The Interactive Fly

Zygotically transcribed genes

Channels

  • Novel functional properties of Drosophila CNS glutamate receptors
  • Functional Coupling of K+-Cl- Cotransporter (KCC) to GABA-Gated Cl- Channels in the Central Nervous System of Drosophila melanogaster leads to altered drug sensitivities
  • A presynaptic glutamate receptor subunit confers robustness to neurotransmission and homeostatic potentiation
  • Drosophila Subdued is a moonlighting transmembrane protein (TMEM16) that transports ions and phospholipids
  • The Drosophila Trpm channel mediates calcium influx during egg activation


    Novel functional properties of Drosophila CNS glutamate receptors

    Phylogenetic analysis reveals AMPA, kainate, and NMDA receptor families in insect genomes, suggesting conserved functional properties corresponding to their vertebrate counterparts. However, heterologous expression of the Drosophila kainate receptor DKaiR1D and the AMPA receptor DGluR1A revealed novel ligand selectivity at odds with the classification used for vertebrate glutamate receptor ion channels (iGluRs). DKaiR1D forms a rapidly activating and desensitizing receptor that is inhibited by both NMDA and the NMDA receptor antagonist AP5; crystallization of the KaiR1D ligand-binding domain reveals that these ligands stabilize open cleft conformations, explaining their action as antagonists. Surprisingly, the AMPA receptor DGluR1A shows weak activation by its namesake agonist AMPA and also by quisqualate. Crystallization of the DGluR1A ligand-binding domain reveals amino acid exchanges that interfere with binding of these ligands. The unexpected ligand-binding profiles of insect iGluRs allows classical tools to be used in novel approaches for the study of synaptic regulation (Li, 2016). Video Abstract

    Glutamate is the major excitatory neurotransmitter in the vertebrate CNS; its actions are mediated largely via three classes of ionotropic glutamate receptors (iGluRs) named AMPA, kainate, and NMDA receptors. The classification of iGluRs into AMPA, kainate, and NMDA receptors was based on the efforts of medicinal chemists who identified subtype selective heterocyclic amino acids such as AMPA, kainate, and quisqualate and amino acid analogs such as NMDA and 2(R)-amino-5-phosphonopentanoic acid (D-AP5) that act as agonists and antagonists. This work was so successful that the selective action of NMDA and D-AP5 formed the corner stone on which the role of NMDA receptors in synaptic plasticity was established (Li, 2016).

    Subsequent cloning of insect iGluRs, which revealed sequence similarity with their vertebrate AMPA, kainate, and NMDA receptor counterparts, suggests that the same series of ligands can be used to investigate their role in CNS function. However, with the exception of the neuromuscular junction (NMJ) of larval Drosophila and the NMJ of adult locusts, the small size and inaccessibility of insect neurons has to date challenged characterization of the functional properties of native insect iGluRs. Sequence analysis of the Drosophila genome identified 14 iGluR genes that resemble vertebrate AMPA, kainate, and NMDA receptors. Transcript profiling revealed that nine of these iGluRs are expressed in the brain, with five expressed at the neuromuscular junction. Very little is known about the structure and functional properties of Drosophila iGluRs and only recently was a functional reconstitution achieved for recombinant Drosophila NMJ iGluRs (Han, 2015). As a result, iGluRs are understudied in model organisms like Drosophila for which powerful genetic techniques have otherwise yielded numerous insights into the molecular neurobiology of synapse development and function (Li, 2016).

    Four presumptive Drosophila kainate receptors (Clumsy, DKaiR1C, DKaiR1D, and CG11155) are functionally required for spectral preference behavior and are thought to mediate excitatory synaptic transmission from the second-order neuron Dm8 to the third-order neuron Tm5c (Karuppudurai, 2014). The eye-enriched kainate receptor (EKAR) is expressed in photoreceptors, receiving feedback glutamatergic signals from amacrine cells, but so far, in vitro reconstitution has not been achieved for any of these presumptive kainate receptors. Instead, functional analysis of their role in CNS glutamatergic circuits relies solely on chronic inactivation using genetic mutants and RNAi-mediated knockdown. This study combined electrophysiological, biochemical, and crystallographic analyses to determine receptor activity and ligand specificity of a Drosophila kainate receptor DKaiR1D and a Drosophila AMPA receptor DGluR1A. DKaiR1D was found to form functional homomeric channels in HEK cells and oocytes with pharmacological properties distinct from vertebrate and Drosophila NMJ iGluRs. Crystal structures of DKaiR1D ligand-binding dimer complexes with glutamate, NMDA, and AP5 revealed that only glutamate triggers domain closure and that NMDA and AP5 are antagonists. DGluR1A receptors respond weakly to AMPA and quisqualate; the crystal structure of DGluR1A revealed that the binding of these ligands is hindered by steric occlusion. Thus, despite structural and sequence similarity between insect and vertebrate iGluRs, insect iGluRs do not conform to the pharmacology-based classification of vertebrate iGluRs. However, the agonist/antagonist binding properties of insect iGluRs we report here provide a new approach for acute inactivation/activation in vivo and for dissecting their functions in complex neural circuits (Li, 2016).

    This study found that DGluR1A and DKaiR1D, similar to vertebrate GluA1-4 AMPA and GluK1-3 kainate receptor subunits, form homomeric calcium-permeable channels. Based on sequence alignments and the lack of RNA-editing of Drosophila iGluRs mRNA at their Q/R sites, it is likely that most insect iGluRs are calcium permeable and that they are inhibited by endogenous cytoplasmic polyamines and by spider venom polyamine toxins. It is noted that homomeric DKaiR1D has a very fast desensitization rate, while for DGluR1A, fit has not yet been possible to achieve sufficient expression to allow recording from outside-out patches with rapid perfusion. Structural analyses revealed that DKaiR1D LBD dimers contain conserved Na+ ion binding sites characteristic of vertebrate kainate receptors, but these appear to not strongly modulate the activation or desensitization of KaiR1D, perhaps because the Cl- binding site found in vertebrate kainate receptors is absent in insect kainate receptors. Sequence analysis revealed that this separation of Na+ and Cl- binding sites in KaiR1D subunits occurs in all insect species examined. Structure-aided sequence analysis also reveals that in the other three groups of fly kainate receptors, different combinations of amino acid substitutions destroy or significantly weaken both the Na+ and Cl− binding sites. Thus, the allosteric modulation by both anions and cations that is characteristic of vertebrate kainate receptors is uncoupled in insect kainate receptors and for the majority of cases both ion binding sites are eliminated (Li, 2016).

    Previous phylogenetic studies suggest that most bilateria, including insects, worms, and vertebrates, have three major classes of cation-selective iGluRs, corresponding to vertebrate AMPA, kainate, and NMDA receptors. The current analysis reveals that in insects, the kainate receptor family is expanded into four groups, while a prior phylogenetic analysis revealed that in Mollusca the AMPA receptor family is expanded. At the neuromuscular junction of Drosophila and the locust Schistocerca gregaria, iGluRs have been extensively studied, in part serving as a surrogate model for CNS iGluRs. Interestingly, this study found that late in evolution, in higher Diptera, the five Drosophila NMJ iGluR subunits, GluRIIA-E, were derived from two separate kainate receptor subtypes, KaiR1C and Clumsy. Thus, despite their unique obligate heterotetrameric subunit stoichiometry and insensitivity to kainate), fly NMJ iGluRs evolved from ancestral kainate-sensitive iGluRs, and it is likely that in other insect species iGluRs related to KaiR1C and Clumsy may function in both the CNS and NMJ (Li, 2016).

    Although phylogenetic analysis supports classification of Drosophila and other insect iGluRs into the familiar AMPA, kainate, and NMDA receptor families, the current results reveal unexpected differences in their ligand-binding properties. The most dramatic change was the conversion of NMDA from an agonist for vertebrate NMDA receptors to an antagonist for Drosophila KaiR1D, a kainate receptor that is also inhibited by both isomers of AP5, while D-AP5, but not L-AP5 acts as a potent vertebrate NMDA receptor antagonist. The crystal structures solved in this study for the DKaiR1D LBD establish that NMDA and AP5 inhibit activation of DKaiR1D by stabilizing an open cleft conformation, similar to the action of competitive antagonists for vertebrate iGluRs from each of the three major families. In addition, NMDA triggered separation of the upper lobes of the DKaiR1D LBD dimer assembly, a conformational change that occurs during desensitization of vertebrate AMPA and kainate receptors, may in addition contribute to the inhibitory action of NMDA on DKaiR1D (Li, 2016).

    AMPA receptors were initially identified by and named in response to their activation by quisqualic acid, a glutamate bioisostere that is nonselective and which activates all of the major vertebrate iGluR subtypes, in addition to acting as a potent agonist for G protein-coupled glutamate receptors. Prior to the cloning of GluA1–4 subunits, the so-called quisqualate receptors were renamed AMPA receptors, following the synthesis of AMPA and the discovery that it was a highly selective agonist, without activity at kainate, NMDA, or G protein-coupled glutamate receptors. These serendipitous events in the history of the development of selective ligands for iGluR subtypes were strongly reinforced when a large family of vertebrate iGluR subunits were cloned, and it was discovered that these encoded discrete families of iGluR subtypes, each with high sequence identity, the ligand-binding properties of which corresponded to the familiar AMPA, kainate, and NMDA receptor subtypes. The current experiments reveal an unexpected breakdown of the classification scheme for Drosophila and most likely other insect species iGluRs (Li, 2016).

    With the plethora of genetic tools and advanced connectome analyses, Drosophila has emerged as a key model organism for studying the circuit basis of behavior. It is now evident that like vertebrates, glutamatergic synapses are abundantly utilized in fly CNS circuits. Functional and structural analyses revealed that Drosophila iGluRs have agonist and antagonist selectivity very different from those of vertebrates, indicating that sequence and structural homology does not confer conserved pharmacological properties. However, the unique pharmacology of Drosophila iGluRs reported in this study has proven of use to reveal the role of KaiR1D in presynaptic homeostasis. It is envisioned that appropriate use of pharmacological tools in combination with powerful fly genetics will greatly aid studies of complex neural circuits in Drosophila (Li, 2016).

    Functional Coupling of K+-Cl- Cotransporter (KCC) to GABA-Gated Cl- Channels in the Central Nervous System of Drosophila melanogaster leads to altered drug sensitivities

    GABAergic signaling is the cornerstone for fast synaptic inhibition of neural signaling in arthropods and mammals and is the molecular target for insecticides and pharmaceuticals, respectively. The K+-Cl- cotransporter (KCC) is the primary mechanism by which mature neurons maintain low intracellular Cl- concentration, yet the fundamental physiology, comparative physiology, and toxicological relevance of insect KCC is understudied. Considering this, electrophysiological, genetic, and pharmacological methods were employed to characterize the physiological underpinnings of KCC function to the Drosophila CNS. The data show that genetic ablation or pharmacological inhibition of KCC results in an increased spike discharge frequency and significantly (P<0.05) reduces the CNS sensitivity to gamma-aminobutyric acid (GABA). Further, simultaneous inhibition of KCC and ligand-gated chloride channel (LGCC) complex results in a significant (P<0.001) increase in CNS spontaneous activity over baseline firing rates that, taken together, supports functional coupling of KCC to LGCC function. Interestingly, 75% reduction in KCC mRNA did not alter basal neurotransmission levels indicating that only a fraction of the KCC population is required to maintain the Cl- ionic gradient when at rest, but prolonged synaptic activity increases the threshold for GABA-mediated inhibition and reduces nerve sensitivity to GABA. These data expand current knowledge regarding the physiological role of KCC in a model insect and provides the necessary foundation to develop KCC as a novel biochemical target of insecticides as well as complements existing research to provide a holistic understanding of the plasticity in mammalian health and disease (Chen, 2019).

    Drosophila Subdued is a moonlighting transmembrane protein (TMEM16) that transports ions and phospholipids

    Transmembrane protein (TMEM16) family members play numerous important physiological roles, ranging from controlling membrane excitability and secretion to mediating blood coagulation and viral infection. These diverse functions are largely due to their distinct biophysical properties. Mammalian TMEM16A and TMEM16B are Ca(2+)-activated Cl(-) channels (CaCCs), whereas mammalian TMEM16F, fungal afTMEM16, and nhTMEM are moonlighting (multifunctional) proteins with both Ca(2+)-activated phospholipid scramblase (CaPLSase) and Ca(2+)-activated, nonselective ion channel (CAN) activities. To further understand the biological functions of the enigmatic TMEM proteins in different organisms, this study combined an improved annexin V-based CaPLSase-imaging assay with inside-out patch clamp technique to thoroughly characterized Subdued, a Drosophila TMEM ortholog. Subdued is also a moonlighting transport protein with both CAN and CaPLSase activities. Using a TMEM16F-deficient HEK293T cell line to avoid strong interference from endogenous CaPLSases, this functional characterization and mutagenesis studies revealed that Subdued is a bona fide CaPLSase. The finding that Subdued is a moonlighting TMEM expands understanding of the molecular mechanisms of TMEM proteins and their evolution and physiology in both Drosophila and humans (Le, 2019).

    The ground-breaking discoveries of TMEM16A and TMEM16B as the long-sought CaCCs advanced the understanding of a novel membrane protein superfamily that includes the TMEM family and its closely related OSCA, TMEM and TMC membrane protein families. TMEM proteins have been found in fungi, amoeboids, insects and vertebrates. The unexpected findings of mammalian TMEM16F as a moonlighting protein, a special type of proteins that can perform two or more distinct functions without gene fusions, multiple RNA splice variants or multiple proteolytic fragments, advanced understanding of the enigmatic TMEM family (10-12,19,20). Serving as a bona fide CaPLSase and a small-conductance CAN (SCAN) channel, TMEM16F has evolved the capability to passively transport phospholipids and ions, two structurally distinct classes of permeants, down their chemical gradients (Le, 2019).

    Upon Ca2+ binding, TMEM16FCaPLSase mediates the rapid flip-flopping of phospholipids across cell membranes and thus dissipates the asymmetric distribution of membrane phospholipids. During platelet activation, TMEM16F-CaPLSase-induced phosphatidylserine (PS) externalization is essential for prothrombinase assembly, subsequent thrombin generation and blood coagulation. Consistent with its importance in blood coagulation, both the Scott syndrome patients who carried TMEM16F loss-of-function mutations and TMEM16F deficient mice exhibited prolonged bleeding phenotype. Despite the known physiological function of TMEM16F-CaPLSase in blood coagulation, it is unclear whether and how TMEM16F's ion channel activity can participate in this process (Le, 2019).

    Recent structural and functional studies elegantly revealed that the fungal nhTMEM16, afTMEM and mammalian TMEM16E were also moonlighting proteins with CaPLSase and channel activities. Interestingly, the mammalian TMEM16A and TMEM16B CaCCs only displayed ion channel activities, while an amoebozoa TMEM homolog from Dictyostelium discoideum only showed CaPLSase activity when heterologously expressed in HEK cells. In order to understand the biological functions of TMEM moonlighting, there is an urgent need to have an in-depth understanding of TMEM evolution and function in different kingdoms ranging from Protozoa, Fungi to Animalia (Le, 2019).

    TMEM moonlighting proteins have not been identified thus far in insects, despite a recent study that clearly demonstrated the physiological importance of CaPLSase in the degeneration of Drosophila sensory neurons (Sapar, 2018). However, the molecular identity of the Drosophila CaPLSase responsible for the observed scramblase activities remains elusive. Among the five Drosophila TMEM homologs, Subdued is the only protein that has been thoroughly characterized using electrophysiological tools (Wong, 2013). When heterologously expressed in HEK293T cells, whole-cell patch clamp recordings suggested that Subdued was a CaCC. Interestingly, Subdued-deficient Drosophila exhibited severe defects in host defense when challenged with the pathogenic bacterium Serratia marcescens. It remains, however, unclear how Subdued CaCC function is involved in Drosophila's immunity (Le, 2019).

    Combining an improved Annexin V-based CaPLSase imaging assay with inside-out patch clamping technique, this study has discovered that Subdued is also a moonlighting TMEM16 protein in Drosophila. Notably, it was also found that Subdued harbors biophysical features that strikingly resembled those of the mammalian TMEM16F, which has been unambiguously shown to function as a CaPLSase and a CAN channel. These results thus support the notion that TMEM moonlighting could be an ancient feature of TMEM family, which is conserved in fungi, insects and vertebrates. These study provided new insights into understanding the evolution of TMEM family, the molecular mechanisms of their ion and phospholipid permeation, as well as TMEM physiological functions in Drosophila (Le, 2019).

    Protein moonlighting as both ion channels and phospholipid scramblases has been observed in mammal and fungal TMEM proteins. By using patch clamp electrophysiology and an improved phospholipid scrambling assay, these studies reveal that Drosophila Subdued, an insect TMEM16, is also a moonlighting protein that can serve as both a CAN channel and a CaPLSase (Le, 2019).

    In this study, it was also shown that the widely used HEK293T cell line had endogenous TMEM16F expression and strong CaPLSase activity. The endogenous CaPLSase activity can interfere with characterization of exogenous TMEM CaPLSases and complicate subsequent interpretation. To circumvent this complication, CRISPR-Cas method was applied to generate a TMEM16F KO HEK293T cell line, which lacks endogenous CaPLSase activity and thereby can serve as an ideal heterologous expression system to characterize CaPLSase activities. When Subdued was heterologously expressed in this KO cell line, robust Subdued-mediated CaPLSase activity was observed. Disrupting a key conserved residue at the extracellular entrance of Subdued abolished its CaPLSase activity. In addition, when one of the conserved Ca2+-binding residues was replaced with a positively charged Arg residue, the mutant Subdued failed to scramble phospholipids\. Collectively, these data show that Subdued is a bona fide CaPLSase (Le, 2019).

    Inside-out excised patch clamp recordings demonstrated that Subdued is a CAN channel with higher cation permeability than chloride (PNa/PCl = 5.83) in μM intracellular Ca2+. This conclusion stands in stark contrast to a previous study, which reported that Subdued functioned as a CaCC (PNa/PCl = 0.16) based on whole-cell patch recordings (Wong, 2013). We postulate that this discrepancy might be derived from the inherent differences between the two patch clamp configurations. First, infusion of pipette solution with high micromolar Ca2+ into cytosol could disrupt intracellular environment, which might subsequently alter channel activity. In the case of Subdued, channel current run-up was observed in whole-cell recording (Wong, 2013). When whole-cell recording was used to measure TMEM16F current, a to 15-minute delay of channel activation has been frequently observed after membrane break-in. Under inside-out configuration, both Subdued and TMEM16F current can be immediately recorded after membrane excision. Without the long delay to obtain stable current, the reversal potential measured using inside-out configuration may reflect the intrinsic channel selectivity. Second, whole-cell patch clamp may suffer from larger leak current during recording, especially when infusing with high micromolar Ca2+ into the cytosol. The potential leak current could confound the reversible potential measurement. Third, measuring the reversal potential requires exchanging solutions with drastically different ionic concentrations. Whole-cell recording usually requires whole-chamber solution exchange, which can induce large liquid junction potential to complicate reversal potential measurement. In the current inside-out patch clamp experiments, a pressurized focal perfusion system was used to achieve rapid solution exchange directly to the excised patch membrane. As this process is fast and only requires a small volume of solution, the impact of liquid junction potential is negligible (Le, 2019).

    This study also found that Subdued ion permeability resembles that of the mammalian TMEM16F-SCAN. Similar to TMEM16F-SCAN (Yang, 2012), common CaCC blockers such as niflumic acid (NFA), flufenamic acid (FFA) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) cannot block Subdued current (Wong, 2013), further supporting that Subdued channel is different from TMEM16A-CaCC. Interestingly, the fungal afTMEM and nhTMEM channels also exhibit substantial cation permeability. The non-selective nature of the moonlighting TMEM proteins towards phospholipids and ions suggests that their ancestors may have experienced low selective pressure during evolution, so that they could mediate simultaneous permeation of different ions and phospholipids without expanding the genome size. Interestingly, TMEM16A and TMEM16B are more selective to anions and lack CaPLSase function. It is hoped that the current findings can shine new lights on understanding TMEM evolution and molecular mechanisms for substrate selectivity. This study also provides new insights into understanding the physiological functions of TMEM moonlighting proteins. Previous studies have shown that Subdued knocked-out or knocked-down Drosophila strains harbored defects in their host defense and exhibited lethality upon ingestion of the pathogenic bacteria S. marcescens (Wong, 2013; Cronin, 2009). It is thus far not clear whether Subdued's CaPLSase function and/or ion channel function play a major role in participating host defense. Interestingly, the moonlighting protein TMEM16F has also been reported to express in immune cells and play an important role in immune responses. Considering the fact that channel activation of both Subdued and TMEM CAN current requires both high Ca2+ and membrane depolarization, both of which conditions are unlikely to be achieved under physiological conditions, it is likely that their CaPLSase functions might play the major role in immunity. A recent study suggested that PS externalization induced by overexpressing mammalian TMEM16F- CaPLSase played an important role in controlling neurite degeneration in Drosophila sensory neurons (Sapar, 2018). The current finding that Subdued as a bona fide CaPLSase might help identify the CaPLSase that is responsible for Drosophila neuronal degeneration (Le, 2019).

    The Drosophila Trpm channel mediates calcium influx during egg activation

    Egg activation is the process in which mature oocytes are released from developmental arrest and gain competency for embryonic development. In Drosophila and other arthropods, eggs are activated by mechanical pressure in the female reproductive tract, whereas in most other species, eggs are activated by fertilization. Despite the difference in the trigger, Drosophila shares many conserved features with higher vertebrates in egg activation, including a rise of intracellular calcium in response to the trigger. In Drosophila, this calcium rise is initiated by entry of extracellular calcium due to opening of mechanosensitive ion channels and initiates a wave that passes across the egg prior to initiation of downstream activation events. This study combined inhibitor tests, germ-line-specific RNAi knockdown, and germ-line-specific CRISPR/Cas9 knockout to identify the Transient Receptor Potential (TRP) channel subfamily M (Trpm) as a critical channel that mediates the calcium influx and initiates the calcium wave during Drosophila egg activation. A reduction was observed in the proportion of eggs that hatched from trpm germ-line knockout mutant females, although eggs were able to complete some egg activation events including cell cycle resumption. Since a mouse ortholog of Trpm was recently reported also to be involved in calcium influx during egg activation and in further embryonic development, these results suggest that calcium uptake from the environment via TRPM channels is a deeply conserved aspect of egg activation (Hu, 2019).

    In almost all animals, mature oocytes are arrested in meiosis at the end of oogenesis and require an external trigger to be activated and transition to start embryogenesis. This 'egg activation' involves multiple events that transition eggs to embryogenesis, including meiosis resumption and completion, maternal protein modification and/or degradation, maternal mRNA degradation or translation, and egg envelope changes (Hu, 2019).

    Triggers of egg activation vary across species. In vertebrate and some invertebrate species, fertilization triggers egg activation. However, changes in pH, ionic environment, or mechanical pressure can also trigger egg activation in other invertebrate species. A conserved response to these triggers is a rise of intracellular free Ca2+ levels in the oocyte. This calcium rise is due to influx of external calcium and/or release from internal storage, depending on the organism. The elevated Ca2+ concentration is thought to activate Ca2+- dependent kinases and/or phosphatases, which in turn change the phospho-proteome of the activated egg, initiating egg activation events (Hu, 2019).

    Drosophila eggs activate independent of fertilization and the trigger is mechanical pressure. When mature oocytes exit the ovary and enter the lateral oviduct, they experience mechanical pressure from reproductive tract tract. As the oocytes swell due to the influx of oviductal fluid, their envelopes become taut. Drosophila oocytes can be activated in vitro by incubation in a hypotonic buffer, although some egg activation events do not proceed completely normally in vitro. Intracellular calcium levels rise in oocytes occur egg activation, as observed with the calcium sensor GCaMP. This calcium rise takes the form of a wave that starts at the oocyte pole(s) and traverses the entire oocyte. Initiation of this calcium wave requires influx of external Ca2+, as chelating external Ca2+ in in vitro egg activation assays blocks the calcium wave and egg activation. Propagation of the calcium wave relies on the release of internal Ca2+ stores, likely through an Inositol 1,4,5-trisphosphate (IP3) mediated pathway, as knocking down the endoplasmic reticulum (ER) calcium channel IP3 receptor (IP3R) prevents propagation of the calcium wave (Hu, 2019).

    How mechanical forces trigger calcium entry during Drosophila egg activation was unknown. However, the lack of initiation of a calcium wave in the presence of Gd3+, an inhibitor of mechanosensitive ion channels, and N-(p-Amylcinnamoyl) anthranilic acid (ACA), an inhibitor of TRP-family ion channels, suggested that TRP family ion channels are likely involved. Further supporting this idea is the recent discovery that a TRP family channel, TRPM7, is needed for calcium influx that leads to calcium oscillations in activating mouse eggs. The Drosophila genome encodes 13 TRP family channels, but according to RNAseq data, only 3 (Painless, Trpm, and Trpml) are expressed in the ovary. This study used specific inhibitors, existing mutants, germline- specific RNAi knockdown, and new knockouts that were created with CRISPR/Cas9 to screen these 3 candidates for their roles in the initiation of the calcium wave. Trpm, the single Drosophila ortholog of mouse TRPM7, was shown to mediate the calcium wave initiation, whereas the other two TRP channels are not necessary to initiate the calcium wave (Hu, 2019).

    Calcium wave phenotypes are normal in oocytes from pain or trpml null mutants. However, the frequency of the calcium wave is diminished in wildtype oocytes in the presence of Trpm inhibitors and in oocytes from trpm germline knockdown or knockout mutants. These results consistently indicated that Trpm mediates the calcium influx that initiates the calcium wave during Drosophila egg activation. trpm germline knockout females also displayed significantly decreased egg hatchability, due to defects after cell cycle resumption. The reduced hatchability suggested that maternal trpm function or the calcium wave is required for further embryogenesis after egg activation (Hu, 2019).

    TRP family ion channels are non-selective and respond to a wide array of environmental stimuli. Drosophila Trpm has been reported to play multiple roles throughout larval development, including maintaining Mg2+ and Zn2+ homeostasis (Georgiev, 2010; Hofmann, 2010), and sensing noxious cold in larval Class III md neurons (Turner, 2016). However, the role of Trpm in reproduction had not been investigated because of the pupal lethality of trpm null mutants. In this study germline specific RNAi knockdown and CRISPR/Cas9 mediated knockout revealed three novel functions of Drosophila Trpm: supporting early oogenesis, mediating influx of environmental calcium to initiate the calcium wave during egg activation, and maternally supporting embryonic development after egg activation (Hu, 2019).

    A previous study suggested that calcium influx during Drosophila egg activation is mediated through mechanosensitive ion channels (Homer, 2008). Both Drosophila Trpm and its mouse ortholog TRPM7 are reported to be constitutively active and permeable to a wide range of divalent cations (Georgiev, 2010). Mouse TRPM7 is known to respond to mechanical pressure, but further study will be needed to determine whether Drosophila Trpm is similarly responsive to mechanical triggers, such as those that occur during ovulation (Hu, 2019).

    Germline knockout of trpm significantly reduced the frequency of observing calcium waves in in vitro egg activation assays and egg hatchability. However, this reduced egg hatchability was not due to failure of cell cycle resumption during egg activation. There are two possible explanations for the reduced egg hatchability of trpm germline knockout females (Hu, 2019).

    First, it is possible that trpm plays a maternal role, independent of its role in initiating the calcium wave, such that lack of maternally deposited Trpm proteins leads to defects during embryogenesis. In mouse, TRPM7 is also required for normal early embryonic development, apart from its role in calcium oscillations. Inhibition of TRPM7 function impairs pre-implantation embryo development and slows progression to the blastocyst stage. Drosophila trpm mutant lethality had been reported to occur during the pupal stage. However, those homozygous mutants were offspring of heterozygous mothers, and thus did not lack maternal Trpm function. Germline specific depletion of trpm reveals a maternal role for Trpm in embryogenesis (Hu, 2019).

    Alternatively, or in addition, it is possible that oocytes lacking Trpm do not take up sufficient Ca2+ from the environment to form a calcium wave, but that at least some events of egg activation can occur despite this. In mouse, an initial calcium rise is induced by sperm-delivered PLCζ via the IP3 pathway. Yet although sperm from PLCζ null males fails to trigger normal calcium oscillations, some eggs fertilized by those sperm develop. Multiple oscillations following fertilization require influx of external calcium, mediated by TRPM7 and CaV3.2 (Bernhardt, 2018). Even though these oscillations were reported to be needed for multiple post-fertilization events, some TRPM7 and CaV3.2 double-knockout embryos still develop, albeit not completely normally. Together, these data suggest that egg activation can still occur in mouse with diminished intracellular calcium rises, analogous to what is seen in Drosophila in the absence of maternal Trpm function (Hu, 2019).

    Insufficient influx of calcium in the absence of Trpm function could disrupt later (but maternally- dependent) embryogenesis. The oocyte-to-embryo transition involves multiple events. In mouse egg activation, these events take place sequentially as calcium oscillations progress, with developmental progression associated with more oscillations and more total calcium signal. Some of the events start after a certain number of oscillations but require additional oscillations to complete. It is possible that mechanisms critical for Drosophila embryo development also depend on reaching a precise level of calcium. A low-level calcium rise might be sufficient to trigger some egg activation mechanisms such as vitelline membrane crosslinking and cell cycle resumption, but high-levels of calcium may be required for further progression (Hu, 2019).

    Given the importance of calcium in egg activation, it was surprising that although trpm knockout eggs lacked a calcium wave in vitro, in vivo such eggs could progress in cell cycles and even, sometimes, hatch. There may be insufficient calcium influx in the absence of Trpm for full and efficient development, but some egg activation events may still occur. Alternatively, it is possible that redundant mechanisms permit a sufficient calcium-level increase without producing a detectable wave form. Despite being able to trigger a series of egg activation events including meiosis resumption and protein translation, osmotic pressure during in vitro activation may have different properties from mechanical pressure exerted on mature oocytes during ovulation. The latter might allow opening of other calcium channels to initiate a normal calcium rise and complete egg activation. Two channels, TRPM7 and Cav3.2, are needed for the calcium oscillations following mouse fertilization, but the Drosophila ortholog of mouse Cav3.2, Ca-α1T, is not detectably expressed in fly ovaries. Other unknown channels might play this redundant role in vivo. In this light it is noted that levels of basal GCaMP fluorescence varied among oocytes incubated in IB that were inhibited from forming a wave, suggesting the possibility of a calcium increase by a redundant mechanism (Hu, 2019).

    It was intriguing that Drosophila Trpm is essential for the calcium rise at egg activation, and that its mouse ortholog, TRPM7, was recently reported to be required (along with CaV3.2) for the calcium influx needed for post-fertilization calcium oscillations that are in turn required for egg activation events. This apparent conservation in mechanisms in egg activation involving orthologous Trpm channels in a protostome (Drosophila) and a deuterostome (mouse) prompts asking whether Trpm-mediated calcium influx is a very ancient and basal aspect of egg activation, with other more variable aspects such as sperm-triggered calcium rises being more derived, if better known, features. It is interesting in this light that a sperm-delivered TRP channel (TRP-3) has also been reported to mediate calcium influx and a calcium rise in another protostome, C. elegans (Hu, 2019).



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    Wong, X. M., Younger, S., Peters, C. J., Jan, Y. N., and Jan, L. Y. (2013). Subdued, a TMEM16 family Ca(2)(+)-activated Cl(-)channel in Drosophila melanogaster with an unexpected role in host defense. Elife 2: e00862. PubMed ID: 24192034

    Yang, H., Kim, A., David, T., Palmer, D., Jin, T., Tien, J., Huang, F., Cheng, T., Coughlin, S. R., Jan, Y. N., and Jan, L. Y. (2012) TMEM16F forms a Ca2+-activated cation channel required for lipid scrambling in platelets during blood coagulation. Cell 151: 111-122. PubMed ID: 23021219



    Zygotically transcribed genes

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