodine Receptor Channel Provided by the Interaction of K+ Channel N-type Inactivation Peptides

We have investigated the interaction of two peptides (ShB — net charge +3 and ShB:E12KD13K — net charge +7) derived from the NH₂-terminal domain of the Shaker K⁺ channel with purified, ryanodine-modified, cardiac Ca²⁺-release channels (RyR). Both peptides produced well resolved blocking events from the cytosolic face of the channel. At a holding potential of +60 mV the relationship between the probability of block and peptide concentration was described by a single-site binding scheme with 50% saturation occurring at 5.92 ± 1.06 μm for ShB and 0.59 ± 0.14 nm for ShB:E12KD13K. The association rates of both peptides varied with concentration (4.0 ± 0.4 sec⁻¹μm ⁻¹ for ShB and 2000 ± 200 sec⁻¹μm ⁻¹ for ShB:E12KD13K); dissociation rates were independent of concentration. The interaction of both peptides was influenced by applied potential with the bulk of the voltage-dependence residing in K_off. The effectiveness of the inactivation peptides as blockers of RyR is enhanced by an increase in net positive charge. As is the case with inactivation and block of K⁺ channels, this is mediated by a large increase in K_on. These observations are consistent with the proposal that the conduction pathway of RyR contains negatively charged sites which will contribute to the ion handling properties of this channel. Received: 15 December 1997/Revised: 13 March 1998     

Native Kv1.3 Channels are Upregulated by Protein Kinase C

The voltage-gated potassium channel, Kv1.3, which is highly expressed in a number of immune cells, contains concensus sites for phosphorylation by protein kinase C (PKC). In lymphocytes, this channel is involved in proliferation—through effects on membrane potential, Ca²⁺ signalling, and interleukin-2 secretion—and in cytotoxic killing and volume regulation. Because PKC activation (as well as increased intracellular Ca²⁺) is required for T-cell proliferation, we have studied the regulation of Kv1.3 current by PKC in normal (nontransformed) human T lymphocytes. Adding intracellular ATP to support phosphorylation, shifted the voltage dependence of activation by +8 mV and inactivation by +17 mV, resulting in a 230% increase in the window current. Inhibiting ATP production and action with ``death brew' (2-deoxyglucose, adenylylimidodiphosphate, carbonyl cyanide-m-chlorophenyl hydrazone) reduced the K⁺ conductance (G _K ) by 41 ± 2%. PKC activation by 4β-phorbol 12,13-dibutyrate, increased G _K by 69 ± 6%, and caused a positive shift in activation (+9 mV) and inactivation (+9 mV), which resulted in a 270% increase in window current. Conversely, several PKC inhibitors reduced the current. Diffusion into the cell of inhibitory pseudosubstrate or substrate peptides reduced G _K by 43 ± 5% and 38 ± 8%, respectively. The specific PKC inhibitor, calphostin C, potently inhibited Kv1.3 current in a dose- and light-dependent manner (IC₅₀∼ 250 nm). We conclude that phosphorylation by PKC upregulates Kv1.3 channel activity in human lymphocytes and, as a result of shifts in voltage dependence, this enhancement is especially prevalent at physiologically relevant membrane potentials. This increased Kv1.3 current may help maintain a negative membrane potential and a high driving force for Ca²⁺ entry in the presence of activating stimuli. Received: 12 July 1996/Revised: 21 October 1996     

Kinetics of Chloride-Bicarbonate Exchange Across the Human Red Blood Cell Membrane

We had previously shown that an influx of extracellular Ca²⁺ (Ca²⁺ _e ), though it occurs, is not strictly required for aminoethyldextran (AED)-triggered exocytotic membrane fusion in Paramecium. We now analyze, by quenched-flow/freeze-fracture, to what extent Ca²⁺ _e contributes to exocytotic and exocytosis-coupled endocytotic membrane fusion, as well as to detachment of ``ghosts' — a process difficult to analyze by any other method or in any other system. Maximal exocytotic membrane fusion (analyzed within 80 msec) occurs readily in the presence of [Ca<sup>2+</sup>] <sub>e</sub> ≥ 5 × 10<sup>−6</sup> m, while normally a [Ca<sup>2+</sup>] <sub>e</sub> = 0.5 mm is in the medium. A new finding is that exocytosis and endocytosis is significantly stimulated by increasing [Ca<sup>2+</sup>] <sub>e</sub> even beyond levels usually available to cells. Quenching of [Ca<sup>2+</sup>] <sub>e</sub> by EGTA application to levels of resting [Ca<sup>2+</sup>] <sub>i</sub> or slightly below does reduce (by ∼50%) but not block AED-triggered exocytosis (again tested with 80 msec AED application). This effect can be overridden either by increasing stimulation time or by readdition of an excess of Ca<sup>2+</sup> <sub>e</sub> . Our data are compatible with the assumption that normally exocytotic membrane fusion will include a step of rapid Ca<sup>2+</sup>-mobilization from subplasmalemmal pools (``alveolar sacs') and, as a superimposed step, a Ca²⁺-influx, since exocytotic membrane fusion can occur at [Ca²⁺] _e even slightly below resting [Ca²⁺] _i . The other important conclusion is that increasing [Ca²⁺] _e facilitates exocytotic and endocytotic membrane fusion, i.e., membrane resealing. In addition, we show for the first time that increasing [Ca²⁺] _e also drives detachment of ``ghosts' — a novel aspect not analyzed so far in any other system. According to our pilot calculations, a flush of Ca²⁺, orders of magnitude larger than stationary values assumed to drive membrane dynamics, from internal and external sources, drives the different steps of the exo-endocytosis cycle. Received: 27 September 1996/Revised: 11 February 1997     

Activated alumina as an energy source for peptide bond formation: Consequences for mineral-mediated prebiotic processes

Summary. The catalytic properties of various forms of alumina were tested for alanine dimerization reaction. The catalytic efficiency of alumina depends on the structure, as well as on acid/base properties of the catalyst. The highest yields of Ala₂ were achieved on activated alumina with surface of neutral pH (about 3% conversion after 2 weeks). Thermal analysis of Ala + alumina reaction systems shows that the thermal behavior of amino acid changes substantially in contact with the activated surface of the alumina catalyst. The reaction of Ala is detected as being strongly endothermic by differential thermal analysis of pure amino acid (above 250°C). The alanine endothermic reaction is shifted substantially to lower values (below 200°C) and hardly detectable if activated alumina is present. The reaction mechanism of amino acid activation on alumina surface and its significance for mineral-catalyzed prebiotic peptide bond formation are discussed. Received June 6, 2000 Accepted July 27, 2000     

Alkaline pH and Internal Calcium Increase Na+ and K+ Effluxes in LK Sheep Red Blood Cells in Cl−-free Solutions

We examined the effects of pH, internal ionized Ca (Ca²⁺ _i ), cellular ATP, external divalent cations and quinine on Cl-independent ouabain-resistant K⁺ efflux in volume-clamped sheep red blood cells (SRBCs) of normal high (HK) and low (LK) intracellular K⁺ phenotypes. In LK SRBCs the K⁺ efflux was higher at pH 9.0 (350%) than at pHs 7.4 and 6.5, and was inhibited by external divalent cations, quinine, and cellular ATP depletion. The above findings suggest that the increased K⁺ efflux at alkaline pH is due to the opening of ion channels or specific transporters in the cell membrane. In addition, K⁺ efflux was activated (100%) when Ca²⁺ _i was increased (+A23187, +Ca²⁺ _o ) into the μm range. However, in comparison to human red blood cells, the Ca²⁺ _i -induced increase in K⁺ efflux in LK SRBCs was fourfold smaller and insensitive to quinine and charybdotoxin. The Na⁺ efflux was also higher at pH 9.0 than at pH 7.4, and activated (about 40%) by increasing Ca²⁺ _i . In contrast, in HK SRBCs the K⁺ efflux at pH 9.0 was neither inhibited by quinine nor activated by Ca²⁺ _i . These studies suggest the presence in LK SRBCs, of at least two pathways for Cl⁻-independent K⁺ and Na⁺ transport, of which one is unmasked by alkalinization, and the other by a rise in Ca²⁺ _i . Received: 23 May 1996/Revised: 6 December 1996     

Potentiation by Isoproterenol on Carbachol-induced K+ and Cl− Currents and Fluid Secretion in Rat Parotid

Isoproterenol (IPR) and 8-(4-chlorophenylthio)-cyclic AMP (cpt-cAMP) enhanced carbachol (CCh)-induced fluid secretion from rat parotid glands, but had no effect by themselves. The enhancement by IPR was blocked by propranolol. In dispersed parotid acinar cells, IPR and cpt-cAMP potentiated CCh-induced K⁺ and Cl⁻ currents (I _K and I _Cl). IPR at the concentration of 0.1 μm significantly potentiated the CCh-induced increase in intracellular Ca²⁺ concentration ([Ca²⁺] _i ), but 1 mm cpt-cAMP did not. The incidence of the potentiation by IPR in CCh-induced Mn²⁺ entry was 31% and that by cpt-cAMP was 21%. The potentiation by IPR in the ionic currents and the [Ca²⁺] _i was suppressed by propranolol. These results suggest that the CCh-induced fluid secretion from rat parotid glands is enhanced by IPR through the potentiation of I _K and I _Cl mainly by the increased cyclic AMP level and partially by the potentiated Ca²⁺ influx and [Ca²⁺] _i increase, and that IPR is more effective than cpt-cAMP in the enhancement of the CCh-induced [Ca²⁺] _i increase. Received: 6 October 1997/Revised: 16 April 1998     

A Long-Term Blockade of L-Type Calcium Currents Upregulates the Number of Ca2+ Channels in Skeletal Muscle

The effects of a long-term blockade of L-type Ca²⁺ channels on membrane currents and on the number of dihydropyridine binding sites were investigated in skeletal muscle fibers. Ca²⁺ currents (I _Ca) and intramembrane charge movement were monitored using a voltage-clamp technique. The peak amplitude of I _Ca increased by more than 40% in fibers that were previously incubated for 24 hr in solutions containing the organic Ca²⁺ channel blocker nifedipine or in Ca²⁺-free conditions. A similar incubation period with Cd²⁺, an inorganic blocker, produced a moderate increase of 20% in peak I _Ca. The maximum mobilized charge (Q _max) increased by 50% in fibers preincubated in Ca²⁺-free solutions or in the presence of Cd²⁺. Microsomal preparations from frog skeletal muscle were isolated by differential centrifugation. Preincubation with Cd²⁺ prior to the isolation of the microsomal fraction doubled the number of ³H-PN200-110 binding sites and produced a similar increase in the values of the dissociation constant. The increase in the number of binding sites is consistent with the increase in the peak amplitude of I _Ca as well as with the increase in Q _max. Received: 31 August 1998/Revised: 7 December 1998     

Calciseptine, a Ca2+ Channel Blocker, Has Agonist Actions on L-type Ca2+ Currents of Frog and Mammalian Skeletal Muscle

Calciseptine is a natural peptide consisting of 60 amino acids with four disulfide bonds. The peptide is a natural L-type Ca²⁺-channel blocker in heart and other systems, but its actions in skeletal muscle have not been previously described. The aim of this study is to characterize the effects of calciseptine on L-type Ca²⁺ channels of skeletal muscle and on contraction. Whole-cell, patch-clamp experiments were performed to record Ca²⁺ currents (I _Ca) from mouse myotubes, whereas Vaseline-gap voltage-clamp experiments were carried out to record I _Ca from frog skeletal muscle fibers. We found that calciseptine acts as a channel agonist in skeletal muscle, increasing peak I _Ca by 37% and 49% in these two preparations. Likewise, the peptide increased intramembrane charge movement, though it had little effect on contraction. The molecular analysis of the peptide indicated the presence of a local, electrostatic potential that resembles that of the 1,4-dihydropyridine agonist Bay K 8644. These observations suggest that calciseptine shares the properties of 1,4-dihydropyridine derivatives in modulating the permeation of divalent cations through L-type channels. Received: 18 December 2000/Revised: 16 July 2001     

Compositional Correlations in the Chicken Genome

This paper analyses the compositional correlations that hold in the chicken genome. Significant linear correlations were found among the regions studied—coding sequences (and their first, second, and third codon positions), flanking regions (5′ and 3′), and introns—as is the case in the human genome. We found that these compositional correlations are not limited to global GC levels but even extend to individual bases. Furthermore, an analysis of 1037 coding sequences has confirmed a correlation among GC₃, GC₂, and GC₁. The implications of these results are discussed. Received: 9 December 1998 / Accepted: 18 April 1999     

Inositol 1,4,5-Trisphosphate But Not Ryanodine-Receptor Agonists Induces Calcium Release from Rat Liver Golgi Apparatus Membrane Vesicles

We investigated the direct effect of inositol 1,4,5-trisphosphate (IP₃) and ryanodine receptor agonists on Ca²⁺ release from vesicles of a rat liver Golgi apparatus (GA) enriched fraction, which were actively loaded with ⁴⁵Ca²⁺. Results in GA were compared with those obtained in a rat liver endoplasmic reticulum (ER) enriched fraction. The addition of IP₃ at concentrations ranging from 100 nm to 100 μm, in the presence of thapsigargin, a specific inhibitor of sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPases, promoted a rapid decrease in the Ca²⁺ content of GA vesicles. The amount of Ca²⁺ released from the vesicles was a function of IP₃ concentration, reaching about 60% in both GA and ER fractions at 100 μm IP₃. Calcium release was inhibited by heparin, an antagonist of IP₃ receptors. Calcium exhibited a bell-shaped effect on IP₃-dependent Ca²⁺ released from GA vesicles: it activated Ca²⁺ release at concentrations up to 1 μm, and inhibited it at higher concentrations. In contrast to that found in the endoplasmic reticulum fraction, none of the ryanodine receptor agonists tested (cyclic ADP-ribose, caffeine and ryanodine) significantly induced Ca²⁺ release from GA fraction vesicles in the presence of thapsigargin. Our results indicate the presence of an IP₃-sensitive Ca²⁺ release mechanism in the Golgi apparatus membrane analogous to that of the ER. However, a Ca²⁺ release mechanism sensitive to ryanodine receptor agonists like that of ER is not evident in the GA membrane. Received: 13 March 2000/Revised: 13 July 2000     

New Molecular Determinant for Inactivation of the Human L-Type α1C Ca2+ Channel

Molecular cloning of the human fibroblast Ca²⁺ channel pore-forming α_1C subunit revealed (Soldatov, 1992. Proc. Natl. Acad. Sci. USA 89:4628-4632) a naturally occurring mutation g²²⁵⁴→ a that causes the replacement of the conservative alanine for threonine at the position 752 at the cytoplasmic end of transmembrane segment IIS6. Using stably transfected HEK293 cell lines, we have compared electrophysiological properties of the conventional α_1C,77 human recombinant L-type Ca²⁺ channel with those of its mutated isoform α_1C,94 containing the A752T replacement. Comparative quantification of steady-state availability of the current carried by α_1C,94 and α_1C,77 showed that A752T mutation prevented a large (≈25%) fraction of the current carried by Ca²⁺ or Ba²⁺ from fully inactivating. This mutation, however, did not appear to alter significantly the Ca²⁺-dependence and kinetics of decay of the inactivating fraction of the current or its voltage-dependence. The data suggests that Ala752 at the cytoplasmic end of IIS6 might serve as a molecular determinant of the Ca²⁺ channel inactivation, possibly regulating the voltage-dependence of its availability. Received: 14 January 2000/Revised: 20 June 2000     

Mechanically Induced Calcium Movements in Astrocytes, Bovine Aortic Endothelial Cells and C6 Glioma Cells

Forces applied to resting primary astrocytes, bovine aortic endothelial cells and C6 glioma cells with collagen-coated magnetite particles produce a fast transient change of intracellular Ca²⁺. It peaks in the micromolar range as measured by Fura-2. This mechanical response adapts within seconds so that repeated stimulation causes smaller responses requiring >10 min for recovery. When cytoplasmic Ca²⁺ is high after treating with ATP, cyclopiazonic acid and thapsigargin, stimulation causes a transient decrease in Ca²⁺. In these three cell types, no influx of ions is required for Ca²⁺ elevation showing the response is not caused by activation of plasmalemmal mechanosensitive channels. Approximately half the cells tested showed similar behavior, while the other half, such as fibroblasts, required extracellular Ca²⁺. The Ca²⁺ response is not temperature sensitive suggesting the possible involvement of intracellular mechanosensitive channels. We tested a number of second messenger reagents and were only able to block the response in BAECs, but not C6 glioma cells, with Xestospongin C, a blocker of IP₃-activated channels. Despite the lack of a causal involvement of plasmalemmal mechanosensitive channels, mechanical stimulation immediately activates a persistent Mn²⁺ influx pathway. This Mn²⁺ pathway may be mechanosensitive channels, Ca²⁺-activated cation channels or depletion-activated Ca²⁺ channels. Received: 7 July 1999/Revised: 12 November 1999     

A Kinetic Model with Ordered Cytoplasmic Dissociation for SUC1, an Arabidopsis H+/Sucrose Cotransporter Expressed in Xenopus Oocytes

To elucidate the kinetic properties of the Arabidopsis H⁺/sucrose cotransporter, SUC1, with respect to transmembrane voltage and ligand concentrations, the transport system was heterologously expressed in Xenopus laevis oocytes. Steady-state plasma membrane currents associated with transport of sucrose were measured with two-electrode voltage clamp over the voltage range −180 to +40 mV as a function of extracellular pH and sugar concentrations. At any given voltage, currents exhibited hyperbolic kinetics with respect to extracellular H⁺ and sugar concentrations, and this enabled determination of values for the maximum currents in the presence of each ligand (i ^H _max , i ^S _max for H⁺ and sucrose) and of the ligand concentrations eliciting half-maximal currents (K ^H _m , K ^S _m ). The i ^H _max and i ^S _max exhibited marked and statistically significant increases as a function of increasingly negative membrane potential. However, the K ^H _m and K ^S _m decreased with increasingly negative membrane potential. Furthermore, at any given voltage, i ^S _max increased and K ^S _m decreased as a function of the external H⁺ concentration. Eight six-state carrier models—which comprised the four possible permutations of intracellular and extracellular ligand binding order, each with charge translocation on the sugar-loaded or -unloaded forms of the carrier—were analyzed algebraically with respect to their competence to account for the ensemble of kinetic observations. Of these, two models (first-on, first-off and last-on, first-off with respect to sucrose binding as it passes from outside to inside the cell and with charge translocation on the loaded form of the carrier) exhibit sufficient kinetic flexibility to describe the observations. Combining these two, a single model emerges in which the binding on the external side can be random, but it can only be ordered on the inside, with the sugar dissociating before the proton. Received: 23 January 1996/Revised: 16 April 1997     

Complex and Tandem Repeat Structure of Subtelomeric Regions in the Taiwan Cricket, <Emphasis Type="BoldItalic">Teleogryllus taiwanemma</Emphasis>

Telomeres of most insects are composed of simple (TTAGG) _n repeats that are synthesized by telomerase. However, in some dipteran insects such as Drosophila melanogaster, (TTAGG) _n repeats or telomerase activity has not been detected. Although telomere structure is well documented in Diptera and Lepidoptera, very limited information is available on lower insect groups. To understand general aspects of telomere function and evolution in insects, we endeavored to characterize structures of the telomeric and subtelomeric regions in a lower insect, the Taiwan cricket, Teleogryllus taiwanemma. FISH analysis of this insect's chromosomes demonstrated (TTAGG) _n repeat elements in all distal ends. Just proximal to the telomeric repeats, the highly conserved 9-kb long terminal unit (LTU) sequences are tandemly repeated. These were observed in four of six chromosomes, three autosomal ends, and one X-chromosomal end. LTU sequences represent about 0.2% of the T. taiwanemma genome. Each LTU contains a core (TTAGG)₈-like sequence (TRLS) and five types of conserved sequences—ST (short telomere associated), J (joint), X, SR (satellite sequence rich), and Y—which vary in length from about 150 bp to 2.7 kb. The LTU sequence is defined as ST–J–TRLS–SR–X–Y–X–Y–X. Most LTU regions may be derived from the ancestral common sequence, which is observed in ST regions six times and at many other LTU sites. We could not find the LTU-like sequence in three other crickets including the closest species, T. emma, suggesting that the LTU in T. taiwanemma has been rapidly amplified in subtelomeric regions through recent evolutional events. It is also suggested that the highly conserved structure of the LTU is maintained by recombination and may contribute to telomere elongation, as seen in dipteran insects. Received: 6 August 2001/Accepted: 10 October 2001     

``Frustrated Exocytosis' — A Novel Phenomenon: Membrane Fusion without Contents Release, Followed by Detachment and Reattachment of Dense Core Vesicles in Paramecium Cells

The lipophilic fluorescent dye, FM1-43, as now frequently used to stain cell membranes and to monitor exo-endocytosis and membrane recycling, induces a cortical [Ca²⁺] _i transient and exocytosis of dense core vesicles (``trichocysts') in Paramecium cells, when applied at usual concentrations (≤10 μm) in presence of extracellular Ca<sup>2+</sup> ([Ca<sup>2+</sup>] <sub>o</sub> = 50 μm). When [Ca<sup>2+</sup>] <sub>o</sub> is kept at 30 nm (<[Ca<sup>2+</sup>]<sup>rest</sup> <sub>i</sub> ), in about one third of the population of extrudable trichocysts docked at the cell membrane, FM1-43 induces membrane fusion, visible by FM1-43 fluorescence of the vesicle membrane. However, in this system extrusion of secretory contents cannot occur in absence of any sufficient Ca<sup>2+</sup> <sub>o</sub> . Upon readdition of Ca<sup>2+</sup> <sub>o</sub> or some other appropriate Me<sup>2+</sup> <sub>o</sub> at 90 μm, secretory contents can be released (complete exocytosis). Resulting ghosts formed in presence of Ca<sup>2+</sup>, Sr<sup>2+</sup> or Mn<sup>2+</sup> are vesicular, but when formed in presence of Mg<sup>2+</sup>, for reasons to be elucidated, they are tubular, though both types are endocytosed and lose their FM1-43 stain. In contrast, in presence of [Mg<sup>2+</sup>] <sub>o</sub> = 3 mm (which inhibits contents release), the exocytotic openings reseal and intact trichocysts with labeled membranes and with still condensed contents are detached from the cell surface (``frustrated exocytosis') within ∼15 min. They undergo cytoplasmic streaming and saltatory redocking, with a half-time of ∼35 min. During this time, the population of redocked trichocysts amenable to exocytosis upon a second stimulus increases with a half-time of ∼35 min. Therefore, acquirement of competence for exocytotic membrane fusion may occur with only a small delay after docking, and this maturation process may last only a short time. A similar number of trichocysts can be detached by merely increasing [Mg²⁺] _o to 3 mm, or by application of the anti-calmodulin drug, R21547 (calmidazolium). Essentially we show (i) requirement of calmodulin and appropriate [Me²⁺] to maintain docking sites in a functional state, (ii) requirement of Ca²⁺ _o or of some other Me²⁺ _o to drive membrane resealing during exo-endocytosis, (iii) requirement of an ``empty' signal to go to the regular endocytotic pathway (with fading fluorescence), and (iv) occurrence of a ``filled' signal for trichocysts to undergo detachment and redocking (with fluorescence) after ``frustrated exocytosis'. Received: 20 January 2000/Revised: 5 May 2000