Localized TPC1-mediated Ca2+ release from endolysosomes contributes to myoseptal junction development in zebrafish.

In the trunk of developing zebrafish embryos, adjacent myotome blocks transmit contractile force via myoseptal junctions (MJs), which are dynamic structures that connect the actin cytoskeleton of skeletal muscle cells to extracellular matrix components via transmembrane protein complexes in the sarcolemma. Here, we report that the endolysosomal ion channel, two-pore channel type 1 (TPC1, encoded by tpcn1), generates highly localized non-propagating Ca2+ transients that play a distinct and required role in the capture and attachment of superficial slow skeletal muscle cells at MJs. Use of antisense morpholinos or CRISPR/Cas9 gene editing to disrupt tpcn1 gene expression resulted in abnormal MJ phenotypes, including slow skeletal muscle cells detaching from or crossing the myosepta. We also report that TPC1-decorated endolysosomes are dynamically associated with MJs in a microtubule-dependent manner, and that attenuating tpcn1 expression or TPC1 function disrupted endolysosomal trafficking and resulted in an abnormal distribution of ß-dystroglycan (encoded by dag1; a key transmembrane component of the dystrophin-associated protein complex). Taken together, our data suggest that localized TPC1-generated Ca2+ signals facilitate essential endolysosomal trafficking and membrane contact events, which help form and maintain MJs following the onset of slow skeletal muscle cell contractile activity. This article has an associated First Person interview with the first author of the paper.

Computational Docking as a Tool in Guiding the Drug Design of Rutaecarpine Derivatives as Potential SARS-CoV-2 Inhibitors.

COVID-19 continues to spread around the world. This is mainly because new variants of the SARS-CoV-2 virus emerge due to genomic mutations, evade the immune system and result in the effectiveness of current therapeutics being reduced. We previously established a series of detection platforms, comprising computational docking analysis, S-protein-based ELISA, pseudovirus entry, and 3CL protease activity assays, which allow us to screen a large library of phytochemicals from natural products and to determine their potential in blocking the entry of SARS-CoV-2. In this new screen, rutaecarpine (an alkaloid from Evodia rutaecarpa) was identified as exhibiting anti-SARS-CoV-2 activity. Therefore, we conducted multiple rounds of structure-activity-relationship (SAR) studies around this phytochemical and generated several rutaecarpine analogs that were subjected to in vitro evaluations. Among these derivatives, RU-75 and RU-184 displayed remarkable inhibitory activity when tested in the 3CL protease assay, S-protein-based ELISA, and pseudovirus entry assay (for both wild-type and omicron variants), and they attenuated the inflammatory response induced by SARS-CoV-2. Interestingly, RU-75 and RU-184 both appeared to be more potent than rutaecarpine itself, and this suggests that they might be considered as lead candidates for future pharmacological elaboration.

Neuromasts and Olfactory Organs of Zebrafish Larvae Represent Possible Sites of SARS-CoV-2 Pseudovirus Host Cell Entry.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the acute respiratory disease coronavirus disease 2019 (COVID-19), which has resulted in millions of deaths globally. Here, we explored the mechanism of host cell entry of a luciferase-ZsGreen spike (SARS-CoV-2)-pseudotyped lentivirus using zebrafish embryos/larvae as an in vivo model. Successful pseudovirus entry was demonstrated via the expression of the luciferase (luc) gene, which was validated by reverse transcription-PCR (RT-PCR). Treatment of larvae with chloroquine (a broad-spectrum viral inhibitor that blocks membrane fusion) or bafilomycin A1 (a specific inhibitor of vacuolar proton ATPases, which blocks endolysosomal trafficking) significantly reduced luc expression, indicating the possible involvement of the endolysosomal system in the viral entry mechanism. The pharmacological inhibition of two-pore channel (TPC) activity or use of the tpcn2dhkz1a mutant zebrafish line also led to diminished luc expression. The localized expression of ACE2 and TPC2 in the anterior neuromasts and the forming olfactory organs was demonstrated, and the occurrence of endocytosis in both locations was confirmed. Together, our data indicate that zebrafish embryos/larvae are a viable and tractable model to explore the mechanism of SARS-CoV-2 host cell entry, that the peripheral sense organs are a likely site for viral host cell entry, and that TPC2 plays a key role in the translocation of the virus through the endolysosomal system. IMPORTANCE Despite the development of effective vaccines to combat the COVID-19 pandemic, which help prevent the most life-threatening symptoms, full protection cannot be guaranteed, especially with the emergence of new viral variants. Moreover, some resistance to vaccination remains in certain age groups and cultures. As such, there is an urgent need for the development of new strategies and therapies to help combat this deadly disease. Here, we provide compelling evidence that the peripheral sensory organs of zebrafish possess several key components required for SARS-CoV-2 host cell entry. The nearly transparent larvae provide a most amenable complementary platform to investigate the key steps of viral entry into host cells, as well as its spread through the tissues and organs. This will help in the identification of key viral entry steps for therapeutic intervention, provide an inexpensive model for screening novel antiviral compounds, and assist in the development of new and more effective vaccines.

Probing the Pulsar Explanation of the Galactic-Center GeV Excess Using Continuous Gravitational-Wave Searches.

Over 10 years ago, Fermi observed an excess of GeV gamma rays from the Galactic Center whose origin is still under debate. One explanation for this excess involves annihilating dark matter, another requires an unresolved population of millisecond pulsars concentrated at the Galactic Center. In this work, we use the results from LIGO and Virgo's most recent all-sky search for quasimonochromatic, persistent gravitational-wave signals from isolated neutron stars, which is estimated to be about 20%-50% of the population, to determine whether unresolved millisecond pulsars could actually explain this excess. First, we choose a luminosity function that determines the number of millisecond pulsars required to explain the observed excess. Then, we consider two models for deformations on millisecond pulsars to determine their ellipticity distributions, which are directly related to their gravitational-wave radiation. Lastly, based on null results from the O3 frequency-Hough all-sky search for continuous gravitational waves, we find that a large set of the parameter space in the pulsar luminosity function can be excluded. We also evaluate how these exclusion regions may change with respect to various model choices. Our results are the first of their kind and represent a bridge between gamma-ray astrophysics, gravitational-wave astronomy, and dark-matter physics.

Actin-mediated endocytosis in the E-YSL helps drive epiboly in zebrafish.

In zebrafish, a punctate band of F-actin is reported to develop in the external yolk syncytial layer (E-YSL) during the latter part of epiboly in zebrafish embryos. Here, electron microscopy (EM) and fluorescence confocal microscopy were conducted to investigate dynamic changes in the E-YSL membrane during epiboly. Using scanning EM, we report that the surface of the E-YSL is highly convoluted, consisting of a complex interwoven network of branching membrane surface microvilli-like protrusions. The region of membrane surface protrusions was relatively wide at 30% epiboly but narrowed as epiboly progressed. This narrowing was coincident with the formation of the punctate actin band. We also demonstrated using immunogold transmission EM that actin clusters were localized at the membrane surface mainly within the protrusions as well as in deeper locations of the E-YSL. Furthermore, during the latter part of epiboly, the punctate actin band was coincident with a region of highly dynamic endocytosis. Treatment with cytochalasin B led to the disruption of the punctate actin band and the membrane surface protrusions, as well as the attenuation of endocytosis. Together, our data suggest that, in the E-YSL, the region encompassing the membrane surface protrusions and its associated punctate actin band are likely to be an integral part of the localized endocytosis, which is important for the progression of epiboly in zebrafish embryos.

The Ethanol Extract of Evodiae Fructus and Its Ingredient, Rutaecarpine, Inhibit Infection of SARS-CoV-2 and Inflammatory Responses.

COVID-19, derived from SARS-CoV-2, has resulted in millions of deaths and caused unprecedented socioeconomic damage since its outbreak in 2019. Although the vaccines developed against SARS-CoV-2 provide some protection, they have unexpected side effects in some people. Furthermore, new viral mutations reduce the effectiveness of the current vaccines. Thus, there is still an urgent need to develop potent non-vaccine therapeutics against this infectious disease. We recently established a series of detecting platforms to screen a large library of Chinese medicinal herbs and phytochemicals. Here, we reveal that the ethanolic extract of Evodiae Fructus and one of its components, rutaecarpine, showed promising potency in inhibiting the activity of 3C-like (3CL) protease, blocking the entry of the pseudo-typed SARS-CoV-2 (including wild-type and omicron) into cultured cells. In addition, inflammatory responses induced by pseudo-typed SARS-CoV-2 were markedly reduced by Evodiae Fructus extract and rutaecarpine. Together our data indicate that the herbal extract of Evodiae Fructus and rutaecarpine are potent anti-SARS-CoV-2 agents, which might be considered as a treatment against COVID-19 in clinical applications.

TPC2-mediated Ca2+ signaling is required for axon extension in caudal primary motor neurons in zebrafish embryos.

The role of two-pore channel type 2 (TPC2, encoded by tcpn2)-mediated Ca2+ release was recently characterized in zebrafish during establishment of the early spinal circuitry, one of the key events in the coordination of neuromuscular activity. Here, we extend our study to investigate the in vivo role of TPC2 in the regulation of caudal primary motor neuron (CaP) axon extension. We used a combination of TPC2 knockdown with a translation-blocking morpholino antisense oligonucleotide (MO), TPC2 knockout via the generation of a tpcn2dhkz1a mutant line of zebrafish using CRISPR/Cas9 gene-editing and pharmacological inhibition of TPC2 via incubation with bafilomycin A1 (an H+-ATPase inhibitor) or trans-ned-19 (an NAADP receptor antagonist), and showed that these treatments attenuated CaP Ca2+ signaling and inhibited axon extension. We also characterized the expression of an arc1-like transcript in CaPs grown in primary culture. MO-mediated knockdown of ARC1-like in vivo led to attenuation of the Ca2+ transients in the CaP growth cones and an inhibition of axon extension. Together, our new data suggest a link between ARC1-like, TPC2 and Ca2+ signaling during axon extension in zebrafish.

Transmembrane H+ fluxes and the regulation of neural induction in Xenopus laevis.

It has previously been reported that in ex vivo planar explants prepared from Xenopus laevis embryos, the intracellular pH (pHi) increases in cells of the dorsal ectoderm from stage 10.5 to 11.5 (i.e. 11-12.5 hpf). It was proposed that such increases (potentially due to H+ being extruded, sequestered, or buffered in some manner), play a role in regulating neural induction. Here, we used an extracellular ion-selective electrode to non-invasively measure H+ fluxes at eight locations around the equatorial circumference of intact X. laevis embryos between stages 9-12 (˜7-13.25 hpf). We showed that at stages 9-11, there was a small H+ efflux recorded from all the measuring positions. At stage 12 there was a small, but significant, increase in the efflux of H+ from most locations, but the efflux from the dorsal side of the embryo was significantly greater than from the other positions. Embryos were also treated from stages 9-12 with bafilomycin A1, to block the activity of the ATP-driven H+ pump. By stage 22 (24 hpf), these embryos displayed retarded development, arresting before the end of gastrulation and therefore did not display the usual anterior and neural structures, which were observed in the solvent-control embryos. In addition, expression of the early neural gene, Zic3, was absent in treated embryos compared with the solvent controls. Together, our new in vivo data corroborated and extended the earlier explant-derived report describing changes in pHi that were suggested to play a role during neural induction in X. laevis embryos.

The Extracts of Polygonum cuspidatum Root and Rhizome Block the Entry of SARS-CoV-2 Wild-Type and Omicron Pseudotyped Viruses via Inhibition of the S-Protein and 3CL Protease.

COVID-19, resulting from infection by the SARS-CoV-2 virus, caused a contagious pandemic. Even with the current vaccines, there is still an urgent need to develop effective pharmacological treatments against this deadly disease. Here, we show that the water and ethanol extracts of the root and rhizome of Polygonum cuspidatum (Polygoni Cuspidati Rhizoma et Radix), a common Chinese herbal medicine, blocked the entry of wild-type and the omicron variant of the SARS-CoV-2 pseudotyped virus into fibroblasts or zebrafish larvae, with IC50 values ranging from 0.015 to 0.04 mg/mL. The extracts were shown to inhibit various aspects of the pseudovirus entry, including the interaction between the spike protein (S-protein) and the angiotensin-converting enzyme II (ACE2) receptor, and the 3CL protease activity. Out of the chemical compounds tested in this report, gallic acid, a phytochemical in P. cuspidatum, was shown to have a significant anti-viral effect. Therefore, this might be responsible, at least in part, for the anti-viral efficacy of the herbal extract. Together, our data suggest that the extracts of P. cuspidatum inhibit the entry of wild-type and the omicron variant of SARS-CoV-2, and so they could be considered as potent treatments against COVID-19.

Has stocking contributed to an increase in the rod catch of anadromous trout (Salmo trutta L.) in the Shetland Islands, UK?

The stocking of hatchery-origin fish into rivers and lakes has long been used in fisheries management to try to enhance catches, especially for trout and salmon species. Frequently, however, the long-term impacts of stocking programmes have not been evaluated. In this study, the authors investigate the contribution of a stocking programme undertaken to support the rod catch of sea trout in the Shetland Islands, UK. Once a highly productive recreational fishery, Shetland sea trout catches crashed in the mid-1990s. Around the time that stocking began, increases in rod catches were also reported, with advocates of the stocking highlighting the apparent success of the programme. Using a suite of genetic markers (microsatellites), this study explores the contribution of the stocking programme to the Shetland sea trout population. The authors found that the domesticated broodstock and wild spawned brown trout from seven streams were genetically distinct. Despite extensive stocking, wild spawned brown trout dominated, even in those streams with a long history of supplementation. The majority of sea trout caught and analysed were of wild origin - only a single individual was of pure stocked origin, with a small number of fish being of wild × stocked origins. This study suggests that stocking with a domesticated strain of brown trout has made only a very limited contribution to the Shetland Islands rod catch, and that the revival of sea trout numbers appears to be driven almost exclusively by recovery of trout spawned in the wild.

Short-term homeostatic regulation of blood/interstitial fluid Ca2+ concentration by the scales of anadromous sea trout Salmo trutta L. during smoltification and migration.

The elasmoid scales of anadromous sea trout Salmo trutta L. represent a significant internal reservoir of Ca2+ . Although more is known about long-term remodelling of scales in response to calciotropic challenges encountered during smoltification and migration, very little is known about the contribution made by scales to the short-term, minute-to-minute regulation of Ca2+ homeostasis in the extracellular fluid (ECF) during these phases of the life cycle. This gap in the knowledge is partly due to the technical challenges involved in measuring small Ca2+ fluxes around the scales of live fish in real time. Here, this study describes exfoliating, mounting and culturing scales and their resident cells from parr, smolt and adult sea trout from a freshwater environment, as well as from adult sea trout caught in sea or brackish water. All the scales were then examined using an extracellular, non-invasive, surface-scanning Ca2+ -sensitive microelectrode. The authors quantified the Ca2+ fluxes, in the absence of any systemic or local regulators, into and out of scales on both the episquamal and hyposquamal sides under different extracellular calcemic challenges set to mimic a variety of ECF-Ca2+ concentrations. Scales from the life-cycle stages as well as from adult fish taken from sea, brackish or fresh water all showed a consistent efflux or influx of Ca2+ under hypo- or hypercalcemic conditions, respectively. What were considered to be isocalcemic conditions resulted in minimal flux of Ca2+ in either direction, or in the case of adult scales, a consistent but small influx. Indeed, adult scales appeared to display the largest flux densities in either direction. These new data extend the current understanding of the role played by fish scales in the short-term, minute-to-minute homeostatic regulation of ECF-Ca2+ concentration, and are similar to those recently reported from zebrafish Danio rerio scales. This suggests that this short-term regulatory response might be a common feature of teleost scales.

Characterization of ADP-ribosyl cyclase 1-like (ARC1-like) activity and NAADP signaling during slow muscle cell development in zebrafish embryos.

We recently demonstrated the requirement of two-pore channel type 2 (TPC2)-mediated Ca2+ release during slow muscle cell differentiation and motor circuit maturation in intact zebrafish embryos. However, the upstream trigger(s) of TPC2/Ca2+ signaling during these developmental processes remains unclear. Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+ mobilizing messenger, which is suggested to target TPC2 in mediating the release of Ca2+ from acidic vesicles. Here, we report the molecular cloning of the zebrafish ADP ribosyl cyclase (ARC) homolog (i.e., ARC1-like), which is a putative enzyme for generating NAADP. We characterized the expression of the arc1-like transcript and the NAADP levels between ~ 16 h post-fertilization (hpf) and ~ 48 hpf in whole zebrafish embryos. We showed that if ARC1-like (when fused with either EGFP or tdTomato) was overexpressed it localized in the plasma membrane, and associated with intracellular organelles, such as the acidic vesicles, Golgi complex and sarcoplasmic reticulum, in primary muscle cell cultures. Morpholino (MO)-mediated knockdown of arc1-like or pharmacological inhibition of ARC1-like (via treatment with nicotinamide), led to an attenuation of Ca2+ signaling and disruption of slow muscle cell development. In addition, the injection of arc1-like mRNA into ARC1-like morphants partially rescued the Ca2+ signals and slow muscle cell development. Together, our data might suggest a link between ARC1-like, NAADP, TPC2 and Ca2+ signaling during zebrafish myogenesis.

TPC2-mediated Ca2+ signaling is required for the establishment of synchronized activity in developing zebrafish primary motor neurons.

During the development of the early spinal circuitry in zebrafish, spontaneous Ca2+ transients in the primary motor neurons (PMNs) are reported to transform from being slow and uncorrelated, to being rapid, synchronized and patterned. In this study, we demonstrated that in intact zebrafish, Ca2+ release via two-pore channel type 2 (TPC2) from acidic stores/endolysosomes is required for the establishment of synchronized activity in the PMNs. Using the SAIGFF213A;UAS:GCaMP7a double-transgenic zebrafish line, Ca2+ transients were visualized in the caudal PMNs (CaPs). TPC2 inhibition via molecular, genetic or pharmacological means attenuated the CaP Ca2+ transients, and decreased the normal ipsilateral correlation and contralateral anti-correlation, indicating a disruption in normal spinal circuitry maturation. Furthermore, treatment with MS-222 resulted in a complete (but reversible) inhibition of the CaP Ca2+ transients, as well as a significant decrease in the concentration of the Ca2+ mobilizing messenger, nicotinic acid adenine diphosphate (NAADP) in whole embryo extract. Together, our new data suggest a novel function for NAADP/TPC2-mediated Ca2+ signaling in the development, coordination, and maturation of the spinal network in zebrafish embryos.

Ca2+ release via two-pore channel type 2 (TPC2) is required for slow muscle cell myofibrillogenesis and myotomal patterning in intact zebrafish embryos.

We recently demonstrated a critical role for two-pore channel type 2 (TPC2)-mediated Ca2+ release during the differentiation of slow (skeletal) muscle cells (SMC) in intact zebrafish embryos, via the introduction of a translational-blocking morpholino antisense oligonucleotide (MO). Here, we extend our study and demonstrate that knockdown of TPC2 with a non-overlapping splice-blocking MO, knockout of TPC2 (via the generation of a tpcn2dhkz1a mutant line of zebrafish using CRISPR/Cas9 gene-editing), or the pharmacological inhibition of TPC2 action with bafilomycin A1 or trans-ned-19, also lead to a significant attenuation of SMC differentiation, characterized by a disruption of SMC myofibrillogenesis and gross morphological changes in the trunk musculature. When the morphants were injected with tpcn2-mRNA or were treated with IP3/BM or caffeine (agonists of the inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RyR), respectively), many aspects of myofibrillogenesis and myotomal patterning (and in the case of the pharmacological treatments, the Ca2+ signals generated in the SMCs), were rescued. STED super-resolution microscopy revealed a close physical relationship between clusters of RyR in the terminal cisternae of the sarcoplasmic reticulum (SR), and TPC2 in lysosomes, with a mean estimated separation of ~52-87nm. Our data therefore add to the increasing body of evidence, which indicate that localized Ca2+ release via TPC2 might trigger the generation of more global Ca2+ release from the SR via Ca2+-induced Ca2+ release.

Dual Functional Roles of Molecular Beacon as a MicroRNA Detector and Inhibitor.

MicroRNAs are essential in many cellular processes. The ability to detect microRNAs is important for understanding its function and biogenesis. This study is aimed at using a molecular beacon to detect miR-430 in developing zebrafish embryos as a proof of principle. miR-430 is crucial for the clearance of maternal mRNA during maternal zygotic transition in embryonic development. Despite its known function, the temporal and spatial expression of miR-430 remains unclear. We used various imaging techniques, including laser scanning confocal microscopy, spinning disk, and lightsheet microscopy, to study the localization of miR-430 and any developmental defects possibly caused by the molecular beacon. Our results show that miR-430 is expressed early in development and is localized in distinct cytoplasmic granules where its target mRNA can be detected. We also show that the designed molecular beacon can inhibit the function of miR-430 and cause developmental defect in the brain, notochord, heart, and kidney, depending on the delivery site within the embryo, suggesting that miR-430 plays a diverse role in embryonic morphogenesis. When compared with morpholino, molecular beacon is 2 orders of magnitude more potent in inhibiting miR-430. Thus, our results reveal that in addition to being used as a valuable tool for the detection of microRNAs in vivo, molecular beacons can also be employed to inhibit microRNAs in a specific manner.

Calcium signaling orchestrates glioblastoma development: Facts and conjunctures.

While it is a relatively rare disease, glioblastoma multiform (GBM) is one of the more deadly adult cancers. Following current interventions, the tumor is never eliminated whatever the treatment performed; whether it is radiotherapy, chemotherapy, or surgery. One hypothesis to explain this poor outcome is the "cancer stem cell" hypothesis. This concept proposes that a minority of cells within the tumor mass share many of the properties of adult neural stem cells and it is these that are responsible for the growth of the tumor and its resistance to existing therapies. Accumulating evidence suggests that Ca(2+) might also be an important positive regulator of tumorigenesis in GBM, in processes involving quiescence, maintenance, proliferation, or migration. Glioblastoma tumors are generally thought to develop by co-opting pathways that are involved in the formation of an organ. We propose that the cells initiating the tumor, and subsequently the cells of the tumor mass, must hijack the different checkpoints that evolution has selected in order to prevent the pathological development of an organ. In this article, two main points are discussed. (i) The first is the establishment of a so-called "cellular society," which is required to create a favorable microenvironment. (ii) The second is that GBM can be considered to be an organism, which fights to survive and develop. Since GBM evolves in a limited space, its only chance of development is to overcome the evolutionary checkpoints. For example, the deregulation of the normal Ca(2+) signaling elements contributes to the progression of the disease. Thus, by manipulating the Ca(2+) signaling, the GBM cells might not be killed, but might be reprogrammed toward a new fate that is either easy to cure or that has no aberrant functioning. This article is part of a Special Issue entitled: Calcium and Cell Fate. Guest Editors: Jacques Haiech, Claus Heizmann, Joachim Krebs, Thierry Capiod and Olivier Mignen.

Ca2+ Signalling and Membrane Dynamics During Cytokinesis in Animal Cells.

Interest in the role of Ca2+ signalling as a possible regulator of the combinatorial processes that result in the separation of the daughter cells during cytokinesis, extend back almost a 100 years. One of the key processes required for the successful completion of cytokinesis in animal cells (especially in the large holoblastically and meroblastically dividing embryonic cells of a number of amphibian and fish species), is the dynamic remodelling of the plasma membrane. Ca2+ signalling was subsequently demonstrated to regulate various different aspects of cytokinesis in animal cells, and so here we focus specifically on the role of Ca2+ signalling in the remodelling of the plasma membrane. We begin by providing a brief history of the animal models used and the research accomplished by the early twentieth century investigators, with regards to this aspect of animal cell cytokinesis. We then review the most recent progress made (i.e., in the last 10 years), which has significantly advanced our current understanding on the role of cytokinetic Ca2+ signalling in membrane remodelling. To this end, we initially summarize what is currently known about the Ca2+ transients generated during animal cell cytokinesis, and then we describe the latest findings regarding the source of Ca2+ generating these transients. Finally, we review the current evidence about the possible targets of the different cytokinetic Ca2+ transients with a particular emphasis on those that either directly or indirectly affect plasma membrane dynamics. With regards to the latter, we discuss the possible role of the early Ca2+ signalling events in the deformation of the plasma membrane at the start of cytokinesis (i.e., during furrow positioning), as well as the role of the subsequent Ca2+ signals in the trafficking and fusion of vesicles, which help to remodel the plasma membrane during the final stages of cell division. As it is becoming clear that each of the cytokinetic Ca2+ transients might have multiple, integrated targets, deciphering the precise role of each transient represents a significant (and ongoing) challenge.

Molecular insights into the coding region determinant-binding protein-RNA interaction through site-directed mutagenesis in the heterogeneous nuclear ribonucleoprotein-K-homology domains.

The ability of its four heterogeneous nuclear RNP-K-homology (KH) domains to physically associate with oncogenic mRNAs is a major criterion for the function of the coding region determinant-binding protein (CRD-BP). However, the particular RNA-binding role of each of the KH domains remains largely unresolved. Here, we mutated the first glycine to an aspartate in the universally conserved GXXG motif of the KH domain as an approach to investigate their role. Our results show that mutation of a single GXXG motif generally had no effect on binding, but the mutation in any two KH domains, with the exception of the combination of KH3 and KH4 domains, completely abrogated RNA binding in vitro and significantly retarded granule formation in zebrafish embryos, suggesting that any combination of at least two KH domains cooperate in tandem to bind RNA efficiently. Interestingly, we found that any single point mutation in one of the four KH domains significantly impacted CRD-BP binding to mRNAs in HeLa cells, suggesting that the dynamics of the CRD-BP-mRNA interaction vary over time in vivo. Furthermore, our results suggest that different mRNAs bind preferentially to distinct CRD-BP KH domains. The novel insights revealed in this study have important implications on the understanding of the oncogenic mechanism of CRD-BP as well as in the future design of inhibitors against CRD-BP function.

CD38 Is Required for Neural Differentiation of Mouse Embryonic Stem Cells by Modulating Reactive Oxygen Species.

CD38 is a multifunctional membrane enzyme and the main mammalian ADP-ribosyl cyclase, which catalyzes the synthesis and hydrolysis of cADPR, a potent endogenous Ca(2+) mobilizing messenger. Here, we explored the role of CD38 in the neural differentiation of mouse embryonic stem cells (ESCs). We found that the expression of CD38 was decreased during the differentiation of mouse ESCs initiated by adherent monoculture. Perturbing the CD38/cADPR signaling by either CD38 knockdown or treatment of cADPR antagonists inhibited the neural commitment of mouse ESCs, whereas overexpression of CD38 promoted it. Moreover, CD38 knockdown dampened reactive oxygen species (ROS) production during neural differentiation of ESCs by inhibiting NADPH oxidase activity, while CD38 overexpression enhanced it. Similarly, application of hydrogen peroxide mitigated the inhibitory effects of CD38 knockdown on neural differentiation of ESCs. Taken together, our data indicate that the CD38 signaling pathway is required for neural differentiation of mouse ESCs by modulating ROS production.

SOCE proteins, STIM1 and Orai1, are localized to the cleavage furrow during cytokinesis of the first and second cell division cycles in zebrafish embryos.

In zebrafish embryos, distinct Ca2+ transients are localized to the early cleavage furrows during the first few cell division cycles. These transients are generated mainly by release via IP3Rs in the endoplasmic reticulum, and they are necessary for furrow positioning, propagation, deepening and apposition. We previously showed, via the use of inhibitors, that store-operated Ca2+ entry (SOCE) also appears to be essential for maintaining the IP3R-mediated elevated levels of [Ca2+]i for the extended periods required for the completion of successful furrow deepening and daughter cell apposition in these large embryonic cells. Here, newly fertilized, dechorionated embryos were fixed at various times during the first and second cell division cycles and immunolabelled with antibodies to STIM1 and/or Orai1 (key components of SOCE). We show that both of these proteins have a dynamic pattern of localization during cytokinesis of the first two cell division cycles. These new data help to support our previous inhibitor results, and provide additional evidence that SOCE contributes to the maintenance of the sustained elevated Ca2+ that is required for the successful completion of cytokinesis in the large cells of embryonic zebrafish.