Luigia Santella

Generation, control, and processing of cellular calcium signals.

Referee: Guiseppe Inesi, M.D., Ph.D., Professor and Chairman, School of Medicine, Dept. of Biochemistry and Molecular Biology, Univeristy of Maryland, Baltimore.

EFFECTS OF 1-METHYLADENINE ON NUCLEAR CA2+ TRANSIENTS AND MEIOSIS RESUMPTION IN STARFISH OOCYTES ARE MIMICKED BY THE NUCLEAR INJECTION OF INOSITOL 1,4 ,5-TRISPHOSPHATE AND CADP-RIBOSE

The treatment of prophase-arrested starfish oocytes with the hormone 1-methyladenine (1-MA) induces the elevation of Ca2+ in both the cytoplasm and the germinal vesicle (nucleus), and is followed by the resumption of meiosis. The injection of the modulators of the intracellular Ca2+ channels inositol 1,4,5-trisphosphate (InsP3) or cyclic adenosine diphosphate ribose (cADPr) into the germinal vesicle promoted meiosis resumption in the absence of 1-MA in about 50% of the oocytes. Caged InsP3 or caged cADPr were injected into the nuclei of oocytes together with the Ca2+ indicator calcium green dextran; their photoreleasing elicited nuclear calcium spikes which, in the case of cADPr, had repetitive behaviour. The spikes were abolished by the nuclear injection of antagonists or antibodies to the InsP3 or cADPr-sensitive Ca2+ channels. cADPr-modulated channels were localized on the membranes of the nuclear envelope using specific antibodies conjugated with IgG-gold complexes.

Calcium, protease action, and the regulation of the cell cycle.

Proteolysis is a key event in the control of the cell cycle. Most of the proteins which are degraded at specific cycle points, e.g. cyclins A, B, and E, are substrates of the ubiquitin/proteasome pathway. The Ca2+ dependent neutral protease calpain also cleaves cell cycle proteins, among them cyclin D1 and the c-mos proto-oncogene product which is a component of the CSF. The proteasome itself, however, may be under Ca2+ control through the binding of Ca2+ to its 29 kDa regulatory subunit. Calpain undergoes relocation among cell compartments during the various steps of the mitotic and meitotic cycles. It promotes the initiation and the progression of mitosis when injected into the perinuclear space of synchronized PtK1 cells, and the resumption of meiosis when directly injected into the nuclei of prophase-arrested starfish oocytes. Apart from the proteins mentioned above, most of the substrates of calpain which become cleaved during mitosis and meiosis are still unknown. Microtubule-associated proteins are likely candidates.

CALCIUM SIGNALLING IN THE CELL NUCLEUS

The First European Conference on Calcium Signalling in the Cell Nucleus took place in Baia Paraelios, Calabria, Italy, from 4-8 October 1997. It was organized by O. Bachs, E. Carafoli, P. Nicotera and L. Santella (local organizers, G. Bagetta and D. Rotiroti) and attended by about 90 specialists. The scientific content was very high and the discussions were particularly intense. Considering that the area is famous for its controversies, one could perhaps have expected aggressive overtones. Instead, in spite of the liveliness of the discussions, the atmosphere was congenial and constructive. The controversies have not disappeared, but a better understanding of some of their origins is now well under way.

NAADP activates a Ca2+ current that is dependent on F-actin cytoskeleton

Nicotinic acid adenine dinucleotide phosphate (NAADP) is involved in the Ca2+ response observed at fertilization in several species, including starfish. In this study, we have employed Ca2+ imaging and the single‐electrode voltage‐clamp technique to investigate whether the NAADP‐mediated Ca2+ entry discovered in our laboratory in starfish oocytes was underlain by a membrane current and whether the response to NAADP required an intact cytoskeleton. Uncaging of preinjected NAADP evoked a cortical Ca2+ flash that was followed by the spreading of the wave to the remainder of the cell. No Ca2+ increase was detected in Ca2+‐free sea water. Under voltage‐clamp conditions, the photoliberation of NAADP activated an inward rectifying membrane current, which reversed at potentials more positive than +50 mV and was abolished by removal of Ca2+ but not of Na+. The current was affected by preincubation with verapamil, SK&F 96356, and thapsigargin but not by preinjection of heparin, 8‐NH2‐ cyclic ADP‐ribose, or both antagonists. The membrane current and the Ca2+ wave were inhibited by latrunculin‐A and jasplakinolide, which depolymerize and stabilize actin cytoskeleton, respectively. These data offer the first demonstration that NAADP initiates a Ca2+ sweep by activating a Ca2+‐permeable membrane current that requires an intact F‐actin cytoskeleton as other Ca2+‐permeable currents, such as ICRAC and IARC.

The cell cycle: a new entry in the field of Ca2+ signaling

Abstract.Ca2+ signaling plays a crucial role in virtually all cellular processes, from the origin of new life at fertilization to the end of life when cells die. Both the influx of external Ca2+ through Ca2+-permeable channels and its release from intracellular stores are essential to the signaling function. Intracellular Ca2+ is influenced by mitogenic factors which control the entry and progression of the cell cycle; this is a strong indication for a role of Ca2+ in the control of the cycle, but surprisingly, the possibility of such a role has only been paid scant attention in the literature. Substantial progress has nevertheless been made in recent years in relating Ca2+ and the principal decoder of its information, calmodulin, to the modulation of various cycle steps. The aim of this review is to critically discuss the evidence for a role of Ca2+ in the cell cycle and to discuss Ca2+-dependent pathways regulating cell growth and differentiation.

Alteration of the Cortical Actin Cytoskeleton Deregulates Ca2+ Signaling, Monospermic Fertilization, and Sperm Entry

Background When preparing for fertilization, oocytes undergo meiotic maturation during which structural changes occur in the endoplasmic reticulum (ER) that lead to a more efficient calcium response. During meiotic maturation and subsequent fertilization, the actin cytoskeleton also undergoes dramatic restructuring. We have recently observed that rearrangements of the actin cytoskeleton induced by actin-depolymerizing agents, or by actin-binding proteins, strongly modulate intracellular calcium (Ca2+) signals during the maturation process. However, the significance of the dynamic changes in F-actin within the fertilized egg has been largely unclear. Methodology/Principal Findings We have measured changes in intracellular Ca2+ signals and F-actin structures during fertilization. We also report the unexpected observation that the conventional antagonist of the InsP3 receptor, heparin, hyperpolymerizes the cortical actin cytoskeleton in postmeiotic eggs. Using heparin and other pharmacological agents that either hypo- or hyperpolymerize the cortical actin, we demonstrate that nearly all aspects of the fertilization process are profoundly affected by the dynamic restructuring of the egg cortical actin cytoskeleton. Conclusions/Significance Our findings identify important roles for subplasmalemmal actin fibers in the process of sperm-egg interaction and in the subsequent events related to fertilization: the generation of Ca2+ signals, sperm penetration, cortical granule exocytosis, and the block to polyspermy.

Activation of oocytes by latrunculin A

Actin depolymerization by latrunculin A (LAT‐A) in mature starfish oocytes induces a massive calcium mobilization that results in the discharge of the cortical granules and in the elevation of the fertilization envelope. The Ca2+ liberation starts as a circumscribed subplasma membrane hotspot, which is followed by a flash of Ca2+ increase restricted to the cortical layer. Ca2+ propagates rapidly from these peripheral regions to the center of the oocyte, initiating calcium oscillations. Blockade of the inositol 1,4,5‐trisphosphate receptors with heparin does not affect the liberation of Ca2+ at the initial hotspot or the cortical flash, but abolishes the centripetal spreading of the wave and the Ca2+ oscillations. In Ca2+‐free medium, LAT‐A also initiates Ca2+ release at a discrete cortical point, but then propagates throughout the cell without first forming the uniform cortical flash. The latter is thus linked to the influx of external Ca2+, somehow promoted by the depolymerization of cortical (microvillar) actin. The Ca2+ response to spermatozoa (i.e., peripheral hotspot, cortical flash, globalization of the signal) closely mimics that promoted by LAT‐A. Thus, the initial cortical release of Ca2+ promoted by the sperm may be due to the depolymerization of actin..—Lim, D., Lange, K., Santella, L. Activation of oocytes by latrunculin A. FASEB J. 16, 1050–1056 (2002)

Actin cytoskeleton modulates calcium signaling during maturation of starfish oocytes.

Before successful fertilization can occur, oocytes must undergo meiotic maturation. In starfish, this can be achieved in vitro by applying 1-methyladenine (1-MA). The immediate response to 1-MA is the fast Ca2+ release in the cell cortex. Here, we show that this Ca2+ wave always initiates in the vegetal hemisphere and propagates through the cortex, which is the space immediately under the plasma membrane. We have observed that alteration of the cortical actin cytoskeleton by latrunculin-A and jasplakinolide can potently affect the Ca2+ waves triggered by 1-MA. This indicates that the cortical actin cytoskeleton modulates Ca2+ release during meiotic maturation. The Ca2+ wave was inhibited by the classical antagonists of the InsP(3)-linked Ca2+ signaling pathway, U73122 and heparin. To our surprise, however, these two inhibitors induced remarkable actin hyper-polymerization in the cell cortex, suggesting that their inhibitory effect on Ca2+ release may be attributed to the perturbation of the cortical actin cytoskeleton. In post-meiotic eggs, U73122 and jasplakinolide blocked the elevation of the vitelline layer by uncaged InsP(3), despite the massive release of Ca2+, implying that exocytosis of the cortical granules requires not only a Ca2+ rise, but also regulation of the cortical actin cytoskeleton. Our results suggest that the cortical actin cytoskeleton of starfish oocytes plays critical roles both in generating Ca2+ signals and in regulating cortical granule exocytosis.

Fertilization in echinoderms

For more than 150 years, echinoderm eggs have served as overly favored experimental model systems in which to study fertilization. Sea urchin and starfish belong to the same phylum and thus share many similarities in their fertilization patterns. However, several subtle but fundamental differences do exist in the fertilization of sea urchin and starfish, reflecting their phylogenetic bifurcation approximately 500 million years ago. In this article we review some of the seminal and recent findings that feature similarities and differences in sea urchin and starfish at fertilization.