Role of elementary Ca(2+) puffs in generating repetitive Ca(2+) oscillations

Inositol (1,4,5)-trisphosphate (IP(3)) liberates intracellular Ca(2+) both as localized 'puffs' and as repetitive waves that encode information in a frequency-dependent manner. Using video-rate confocal imaging, together with photorelease of IP(3) in Xenopus oocytes, we investigated the roles of puffs in determining the periodicity of global Ca(2+) waves. Wave frequency is not delimited solely by cyclical recovery of the cell's ability to support wave propagation, but further involves sensitization of Ca(2+)-induced Ca(2+) release by progressive increases in puff frequency and amplitude at numerous sites during the interwave period, and accumulation of pacemaker Ca(2+), allowing a puff at a 'focal' site to trigger a subsequent wave. These specific 'focal' sites, distinguished by their higher sensitivity to IP(3) and close apposition to neighboring puff sites, preferentially entrain both the temporal frequency and spatial directionality of Ca(2+) waves. Although summation of activity from many stochastic puff sites promotes the generation of regularly periodic global Ca(2+) signals, the properties of individual Ca(2+) puffs control the kinetics of Ca(2+) spiking and the (higher) frequency of subcellular spikes in their local microdomain.     

Increased sensitivity and clustering of elementary Ca2+ release events during oocyte maturation

The universal signal for egg activation at fertilization is a rise in cytoplasmic Ca(2+) with defined spatial and temporal kinetics. Mammalian and amphibian eggs acquire the ability to produce such Ca(2+) signals during a maturation period that precedes fertilization and encompasses resumption of meiosis and progression to metaphase II. In Xenopus, immature oocytes produce fast, saltatory Ca(2+) waves that can be oscillatory in nature in response to IP(3). In contrast, mature eggs produce a single continuous, sweeping Ca(2+) wave in response to IP(3) or sperm fusion. The mechanisms mediating the differentiation of Ca(2+) signaling during oocyte maturation are not well understood. Here, I characterized elementary Ca(2+) release events (Ca(2+) puffs) in oocytes and eggs and show that the sensitivity of IP(3)-dependent Ca(2+) release is greatly enhanced during oocyte maturation. Furthermore, Ca(2+) puffs in eggs have a larger spatial fingerprint, yet are short lived compared to oocyte puffs. Most interestingly, Ca(2+) puffs cluster during oocyte maturation resulting in a continuum of Ca(2+) release sites over space in eggs. These changes in the spatial distribution of elementary Ca(2+) release events during oocyte maturation explain the continuous nature and slower speed of the fertilization Ca(2+) wave.     

Mitochondrial respiration and Ca2+ waves are linked during fertilization and meiosis completion

Fertilization increases both cytosolic Ca(2+) concentration and oxygen consumption in the egg but the relationship between these two phenomena remains largely obscure. We have measured mitochondrial oxygen consumption and the mitochondrial NADH concentration on single ascidian eggs and found that they increase in phase with each series of meiotic Ca(2+) waves emitted by two pacemakers (PM1 and PM2). Oxygen consumption also increases in response to Ins(1,4,5)P(3)-induced Ca(2+) transients. Using mitochondrial inhibitors we show that active mitochondria sequester cytosolic Ca(2+) during sperm-triggered Ca(2+) waves and that they are strictly necessary for triggering and sustaining the activity of the meiotic Ca(2+) wave pacemaker PM2. Strikingly, the activity of the Ca(2+) wave pacemaker PM2 can be restored or stimulated by flash photolysis of caged ATP. Taken together our observations provide the first evidence that, in addition to buffering cytosolic Ca(2+), the egg's mitochondria are stimulated by Ins(1,4,5)P(3)-mediated Ca(2+) signals. In turn, mitochondrial ATP production is required to sustain the activity of the meiotic Ca(2+) wave pacemaker PM2.     

On the roles of Ca2+ diffusion, Ca2+ buffers, and the endoplasmic reticulum in IP3-induced Ca2+ waves.

We have investigated the effects of Ca2+ diffusion, mobile and stationary Ca2+ buffers in the cytosol, and Ca2+ handling by the endoplasmic reticulum on inositol 1,4,5-trisphosphate-induced Ca2+ wave propagation. Rapid equilibration of free and bound Ca2+ is used to describe Ca2+ sequestration by buffers in both the cytosol and endoplasmic reticulum (ER) lumen. Cytosolic Ca2+ regulation is based on a kinetic model of the inositol 1,4,5-trisphosphate (IP3) receptor of De Young and Keizer that includes activation and inhibition of the IP3 receptor Ca2+ channel in the ER membrane and SERCA Ca2+ pumps in the ER. Diffusion of Ca2+ in the cytosol and the ER and the breakdown and diffusion of IP3 are also included in our calculations. Although Ca2+ diffusion is severely limited because of buffering, when conditions are chosen just below the threshold for Ca2+ oscillations, a pulse of IP3 or Ca2+ results in a solitary trigger wave that requires diffusion of Ca2+ for its propagation. In the oscillatory regime repetitive wave trains are observed, but for this type of wave neither the wave shape nor the speed is strongly dependent on the diffusion of Ca2+. Local phase differences lead to waves that are predominately kinematic in nature, so that the wave speed (c) is related to the wavelength (lambda) and the period of the oscillations (tau) approximately by the formula c = lambda/tau. The period is determined by features that control the oscillations, including [IP3] and pump activity, which are related to recent experiments. Both solitary waves and wave trains are accompanied by a Ca2+ depletion wave in the ER lumen, similar to that observed in cortical preparations from sea urchin eggs. We explore the effect of endogenous and exogenous Ca2+ buffers on wave speed and wave shape, which can be explained in terms of three distinct effects of buffering, and show that exogenous buffers or Ca2+ dyes can have considerable influence on the amplitude and width of the waves.     

The probability of triggering calcium puffs is linearly related to the number of inositol trisphosphate receptors in a cluster

Puffs are local Ca(2+) signals that arise by Ca(2+) liberation from the endoplasmic reticulum through concerted opening of tightly clustered inositol trisphosphate receptor/channels (IP(3)R). They serve both local signaling functions and trigger global Ca(2+) waves. The numbers of functional IP(3)R within clusters differ appreciably between different puff sites, and we investigated how the probability of puff occurrence varies with cluster size. We imaged puffs in SH-SY5Y cells using total internal fluorescence microscopy, and estimated cluster sizes from the magnitude of the largest puff observed at each site relative to the signal from a single channel. We find that the initial triggering rate of puffs following photorelease of IP(3), and the average frequency of subsequent repetitive puffs, vary about linearly with cluster size. These data accord well with stochastic simulations in which opening of any individual IP(3)R channel within a cluster triggers a puff via Ca(2+)-induced Ca(2+) release. An important consequence is that the signaling power of a puff site (average amount of Ca(2+) released per puff × puff frequency) varies about the square of cluster size, implying that large clusters contribute disproportionately to cellular signaling and, because of their higher puff frequency, preferentially act as pacemakers to initiate Ca(2+) waves.     

Localised and rapid Ca2+ micro-events in human neutrophils: conventional Ca2+ puffs and global waves without peripheral-restriction or wave cycling

Ultra-localised and peripherally restricted zones of elevated Ca2+ (z-waves) have been reported to cycle around the periphery of neutrophils at low frequency (1/20s) in the absence of conventional localised Ca2+ (puffs) and global Ca2+ (waves) signals. However, we report here that fast confocal laser scanning of human neutrophils loaded with either cytosolic fluo4 or its membrane associated analogue, MOMO reports both "conventional" stationary Ca2+ "puffs" (diameter c.3 microm) and global Ca2+ waves that sweep across the cell. The Ca2+ puff size and frequency of detection suggests that each neutrophil contained only a single release site and that its detection was limited by the location of the confocal plane relative to the event. Both formylated peptide receptor stimulation and cytosolic IP3 uncaging generated Ca2+ puffs (c.6% of cells) and global Ca2+ signals (c.75% of cells). The Ca2+ puffs peaked at approx. 250 nM and had a duration of approx. 235 msec and remained at a single locus. This was similar to other Ca2+ events in other cell types but in direct contrast to the reported z-waves. It was concluded that the micro-events which underlie Ca2+ signalling in neutrophils are conventional and that the existence of novel Ca2+z-waves is doubtful.     

Simulation of the fertilization Ca2+ wave in Xenopus laevis eggs.

In the preceding paper Fontanilla and Nuccitelli (Biophysical Journal 75:2079-2087 (1998)) present detailed measurements of the shape and speed of the fertilization Ca2+ wave in Xenopus laevis eggs. In order to help interpret their results, we develop here a computational technique based on the finite element method that allows us to carry out realistic simulations of the fertilization wave. Our simulations support the hypothesis that the physiological state of the mature egg is bistable, i.e., that its cytoplasm can accommodate two alternative physiological Ca2+ concentrations: a low concentration characteristic of the prefertilization state and a greatly elevated concentration characteristic of the state following the passage of the wave. We explore this hypothesis by assuming that the bistability is due to the release and re-uptake properties of the endoplasmic reticulum (ER) as determined by inositol trisphosphate (IP3) receptor/Ca2+ channels and sarcoendoplasmic reticulum calcium ATPase (SERCA) pumps. When combined with buffered diffusion of Ca2+ in the cytoplasm, our simulations show that inhomogeneities in the Ca2+ release properties near the plasma membrane are required to explain the temporal and spatial dependences of the shape and speed of these waves. Our results are consistent with an elevated IP3 concentration near the plasma membrane in the unfertilized egg that is augmented significantly near the site of fertilization. These gradients are essential in determining the concave shape of the Ca2+ fertilization wave front.     

Mitochondria exert a negative feedback on the propagation of intracellular Ca2+ waves in rat cortical astrocytes.

We have used digital fluorescence imaging techniques to explore the interplay between mitochondrial Ca2+ uptake and physiological Ca2+ signaling in rat cortical astrocytes. A rise in cytosolic Ca2+ ([Ca2+]cyt), resulting from mobilization of ER Ca2+ stores was followed by a rise in mitochondrial Ca2+ ([Ca2+]m, monitored using rhod-2). Whereas [Ca2+]cyt recovered within approximately 1 min, the time to recovery for [Ca2+]m was approximately 30 min. Dissipating the mitochondrial membrane potential (Deltapsim, using the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxy-phenyl-hydrazone [FCCP] with oligomycin) prevented mitochondrial Ca2+ uptake and slowed the rate of decay of [Ca2+]cyt transients, suggesting that mitochondrial Ca2+ uptake plays a significant role in the clearance of physiological [Ca2+]cyt loads in astrocytes. Ca2+ signals in these cells initiated either by receptor-mediated ER Ca2+ release or mechanical stimulation often consisted of propagating waves (measured using fluo-3). In response to either stimulus, the wave traveled at a mean speed of 22.9 +/- 11.2 micrometer/s (n = 262). This was followed by a wave of mitochondrial depolarization (measured using tetramethylrhodamine ethyl ester [TMRE]), consistent with Ca2+ uptake into mitochondria as the Ca2+ wave traveled across the cell. Collapse of Deltapsim to prevent mitochondrial Ca2+ uptake significantly increased the rate of propagation of the Ca2+ waves by 50%. Taken together, these data suggest that cytosolic Ca2+ buffering by mitochondria provides a potent mechanism to regulate the localized spread of astrocytic Ca2+ signals.     

The cortical endoplasmic reticulum (ER) of the mouse egg: localization of ER clusters in relation to the generation of repetitive calcium waves.

The endoplasmic reticulum (ER) of the mature mouse egg consists of a fine tubular network and pronounced accumulations in the cortex. The ER was visualized both in intact eggs and with in vitro preparations of the cortex using the fluorescent lipophilic dye, DiI. Immunofluorescent labeling of the ER in isolated cortical preparations demonstrated that the ER clusters contain inositol 1,4, 5-trisphosphate (IP(3)) receptors, indicating an important involvement in sperm-induced Ca(2+) transients, which are triggered by IP(3). We imaged the ER during fertilization and the subsequent Ca(2+) transients and found that the clusters remained intact throughout this period. Recovery of fluorescence after photobleaching established that the ER clusters are continuous with the reticular ER network and that these structures remain stable and continuous throughout the time of fertilization-induced Ca(2+) transients; continuity also remained during IP(3) injection. These results indicate that, in contrast to echinoderm eggs, the ER of mouse eggs does not become disrupted when it releases Ca(2+)at fertilization. The localization and apparent stability of the cortical ER clusters may be important in generating Ca(2+) oscillations, which are characteristic of fertilized mammalian eggs. Imaging of intracellular Ca(2+) revealed that Ca(2+) transients originate in the hemisphere of the egg that contains abundant ER clusters, thus the mouse contains a stable cortical pacemaker responsible for generating Ca(2+) waves.     

Injection of a sperm extract triggers egg activation in the newt Cynops pyrrhogaster

Unfertilized eggs of the newt Cynops pyrrhogaster are arrested at the second meiotic metaphase. The primary signal for egg activation is a transient increase in [Ca2+](i), which is triggered by the fertilizing sperm and propagates over the egg cortex as a Ca2+ wave. We injected an extract of Cynops sperm (SE) into unfertilized eggs and induced a wave-like [Ca2+](i) increase which resulted in activation and resumption of meiosis. The SE-injected eggs showed degradation of cyclin B1 and DNA replication. When SE was boiled or treated with proteinase K before injection, it was unable to cause egg activation. Preinjection of Ca2+ -chelator BAPTA before SE injection inhibited egg activation. These results indicate that a heat-labile and proteinaceous factor in the sperm cytoplasm induces a transient increase in [Ca2+](i) which is required for egg activation. Injection of IP3 into unfertilized eggs caused an increase in [Ca2+](i) and egg activation, but injection of cADP-ribose did not. These results support the hypothesis that Ca2+ release at fertilization occurs via IP3 receptors.     

Functional mapping of Ca2+ signaling complexes in plasma membrane microdomains of polarized cells

Many cells cluster signaling complexes in plasma membrane microdomains. Polarized secretory cells cluster all Ca2+ signaling proteins, including GPCRs, at the apical pole. The functional significance of such an arrangement is not known because of a lack of techniques for functional mapping of signaling complexes at plasma membrane patches. In the present work, we developed such a technique based on the use of two patch pipettes, a recording and a stimulating pipette (SP). Including 20% glycerol in the SP solution increased the viscosity and the hydrophobicity to prevent leakage and formation of tight seals on the plasma membrane. This allowed moving the SP between sites to stimulate multiple patches of the same cell and with the same agonist concentrations. Functional mapping of Ca2+ signaling in pancreatic acinar cells revealed that the M3, cholecystokinin, and bombesin signaling complexes at the apical pole are much more sensitive to stimulation than those at the basal pole. Furthermore, at physiological agonist concentrations, Ca2+ signals could be evoked only by stimulation of membrane patches at the apical pole. [Ca2+](i) imaging revealed that Ca2+ waves were invariably initiated at the site of apical membrane patch stimulation, suggesting that long range diffusion of second messengers is not obligatory to initiate and propagate apical-to-basal Ca2+ waves. The present studies reveal a remarkable heterogeneity in responsiveness of Ca2+ signaling complexes at membrane microdomains, with the most responsive complexes confined to the apical pole, probably to restrict the Ca2+ signals to the site of exocytosis and allow the polarized functions of secretory cells.     

A wave of IP3 production accompanies the fertilization Ca2+ wave in the egg of the frog, Xenopus laevis: theoretical and experimental support

The fertilization Ca2+ wave in Xenopus laevis is a single, large wave of elevated free Ca2+ that is initiated at the point of sperm-egg fusion and traverses the entire width of the egg. This Ca2+ wave involves an increase in inositol-1,4,5-trisphosphate (IP3) resulting from the interaction of the sperm and egg, which then results in the activation of the endoplasmic reticulum Ca2+ release machinery. The extraordinarily large size of this cell (1.2 mm diameter) together with the small surface region of sperm-receptor activation makes special demands on the IP3-dependent Ca2+ mobilizing machinery. We propose a detailed model of the fertilization Ca2+ wave in Xenopus eggs that requires an accompanying wave of IP3 production. While the Ca2+ wave is initiated by a localized increase of IP3 near the site of sperm-egg fusion, the Ca2+ wave propagates via IP3 production correlated with the Ca2+ wave-possibly via Ca(2+)-mediated PLC activation. Such a Ca(2+)-mediated IP(3) production wave has not been required previously to explain the fertilization Ca2+ wave in eggs; we argue this is necessary to explain the observed IP3 dynamics in Xenopus eggs. To test our hypothesis, we have measured the IP3 levels from 20 nl "sips" of the egg cortex during wave propagation. We were unable to detect the low IP3 levels in unfertilized eggs, but after fertilization, [IP3] ranged from 175 to 430 nM at the sperm entry point and from 120 to 700 nM 90 degrees away once the Ca2+ wave passed that region about 2 min after fertilization. Prior to the Ca2+ wave reaching that region the IP3 levels were undetectable. Since significant IP3 could not diffuse to this region from the sperm entry point within 2 min, this observation is consistent with a regenerative wave of IP3 production.     

Modeling of ATP-mediated signal transduction and wave propagation in astrocytic cellular networks

Astrocytes, a special type of glial cells, were considered to have supporting role in information processing in the brain. However, several recent studies have shown that they can be chemically stimulated by neurotransmitters and use a form of signaling, in which ATP acts as an extracellular messenger. Pathological conditions, such as spreading depression, have been linked to abnormal range of wave propagation in astrocytic cellular networks. Nevertheless, the underlying intra- and inter-cellular signaling mechanisms remain unclear. Motivated by the above, we constructed a model to understand the relationship between single-cell signal transduction mechanisms and wave propagation and blocking in astrocytic networks. The model incorporates ATP-mediated IP3 production, the subsequent Ca2+ release from the ER through IP3R channels and ATP release into the extracellular space. For the latter, two hypotheses were tested: Ca2+- or IP3-dependent ATP release. In the first case, single astrocytes can exhibit excitable behavior and frequency-encoded oscillations. Homogeneous, one-dimensional astrocytic networks can propagate waves with infinite range, while in two dimensions, spiral waves can be generated. However, in the IP3-dependent ATP release case, the specific coupling of the driver ATP-IP3 system with the driven Ca2+ subsystem leads to one- and two-dimensional wave patterns with finite range of propagation.     

Transformation of local Ca2+ spikes to global Ca2+ transients: the combinatorial roles of multiple Ca2+ releasing messengers

In pancreatic acinar cells, low, threshold concentrations of acetylcholine (ACh) or cholecystokinin (CCK) induce repetitive local cytosolic Ca2+ spikes in the apical pole, while higher concentrations elicit global signals. We have investigated the process that transforms local Ca2+ spikes to global Ca2+ transients, focusing on the interactions of multiple intracellular messengers. ACh-elicited local Ca2+ spikes were transformed into a global sustained Ca2+ response by cyclic ADP-ribose (cADPR) or nicotinic acid adenine dinucleotide phosphate (NAADP), whereas inositol 1,4,5-trisphosphate (IP3) had a much weaker effect. In contrast, the response elicited by a low CCK concentration was strongly potentiated by IP3, whereas cADPR and NAADP had little effect. Experiments with messenger mixtures revealed a local interaction between IP3 and NAADP and a stronger global potentiating interaction between cADPR and NAADP. NAADP strongly amplified the local Ca2+ release evoked by a cADPR/IP3 mixture eliciting a vigorous global Ca2+ response. Different combinations of Ca2+ releasing messengers can shape the spatio-temporal patterns of cytosolic Ca2+ signals. NAADP and cADPR are emerging as key messengers in the globalization of Ca2+ signals.     

Long distance communication between muscarinic receptors and Ca2+ release channels revealed by carbachol uncaging in cell-attached patch pipette

We have investigated the characteristics of cytosolic Ca2+ signals induced by muscarinic receptor activation of pancreatic acinar cells that reside within intact pancreatic tissue. We show that these cells exhibit global Ca2+ waves and local apical Ca2+ spikes. This is the first evidence for local Ca2+ signaling in undissociated pancreatic tissue. The mechanism of formation of localized Ca2+ signals was examined using a novel approach involving photolysis of caged carbachol inside a patch pipette attached to the basal surface of an acinar unit. This local activation of basal muscarinic receptors elicited local cytosolic Ca2+ spikes in the apical pole more than 15 microm away from the site of stimulation. In some experiments, local basal receptor activation elicited a Ca2+ wave that started in the apical pole and then spread toward the base. Currently, there are two competing hypotheses for preferential apical Ca2+ signaling. One invokes the need for structural proximity of the cholinergic receptors and the Ca2+ release channels in the apical pole, whereas the other postulates long distance communication between basal receptors and the channels. Our intrapipette uncaging experiments provide definitive evidence for long distance communication between basal muscarinic receptors and apical Ca2+ release channels.     

Spatio-temporal propagation of Ca2+ signals by cyclic ADP-ribose in 3T3 cells stimulated via purinergic P2Y receptors

The role of cyclic ADP-ribose in the amplification of subcellular and global Ca2+ signaling upon stimulation of P2Y purinergic receptors was studied in 3T3 fibroblasts. Either (1) 3T3 fibroblasts (CD38- cells), (2) 3T3 fibroblasts preloaded by incubation with extracellular cyclic ADP-ribose (cADPR), (3) 3T3 fibroblasts microinjected with ryanodine, or (4) 3T3 fibroblasts transfected to express the ADP-ribosyl cyclase CD38 (CD38+ cells) were used. Both preincubation with cADPR and CD38 expression resulted in comparable intracellular amounts of cyclic ADP-ribose (42.3 +/- 5.2 and 50.5 +/- 8.0 pmol/mg protein). P2Y receptor stimulation of CD38- cells yielded a small increase of intracellular Ca2+ concentration and a much higher Ca2+ signal in CD38-transfected cells, in cADPR-preloaded cells, or in cells microinjected with ryanodine. Confocal Ca2+ imaging revealed that stimulation of ryanodine receptors by cADPR or ryanodine amplified localized pacemaker Ca2+ signals with properties resembling Ca2+ quarks and triggered the propagation of such localized signals from the plasma membrane toward the internal environment, thereby initiating a global Ca2+ wave.     

Control of calcium signal propagation to the mitochondria by inositol 1,4,5-trisphosphate-binding proteins

Cytosolic Ca2+ ([Ca2+]c) signals triggered by many agonists are established through the inositol 1,4,5-trisphosphate (IP3) messenger pathway. This pathway is believed to use Ca2+-dependent local interactions among IP3 receptors (IP3R) and other Ca2+ channels leading to coordinated Ca2+ release from the endoplasmic reticulum throughout the cell and coupling Ca2+ entry and mitochondrial Ca2+ uptake to Ca2+ release. To evaluate the role of IP3 in the local control mechanisms that support the propagation of [Ca2+]c waves, store-operated Ca2+ entry, and mitochondrial Ca2+ uptake, we used two IP3-binding proteins (IP3BP): 1) the PH domain of the phospholipase C-like protein, p130 (p130PH); and 2) the ligand-binding domain of the human type-I IP3R (IP3R224-605). As expected, p130PH-GFP and GFP-IP3R224-605 behave as effective mobile cytosolic IP3 buffers. In COS-7 cells, the expression of IP3BPs had no effect on store-operated Ca2+ entry. However, the IP3-linked [Ca2+]c signal appeared as a regenerative wave and IP3BPs slowed down the wave propagation. Most importantly, IP3BPs largely inhibited the mitochondrial [Ca2+] signal and decreased the relationship between the [Ca2+]c and mitochondrial [Ca2+] signals, indicating disconnection of the mitochondria from the [Ca2+]c signal. These data suggest that IP3 elevations are important to regulate the local interactions among IP3Rs during propagation of [Ca2+]c waves and that the IP3-dependent synchronization of Ca2+ release events is crucial for the coupling between Ca2+ release and mitochondrial Ca2+ uptake.     

Cortically restricted production of IP3 leads to propagation of the fertilization Ca2+ wave along the cell surface in a model of the Xenopus egg

The fertilization Ca2+ wave in Xenopus laevis is a single, large wave of elevated free cytosolic Ca2+ concentration that emanates from the point of sperm-egg fusion and traverses the entire diameter of the egg. This phenomenon appears to involve an increase in inositol-1,4,5-trisphosphate (IP3) resulting from interaction of the sperm and egg, which then results in the activation of the endoplasmic reticulum Ca2+ release machinery. We have proposed models based on a static elevated distribution of IP3, and dynamic [IP3], however, these models have suggested that the fertilization wave passes through the center of the egg. Complementing these earlier models, we propose a more detailed model of the fertilization Ca2+ wave in Xenopus eggs to explore the hypothesis that IP3 is produced only at or near the plasma membrane. In this case, we find that the wave propagates primarily through the cortex of the egg, and that Ca2+ -induced production of IP3 at the plasma membrane allows IP3 to propagate in advance of the wave. Our model includes Ca2+ -dependent production of IP3 at the plasma membrane and IP3 degradation. Simulations in 1 dimension and axi-symmetric 3 dimensions illustrate the basic features of the wave.     

Mechanisms of calcium elevation in the micromeres of sea urchin embryos

The micromeres, the first cells to be specified in sea urchin embryos, are generated by unequal cleavage at the fourth cell division. The micromeres differentiate autonomously to form spicules and dispatch signals to induce endomesoderm in the neighbouring macromeres cells in the embryo. Using a calcium indicator Fura-2/AM and a mixture of dextran conjugated Oregon green-BAPTA 488 and Rhodamine red, the intracellular calcium ion concentration ([Ca2+]i) was studied in embryos at the 16-cell stage. [Ca2+]i was characteristically elevated in the micromeres during furrowing at the 4th cleavage. Subsequently, Ca2+ oscillated for about 10 min in the micromeres, resulting in episodic high levels of [Ca2+]i. High [Ca2+]i regions were associated with regional localizations of the endoplasmic reticulum (ER), though not with ER accumulated at the vegetal pole of the micromeres during the 4th division. Pharmacological studies, using a blocker of IP3-mediated Ca2+ release (Xestospongin), a store-operated Ca2+ entry inhibitor (2 aminoethoxydiphenyl borate (2-APB)) and an inhibitor of stretch-dependent ion channels (gadolinium), suggest that the high [Ca2+]i and oscillations in the micromeres are triggered by calcium influx caused by the activation of stretch-dependent calcium channels, followed by the release of calcium ions from the endoplasmic reticulum. On the basis of these new findings, a possible mechanism for autonomous formation of the micromeres is discussed.