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.

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.

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.

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.

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.

Retrospective on the development of aequorin and aequorin-based imaging to visualize changes in intracellular free [Ca(2+) ].

In this review, we take a retrospective look at the discovery and utilization of the Ca(2+) -sensitive bioluminescent protein complex, aequorin. We do consider the contribution it has made to our understanding of the natural phenomenon of bioluminescence, but it is in the application of extracted and purified aequorin as a reporter of Ca(2+) dynamics in living cells, which is arguably its major contribution to biological and biomedical science. Following its extraction, purification, and subsequent availability in the mid-1960s, aequorin became the intracellular reporter of choice until it was replaced in the late 1970s by easier-to-use fluorescence-based reporters. From the mid-1980s onwards, however, aequorin-based Ca(2+) imaging underwent a renaissance following the cloning of the aequorin gene and the emergence of routine techniques to target and express it exogenously in plant and animal systems. The development of aequorin as a tool continues as spectral varieties are being developed that allow simultaneous imaging of Ca(2+) dynamics in different cellular organelles and microdomains. We predict that further developments in the use of aequorin, as well as other bioluminescent proteins, will continue, especially in the areas of regenerative medicine and whole organism imaging.

Characterization of Ca(2+) signaling in the external yolk syncytial layer during the late blastula and early gastrula periods of zebrafish development.

Preferential loading of the complementary bioluminescent (f-aequorin) and fluorescent (Calcium Green-1 dextran) Ca(2+) reporters into the yolk syncytial layer (YSL) of zebrafish embryos, revealed the generation of stochastic patterns of fast, short-range, and slow, long-range Ca(2+) waves that propagate exclusively through the external YSL (E-YSL). Starting abruptly just after doming (~4.5h post-fertilization: hpf), and ending at the shield stage (~6.0hpf) these distinct classes of waves propagated at mean velocities of ~50 and ~4µm/s, respectively. Although the number and pattern of these waves varied between embryos, their initiation site and arcs of propagation displayed a distinct dorsal bias, suggesting an association with the formation and maintenance of the nascent dorsal-ventral axis. Wave initiation coincided with a characteristic clustering of YSL nuclei (YSN), and their associated perinuclear ER, in the E-YSL. Furthermore, the inter-YSN distance (IND) appeared to be critical such that Ca(2+) wave propagation occurred only when this was <~8µm; an IND >~8µm was coincidental with wave termination at shield stage. Treatment with the IP3R antagonist, 2-APB, the Ca(2+) buffer, 5,5'-dibromo BAPTA, and the SERCA-pump inhibitor, thapsigargin, resulted in a significant disruption of the E-YSL Ca(2+) waves, whereas exposure to the RyR antagonists, ryanodine and dantrolene, had no significant effect. These findings led us to propose that the E-YSL Ca(2+) waves are generated mainly via Ca(2+) release from IP3Rs located in the perinuclear ER, and that the clustering of the YSN is an essential step in providing a CICR pathway required for wave propagation. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.

Biphasic assembly of the contractile apparatus during the first two cell division cycles in zebrafish embryos.

The large and optically clear embryos of the zebrafish provide an excellent model system in which to study the dynamic assembly of the essential contractile band components, actin and myosin, via double fluorescent labelling in combination with confocal microscopy. We report the rapid appearance (i.e. within <2 min) of a restricted arc of F-actin patches along the prospective furrow plane in a central, apical region of the blastodisc cortex. These patches then fused with each other end-to-end forming multiple actin cables, which were subsequently bundled together forming an F-actin band. During this initial assembly phase, the F-actin-based structure did not elongate laterally, but was still restricted to an arc extending ~15° either side of the blastodisc apex. This initial assembly phase was then followed by an extension phase, where additional F-actin patches were added to each end of the original arc, thus extending it out to the edges of the blastodisc. The dynamics of phosphorylated myosin light chain 2 (MLC2) recruitment to this F-actin scaffold also reflect the two-phase nature of the contractile apparatus assembly. MLC2 was not associated with the initial F-actin arc, but MLC2 clusters were recruited and assembled into the extending ends of the band. We propose that the MLC2-free central region of the contractile apparatus acts to position and then extend the cleavage furrow in the correct plane, while the actomyosin ends alone generate the force required for furrow ingression. This biphasic assembly strategy may be required to successfully divide the early cells of large embryos.

Aequorin-based genetic approaches to visualize Ca2+ signaling in developing animal systems.

BACKGROUND: In recent years, as our understanding of the various roles played by Ca2+ signaling in development and differentiation has expanded, the challenge of imaging Ca2+ dynamics within living cells, tissues, and whole animal systems has been extended to include specific signaling activity in organelles and non-membrane bound sub-cellular domains. SCOPE OF REVIEW: In this review we outline how recent advances in genetics and molecular biology have contributed to improving and developing current bioluminescence-based Ca2+ imaging techniques. Reporters can now be targeted to specific cell types, or indeed organelles or domains within a particular cell. MAJOR CONCLUSIONS: These advances have contributed to our current understanding of the specificity and heterogeneity of developmental Ca2+ signaling. The improvement in the spatial resolution that results from specifically targeting a Ca2+ reporter has helped to reveal how a ubiquitous signaling messenger like Ca2+ can regulate coincidental but different signaling events within an individual cell; a Ca2+ signaling paradox that until now has been hard to explain. GENERAL SIGNIFICANCE: Techniques used to target specific reporters via genetic means will have applications beyond those of the Ca2+ signaling field, and these will, therefore, make a significant contribution in extending our understanding of the signaling networks that regulate animal development. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signalling.

A Role for Two-Pore Channel Type 2 (TPC2)-Mediated Regulation of Membrane Contact Sites During Zebrafish Notochord Biogenesis?

We have previously shown that in the developing trunk of zebrafish embryos, two-pore channel type 2 (TPC2)-mediated Ca2+ release from endolysosomes plays a role in the formation of the skeletal slow muscle. In addition, TPC2-mediated Ca2+ signaling is required for axon extension and the establishment of synchronized activity in the primary motor neurons. Here, we report that TPC2 might also play a role in the development of the notochord of zebrafish embryos. For example, when tpcn2 was knocked down or out, increased numbers of small vacuoles were formed in the inner notochord cells, compared with the single large vacuole in the notochord of control embryos. This abnormal vacuolation was associated with embryos displaying attenuated body axis straightening. We also showed that TPC2 has a distinct pattern of localization in the notochord in embryos at ∼24 hpf. Finally, we conducted RNAseq to identify differentially expressed genes in tpcn2 mutants compared to wild-type controls, and found that those involved in actin filament severing, cellular component morphogenesis, Ca2+ binding, and structural constituent of cytoskeleton were downregulated in the mutants. Together, our data suggest that TPC2 activity plays a key role in notochord biogenesis in zebrafish embryos.