Modulation of Calcium signalling by F-actin during maturation and fertilization of starfish oocytes

The Calcium ion (Ca2+) is a universal intracellular messenger that is implicated in the regulation of a great variety of intracellular processes. Among them, there is the meiotic maturation of oocytes from several species and their fertilization. The cell model system chosen for the work of this thesis has been the starfish oocytes. They constitute an exceptional model to study the properties of the dynamic release of Ca2+. First, the cells are large and nearly transparent, making them suitable for imaging experiments after microinjection of fluorescent markers. Immature oocytes extracted from the gonad are arrested at the prophase stage of the first meiotic division. Overcoming the meiotic arrest (maturation), is induced by the application of the maturing hormone 1-Methyladenine (1-MA), whose action involves intracellular Ca2+ transients. During the maturation process, the oocytes develop their ability to be successfully activated by a fertilizing spermatozoon by liberating higher levels of calcium. 1-MA also induces dramatic actin cytoskeleton rearrangements. In the present work, the relevance of the actin cytoskeleton in the modulation of the Ca2+ signals generated by hormonal stimulation and by the sperm has been studied. For this purpose, actin-specific disrupting agents and a specific antibody targeting the actin binding protein depactin have been delivered into living cells by needle microinjection, to study their effects using confocal laser fluorescent microscopy. Thus, the Ca2+ increases induced either by 1-MA, by the fertilizing sperm or by the injection of exogenous InsP3. which is the canonical Ca2+ mobilizer second messenger, have been monitored with video fluorescent microscopy. The results described indicate that actin filaments play direct and indirect roles in modulating the intracellular Ca2+ mobilization induced by the maturation process triggered by 1-MA as well as in the Ca2+ responses induced by the sperm

The role of the actin cytoskeleton in calcium signaling in starfish oocytes

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MicroRNA-Restricted Transgene Expression in the Retina

Background: Gene transfer using adeno-associated viral (AAV) vectors has been successfully applied in the retina for the treatment of inherited retinal dystrophies. Recently, microRNAs have been exploited to fine-tune transgene expression improving therapeutic outcomes. Here we evaluated the ability of retinal-expressed microRNAs to restrict AAV-mediated transgene expression to specific retinal cell types that represent the main targets of common inherited blinding conditions. Methodology/Principal Findings: To this end, we generated AAV2/5 vectors expressing EGFP and containing four tandem copies of miR-124 or miR-204 complementary sequences in the 39UTR of the transgene expression cassette. These vectors were administered subretinally to adult C57BL/6 mice and Large White pigs. Our results demonstrate that miR-124 and miR-204 target sequences can efficiently restrict AAV2/5-mediated transgene expression to retinal pigment epithelium and photoreceptors, respectively, in mice and pigs. Interestingly, transgene restriction was observed at low vector doses relevant to therapy. Conclusions: We conclude that microRNA-mediated regulation of transgene expression can be applied in the retina to either restrict to a specific cell type the robust expression obtained using ubiquitous promoters or to provide an additiona

The biphasic increase of PIP2 in the fertilized eggs of starfish: new roles in actin polymerization and Ca2+ signaling.

BACKGROUND: Fertilization of echinoderm eggs is accompanied by dynamic changes of the actin cytoskeleton and by a drastic increase of cytosolic Ca(2+). Since the plasma membrane-enriched phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) serves as the precursor of inositol 1,4,5 trisphosphate (InsP(3)) and also regulates actin-binding proteins, PIP2 might be involved in these two processes. METHODOLOGY/PRINCIPAL FINDINGS: In this report, we have studied the roles of PIP2 at fertilization of starfish eggs by using fluorescently tagged pleckstrin homology (PH) domain of PLC-δ1, which has specific binding affinity to PIP2, in combination with Ca(2+) and F-actin imaging techniques and transmission electron microscopy. During fertilization, PIP2 increased at the plasma membrane in two phases rather than continually decreasing. The first increase was quickly followed by a decrease about 40 seconds after sperm-egg contact. However, these changes took place only after the Ca(2+) wave had already initiated and propagated. The fertilized eggs then displayed a prolonged increase of PIP2 that was accompanied by the appearance of numerous spikes in the perivitelline space during the elevation of the fertilization envelope (FE). These spikes, protruding from the plasma membrane, were filled with microfilaments. Sequestration of PIP2 by RFP-PH at higher doses resulted in changes of subplasmalemmal actin networks which significantly delayed the intracellular Ca(2+) signaling, impaired elevation of FE, and increased occurrences of polyspermic fertilization. CONCLUSIONS/SIGNIFICANCE: Our results suggest that PIP2 plays comprehensive roles in shaping Ca(2+) waves and guiding structural and functional changes required for successful fertilization. We propose that the PIP2 increase and the subsequent formation of actin spikes not only provide the mechanical supports for the elevating FE, but also accommodate increased membrane surfaces during cortical granule exocytosis

Microvilli on the surface of starfish oocytes

<div>A short movie clip of video animation showing the</div><div>fluctuating microvilli on the surface of a starfish oocyte (<i>Astropecten aranciacus</i>). Following microinjection with PH-GFP, a serial of 10 sequential images were taken by confocal microscopy (47 sec intervals) and composited in MetaMorph.</div><div><br></div><div><br></div

Recombinant Vectors Based on Porcine Adeno-Associated Viral Serotypes Transduce the Murine and Pig Retina

<div><p>Recombinant adeno-associated viral (AAV) vectors are known to safely and efficiently transduce the retina. Among the various AAV serotypes available, AAV2/5 and 2/8 are the most effective for gene transfer to photoreceptors (PR), which are the most relevant targets for gene therapy of inherited retinal degenerations. However, the search for novel AAV serotypes with improved PR transduction is ongoing. In this work we tested vectors derived from five AAV serotypes isolated from porcine tissues (referred to as porcine AAVs, four of which are newly identified) for their ability to transduce both the murine and the cone-enriched pig retina. Porcine AAV vectors expressing EGFP under the control of the CMV promoter were injected subretinally either in C57BL/6 mice or Large White pigs. The resulting retinal tropism was analyzed one month later on histological sections, while levels of PR transduction were assessed by Western blot. Our results show that all porcine AAV transduce murine and porcine retinal pigment epithelium and PR upon subretinal administration. AAV2/po1 and 2/po5 are the most efficient porcine AAVs for murine PR transduction and exhibit the strongest tropism for pig cone PR. The levels of PR transduction obtained with AAV2/po1 and 2/po5 are similar, albeit not superior, to those obtained with AAV2/5 and AAV2/8, which evinces AAV2/po1 and 2/po5 to be promising vectors for retinal gene therapy.</p> </div

Phylogenetic tree obtained through the alignment of the <i>cap</i> sequences of the newly isolated porcine serotypes and other preexisting AAVs.

<p>Phylogenetic tree obtained through the alignment of the <i>cap</i> sequences of the newly isolated porcine serotypes and other preexisting AAVs.</p

Quantification of PR-specific EGFP expression following subretinal delivery of AAV vectors.

<p><b>A:</b> Representative Western blot out of five, each containing lysates (50 μg/lane) from retinas injected subretinally one month before with the various AAV vectors (indicated above each lane) containing the Rho promoter (6E9 GC/eye). Each blot was incubated with anti-EGFP (upper panel, α-EGFP) and anti-β-tubulin (lower panel, for normalization, α-β-tubulin) antibodies. The molecular weight is indicated on the left. <b>B:</b> Quantification of PR-specific EGFP expression mediated by each AAV vector (indicated below each bar), compared to AAV2/5. The intensity of the EGFP and β-tubulin bands was quantified and the resulting EGFP/β-tubulin ratio values were divided by those of AAV2/5. Averages (indicated above or inside each bar) ± SE for the 5 retinas injected with each AAV have been plotted. Statistical differences were calculated using one-way ANOVA (p-value  = 1,87E-5). * corresponds to p≤0.05, ** to p≤0.01, *** to p≤0.001 and **** to p≤0.0001.</p