Puffing Activity Research Articles

KRAP is required for diffuse and punctate IP3-mediated Ca2+ liberation and determines the number of functional IP3R channels within clusters

KRas-induced actin-interacting protein (KRAP) has been identified as crucial for the appropriate localization and functioning of the inositol trisphosphate receptors (IP3Rs) that mediate Ca2+ release from the endoplasmic reticulum. Here, we used siRNA knockdown of KRAP expression in HeLa and HEK293 cells to examine the roles of KRAP in the generation of IP3-mediated local Ca2+ puffs and global, cell-wide Ca2+ signals. High resolution Ca2+ imaging revealed that the mean amplitude of puffs was strongly reduced by KRAP knockdown, whereas the Ca2+ flux during openings of individual IP3R channels was little affected. In both control and KRAP knockdown cells the numbers of functional channels in the clusters underlying puff sites were stochastically distributed following a Poisson relationship, but the mean number of functional channels per site was reduced by about two thirds by KRAP knockdown. We conclude that KRAP is required for activity of IP3R channels at puff sites and stochastically ‘licenses’ the function of individual channels on a one-to-one basis, rather than determining the functioning of the puff site as a whole. In addition to puff activity (‘punctate’ Ca2+ release), global, cell-wide Ca2+ signals evoked by higher levels of IP3 are further composed from a discrete ‘diffuse’ mode of Ca2+ release. By applying fluctuation analysis to isolate the punctate component during global Ca2+ signals, we find that KRAP knockdown suppresses to similar extents punctate and diffuse Ca2+ release in wild-type cells and in HEK293 cells exclusively expressing type 1 and type 3 IP3Rs. Thus, KRAP appears essential for the functioning of the IP3Rs involved in diffuse Ca2+ release as well as the clustered IP3Rs that generate local Ca2+ puffs.

Termination of Ca2+ puffs during IP3-evoked global Ca2+ signals

We previously described that cell-wide cytosolic Ca2+ transients evoked by inositol trisphosphate (IP3) are generated by two modes of Ca2+ liberation from the ER; ‘punctate’ release via an initial flurry of transient Ca2+ puffs from local clusters of IP3 receptors, succeeded by a spatially and temporally ‘diffuse’ Ca2+ liberation. Those findings were derived using statistical fluctuation analysis to monitor puff activity which is otherwise masked as global Ca2+ levels rise. Here, we devised imaging approaches to resolve individual puffs during global Ca2+ elevations to better investigate the mechanisms terminating the puff flurry. We find that puffs contribute about 40% (∼90 attomoles) of the total Ca2+ liberation, largely while the global Ca2+ signal rises halfway to its peak. The major factor terminating punctate Ca2+ release is an abrupt decline in puff frequency. Although the amplitudes of large puffs fall during the flurry, the amplitudes of more numerous small puffs remain steady, so overall puff amplitudes decline only modestly (∼30%). The Ca2+ flux through individual IP3 receptor/channels does not measurably decline during the flurry, or when puff activity is depressed by pharmacological lowering of Ca2+ levels in the ER lumen, indicating that the termination of punctate release is not a simple consequence of reduced driving force for Ca2+ liberation. We propose instead that the gating of IP3 receptors at puff sites is modulated such that their openings become suppressed as the bulk [Ca2+] in the ER lumen falls during global Ca2+ signals.

Changes in Puffing Pattern ofDrosophila melanogaster(Diptera: Drosophilidae) Polytene Chromosomes after Egg Exposure to Microwave Radiation and Magnetic Field1

Abstract We determined the effects of microwave radiation and static magnetic field on the gene activity in Drosophila by measuring the dimensions of puffing the salivary gland polytene chromosomes of Drosophila melanogaster Meigen following exposure of eggs to the radiation and magnetic field. Drosophila eggs were exposed to either microwave radiation of 36.64 GHz frequency and 1 W/m2 intensity for 30 s, a static magnetic field of 25 mT for 5 min, or both. The diameter of puffs was measured in squashed salivary gland preparations extracted from larvae from those eggs as they entered the prepupal stage. The puffs measured were 50CD, 63F, 71CE, 72CD (chromosome 3L) and 82EF, 83E, 93D (chromosome 3R). Results demonstrated that (a) microwave radiation exposure decreased puffing activity in puffs 63F, 71CE, and 82 EF but had no effect on puffing at 72CD, 93D and 50CD; (b) exposure to magnetic fields only did not change puff activity, but magnetic field exposure appeared to enhance the impact of microwave rad...

Aluminum effects on embryo suspensor polytene chromosomes of Phaseolus coccineus L

Aluminum (Al) represents a widespread environmental pollutant, with severe toxic impacts on plants. In this study, we documented for the first time the structural and functional responses induced by two concentrations of AlCl3 (10−2 M and 10−1 M) in the polytene chromosomes that characterize the chromatin organization in the embryo suspensor cells of Phaseolus coccineus. Polytene chromosomes showed signs of dose-dependent genotoxicity following AlCl3 treatments with a significant increase in both chromatin stickiness and chromatin fragmentation. Polytene chromosomes specifically reacted to AlCl3 also in terms of DNA and RNA puffing activity: with respect to the control, the treatments promoted ex-novo and/or inhibited puff formation along chromosome arms, suggesting a fine modulation of the differential genome activity in response to the treatments. The nuclei of suspensors from control and treated seeds showed nucleoli mainly arranged by more than one NOR-bearing chromosome. In addition, AlCl3 treatments affected the frequency of nucleoli organized by singular organizer chromosomes, with an increase in the frequencies of nucleoli organized by chromosome II and a reduction in the frequencies of those organized by chromosomes I or V. These results confirm that, also in our system, nucleolus may react as stress response organelle.

Exploring the explosive‐effusive transition using permanent ultraviolet cameras

AbstractUnderstanding the mechanisms that cause effusive eruptions is the key to mitigating their associated hazard. Here we combine results from permanent ultraviolet (UV) cameras, and from other geophysical observations (seismic very long period, thermal, and infrasonic activity), to characterize volcanic SO2 flux regime in the period prior, during, and after Stromboli's August–November 2014 effusive eruption. We show that, in the 2 months prior to effusion onset, the SO2 flux levels are 2 times average level. We explain this anomalously high SO2 regime as primarily determined by venting of rapidly rising, pressurized SO2‐rich gas pockets produced by strombolian explosions being more frequent and intense than usual. We develop a procedure to track (and count), in the UV camera record, the SO2 flux pulses produced by individual explosions and puffing activity (active degassing). We find that these SO2 pulses are far more numerous (67 ± 47 events per hour) before the effusion onset than during normal activity (20 ± 15 events per hour). This observation, combined with geophysical evidence, demonstrates an elevated gas bubble supply to the shallow conduits, causing elevated explosive and puffing activity. This increase (≥0.1 m3 s−1) in magma transport rate in the north‐east feeding conduits finally triggers effusion onset. Active degassing remains elevated also during the effusive phase, supporting the persistence of explosive and puffing activity during the effusive eruption, deep in the volcanic conduit. Our results demonstrate that permanent UV cameras can valuably contribute to monitoring at high‐sampling frequency gas dynamics and fluxes, thus opening the way to direct comparison with more established geophysical observations.

Integrating puffing and explosions in a general scheme for Strombolian‐style activity

AbstractStrombolian eruptions are among the most common subaerial styles of explosive volcanism worldwide. Distinctive features of each volcano lead to a correspondingly wide range of variations of magnitude and erupted products, but most papers focus on a single type of event at a single volcano. Here, in order to emphasize the common features underlying this diversity of styles, we scrutinize a database from 35 different erupting vents, including 21 thermal infrared videos from Stromboli (Italy), Etna (Italy), Yasur (Vanuatu), and Batu Tara (Indonesia), from puffing, through rapid explosions to normal explosions, with variable ejection parameters and relative abundance of gas, ash, and bombs. Using field observations and high‐speed thermal infrared videos processed by a new algorithm, we identify the distinguishing characteristics of each type of activity and how they may relate and interact. In particular, we record that ash‐poor normal explosions may be preceded and followed by the onset or the increase of the puffing activity, while ash‐rich explosions are emergent, i.e., with inflation of the free surface followed directly by emission of increasingly large gas pockets. Overall, we see that all Strombolian activities form a continuum arising from a common mechanism and are modulated by the combination of two well‐established controls: (1) the length of the bursting gas pocket with respect to the vent diameter and (2) the presence and thickness of a high‐viscosity layer in the uppermost part of the volcanic conduit.

Characteristics of puffing activity revealed by ground-based, thermal infrared imaging: the example of Stromboli Volcano (Italy)

Puffing, i.e., the frequent (1 s ca.) release of small (0.1–10 m3), over-pressurized pockets of magmatic gases, is a typical feature of open-conduit basaltic volcanoes worldwide. Despite its non-trivial contribution to the degassing budget of these volcanoes and its recognized role in volcano monitoring, detection and metering tools for puffing are still limited. Taking advantage of the recent developments in high-speed thermal infrared imaging, we developed a specific processing algorithm to detect the emission of individual puffs and measure their duration, size, volume, and apparent temperature at the vent. As a test case, we applied our method at Stromboli Volcano (Italy), studying “snapshots” of 1 min collected in the years 2012, 2013, and 2014 at several vents. In all 3 years, puffing occurred simultaneously at three or more vents with variable features. At the scale of the single vent, a direct relationship links puff temperature and radius, suggesting that the apparent temperature is mostly a function of puff thickness, while the real gas temperature is constant for all puffs. Once released in the atmosphere, puffs dissipate in less than 20 m. On a broader scale, puffing activity is highly variable from vent to vent and year to year, with a link between average frequency, temperature, and volume from 136 puffs per minute, 600 K above ambient temperature, 0.1 m3, and the occasional ejection of pyroclasts to 20 puffs per minute, 3 K above ambient, 20 m3, and no pyroclasts. Frequent, small, hot puffs occur at random intervals, while as the frequency decreases and size increases, an increasingly longer minimum interval between puffs, up to 0.5 s, appears. These less frequent and smaller puffs also display a positive correlation between puff volume and the delay from the previous puff. Our results suggest an important role of shallow bubble coalescence in controlling puffing activity. The smaller and more frequent puffing at “hotter” vents is in agreement with the rapid rise of small gas pockets with only limited coalescence, while slower rise, allowing more time for coalescence, leads to larger but less frequent puffing at “colder” vents. This link between puffing style and vent thermal state points to a feedback between gas flux and magma temperature (and viscosity), where higher gas flux stirs and heats the magma, which, by getting less viscous, becomes a preferential way for bubble rise. Such a link has implications for the monitoring of the state of the shallow conduit at open-vent volcanoes as well as for determination of their total gas budget, relevant for hazard forecast and environmental studies.

Ip3 Receptor Mediated Ca Release in Atrial Cells from Normal and Failing Hearts

We studied excitation-contraction coupling (ECC) and inositol-1,4,5-triphosphate (IP3)-dependent Ca release in atrial cells from normal and heart failure (HF) rabbit hearts. Elementary Ca release events (Ca puffs) from IP3 receptors (IP3Rs) were detected in permeabilized myocytes after inhibition of ryanodine-receptor mediated Ca spark activity with tetracaine. Puff activity was induced by IP3 exposure and blocked by 2-APB, identifying their origin from IP3Rs. In normal myocytes puff activity was low and restricted to the cell periphery, whereas in HF myocytes their overall frequency was increased and puffs were more prominent in the cell center. Compared to normal cells, in HF atrial myocytes diastolic [Ca]i was increased, action potential (AP)-induced Ca transients (CaTs) were larger in amplitude (primarily due to enhanced Ca release from central non-junctional sarcoplasmic reticulum, SR), propagation of activation from cell periphery to the center was accelerated, and fractional SR Ca release was increased, whereas no difference in SR Ca load was observed. These differences were largely abolished by exposure to 2-APB, suggesting an increased basal IP3 activity in HF. Angiotensin II (AngII) increased diastolic [Ca]i in normal and HF myocytes whereas the effect was more pronounced in HF. The amplitude of AP-dependent CaTs was increased in the presence of AngII or after photolytical release of caged IP3 in normal cells, whereas the opposite effect was observed in HF. The frequency of spontaneous Ca waves was increased in HF, and returned to levels comparable to normal myocytes after 2-APB exposure. In conclusion, HF atrial myocytes revealed enhanced Ca release during ecc and more frequent spontaneous Ca waves, presumably resulting from increased basal IP3R-mediated Ca release. However, additional neurohumoral stimulation with AngII, as it might occur in-vivo in cardiac disease, negatively affected atrial Ca signaling during ECC.

New insights into volcanic processes at Stromboli from Cerberus, a remote-controlled open-path FTIR scanner system

The ordinary, low intensity activity of Stromboli volcano is sporadically interrupted by more energetic events termed, depending on their intensity, “major explosions” and “paroxysms”. These short-lived energetic episodes represent a potential risk to visitors to the highly accessible summit of Stromboli. Observations made at Stromboli over the last decade have shown that the composition of gas emitted from the summit craters may change prior to such explosions, allowing the possibility that such changes may be used to forecast these potentially dangerous events.In 2008 we installed a novel, remote-controlled, open-path FTIR scanning system called Cerberus at the summit of Stromboli, with the objective of measuring gas compositions from individual vents within the summit crater terrace of the volcano with high temporal resolution and for extended periods. In this work we report the first results from the Cerberus system, collected in August–September 2009, November 2009 and May–June 2010.We find significant, fairly consistent intra-crater variability for CO2/SO2 and H2O/CO2 ratios, and relatively homogeneous SO2/HCl ratios. In general, the southwest crater is richest in CO2, and the northeast crater poorest, while the central crater is richest in H2O. It thus appears that during the measurement period the southwest crater had somewhat more direct connection to a primary, deep degassing system while the central and northeast craters reflect a slightly more secondary degassing nature, with a supplementary, shallow H2O source for the central crater, probably related to puffing activity. Such water-rich emissions from the central crater can account for the lower crystal content of its eruption products, and emphasise the role of continual magma supply to the shallowest levels of Stromboli's plumbing system.Our observations of heterogeneous crater gas emissions and high H2O/CO2 ratios do not agree with models of CO2-flushing, and we show that simple depressurisation during magma ascent to the surface is a more likely model for H2O loss at Stromboli. We highlight that alternative explanations other than CO2 flushing are required to explain distributions of H2O and CO2 amounts dissolved in melt inclusions.We detected fairly systematic increases in CO2/SO2 ratio some weeks prior to major explosions, and some evidence of a decrease in this ratio in the days immediately preceding the explosions, with periods of low, stable CO2/SO2 ratios between explosions otherwise. Our measurements, therefore, confirm the medium term (~weeks) precursory increases previously observed with MultiGas instruments, and, in addition, reveal new short-term precursory decreases in CO2/SO2 ratios immediately prior to the major explosions. Such patterns, if shown to be systematic, may be of great utility for hazard management at Stromboli's summit.Our results suggest that intra-crater CO2/SO2 variability may produce short-term peaks and troughs in CO2/SO2 time series measured with in-situ MultiGas instruments, due simply to variations in wind direction.

Timescales of IP3-Evoked Ca2+ Spikes Emerge from Ca2+ Puffs Only at the Cellular Level

The behavior of biological systems is determined by the properties of their component molecules, but the interactions are usually too complex to understand fully how molecular behavior generates cellular behavior. Ca2+ signaling by inositol trisphosphate receptors (IP3R) offers an opportunity to understand this relationship because the cellular behavior is defined largely by Ca2+-mediated interactions between IP3R. Ca2+ released by a cluster of IP3R (giving a local Ca2+ puff) diffuses and ignites the behavior of neighboring clusters (to give repetitive global Ca2+ spikes). We use total internal reflection fluorescence microscopy of two mammalian cell lines to define the temporal relationships between Ca2+ puffs (interpuff intervals, IPI) and Ca2+ spikes (interspike intervals) evoked by flash photolysis of caged IP3. We find that IPI are much shorter than interspike intervals, that puff activity is stochastic with a recovery time that is much shorter than the refractory period of the cell, and that IPI are not periodic. We conclude that Ca2+ spikes do not arise from oscillatory dynamics of IP3R clusters, but that repetitive Ca2+ spiking with its longer timescales is an emergent property of the dynamics of the whole cluster array.

Effects of microwaves on the puffing pattern of D. melanogaster

Abstract The influence of electromagnetic field exposure on puffing pattern of salivary gland polythene chromosomes, viability and fertility of Drosophila melanogaster of the wild type Canton-S line was studied. Experimental conditions: Electromagnetic field characteristics: frequency — 36.64 GHz, power density — 0.4 W/m2, exposure time −10 seconds. Electromagnetic field exposure was conducted on the egg stage. Results: in larvae developed from the exposed eggs 3 of 8 chromosomal puffs tested (71CE, 82EF, and 83E) had significantly smaller dimensions than these in control at the prepupal stage. Viability of Drosophila estimated by the number of adult flies hatched from exposed eggs decreased, while the number of dominant lethal mutations increased. Conclusion: the exposure to a low-level microwave irradiation suppressed puffing activity at ecdysone-inducible loci of Drosophila polythene chromosomes, increased frequency of dominant lethal mutations and decreased Drosophila viability but did not influence Drosophila fertility.

Integrated petrochemical and geophysical data reveals thermal distribution of the feeding conduits at Stromboli volcano, Italy

[1] Samples of scoriae erupted at Stromboli volcano during its persistent strombolian activity were collected between 2005 and 2008. Chemical and mineralogical compositions were obtained on products erupted from the three main crater sectors (SW, Central and NE). Small chemical variations indicate a different degree of evolution coupled with small difference of magma temperature <10°C. Analysis of the acoustic data for the same time period as the scoria sampling, indicates that puffing (a persistent overpressurized bubble degassing) was, on average, mainly observed at the central craters and at times moved to the NE sector. The cross-check of the two independent data sets allowed us to assess correlation between composition of products and puffing activity at vents. The hotter products are always erupted from the vents where puffing occurs indicating that slightly higher temperature can be the expression of an enhanced two-phase bubble flow dynamics.

Localization of puff sites adjacent to the plasma membrane: Functional and spatial characterization of Ca 2+ signaling in SH-SY5Y cells utilizing membrane-permeant caged IP 3

The Xenopus oocyte has been a favored model system in which to study spatio-temporal mechanisms of intracellular Ca2+ dynamics, in large part because this giant cell facilitates intracellular injections of Ca2+ indicator dyes, buffers and caged compounds. However, the recent commercial availability of membrane-permeant ester forms of caged IP3 (ci-IP3) and EGTA, now allows for facile loading of these compounds into smaller mammalian cells, permitting control of [IP3]i and cytosolic Ca2+ buffering. Here, we establish the human neuroblastoma SH-SY5Y cell line as an advantageous experimental system for imaging Ca2+ signaling, and characterize IP3-mediated Ca2+ signaling mechanisms in these cells. Flash photo-release of increasing amounts of i-IP3 evokes Ca2+ puffs that transition to waves, but intracellular loading of EGTA decouples release sites, allowing discrete puffs to be studied over a wide range of [IP3]. Puff activity persists for minutes following a single photo-release, pointing to a slow rate of i-IP3 turnover in these cells and suggesting that repetitive Ca2+ spikes with periods of 20-30s are not driven by oscillations in [IP3]. Puff amplitudes are independent of [IP3], whereas their frequencies increase with increasing photo-release. Puff sites in SH-SY5Y cells are not preferentially localized near the nucleus, but instead are concentrated close to the plasma membrane where they can be visualized by total internal reflection microscopy, offering the potential for unprecedented spatio-temporal resolution of Ca2+ puff kinetics.

Temperature and dynamics of degassing at Stromboli

Between 1999 and 2004 we deployed a series of thermal infrared experiments at Stromboli volcano (Italy) to thermally define the dynamics of gas puffing. These experiments involved targeting degassing vents with thermal infrared thermometers and thermal imagers from a distance of ∼300 m. The data showed that puffs involved a short‐lived gas thrust phase (velocities up to ∼20 m/s) followed by a dominant phase of buoyant rise with constant ascent velocities (∼7 m/s), until cooling caused the puff to stall and disperse. The puff could best be described as a starting plume that rapidly developed into a thermal. Ascent velocities, puff volumes and frequencies were extremely stable over the 6‐year‐long study period, typically being 10 ± 5 m/s, 50–190 m3 per burst and 0.5–1 Hz, respectively. These volumes convert to puff masses of 10–40 kg and give a total gas flux of 7–26 kg/s. Although stable at the system as a whole, at a single vent, gas puffing showed short‐term variation and instability. Gas puffing would wax and wane at individual vents, shifting to other linked vents so that for the system has a whole, puffing activity was maintained. Variations in vent temperature can thus be tied to variations in gas flux, where termination of gas puff activity (a reduction in the flux) at a particular vent results in decreased vent temperature. We suggest that gas puffing is related to a rapid and repeated release (bursting) of gas slugs at the head of the magma column, at a rate of one every 1–2 s. Released gas packets then ascend the empty conduit section to be emitted as the puffing plume. This style of degassing activity persists between Strombolian explosions and operates alongside passive degassing; but has a very different source mechanism. It appears to represent a highly stable and repetitive, but extremely mild, stream of explosive bursts. This style of nonpassive degassing may account for up to 45% of Stromboli's total gas flux.

Sources, size distribution, and downwind grounding of aerosols from Mount Etna

The number concentrations and size distributions of aerosol particles &gt;0.3 μm diameter were measured at the summit of Mount Etna and up to 10 km downwind from the degassing vents during July and August 2004. Aerosol number concentrations reached in excess of 9 × 106 L−1 at summit vents, compared to 4–8 × 104 L−1 in background air. Number concentrations of intermediate size particles were higher in emissions from the Northeast crater compared to other summit crater vents, and chemical composition measurements showed that Northeast crater aerosols contained a higher mineral cation content compared to those from Voragine or Bocca Nuova, attributed to Strombolian or gas puffing activity within the vent. Downwind from the summit the airborne plume was located using zenith sky ultraviolet spectroscopy. Simultaneous measurements indicated a coincidence of elevated ground level aerosol concentrations with overhead SO2, demonstrating rapid downward mixing of the plume onto the lower flanks of the volcano under certain meteorological conditions. At downwind sites the ground level particle number concentrations were elevated in all size fractions, notably in the 2.0–7.5 μm size range. These findings are relevant for assessing human health hazard and suggest that aerosol size distribution measurements may aid volcanic risk management.

Structural and functional relationships between Ca2+ puffs and mitochondria in Xenopus oocytes.

Ca2+ uptake and release from endoplasmic reticulum (ER) and mitochondrial Ca2+ stores play important physiological and pathological roles, and these processes are shaped by interactions that depend on the structural intimacy between these organelles. Here we investigate the morphological and functional relationships between mitochondria, ER, and the sites of intracellular Ca2+ release in Xenopus laevis oocytes by combining confocal imaging of local Ca2+ release events ("Ca2+ puffs") with mitochondrial localization visualized using vital dyes and subcellularly targeted fluorescent proteins. Mitochondria and ER are localized in cortical bands approximately 6-8 microm wide, with the mitochondria arranged as densely packed "islands" interconnected by discrete strands. The ER is concentrated more superficially than mitochondria, and the mean separation between Ca2+ puff sites and mitochondria is approximately 2.3 microm. However, a subpopulation of Ca2+ puff sites is intimately associated with mitochondria (approximately 28% within <600 nm), a greater number than expected if Ca2+ puff sites were randomly distributed. Ca2+ release sites close to mitochondria exhibit lower Ca2+ puff activity than Ca2+ puff sites in regions with lower mitochondrial density. Furthermore, Ca2+ puff sites in close association with mitochondria rarely serve as the sites for Ca2+ wave initiation. We conclude that mitochondria play important roles in regulating local ER excitability, Ca2+ wave initiation, and, thereby, spatial patterning of global Ca2+ signals.

Ecdysone-regulated chromosome puffing in the salivary glands of the Mediterranean fruit fly, Ceratitis capitata

The effect of ecdysone on the puffing activity of the polytene chromosomes of Ceratitis capitata has been studied in organ cultures of late-larval salivary glands. Culture of glands from 120-h-old larvae (puff stage 1) in the presence of ecdysone resulted in the initiation of the late-larval puffing cycle that is normally observed in 145-h-old larvae (puff stage 4). During a 7-h period in the presence of ecdysone, the puffing patterns of most loci resembled the in vivo patterns observed in the period between puff stages 4 and 10, indicating that the first puffing cycle can be initiated by the hormone and proceed almost to completion, in vitro. Culture of salivary glands in the presence of ecdysone and a protein-synthesis inhibitor, as well as ecdysone withdrawal and readdition experiments, indicated that most of the ecdysone-regulated puffs could be categorized into three classes: (i) the puffs that were suppressed immediately by ecdysone, even in the absence of protein synthesis; (ii) the puffs that were induced directly by ecdysone; and (iii) the puffs that were induced indirectly by ecdysone, that is, they were induced after a lag period of a few hours and required protein synthesis for their induction.

Control of IP(3)-mediated Ca2+ puffs in Xenopus laevis oocytes by the Ca2+-binding protein parvalbumin.

1. Elementary events of Ca2+ release (Ca2+ puffs) can be elicited from discrete clusters of inositol 1,4,5 trisphosphate receptors (IP(3)Rs) at low concentrations of IP(3). Ca(2+) puffs have rarely been observed unless elicited by either hormone treatment or introduction of IP(3) into the cell. However, cells appear to have sufficient concentrations of IP(3) (0.1-3.0 microM) to induce Ca2+ release under resting conditions. 2. Here, we investigated Ca2+ puff activity in non-stimulated Xenopus oocytes using confocal microscopy. The fluorescent Ca2+ dye indicators Calcium Green 1 and Oregon Green 488 BAPTA-2 were injected into oocytes to monitor basal Ca2+ activity. 3. In this preparation, injection or overexpression of parvalbumin, an EF-hand Ca(2+)-binding protein (CaBP), induced Ca2+ puffs in resting Xenopus oocytes. This activity was inhibited by heparin, an IP(3)R channel blocker, and by mutation of the Ca(2+)-binding sites in parvalbumin. 4. Ca2+ puff activity was also evoked by injection of low concentrations of the Ca2+ chelator EGTA, but not by calbindin D(28k), another member of the EF-hand CaBP superfamily. 5. BAPTA and the Ca2+ indicator dye Oregon Green 488 BAPTA-1 evoked Ca2+ puff activity, while the dextran conjugate of Oregon Green 488 BAPTA-1 did not. These data indicate that a Ca(2+) buffer must be mobile in order to increase Ca2+ puff activity. 6. Together, the data indicate that some IP(3)Rs spontaneously release Ca2+ under resting concentrations of IP(3). These elementary Ca2+ events appear to be below the level of detection of current imaging techniques. We suggest that parvalbumin evokes Ca2+ puffs by coordinating the activity of elementary IP(3)R channel openings. 7. We conclude that Ca2+ release can be evoked not only by hormone-induced increases in IP(3), but also by expression of mobile cytosolic CaBPs under resting concentrations of IP(3).

The 62E early-late puff of Drosophila contains D-spinophilin, an ecdysone-inducible PDZ-domain protein dynamically expressed during metamorphosis

At the onset of metamorphosis in Drosophila melanogaster, the steroid hormone 20-OH ecdysone induces a small number of early and early-late puffs in the polytene chromosomes of the third-instar larval salivary gland whose activity is required for regulating the activity of a larger set of late puffs. Most of the corresponding early and early-late genes have been found to encode transcription factors that regulate a much larger set of late genes. In contrast, we describe here the identification of an ecdysone-regulated gene in the 62E early-late puff, denoted D-spinophilin, that encodes a protein similar to the mammalian protein spinophilin/neurabin II. The D-spinophilin protein is predicted to contain a highly conserved PP1-binding domain and adjacent PDZ domain, as well as a coiled-coil domain and SAM domain, and belongs to a family of related proteins from diverse organisms. Transcription of D-spinophilin is correlated with 62E puff activity during the early stages of metamorphosis and is ecdysone-dependent, making this the first member of this gene family shown to be regulated by a steroid hormone. Examination of the dynamic patterns of D-spinophilin expression during the early stages of metamorphosis are consistent with a role in central nervous system metamorphosis as well as a more general role in other tissues. As D-spinophilin appears to be the only member of this gene family in Drosophila, its study provides an excellent opportunity to elucidate the role of an important adaptor protein in a genetic model organism.

A dual role of 20–hydroxyecdysone in the control of protein synthesis related to DNA puff activity in the anterior region of Bradysia hygida (Diptera, Sciaridae) salivary gland

During the last 30 h of the larval stage, the salivary glands of Bradysia hygida show the amplification of some genes, resulting in the formation of two successive groups of DNA puffs, which direct the synthesis of two different sets of polypeptides. Incubation of anterior (S1) salivary gland regions, at age E7, beginning of first group of DNA puffs activity, in culture medium for 2 to 10 h results in a decrease in the synthesis of the polypeptides characteristic of this period. However, during subsequent incubation (from E7 to E7+12 h–24 h), when the second group of DNA puffs is active, S1 regions were able to synthesize some polypeptides characteristic of this period. The role of 20–OH ecdysone was studied, in vitro and in vivo, during these two periods of protein synthesis in S1 regions. The presence of the hormone was shown to be necessary to maintain, in vitro, the synthesis of the first set of polypeptides and was strongly inhibitory, in vitro and in vivo, to the synthesis of the second set of polypeptides. Thus, it is likely that the activity of the two distinct groups of DNA puffs is under opposite 20–OH–ecdysone control mechanisms.