New Roles for Actin-Binding Proteins and PIP₂ in Intracellular Calcium Homeostasis

Spatiotemporal increase of the intracellular Ca2+ is the most universal way to regulate the function of a eukaryotic cell. Owing to a host of actin-binding proteins and enzymes whose activities are modulated by the local concentration of Ca2+, free Ca2+ in cytosol serves as a pivotal second messenger in a variety of cell functions. The rise and fall of intracellular Ca2+ wave has been best illustrated in eggs at fertilization. However, the molecular mechanism by which intracellular Ca2+ is increased in the fertilized egg is largely unknown despite the discoveries of the distinct Ca2+-mobilizing second messengers in the past 30 years. In this thesis, I have used the starfish oocytes to study how Ca2+ signaling can be modulated by the actin cytoskeleton, which is known to be dynamically remodelled during meiotic maturation and fertilization of the egg. The principal issues of my experimental work are: (i) to establish the role of actin-binding proteins and PIP2 in the regulation of the Ca2+ signaling; (ii) to study the effect of the Ca2+-store depletion on Ca2+ signaling and on the structure and function of the actin cytoskeleton, and (iii) to study the role of the actin-cytoskeleton in establishing the block to polyspermy. Microinjected into starfish eggs, actin-binding protein gelsolin, function-blocking antibody to depactin, and the PHVsequestering fusion protein that indirectly alters the actin cytoskeleton, all changed a certain aspect of Ca2+ signaling. Depletion of the Ca2+ store with ionomycin in turn drastically changed the cortical structure and the actin cytoskeleton of the eggs, eventually leading to a deleterious effect on egg activation and early development. Finally, the alteration of the actin cytoskeleton led to failure to establish a fast and slow block to polyspermy. Taken together, this study indicated that the actin cytoskeleton is an important factor that optimizes the Ca2+ response at egg activation and guides monospermic fertilization

Disassembly of Subplasmalemmal Actin Filaments Induces Cytosolic Ca2+ Increases in Astropecten aranciacus Eggs

Background/Aims: Eggs of all animal species display intense cytoplasmic Ca2+ increases at fertilization. Previously, we reported that unfertilized eggs of Astropecten aranciacus exposed to an actin drug latrunculin A (LAT-A) exhibit similar Ca2+ waves and cortical flashes after 5-10 min time lag. Here, we have explored the molecular mechanisms underlying this unique phenomenon. Methods: Starfish eggs were pretreated with various agents such as other actin drugs or inhibitors of phospholipase C (PLC), and the changes of the intracellular Ca2+ levels were monitored by use of Calcium Green in the presence or absence of LAT-A. The concomitant changes of the actin cytoskeleton were visualized with fluorescent F-actin probes in confocal microscopy. Results: We have shown that the LAT-A-induced Ca2+ increases are related to the disassembly of actin flaments: i) not only LAT-A but also other agents depolymerizing F-actin (i.e. cytochalasin B and mycalolide B) induced similar Ca2+ increases, albeit with slightly lower efficiency; ii) drugs stabilizing F-actin (i.e. phalloidin and jasplakinolide) either blocked or significantly delayed the LAT-A-induced Ca2+ increases. Further studies utilizing pharmacological inhibitors of PLC (U-73122 and neomycin), dominant negative mutant of PLC-ɣ, specific sequestration of PIP2 (RFP-PH), InsP3 uncaging, and quantitation of endogenous InsP3 all indicated that LAT-A induces Ca2+ increases by stimulating PLC rather than sensitizing InsP3 receptors. In support of the idea, it bears emphasis that LAT-A timely increased intracellular contents of InsP3 with concomitant decrease of PIP2 levels in the plasma membrane. Conclusion: Taken together, our results suggest that suboolemmal actin filaments may serve as a scaffold for cell signaling and modulate the activity of the key enzyme involved in intracellular Ca2+ signaling

Towards a Map of the European Tree Cover based on Sentinel-2

Many areas of science and policy depend on knowledge of the tree cover in Europe. Sentinel-2 is a new (launched in 2015) satellite with a higher spatial resolution compared to previous satellites. In the present study a new algorithm for mapping tree cover from Sentinel-2 is developed, an analysis of which bands should be used for tree cover mapping is made, the accuracy of the mapping is assessed, and the tree cover from the present approach is compared with previous estimates. Firstly, the feasibility of the present algorithm is demonstrated. Secondly, it is shown that only ten band combinations have good performance in four selected Sentinel-2 tiles and that the bands 3, 5, 6, 12 appear in most combinations. Thirdly, the accuracy is assessed to be high, and lastly it is shown that the relative difference between the tree cover of the present study and the tree cover of previous studies is between -14% and 68

Correlations in STAR: interferometry and event structure

STAR observes a complex picture of RHIC collisions where correlation effects of different origins -- initial state geometry, semi-hard scattering, hadronization, as well as final state interactions such as quantum intensity interference -- coexist. Presenting the measurements of flow, mini-jet deformation, modified hadronization, and the Hanbury Brown and Twiss effect, we trace the history of the system from the initial to the final state. The resulting picture is discussed in the context of identifying the relevant degrees of freedom and the likely equilibration mechanism.Comment: 8 pages, 6 figures, plenary talk at the 5th International Conference on Physics and Astrophysics of Quark Gluon Plasma, to appear in Journal of Physics G (http://www.iop.org

Neutral Kaon Interferometry in Au+Au collisions at sqrt(s_NN) = 200 GeV

We present the first statistically meaningful results from two-K0s interferometry in heavy-ion collisions. A model that takes the effect of the strong interaction into account has been used to fit the measured correlation function. The effects of single and coupled channel were explored. At the mean transverse mass m_T = 1.07 GeV, we obtain the values R = 4.09 +/- 0.46 (stat.) +/- 0.31 (sys) fm and lambda = 0.92 +/- 0.23 (stat) +/- 0.13 (sys), where R and lambda are the invariant radius and chaoticity parameters respectively. The results are qualitatively consistent with m_T systematics established with pions in a scenario characterized by a strong collective flow.Comment: 11 pages, 10 figure

Strangelet search at RHIC

Two position sensitive Shower Maximum Detector (SMDs) for Zero-Degree Calorimeters (ZDCs) were installed by STAR before run 2004 at both upstream and downstream from the interaction point along the beam axis where particles with small rigidity are swept away by strong magnetic field. The ZDC-SMDs provides information about neutral energy deposition as a function of transverse position in ZDCs. We report the preliminary results of strangelet search from a triggered data-set sampling 100 million Au+Au collisions at top RHIC energy.Comment: Strange Quark Matter 2004 conference proceedin

Azimuthal anisotropy and correlations at large transverse momenta in p+p and Au+Au collisions at sqrt[sNN] = 200 GeV

Results on high transverse momentum charged particle emission with respect to the reaction plane are presented for Au+Au collisions at sqrt[sNN]=200 GeV. Two- and four-particle correlations results are presented as well as a comparison of azimuthal correlations in Au+Au collisions to those in p+p at the same energy. The elliptic anisotropy v2 is found to reach its maximum at pt~3 GeV/c, then decrease slowly and remain significant up to pt ~ 7-10 GeV/c. Stronger suppression is found in the back-to-back high-pt particle correlations for particles emitted out of plane compared to those emitted in plane. The centrality dependence of v2 at intermediate pt is compared to simple models based on jet quenching

Spintronics: Fundamentals and applications

Spintronics, or spin electronics, involves the study of active control and manipulation of spin degrees of freedom in solid-state systems. This article reviews the current status of this subject, including both recent advances and well-established results. The primary focus is on the basic physical principles underlying the generation of carrier spin polarization, spin dynamics, and spin-polarized transport in semiconductors and metals. Spin transport differs from charge transport in that spin is a nonconserved quantity in solids due to spin-orbit and hyperfine coupling. The authors discuss in detail spin decoherence mechanisms in metals and semiconductors. Various theories of spin injection and spin-polarized transport are applied to hybrid structures relevant to spin-based devices and fundamental studies of materials properties. Experimental work is reviewed with the emphasis on projected applications, in which external electric and magnetic fields and illumination by light will be used to control spin and charge dynamics to create new functionalities not feasible or ineffective with conventional electronics.Comment: invited review, 36 figures, 900+ references; minor stylistic changes from the published versio

Effects of Ionomycin on Egg Activation and Early Development in Starfish

Ionomycin is a Ca2+-selective ionophore that is widely used to increase intracellular Ca2+ levels in cell biology laboratories. It is also occasionally used to activate eggs in the clinics practicing in vitro fertilization. However, neither the precise molecular action of ionomycin nor its secondary effects on the eggs' structure and function is well known. In this communication we have studied the effects of ionomycin on starfish oocytes and zygotes. By use of confocal microscopy, calcium imaging, as well as light and transmission electron microscopy, we have demonstrated that immature oocytes exposed to ionomycin instantly increase intracellular Ca2+ levels and undergo structural changes in the cortex. Surprisingly, when microinjected into the cells, ionomycin produced no Ca2+ increase. The ionomycin-induced Ca2+ rise was followed by fast alteration of the actin cytoskeleton displaying conspicuous depolymerization at the oocyte surface and in microvilli with concomitant polymerization in the cytoplasm. In addition, cortical granules were disrupted or fused with white vesicles few minutes after the addition of ionomycin. These structural changes prevented cortical maturation of the eggs despite the normal progression of nuclear envelope breakdown. At fertilization, the ionomycin-pretreated eggs displayed reduced Ca2+ response, no elevation of the fertilization envelope, and the lack of orderly centripetal translocation of actin fibers. These alterations led to difficulties in cell cleavage in the monospermic zygotes and eventually to a higher rate of abnormal development. In conclusion, ionomycin has various deleterious impacts on egg activation and the subsequent embryonic development in starfish. Although direct comparison is difficult to make between our findings and the use of the ionophore in the in vitro fertilization clinics, our results call for more defining investigations on the issue of a potential risk in artificial egg activation

De novo assembly of a transcriptome from the eggs and early embryos of Astropecten aranciacus

Starfish have been instrumental in many fields of biological and ecological research. Oocytes of Astropecten aranciacus, a common species native to the Mediterranean Sea and the East Atlantic, have long been used as an experimental model to study meiotic maturation, fertilization, intracellular Ca2+ signaling, and cell cycle controls. However, investigation of the underlying molecular mechanisms has often been hampered by the overall lack of DNA or protein sequences for the species. In this study, we have assembled a transcriptome for this species from the oocytes, eggs, zygotes, and early embryos, which are known to have the highest RNA sequence complexity. Annotation of the transcriptome identified over 32,000 transcripts including the ones that encode 13 distinct cyclins and as many cyclin-dependent kinases (CDK), as well as the expected components of intracellular Ca2+ signaling toolkit. Although the mRNAs of cyclin and CDK families did not undergo significant abundance changes through the stages from oocyte to early embryo, as judged by real-time PCR, the transcript encoding Mos, a negative regulator of mitotic cell cycle, was drastically reduced during the period of rapid cleavages. Molecular phylogenetic analysis using the homologous amino acid sequences of cytochrome oxidase subunit I from A. aranciacus and 30 other starfish species indicated that Paxillosida, to which A. aranciacus belongs, is not likely to be the most basal order in Asteroidea. Taken together, the first transcriptome we assembled in this species is expected to enable us to perform comparative studies and to design gene-specific molecular tools with which to tackle long-standing biological questions