Cyclic nucleotide-gated channel 18 is an essential Ca2+ channel in pollen tube tips for pollen tube guidance to ovules in Arabidopsis

In flowering plants, pollen tubes are guided into ovules by multiple attractants from female gametophytes to release paired sperm cells for double fertilization. It has been well-established that Ca²⁺ gradients in the pollen tube tips are essential for pollen tube guidance and that plasma membrane Ca²⁺ channels in pollen tube tips are core components that regulate Ca²⁺ gradients by mediating and regulating external Ca²⁺ influx. Therefore, Ca²⁺ channels are the core components for pollen tube guidance. However, there is still no genetic evidence for the identification of the putative Ca²⁺ channels essential for pollen tube guidance. Here, we report that the point mutations R491Q or R578K in cyclic nucleotide-gated channel 18 (CNGC18) resulted in abnormal Ca²⁺ gradients and strong pollen tube guidance defects by impairing the activation of CNGC18 in Arabidopsis. The pollen tube guidance defects of cngc18-17 (R491Q) and of the transfer DNA (T-DNA) insertion mutant cngc18-1 (+/−) were completely rescued by CNGC18. Furthermore, domain-swapping experiments showed that CNGC18’s transmembrane domains are indispensable for pollen tube guidance. Additionally, we found that, among eight Ca²⁺ channels (including six CNGCs and two glutamate receptor-like channels), CNGC18 was the only one essential for pollen tube guidance. Thus, CNGC18 is the long-sought essential Ca²⁺ channel for pollen tube guidance in Arabidopsis.Pollen tubes deliver paired sperm cells into ovules for double fertilization, and signaling communication between pollen tubes and female reproductive tissues is required to ensure the delivery of sperm cells into the ovules (1). Pollen tube guidance is governed by both female sporophytic and gametophytic tissues (2, 3) and can be separated into two categories: preovular guidance and ovular guidance (1). For preovular guidance, diverse signaling molecules from female sporophytic tissues have been identified, including the transmitting tissue-specific (TTS) glycoprotein in tobacco (4), γ-amino butyric acid (GABA) in Arabidopsis (5), and chemocyanin and the lipid transfer protein SCA in Lilium longiflorum (6, 7). For ovular pollen tube guidance, female gametophytes secrete small peptides as attractants, including LUREs in Torenia fournieri (8) and Arabidopsis (9) and ZmEA1 in maize (10, 11). Synergid cells, central cells, egg cells, and egg apparatus are all involved in pollen tube guidance, probably by secreting different attractants (9–15). Additionally, nitric oxide (NO) and phytosulfokine peptides have also been implicated in both preovular and ovular pollen tube guidance (16–18). Thus, pollen tubes could be guided by diverse attractants in a single plant species.Ca²⁺ gradients at pollen tube tips are essential for both tip growth and pollen tube guidance (19–27). Spatial modification of the Ca²⁺ gradients leads to the reorientation of pollen tube growth in vitro (28, 29). The Ca²⁺ gradients were significantly increased in pollen tubes attracted to the micropyles by synergid cells in vivo, compared with those not attracted by ovules (30). Therefore, the Ca²⁺ gradients in pollen tube tips are essential for pollen tube guidance. The Ca²⁺ gradients result from external Ca²⁺ influx, which is mainly mediated by plasma membrane Ca²⁺ channels in pollen tube tips. Thus, the Ca²⁺ channels are the key components for regulating the Ca²⁺ gradients and are consequently essential for pollen tube guidance. Using electrophysiological techniques, inward Ca²⁺ currents were observed in both pollen grain and pollen tube protoplasts (31–36), supporting the presence of plasma membrane Ca²⁺ channels in pollen tube tips. Recently, a number of candidate Ca²⁺ channels were identified in pollen tubes, including six cyclic nucleotide-gated channels (CNGCs) and two glutamate receptor-like channels (GLRs) in Arabidopsis (37–40). Three of these eight channels, namely CNGC18, GLR1.2, and GLR3.7, were characterized as Ca²⁺-permeable channels (40, 41) whereas the ion selectivity of the other five CNGCs has not been characterized. We hypothesized that the Ca²⁺ channel essential for pollen tube guidance could be among these eight channels.In this research, we first characterized the remaining five CNGCs as Ca²⁺ channels. We further found that CNGC18, out of the eight Ca²⁺ channels, was the only one essential for pollen tube guidance in Arabidopsis and that its transmembrane domains were indispensable for pollen tube guidance.     

Loss of Miro1-directed mitochondrial movement results in a novel murine model for neuron disease

Defective mitochondrial distribution in neurons is proposed to cause ATP depletion and calcium-buffering deficiencies that compromise cell function. However, it is unclear whether aberrant mitochondrial motility and distribution alone are sufficient to cause neurological disease. Calcium-binding mitochondrial Rho (Miro) GTPases attach mitochondria to motor proteins for anterograde and retrograde transport in neurons. Using two new KO mouse models, we demonstrate that Miro1 is essential for development of cranial motor nuclei required for respiratory control and maintenance of upper motor neurons required for ambulation. Neuron-specific loss of Miro1 causes depletion of mitochondria from corticospinal tract axons and progressive neurological deficits mirroring human upper motor neuron disease. Although Miro1-deficient neurons exhibit defects in retrograde axonal mitochondrial transport, mitochondrial respiratory function continues. Moreover, Miro1 is not essential for calcium-mediated inhibition of mitochondrial movement or mitochondrial calcium buffering. Our findings indicate that defects in mitochondrial motility and distribution are sufficient to cause neurological disease.Motor neuron diseases (MNDs), including ALS and spastic paraplegia (SP), are characterized by the progressive, length-dependent degeneration of motor neurons, leading to muscle atrophy, paralysis, and, in some cases, premature death. There are both inherited and sporadic forms of MNDs, which can affect upper motor neurons, lower motor neurons, or both. Although the molecular and cellular causes of most MNDs are unknown, many are associated with defects in axonal transport of cellular components required for neuron function and maintenance (1–6).A subset of MNDs is associated with impaired mitochondrial respiration and mitochondrial distribution. This observation has led to the hypothesis that neurodegeneration results from defects in mitochondrial motility and distribution, which, in turn, cause subcellular ATP depletion and interfere with mitochondrial calcium ([Ca²⁺]_m) buffering at sites of high synaptic activity (reviewed in ref. 7). It is not known, however, whether mitochondrial motility defects are a primary cause or a secondary consequence of MND progression. In addition, it has been difficult to isolate the primary effect of mitochondrial motility defects in MNDs because most mutations that impair mitochondrial motility in neurons also affect transport of other organelles and vesicles (1, 8–11).In mammals, the movement of neuronal mitochondria between the cell body and the synapse is controlled by adaptors called trafficking kinesin proteins (Trak1 and Trak2) and molecular motors (kinesin heavy chain and dynein), which transport the organelle in the anterograde or retrograde direction along axonal microtubule tracks (7, 12–24). Mitochondrial Rho (Miro) GTPase proteins are critical for transport because they are the only known surface receptors that attach mitochondria to these adaptors and motors (12–15, 18, 25, 26). Miro proteins are tail-anchored in the outer mitochondrial membrane with two GTPase domains and two predicted calcium-binding embryonic fibroblast (EF) hand motifs facing the cytoplasm (12, 13, 25, 27, 28). A recent Miro structure revealed two additional EF hands that were not predicted from the primary sequence (29). Studies in cultured cells suggest that Miro proteins also function as calcium sensors (via their EF hands) to regulate kinesin-mediated mitochondrial “stopping” in axons (15, 16, 26). Miro-mediated movement appears to be inhibited when cytoplasmic calcium is elevated in active synapses, effectively recruiting mitochondria to regions where calcium buffering and energy are needed. Despite this progress, the physiological relevance of these findings has not yet been tested in a mammalian animal model. In addition, mammals ubiquitously express two Miro orthologs, Miro1 and Miro2, which are 60% identical (12, 13). However, the individual roles of Miro1 and Miro2 in neuronal development, maintenance, and survival have no been evaluated.We describe two new mouse models that establish the importance of Miro1-mediated mitochondrial motility and distribution in mammalian neuronal function and maintenance. We show that Miro1 is essential for development/maintenance of specific cranial neurons, function of postmitotic motor neurons, and retrograde mitochondrial motility in axons. Loss of Miro1-directed retrograde mitochondrial transport is sufficient to cause MND phenotypes in mice without abrogating mitochondrial respiratory function. Furthermore, Miro1 is not essential for calcium-mediated inhibition of mitochondrial movement or [Ca²⁺]_m buffering. These findings have an impact on current models for Miro1 function and introduce a specific and rapidly progressing mouse model for MND.     

A ribosome-inactivating protein in a Drosophila defensive symbiont

Vertically transmitted symbionts that protect their hosts against parasites and pathogens are well known from insects, yet the underlying mechanisms of symbiont-mediated defense are largely unclear. A striking example of an ecologically important defensive symbiosis involves the woodland fly Drosophila neotestacea, which is protected by the bacterial endosymbiont Spiroplasma when parasitized by the nematode Howardula aoronymphium. The benefit of this defense strategy has led to the rapid spread of Spiroplasma throughout the range of D. neotestacea, although the molecular basis for this protection has been unresolved. Here, we show that Spiroplasma encodes a ribosome-inactivating protein (RIP) related to Shiga-like toxins from enterohemorrhagic Escherichia coli and that Howardula ribosomal RNA (rRNA) is depurinated during Spiroplasma-mediated protection of D. neotestacea. First, we show that recombinant Spiroplasma RIP catalyzes depurination of 28S rRNAs in a cell-free assay, as well as Howardula rRNA in vitro at the canonical RIP target site within the α-sarcin/ricin loop (SRL) of 28S rRNA. We then show that Howardula parasites in Spiroplasma-infected flies show a strong signal of rRNA depurination consistent with RIP-dependent modification and large decreases in the proportion of 28S rRNA intact at the α-sarcin/ricin loop. Notably, host 28S rRNA is largely unaffected, suggesting targeted specificity. Collectively, our study identifies a novel RIP in an insect defensive symbiont and suggests an underlying RIP-dependent mechanism in Spiroplasma-mediated defense.Symbiosis is now recognized to be a key driver of evolutionary novelty and complexity (1, 2), and symbioses between microbes and multicellular hosts are understood as essential to the health and success of diverse lineages, from plants to humans (3). Insects, in particular, have widespread associations with symbiotic bacteria, with most insect species infected by maternally transmitted endosymbionts (4, 5). Although many insect symbionts perform roles essential for host survival, such as supplementing nutrition, others are facultative and not strictly required by their hosts. These facultative symbionts have evolved diverse and intriguing strategies to maintain themselves in host populations despite loss from imperfect maternal transmission and metabolic costs to the host. These range from manipulating host reproduction to increase their own transmission (6, 7), such as by killing male hosts, to providing context-dependent fitness benefits (8). Recently, it has become clear that different insect endosymbionts have independently evolved to protect their hosts against diverse natural enemies that so far include pathogenic fungi (9), RNA viruses (10, 11), parasitoid wasps (12), parasitic nematodes (13), and predatory spiders (14, 15). This suggests that defense might be a common aspect of many insect symbioses and demonstrates that symbionts can serve as dynamic and heritable sources of protection against natural enemies (8).Despite a growing appreciation of the importance of symbiont-mediated defense in insects, key questions remain. Most demonstrations of defense have been under laboratory conditions, and the importance of symbiont-mediated protection in natural systems is unclear in most cases (16). At the same time, the proximate causes of defense are largely unknown, although recent studies have provided some intriguing early insights: A Pseudomonas symbiont of rove beetles produces a polyketide toxin thought to deter predation by spiders (14), Streptomyces symbionts of beewolves produce antibiotics to protect the host from fungal infection (17), and bacteriophages encoding putative toxins are required for Hamiltonella defensa to protect its aphid host from parasitic wasps (18), whereas the causes of other naturally occurring defensive symbioses are unresolved. From an applied perspective, the ongoing goal of exploiting insect symbioses to arrest disease transmission to humans from insect vectors (19) makes a deeper understanding of the factors contributing to ecologically relevant and evolutionarily durable defensive symbioses urgently needed.Here, we investigate the mechanism underlying one of the most striking examples of an ecologically important defensive symbiosis. Drosophila neotestacea is a woodland fly that is widespread across North America and is commonly parasitized by the nematode Howardula aoronymphium. Infection normally sterilizes flies (20); however, when flies harbor a strain of the inherited symbiont Spiroplasma—a Gram-positive bacterium in the class Mollicutes—they remarkably tolerate Howardula infection without loss of fecundity, and infection intensity is substantially reduced (13). The benefit conferred by this protection lends a substantial selective advantage to Spiroplasma-infected flies and has led to Spiroplasma’s recent spread across North America, with symbiont-infected flies rapidly replacing uninfected ones (21). Spiroplasma is a diverse and widespread lineage of arthropod-associated bacteria that can be commensal, pathogenic, or mutualistic (22). Maternal transmission has arisen numerous times in Spiroplasma, including strains that are well known as male-killers (22). In addition to defense against nematodes in D. neotestacea, other strains of Spiroplasma have recently been shown to protect flies and aphids against parasitic wasps and pathogenic fungi, respectively (23–25), but in no case is the mechanism of defense understood.In theory, there are multiple avenues by which a symbiont may protect its host that include competing with parasites for limiting resources, priming host immunity, or producing factors to directly attack parasites (26). We previously assessed these possibilities in the defensive Spiroplasma from D. neotestacea (27); our findings best supported a role for toxins in defense, with Spiroplasma encoding a highly expressed putative ribosome-inactivating protein (RIP). RIPs are widespread across plants and some bacteria and include well-known plant toxins of particular human concern such as ricin, as well as important virulence factors in human toxigenic strains of Escherichia coli and Shigella (28, 29). RIPs characteristically exert their cytotoxic effects through depurination of eukaryotic 28S ribosomal RNAs (rRNAs) at a highly conserved adenine in the α-sarcin/ricin loop (SRL) of the rRNA by cleaving the N-glycosidic bond between the rRNA backbone and adenine (30, 31). The proliferation of RIPs across different lineages implies functional significance, but their ecological roles are unclear, although they often appear to have antiviral or other defensive roles (29, 32). Here, we find that Spiroplasma expresses a functional RIP distinct from previously characterized toxins that appears to specifically affect Howardula rRNA in flies coinfected with Spiroplasma and Howardula. This work suggests the mechanisms used in defensive associations to protect the host from disease as well as intriguing ecological roles for RIPs in a tripartite defensive symbiosis.     

Reducing the genetic code induces massive rearrangement of the proteome

Expanding the genetic code is an important aim of synthetic biology, but some organisms developed naturally expanded genetic codes long ago over the course of evolution. Less than 1% of all sequenced genomes encode an operon that reassigns the stop codon UAG to pyrrolysine (Pyl), a genetic code variant that results from the biosynthesis of Pyl-tRNA^Pyl. To understand the selective advantage of genetically encoding more than 20 amino acids, we constructed a markerless tRNA^Pyl deletion strain of Methanosarcina acetivorans (ΔpylT) that cannot decode UAG as Pyl or grow on trimethylamine. Phenotypic defects in the ΔpylT strain were evident in minimal medium containing methanol. Proteomic analyses of wild type (WT) M. acetivorans and ΔpylT cells identified 841 proteins from >7,000 significant peptides detected by MS/MS. Protein production from UAG-containing mRNAs was verified for 19 proteins. Translation of UAG codons was verified by MS/MS for eight proteins, including identification of a Pyl residue in PylB, which catalyzes the first step of Pyl biosynthesis. Deletion of tRNA^Pyl globally altered the proteome, leading to >300 differentially abundant proteins. Reduction of the genetic code from 21 to 20 amino acids led to significant down-regulation in translation initiation factors, amino acid metabolism, and methanogenesis from methanol, which was offset by a compensatory (100-fold) up-regulation in dimethyl sulfide metabolic enzymes. The data show how a natural proteome adapts to genetic code reduction and indicate that the selective value of an expanded genetic code is related to carbon source range and metabolic efficiency.Synthesizing whole genomes (1) and eliminating codons (2) are novel methods for rewriting the genetic code that may dramatically alter the repertoire of genetically encoded amino acids. Expansion of the genetic code has led to exciting technologies, including site-directed protein labeling and production of proteins with hardwired posttranslational modifications (3). The current approaches to cotranslationally insert noncanonical amino acids (ncAAs) into proteins rely on the reassigning of one of three stop codons (4).Although these approaches were highly successful in incorporating over 100 ncAAs into proteins (3), they limit the expansion of the code to no more than 2 additional amino acids at a time and significantly challenge the cellular production host by unnaturally extending proteins and reducing growth rate (5). Alternate methods focus on quadruplet codons (6, 7) and recoding (8) or reassigning sense codons (9–13). Attempts to reassign a sense codon in Mycoplasma capricolum were defied by tRNA misacylation by endogenous aminoacyl-tRNA synthetases (9). This result indicates that, although extensively rewriting the genetic code may be possible, it comes with unexpected challenges related to cellular fitness and translation fidelity. These considerations will impact efforts to engineer cells to synthesize proteins with multiple ncAAs or create biologically contained strains that require an expanded code for survival (14).Opening codons by reducing the genetic code is highly promising, but it is unknown how removing 1 amino acid from the genetic code might impact the proteome or cellular viability. Many genetic code variations are found in nature (15), including stop or sense codon reassignments, codon recoding, and natural code expansion (16). Pyrrolysine (Pyl) is a rare example of natural genetic code expansion. Evidence for genetically encoded Pyl is found in <1% of all sequenced genomes (17). In these organisms, Pyl is encoded by the UAG codon, which requires tRNA^Pyl, pyrrolysyl-tRNA synthetase (PylRS), and the products of three genes (pylBCD) that synthesize Pyl from two molecules of lysine (18). The PylRS enzyme was engineered to genetically encode >100 ncAAs (19). The Pyl encoding system has already been used to expand the genetic codes of Escherichia coli (20–22), mammalian cells, and animals (23).Despite the use of Pyl in synthetic biology, little is known about the role of Pyl in its native environment or the evolutionary pressures that sustain expanded genetic codes in nature. The Pyl-decoding trait is found in methanogenic archaea of the orders Methanosarcinales and Methanomassiliicoccales (24) and certain anaerobic bacteria (17). In addition to producing 74% of global methane emissions, methanogens are remarkable for their ability to survive with only the most basic carbon and energy sources (25). Methanosarcina shows the greatest substrate range among methanogens and survives on acetate, carbon monoxide, methylamines, methanol, or dimethyl sulfide (DMS). Their broad substrate range depends, in part, on the presence of Pyl in the active site of several methylamine methyltransferases (26). Hundreds of Methanosracina genes contain in-frame TAG codons (27), but natural Pyl incorporation was only shown in methylamine methyltransferases (17, 28) and tRNA^His guanylyltransferase (Thg1) (29).Methanosarcina acetivorans provides an ideal model system to identify Pyl-containing proteins and study the impact of genetic code reduction on the proteome and physiology of the cell. We constructed a markerless tRNA^Pyl deletion (ΔpylT) strain of M. acetivorans C2A and used three independent mass spectrometry (MS) approaches to characterize soluble proteomes from M. acetivorans grown on minimal medium containing trimethylamine (TMA) or methanol and ΔpylT cells grown on methanol. The data reveal previously unidentified biochemical roles for Pyl and Pyl-containing proteins and indicate that the expanded genetic code of Methanosarcina is intricately linked with cellular metabolism and the composition of the proteome.     

Targeting glutamine metabolism rescues mice from late-stage cerebral malaria

The most deadly complication of Plasmodium falciparum infection is cerebral malaria (CM) with a case fatality rate of 15–25% in African children despite effective antimalarial chemotherapy. There are no adjunctive treatments for CM, so there is an urgent need to identify new targets for therapy. Here we show that the glutamine analog 6-diazo-5-oxo-l-norleucine (DON) rescues mice from CM when administered late in the infection a time at which mice already are suffering blood–brain barrier dysfunction, brain swelling, and hemorrhaging accompanied by accumulation of parasite-specific CD8⁺ effector T cells and infected red blood cells in the brain. Remarkably, within hours of DON treatment mice showed blood–brain barrier integrity, reduced brain swelling, decreased function of activated effector CD8⁺ T cells in the brain, and levels of brain metabolites that resembled those in uninfected mice. These results suggest DON as a strong candidate for an effective adjunctive therapy for CM in African children.The World Health Organization estimates that there are nearly 200 million clinical cases of Plasmodium falciparum malaria annually (1). For most individuals living in endemic areas, malaria is uncomplicated and resolves with time. However, in about 1% of cases, almost exclusively among young children, malaria becomes severe and life threatening, resulting in 525,000 deaths each year in Africa alone. One of the most deadly complications of P. falciparum infection in humans is cerebral malaria (HCM) characterized by the onset of severe neurological signs such as altered consciousness, seizures, and coma (2). Autopsy and MRI analyses of brains of children with HCM indicate sequestration of infected red blood cells (iRBCs), microhemorrhaging, breakdown of the blood–brain barrier (BBB) (3), and a fatal increase in intracranial pressure resulting from edema (4, 5). At present, despite effective antimalarial drug treatment, mortality for children presenting with HCM remains high, at 15–25%. HCM takes a second toll on African children, leaving survivors at risk for debilitating neurological defects (6). Thus, there is an urgent need for the development of effective adjunctive therapies that can be used in conjunction with antimalarials to treat children with HCM.Experimental cerebral malaria (ECM) in mice is a widely used model of HCM and provides a valuable tool for elucidating the mechanisms involved in CM pathogenesis and identifying cellular and molecular targets for adjunctive therapy (7). In ECM, 6–7 d after infection with Plasmodium berghei ANKA (PbA), mice of susceptible strains, such as C57BL/6, develop ataxia, paralysis, seizures, and coma and ultimately die (8). ECM displays key features of HCM, including BBB breakdown, focal hemorrhaging, and brain swelling (9–11). ECM’s pathology also requires sequestration of iRBCs in the brain vasculature (12), a hallmark of HCM (3). Histological analysis of the brains of children who died of HCM showed leukocytes, primarily monocytes with phagocytized hemozoin and platelets but also intravasculature leukocytes, including CD8⁺ T cells, sequestered in the brain vessels (13, 14). In ECM monocytes and both CD4⁺ and CD8⁺ T cells have been shown to accumulate in the brain by both flow cytometry and by intravital imaging (15). Current evidence indicates that CD8⁺ T cells are the major mediators of death in ECM (16) and that antigen-specific CD8⁺ T cells engage parasite antigens cross-presented on MHC class I molecules on brain endothelium, resulting in endothelial cell dysfunction by a perforin-dependent mechanism (17).A critical role for metabolic reprogramming in regulating immune responses is becoming increasingly appreciated. Upon activation, T cells undergo metabolic reprogramming to meet the increased energetic and biosynthetic demands of growth and effector T-cell functions (18–20). Reprogramming involves a shift to aerobic glycolysis and increased glutaminolysis. Activated T cells import large quantities of Gln and increase their expression of glutaminase (21–23). Because the pathology leading to death in CM is believed to be in part immune mediated, we hypothesized that blocking T-cell metabolism might effectively mitigate the pathology leading to death in HCM. To this end, in the present study we focus on targeting Gln metabolism for an adjunctive therapy for CM using the Gln analog 6-diazo-5-oxo-l-norleucine (DON). DON broadly inhibits Gln metabolism, in part by blocking Gln transport and inhibiting all three isoforms of glutaminase as well as other Gln-using enzymes such as the amidotransferases and glutamine synthetase (24). Consequently, DON has been shown to be a potent inhibitor of T-cell proliferation (22).Here we show that DON treatment rescues PbA-infected mice from ECM at late stages in the disease, at a time when the animals show clinical signs of neurological damage and physical loss of BBB integrity, brain swelling, and hemorrhaging. The ability of DON to arrest disease is concomitant with a decrease in the effector function of parasite-specific CD8⁺ T cells in the brains of treated mice. However, the striking ability of DON treatment to arrest pathology and promote survival so late in the disease suggests a fundamental and potentially direct role for Gln metabolism in promoting neuropathology. Overall, these results suggest DON as a candidate for an adjunctive therapy for HCM in African children.     

Conformational cycle and ion-coupling mechanism of the Na+/hydantoin transporter Mhp1

Ion-dependent transporters of the LeuT-fold couple the uptake of physiologically essential molecules to transmembrane ion gradients. Defined by a conserved 5-helix inverted repeat that encodes common principles of ion and substrate binding, the LeuT-fold has been captured in outward-facing, occluded, and inward-facing conformations. However, fundamental questions relating to the structural basis of alternating access and coupling to ion gradients remain unanswered. Here, we used distance measurements between pairs of spin labels to define the conformational cycle of the Na⁺-coupled hydantoin symporter Mhp1 from Microbacterium liquefaciens. Our results reveal that the inward-facing and outward-facing Mhp1 crystal structures represent sampled intermediate states in solution. Here, we provide a mechanistic context for these structures, mapping them into a model of transport based on ion- and substrate-dependent conformational equilibria. In contrast to the Na⁺/leucine transporter LeuT, our results suggest that Na⁺ binding at the conserved second Na⁺ binding site does not change the energetics of the inward- and outward-facing conformations of Mhp1. Comparative analysis of ligand-dependent alternating access in LeuT and Mhp1 lead us to propose that different coupling schemes to ion gradients may define distinct conformational mechanisms within the LeuT-fold class.Secondary active transporters harness the energy of ion gradients to power the uphill movement of solutes across membranes. Mitchell (1) and others (2, 3) proposed and elaborated “alternating access” mechanisms wherein the transporter transitions between two conformational states that alternately expose the substrate binding site to the two sides of the membrane. The LeuT class of ion-coupled symporters consists of functionally distinct transporters that share a conserved scaffold of two sets of five transmembrane helices related by twofold symmetry around an axis nearly parallel to the membrane (4). Ions and substrates are bound near the middle of the membrane stabilized by electrostatic interactions with unwound regions of transmembrane helix (TM) 1 and often TM6 (4). The recurrence of this fold in transporters that play critical roles in fundamental physiological processes (5, 6) has spurred intense interest in defining the principles of alternating access.Despite rapid progress in structure determination of ion-coupled LeuT-fold transporters (7–11), extrapolation of these static snapshots to a set of conformational steps underlying alternating access (4, 7, 9–12) remains incomplete, often hindered by uncertainties in the mechanistic identities of crystal structures. Typically, transporter crystal structures are classified as inward-facing, outward-facing, or occluded on the basis of the accessibility of the substrate binding site (7–11). In a recent spectroscopic analysis of LeuT, we demonstrated that detergent selection and mutations of conserved residues appeared to stabilize conformations that were not detected in the wild-type (WT) LeuT and concurrently inhibited movement of structural elements involved in ligand-dependent alternating access (13). Therefore, although crystal structures define the structural context and identify plausible pathways of substrate binding and release, development of transport models requires confirming or assigning the mechanistic identity of these structures and framing them into ligand-dependent equilibria (14).Mhp1, an Na⁺-coupled symporter of benzyl-hydantoin (BH) from Microbacterium liquefaciens, was the first LeuT-fold member to be characterized by crystal structures purported to represent outward-facing, inward-facing, and outward-facing/occluded conformations of an alternating access cycle (8, 15). In these structures, solvent access to ligand-binding sites is defined by the relative orientation between a 4-helix bundle motif and a 4-helix scaffold motif (8). In Mhp1, alternating access between inward- and outward-facing conformations, was predicted from a computational analysis based on the inverted repeat symmetry of the LeuT fold and is referred to as the rocking-bundle model (16). The conservation of the inverted symmetry prompted proposal of the rocking-bundle mechanism as a general model for LeuT-fold transporters (16). Subsequent crystal structures of other LeuT-fold transporters (7, 9, 10) tempered this prediction because the diversity of the structural rearrangements implicit in these structures is seemingly inconsistent with a conserved conformational cycle.Another outstanding question pertains to the ion-coupling mechanism and the driving force of conformational changes. The implied ion-to-substrate stoichiometry varies across LeuT-fold ion-coupled transporters. For instance, LeuT (17) and BetP (18) require two Na⁺ ions that bind at two distinct sites referred to as Na1 and Na2 whereas Mhp1 (15) and vSGLT (19) appear to possess only the conserved Na2 site. Molecular dynamics (MD) simulations (20, 21) and electron paramagnetic resonance (EPR) analysis (13, 22) of LeuT demonstrated that Na⁺ binding favors an outward-facing conformation although it is unclear which Na⁺ site (or both) is responsible for triggering this conformational transition. Similarly, a role for Na⁺ in conformational switching has been uncovered in putative human LeuT-fold transporters, including hSGLT (23). In Mhp1, the sole Na2 site has been shown to modulate substrate affinity (15); however, its proposed involvement in gating of the intracellular side (12, 21) lacks experimental validation.Here, we used site-directed spin labeling (SDSL) (24) and double electron-electron resonance (DEER) spectroscopy (25) to elucidate the conformational changes underlying alternating access in Mhp1 and define the role of ion and substrate binding in driving transition between conformations. This methodology has been successfully applied to define coupled conformational cycles for a number of transporter classes (13, 26–32). We find that patterns of distance distributions between pairs of spin labels monitoring the intra- and extracellular sides of Mhp1 are consistent with isomerization between the crystallographic inward- and outward-facing conformations. A major finding is that this transition is driven by substrate but not Na⁺ binding. Although the amplitudes of the observed distance changes are in overall agreement with the rocking-bundle model deduced from the crystal structures of Mhp1 (8, 15) and predicted computationally (16), we present evidence that relative movement of bundle and scaffold deviate from strict rigid body. Comparative analysis of LeuT and Mhp1 alternating access reveal how the conserved LeuT fold harnesses the energy of the Na⁺ gradient through two distinct coupling mechanisms and supports divergent conformational cycles to effect substrate binding and release.     

Short and long-lasting behavioral consequences of agonistic encounters between male Drosophila melanogaster

In many animal species, learning and memory have been found to play important roles in regulating intra- and interspecific behavioral interactions in varying environments. In such contexts, aggression is commonly used to obtain desired resources. Previous defeats or victories during aggressive interactions have been shown to influence the outcome of later contests, revealing loser and winner effects. In this study, we asked whether short- and/or long-term behavioral consequences accompany victories and defeats in dyadic pairings between male Drosophila melanogaster and how long those effects remain. The results demonstrated that single fights induced important behavioral changes in both combatants and resulted in the formation of short-term loser and winner effects. These decayed over several hours, with the duration depending on the level of familiarity of the opponents. Repeated defeats induced a long-lasting loser effect that was dependent on de novo protein synthesis, whereas repeated victories had no long-term behavioral consequences. This suggests that separate mechanisms govern the formation of loser and winner effects. These studies aim to lay a foundation for future investigations exploring the molecular mechanisms and circuitry underlying the nervous system changes induced by winning and losing bouts during agonistic encounters.Across the animal kingdom, aggression between conspecifics often accompanies the competition for food, mates, and territory. Although an innate behavior, aggression is a highly adaptive trait as well, with animals learning from previous experience and changing their behavior in response to new challenges. In competition for rank, for example, previous fighting experience influences the outcome of subsequent contests: prior defeat decreases whereas prior victory increases the probability of winning later contests. These have been called “loser” and “winner” effects (1). Such effects have been observed in many species, including fish (2), birds (3), and mammals (4). In general, loser effects persist longer than winner effects (5). The durational asymmetry observed between loser and winner effects has been hypothesized to participate in stabilizing social hierarchies among conspecifics (6).Fruit flies (Drosophila melanogaster) exhibit a variety of simple and complex social behaviors, including aggregation (7), courtship (8), and aggression (9) in which learning and memory have been demonstrated or postulated to serve important roles (10–12). Thus, characterizing the molecular basis of memory formation, retention, and retrieval is crucial to ultimately understanding the adaptability of these social behaviors. In Drosophila, a variety of operant and classical training paradigms have been used to subdivide memory into distinct categories. Short-term memory (STM) lasting minutes to hours is induced by a single training session, whereas long-term memory (LTM) lasting days usually requires repeated training sessions and involves de novo protein synthesis (13). A large number of studies have been carried out using olfactory, visual, social, and place memory paradigms. These have allowed the functional and molecular characterization of neuronal circuits and the identification of numerous genes underlying learning and memory (14–16). Included are mutations in rutabaga (rut, type 1 adenylyl cyclase) that interfere with learning and STM formation (17); amnesiac (amn, peptide regulator of adenylyl cyclase) that affect STM retention (18); and crammer (cer, inhibitor of a cathepsin subfamily) that prevent LTM formation (19). Whether rut, amn, and cer serve roles in the learning and memory that accompanies aggression remains unknown.Male–male aggression in fruit flies was first described almost 100 y ago (20). Since then, considerable progress has been made in understanding its expression and regulation (21–26). In competition for food resources and territory, male–male agonistic encounters, composed of stereotyped behavioral patterns, usually result in the formation of dominance relationships (9). During the progression of fights, both flies modify their fighting strategies: The ultimate winners chase and lunge at their opponents to gain sole access to the resources, whereas the losers retreat from the resources after receiving such attacks (9, 10).In second fights (30 min after first fights), losing flies display greater submissive behavior and never win against naïve or experienced opponents, revealing short-term loser effects (10). Evidence for winner effects, however, was not found (11). Recently, in olive fruit flies (Bactrocera olea) it was found that previous losing and winning experiences both increased the aggressiveness of the flies. This suggests that the consequences of losing or winning may vary across species (27).We previously suggested that fights between male flies function as operant learning situations in which males learn to use their most advantageous fighting strategy during fights and then continue to do so in subsequent contests (28). In an attempt to optimize the learning and memory associated with aggression, we designed new “handling-free” behavioral chambers (29). These proved to be more desirable for studying the formation of loser effects (12). By using these experimental arenas and pairing familiar opponents in second fights we previously showed that changes in fighting strategies could be developed by both winning and losing flies. This allowed us to suggest the existence of short-term winner effects along with the previously demonstrated loser effects (12). A more detailed examination of these short-term effects is presented here along with experiments attempting to measure the intrinsic changes in fighting abilities of losing and winning flies.In the present study, we ask (i) whether a single fight can lead to the formation of loser and winner effects and how long these effects persist, (ii) whether flies adopt different fighting strategies in second fights depending on their opponents, (iii) whether longer-lasting behavioral effects result from sequential repeated defeats or victories, (iv) whether protein synthesis is required for the short- or long-term effects observed, and (v) whether mutations in genes involved in learning and memory affect aggressive behavior.     

Ion-binding properties of a K+ channel selectivity filter in different conformations

K⁺ channels are membrane proteins that selectively conduct K⁺ ions across lipid bilayers. Many voltage-gated K⁺ (K_V) channels contain two gates, one at the bundle crossing on the intracellular side of the membrane and another in the selectivity filter. The gate at the bundle crossing is responsible for channel opening in response to a voltage stimulus, whereas the gate at the selectivity filter is responsible for C-type inactivation. Together, these regions determine when the channel conducts ions. The K⁺ channel from Streptomyces lividians (KcsA) undergoes an inactivation process that is functionally similar to K_V channels, which has led to its use as a practical system to study inactivation. Crystal structures of KcsA channels with an open intracellular gate revealed a selectivity filter in a constricted conformation similar to the structure observed in closed KcsA containing only Na⁺ or low [K⁺]. However, recent work using a semisynthetic channel that is unable to adopt a constricted filter but inactivates like WT channels challenges this idea. In this study, we measured the equilibrium ion-binding properties of channels with conductive, inactivated, and constricted filters using isothermal titration calorimetry (ITC). EPR spectroscopy was used to determine the state of the intracellular gate of the channel, which we found can depend on the presence or absence of a lipid bilayer. Overall, we discovered that K⁺ ion binding to channels with an inactivated or conductive selectivity filter is different from K⁺ ion binding to channels with a constricted filter, suggesting that the structures of these channels are different.K⁺ channels are found in all three domains of life, where they selectively conduct K⁺ ions across cell membranes. Specific stimuli trigger the activation of K⁺ channels, which results in a hinged movement of the inner helix bundle (1–7). This opening on the intracellular side of the membrane initiates ion conduction across the membrane by allowing ions to enter into the channel. After a period, many channels spontaneously inactivate to attenuate the response (8–17). The inactivation process is a timer that terminates the flow of ions in the presence of an activator to help shape the response of the system. Two dominant types of inactivation have been characterized in voltage-dependent channels: N-type and C-type (18). N-type inactivation is fast and involves an N-terminal positively charged “ball” physically plugging the pore of the channel when the membrane is depolarized. C-type inactivation, on the other hand, is a slower process involving a conformational change in the selectivity filter that is initiated by a functional link between the intracellular gate and the selectivity filter (10, 19).Several experimental observations indicate a role for the selectivity filter in C-type inactivation. First, mutations in and around the selectivity filter can alter the kinetics of inactivation (20–23). Second, increasing concentrations of extracellular K⁺ ions decrease the rate of inactivation, as if the ions are stabilizing the conductive conformation of the channel to prevent a conformational change in the selectivity filter (14, 16, 17, 22). Finally, a loss of selectivity of K⁺ over Na⁺ has been observed during the inactivation process in Shaker channels, suggesting a role for the selectivity filter (24, 25). Together, these data indicate that channels in their inactivated and conductive conformations interact with K⁺ ions differently, and suggest that C-type inactivation involves a conformational change in the selectivity filter. Although several structures of K⁺ channels in their conductive state have been solved using X-ray crystallography, there is at present no universally accepted model for the C-type inactivated channel (1, 3–5, 9, 19, 26–28) (Fig. 1B).Open in a separate windowFig. 1.Macroscopic recordings and structural models of KcsA K⁺ channel. (A) Macroscopic currents of WT KcsA obtained by a pH jump from pH 8 to pH 4 reveal channel inactivation. Two models representing the conformation of the channel are shown below. (B) Conductive [Left, Protein Data Bank (PDB) ID code 1K4C] and constricted (Right, PDB ID code 1K4D) conformations of the selectivity filter are shown as sticks, and the ion-binding sites are indicated with green spheres. The thermodynamic properties of the conductive, constricted, and inactivated (Middle) conformations are the subject of this study.Inactivation in the K⁺ channel from Streptomyces lividians (KcsA) has many of the same functional properties of C-type inactivation, which has made it a model to understand its structural features (20). KcsA channels transition from their closed to open gate upon changing the intracellular pH from high to low (Fig. 1A). The rapid flux of ions through the channel is then attenuated by channel inactivation, where most open WT channels are not conducting, suggesting that crystal structures of open KcsA channels would reveal the inactivated channel. In some crystal structures of truncated WT KcsA solved with an open gate, the selectivity filter appears in the constricted conformation, similar to the conformation observed in structures of the KcsA channel determined in the presence of only Na⁺ ions or low concentrations of K⁺ ions (3, 10, 29, 30) (Fig. 1B). Solid-state and solution NMR also indicate that the selectivity filter of the KcsA channel is in the constricted conformation when the cytoplasmic gate is open (31–33).However, a recently published study shows that even when the constricted conformation of KcsA’s selectivity filter is prevented by a nonnatural amino acid substitution, the channel inactivates like WT channels, suggesting the constricted filter does not correspond to the functionally observed inactivation in KcsA (28). In this study, we use isothermal titration calorimetry (ITC) to quantify the ion-binding properties of WT and mutant KcsA K⁺ channels with their selectivity filters in different conformations and EPR spectroscopy to determine the conformation of the channels’ intracellular gates. A comparison of these ion-binding properties leads us to conclude that the conductive and inactivated filters are energetically more similar to each other than the constricted and inactivated filters.     

The Two-pore channel (TPC) interactome unmasks isoform-specific roles for TPCs in endolysosomal morphology and cell pigmentation

The two-pore channels (TPC1 and TPC2) belong to an ancient family of intracellular ion channels expressed in the endolysosomal system. Little is known about how regulatory inputs converge to modulate TPC activity, and proposed activation mechanisms are controversial. Here, we compiled a proteomic characterization of the human TPC interactome, which revealed that TPCs complex with many proteins involved in Ca²⁺ homeostasis, trafficking, and membrane organization. Among these interactors, TPCs were resolved to scaffold Rab GTPases and regulate endomembrane dynamics in an isoform-specific manner. TPC2, but not TPC1, caused a proliferation of endolysosomal structures, dysregulating intracellular trafficking, and cellular pigmentation. These outcomes required both TPC2 and Rab activity, as well as their interactivity, because TPC2 mutants that were inactive, or rerouted away from their endogenous expression locale, or deficient in Rab binding, failed to replicate these outcomes. Nicotinic acid adenine dinucleotide phosphate (NAADP)-evoked Ca²⁺ release was also impaired using either a Rab binding-defective TPC2 mutant or a Rab inhibitor. These data suggest a fundamental role for the ancient TPC complex in trafficking that holds relevance for lysosomal proliferative scenarios observed in disease.Two-pore channels (TPCs) are an ancient family of intracellular ion channels and a likely ancestral stepping stone in the evolution of voltage-gated Ca²⁺ and Na⁺ channels (1). Architecturally, TPCs resemble a halved voltage-gated Ca²⁺/Na⁺ channel with cytosolic NH₂ and COOH termini, comprising two repeats of six transmembrane spanning helices with a putative pore-forming domain between the fifth and sixth membrane-spanning regions. Since their discovery in vertebrate systems, many studies have investigated the properties of these channels (2–7) that may support such a lengthy evolutionary pedigree.In this context, demonstration that (i) the two human TPC isoforms (TPC1 and TPC2) are uniquely distributed within the endolysosomal system (2, 3) and that (ii) TPC channel activity is activated by the Ca²⁺ mobilizing molecule nicotinic acid adenine dinucleotide phosphate (NAADP) (4–6) generated considerable excitement that TPCs function as effectors of this mercurial second messenger long known to trigger Ca²⁺ release from “acidic stores.” The spectrum of physiological activities that have been linked to NAADP signaling over the last 25 years (8, 9) may therefore be realized through regulation of TPC activity. However, recent studies have questioned the idea that TPCs are NAADP targets (10, 11), demonstrating instead that TPCs act as Na⁺ channels regulated by the endolysosomal phosphoinositide PI(3,5)P₂. Such controversy (12, 13) underscores how little we know about TPC regulatory inputs and the dynamic composition of TPC complexes within cells.Here, to generate unbiased insight into the cell biology of the TPC complex, we report a proteomic analysis of human TPCs. The TPC interactome establishes a useful community resource as a “rosetta stone” for interrogating the cell biology of TPCs and their regulation. The dataset reveals a predomination of links between TPCs and effectors controlling membrane organization and trafficking, relevant for disease states involving lysosomal proliferation where TPC functionality may be altered (14).     

Tissue nonautonomous effects of fat body methionine metabolism on imaginal disc repair in Drosophila

Regulatory mechanisms for tissue repair and regeneration within damaged tissue have been extensively studied. However, the systemic regulation of tissue repair remains poorly understood. To elucidate tissue nonautonomous control of repair process, it is essential to induce local damage, independent of genetic manipulations in uninjured parts of the body. Herein, we develop a system in Drosophila for spatiotemporal tissue injury using a temperature-sensitive form of diphtheria toxin A domain driven by the Q system to study factors contributing to imaginal disc repair. Using this technique, we demonstrate that methionine metabolism in the fat body, a counterpart of mammalian liver and adipose tissue, supports the repair processes of wing discs. Local injury to wing discs decreases methionine and S-adenosylmethionine, whereas it increases S-adenosylhomocysteine in the fat body. Fat body-specific genetic manipulation of methionine metabolism results in defective disc repair but does not affect normal wing development. Our data indicate the contribution of tissue interactions to tissue repair in Drosophila, as local damage to wing discs influences fat body metabolism, and proper control of methionine metabolism in the fat body, in turn, affects wing regeneration.Tissue repair is the necessary ability of an organism to maintain tissue homeostasis. The self-repair mechanisms of injured tissues have been extensively studied (1, 2). The homeostatic mechanisms of tissue repair, on the other hand, are not confined to the damaged tissues, but rather involve organismal regulation with contributions from multiple systems, including the circulatory, nervous, and endocrine systems. The importance of interactions between damaged tissue and other tissues has been suggested. For example, nerve-derived factor is essential for limb regeneration in the newt and axolotl (3, 4). Studies of parabiosis, the fusion of two organisms that consequently share a vascular system, revealed that regenerative ability was affected by internal environmental conditions based on the circulation of body fluids (5, 6). However, the molecular mechanisms of tissue nonautonomous regulation of the repair process are only beginning to be understood.Drosophila is a useful model animal for studying tissue interactions because of the availability of tissue-specific gene manipulation systems, such as the Gal4/Upstream Activation Sequence (UAS) system (7) or the Q system (8, 9). In Drosophila larvae, the epithelial sheets of imaginal discs are known to have a remarkable ability to repair massive tissue damage. A classic example is their regenerative capacity following surgical ablation (10–12). To overcome the technical difficulties of surgical ablation/regeneration experiments, a system was established for studying imaginal discs repair following genetic ablation, using a Gal4/UAS/Gal80^ts system (13). Despite the great advantages provided by this system, the use of Gal80^ts limits the manipulation of genes with UAS constructs to only the ablating cells at the time of ablation. One strategy for overcoming this problem is to conduct tissue ablation independent of the Gal4/UAS/Gal80^ts system, which allows for utilization of the Gal4/UAS system to manipulate gene expression in uninjured parts of the body. In the present study, we established a cell ablation system using a temperature-sensitive form of the diphtheria toxin A domain (DtA^ts) (14). It has been demonstrated that DtA^ts is active at low temperatures (18 °C) and induces cell death through nuclease activity, but is inactivated at high temperatures (29 °C) (14, 15). Inducing DtA^ts expression by using the Q system enabled us to manipulate Gal4/UAS-mediated gene expression in other organs, independent of the temporal tissue damage in wing discs.Several studies using Drosophila have demonstrated that damaged tissues send signals to surrounding tissues. Cytokine signaling from UV-damaged epidermal cells mediates nociceptive sensitization in larvae (16). Disc injury caused by aseptic wounds leads to production of Unpaired 3 (Upd3), an IL-6 like cytokine in the hemocytes and fat body, which triggers hemocyte proliferation, probably to accelerate clearance of injured cells (17). Hemocyte ablation, however, had no effect on wound closure in the injured larval epidermis (18), raising the question in terms of the functional contribution of hemocytes to tissue repair. In addition, damaged imaginal discs secrete Drosophila insulin-like peptide 8 (Dilp8), which inhibits ecdysone biosynthesis in the ring gland and arrests developmental processes, probably to gain time for repair (19, 20).We previously revealed that local necrosis in adult wings resulted in reduced levels of systemic S-adenosylmethionine (SAM) through the up-regulation of glycine N-methyltransferase (gnmt) in the fat body (21). This report is an example of systemic regulation of SAM metabolism in the fat body on epithelial damage. SAM is a metabolite present in all living cells; it plays various roles, including that of a precursor for transmethylation, transsulfuration, and polyamine biosynthesis (22). It is produced by sole SAM synthase (Sams) from the essential amino acid methionine (Met) and ATP, and SAM levels are strongly regulated by Gnmt in Drosophila fat body (21, 22). Gnmt consumes excess SAM and produces S-adenosylhomocysteine (SAH), which is further metabolized to homocysteine (Hcy). Changes in SAM levels in response to necrotic tissue led us to hypothesize that tissue damage in larval discs also affects methionine metabolism in the fat body, which turned out to be the case in the present study using DtA^ts-ablation system. We further tested whether the modulation of methionine metabolism in the fat body could affect imaginal disc repair in a tissue nonautonomous manner. Our study indicated that proper control of methionine metabolism in the fat body is crucial for repair of wing discs, highlighting the significance of systemic regulation for epithelial tissue repair.