Natural and artificial riboswitches were discussed. Recent advances for the design of mutated polymerases and of chemically modified nucleic acid bases that provide aptamers with new properties were presented. The power of aptamer platforms for multiplex analysis of biomarkers of major human diseases was described. The potential of aptamers for the treatment of cancer or cardiovascular diseases was also presented. Brief summaries of the lectures presented during the symposium are given in this report. A second edition of “Aptamers in Bordeaux” will take place on September 2017 ( Twenty-six years after the publication of three seminal papers on in vitro selection of ligands and enzymes from randomly synthesized oligonucleotide libraries, aptamers are coming to fruition. From these early days, new methodologies have been described, beyond SELEX—the gold standard—for the identification and the optimization of aptamers that can be subsequently converted into biotechnological tools or integrated into various devices. Health and life sciences are still major areas of investigation; indeed, diagnostic and therapeutic applications of aptamers drive the activity of many laboratories and companies. However, other areas  take advantage of aptamer characteristics for analytical purposes. Most of these aspects were covered by about 100 communications presented during the eight sessions of the symposium: riboswitches, selection methodology, aptamer chemistry, biosensors, diagnostics, therapeutics and clinical trials. Over 160 participants—biologists, biophysicists, chemists, engineers, clinicians—either from academy or from industry, coming from 17 countries, contributed to this very stimulating interdisciplinary symposium. Furthermore, this meeting offered the opportunity for 44 PhD students to present their work. The program included not less than six keynote lectures and 26 invited oral presentations which are briefly summarized in this report.Escherichia coli) and one eukaryotic TPP riboswitch regulating alternative splicing in plants . By using a new technique named kinITC, developed in his laboratory, he showed that in contrast to the TPP riboswitch found in plants, the two bacterial TPP riboswitches are kinetically controlled. This regulation requires that the RNA polymerase pauses after synthesis of each riboswitch aptamer to leave time for TPP binding, but only when its concentration is sufficient for binding. Dr. David Horning  described the use of directed evolution to improve the activity of an RNA polymerase ribozyme by selecting variants that can synthesize functional RNA aptamers from an RNA template. Beyond the interest of this work with respect to the RNA-based origin of life, it also shows the potential use of this ribozyme to replicate and amplify short RNA molecules in a way similar to the polymerase chain reaction (riboPCR).The first session of the meeting chaired by Dr. Fabien Darfeuille  was a joint session co-organized with the Bordeaux RNA club symposium and focused on two exciting research domains linking the aptamer field with RNA biology, namely riboswitches and ribozymes. Dr. Dominique Burnouf  introduced the folding dynamics of RNA regulation mediated by two bacterial TPP riboswitches  involved in the activation of virulence genes in response to a temperature shift. She introduced several examples dealing with the regulation mediated by antisense RNA and/or overlapping operons and presented the excludon concept. In this context, she then showed that riboswitches are not only controlling the expression of mRNAs but are also controlling the transcription of noncoding RNAs. In particular, she showed the importance of such regulation in controlling ethanolamine utilization in response to B12, a vitamin required as a co-factor in this metabolic pathway. She finally presented her latest work done in collaboration with Dr. Rotem Sorek and the development of a new approach named Term-seq. Using this new technology, they discovered numerous antibiotic-responsive ribo-regulators that control antibiotic resistance genes in bacterial pathogens. As an example, she described the existence of an attenuation mechanism mediated by antibiotic-stalled ribosomes in L. monocytogenes.The keynote speaker of the first session was Dr. Pascale Cossart . She first started her lecture by playing a movie describing at the molecular level the life cycle of the main bacterial pathogen responsible for foodborne infections, The second morning session of 24 June, chaired by Prof. Eric Peyrin , was dedicated to small chemical ligands binding to RNA that could induce conformational changes or perturb the biological function of an RNA sequence. Dr. Jean-Jacques Toulmé  described biosensors based on the conditional formation of kissing complexes in response to a conformational change of aptamers induced by their cognate ligand. So-called aptaswitches were engineered for the specific recognition of purine analogues: adenosine, theophylline and GTP. For multiplex detection of these three molecules either by surface plasmon resonance in a grafted format by fluorescence, Dr. Toulmé took advantage of a large repertoire of RNA-RNA, RNA-DNA and DNA-DNA kissing motifs. He described the use of aptaswitches for driving the assembly of oligonucleotide scaffolds through kissing interactions. In the second lecture of this session, Dr. Maria Duca  reported on a series of synthetic RNA ligands aimed at preventing the maturation of oncogenic pre-microRNA by DICER. A library of 640 compounds was screened in vitro, allowing the identification of molecules of interest. The best compound inhibited the proliferation of cells over-expressing targeted miRNAs in a specific manner. This effect was correlated to the decrease in the production of oncogenic miRNAs as shown by qRT-PCR and luciferase reporter experiments and to the de-repression of miRNA target proteins. The lecture by Prof. Kazuhiko Nakatani  was dedicated to the regulation of gene expression by ligands inducing a −1 ribosomal frameshifting. Small molecules were designed to bind to mismatched regions in double-stranded RNA (or DNA). Naphthyridine carbamate tetramer (NCTn), one of these molecules, strongly stabilizes mismatched duplexes containing a guanine-rich sequence. Prof. Nakatani described that the NCTn-inducible formation of an RNA pseudoknot can regulate the translation of a target reporter gene by −1 ribosomal frameshifting both in vitro and in cultured cells. Dr. Heiko Sievers  presented the switchSENSE biochip technology that employs DNA nanolevers for real-time measurement of thermodynamic and kinetic parameters of interactions. He demonstrated the potential of this technology for protein-DNA complexes.Bacillus anthracis. The following talk by Dr. Rihe Liu  reported the development of a robust system that allows the direct selection, with high efficiency, of aptamers from a 2′ fully modified fGmH RNA  library transcribed by a variant of the T7 RNA polymerase. Results revealed the higher stability of fGmH RNA aptamers against alkaline hydrolysis, as well as resistance to many of the nucleases present in serum, as compared to 2′ partially modified RNA variants. Further, Dr. Liu reported that the resulting aptamers exhibit high target binding affinity and specificity, thus potentially improving their in vivo applicability as therapeutics and/or as diagnostic tools. The keynote lecture was given by Prof. Philipp Holliger  who closed the session by providing a framework of the extraordinary recent progresses achieved in the last few years exploring the chemical etiology of nucleic acid evolution. Being conceivable that the variety of nucleobases was larger than RNA and DNA at the initial steps of evolution, he presented data on the novel synthetic nucleic acid polymers (XNAs) with fully altered backbone chemistries in which the canonical five-membered ribofuranose ring of DNA and RNA has been replaced by non-natural congeners. Prof. Holliger showed that eight different synthetic polymers, based on nucleic acid architectures, can also mediate genetic information storage and propagation. He demonstrated a capacity for Darwinian evolution by the de novo selection of specific ligands (XNA aptamers) and catalysts (XNAzymes) based on entirely synthetic backbones, and by the in vitro evolution of RNA polymerase ribozymes able to catalyze the synthesis of RNA polymers exceeding their own size.The Saturday afternoon session, chaired by Dr. Vittorio de Franciscis , started with the presentation of Dr. Thomas Shubert  about an innovative method to analyze biomolecular interactions in solution at the microliter scale. MicroScale thermophoresis (MST) is a technique based on the analysis of the movement of molecules through temperature gradients and is easily applicable to measuring interactions between aptamers and their targets. MST assays are provided by 2bind GmbH. The following three scientific lectures were focused on the possibility of expanding the functional potential of nucleic acids by increasing the chemical diversity of functional groups or increasing their stability in serum. Increasing the bio-stability and target interactions of aptamers are topics of particular interest for in vivo applications. In the past decade, many groups have suggested adding functionality to the four standard DNA and RNA building blocks to obtain more efficient aptamers. The first lecture given by Dr. Elisa Biondi  reported research focused on the possibility of adding replicable nucleotides to the DNA alphabet, creating an artificially expanded genetic information system (AEGIS) by developing a new molecular biology to support this expanded laboratory in vitro evolution (LIVE) process and the analytical chemistry needed to sequence AEGIS DNA survivors that might emerge under selective pressure. The speaker presented data from examples of this process in three successful AEGIS-LIVE experiments, two of which were targeted towards whole-living cancer cells, and one towards a specific protein target, the protective antigen from The last session on Friday, chaired by Dr. Günter Mayer , included four talks in different aptamer fields ranging from improved selection methodologies to nanostructure and biosensor applications. The first speaker of the session, Dr. Ichiro Hirao , focused his talk on the genetic alphabet expansion technology that uses artificial base pairs with different chemical and physical properties to improve the affinity of DNA aptamers. Through the use of the unnatural base pair between 7-(2-thienyl)imidazopyridine and 2-nitro-4-propynylpyrrole, DNA aptamers with enhanced affinity were described for various targets including VEGF165, interferon-γ and vWF. Furthermore, Dr. Hirao presented an aptamer stabilization strategy relying on the introduction of a nuclease-resistant mini-hairpin structure into the interferon-γ DNA aptamer, protecting about 70% of this aptamer in human serum (at 37 °C after three days). Next, Dr. Curtis H. Lam , described the non-SELEX bead-based selection of chemically modified aptamers directed against the snake venom myotoxin. The dissociation constant determined by biolayer interferometry was found to be about 40 nM for the highest-affinity sequence. Dr. Lam subsequently reported the results of in vivo anti-hind limb paralysis assays in mice. The addition of 16 equivalents of aptamer resulted in a complete neutralization of the symptoms of the myotoxin, establishing the proof of principle for the use of aptamers as a possible treatment of snakebite. Dr. Chenze Lu  gave a presentation on the use of the split-aptamer strategy to construct DNA assemblies mediated by the presence of a small ligand, i.e., adenosine. Surface plasmon resonance was used to evaluate the ability of the adenosine molecule to trigger the formation of a multiple chain structure. The experimental conditions such as the strand concentration, flow rate and grafting density of probes were optimized to provide a significant adenosine-based SPR signal. The last speaker of this day, Prof. Eric Peyrin , described the recent developments in the fluorescence anisotropy–based aptamer sensing area. The main design strategies and transduction principles were detailed and discussed in relation to the kinds of targets and fluorophores. Some detection examples in real samples were also reported in order to demonstrate the potential applicability of the reported methodologies.Plasmodium falciparum. This parasite causes hundreds of millions of cases of malaria and is responsible for more than a million deaths of children per year alone in Africa. Dr. Ulrich’s research group is currently using these high-affinity aptamers for isolation and identification of their parasite target proteins. Future applications of these aptamers will include novel probes for malaria imaging and diagnostics and new drugs for potential therapeutic applications. Dr. O’Sullivan  reported on two ultrasensitive aptamer-based assays for the detection of β-conglutin. The two approaches were compared in terms of the detection limits and assay time required, and can be adapted (by modifying the aptamer) for the detection of other clinically relevant targets. Dr. Larry Gold  gave a keynote presentation on a novel class of aptamers called SOMAmers (which are slow off-rate modified aptamers), developed at SomaLogic, Inc. Using their SOMAmer-based technology (SOMAScan), Dr. Gold and his team were able to simultaneously quantify over 4000 human proteins in various sample matrices including blood, urine, tissues, and more. To date, they have analyzed over 100,000 human blood samples from a variety of physiological and/or medical conditions—heart disease, Alzheimer’s disease, Duchenne muscular dystrophy, several cancers and diet studies. Dr. Gold concluded his presentation by presenting to the audience what he referred to as the ‘Big Dream at SomaLogic The Wellness Chip’—an ambitious undertaking at SomaLogic to develop a chip capable of monitoring the health profile of every individual over time: the future of personalized medicine.Saturday morning’s session, chaired by Dr. Paloma H Giangrande , included four lectures covering various topics ranging from novel approaches to SELEX  to new aptamers and applications of aptamers for diagnostics. The first speaker of the session, Dr. Günter Mayer , reported on adaptive dynamic artificial poly-ligand targeting (ADAPT) as an ssDNA aptamer-based highly multiplexed biomarker discovery platform. The ADAPT platform identifies and measures thousands of ODNs that form signatures by their association with differentially expressed biomolecules in their native states. Dr. Mayer presented data in breast cancer suggesting that ADAPT may have a promising utility as a breast cancer diagnostic tool that can be easily extended to developing diagnostics for other malignancies. The potential of this ADAPT platform was further described for exosome profiling by Dr. D.B. Spetzler . Dr. Henning Ulrich  reported on DNA aptamer ligands, which bind to proteins secreted into the erythrocyte plasma membrane by the malaria-causing parasite The second session on Saturday, chaired by Dr. B. Sullenger, started with the TRAIL lecture given by Dr. Samie R. Jaffrey . He reported about the selection and utilization of RNA aptamers that bind fluorophores and image RNA in living cells. The aptamers bind small-molecule dyes that become fluorescent only upon specific binding to the aptamer. These fluorescent switches are excellent tags for imaging RNA molecules inside cells. Dr. Jaffrey introduced several aptamer variants termed Spinach, Corn and Red Broccoli which offer a whole spectrum of fluorescent colors analogous to green and red fluorescent proteins. He demonstrated the use of these aptamers as genetically encoded real-time biosensors for cellular metabolites and proteins. For this, a metabolite-sensing RNA  was fused to the Spinach aptamer, allowing fluorescence-based quantification of the metabolite level in the cell. In the second talk of the session, Dr. Beatrix Suess  discussed several strategies for the use of small-molecule binding aptamers for the conditional control of gene expression such as translation initiation, mRNA splicing or mRNA stability and they were shown to efficiently function in all three domains of life. Dr. Frédéric Ducongé  presented PATTERNITY-Seq, a method to improve the identification of aptamers using next-generation sequencing. The method builds dendrograms (named FREDrograms) to visualize the evolution of sequences and their family relationship at each round of SELEX. Re-analyzing data from a cell-SELEX using this approach, improved aptamers were identified against targets such as Annexin-2 compared to those previously published. Finally, Dr. Anna Zamay  presented the ex vivo selection of aptamers against human glioblastoma. These aptamers were conjugated through a biotin/streptavidin linkage to a Brilliant Violet 650 dye and evaluated on freshly resected tumors using a surgical fluorescence microscope. The goal of this approach, named AptaMargin, is to improve the ability of neurosurgeons to specifically and completely resect human brain tumors using in situ fluorescent visualization of tumor margins.In the session chaired by Dr. F. Ducongé , Dr. Maxim Berezovski  described an original application of aptamers targeting the VSV (vesicular stomatitis virus) oncolytic virus in order to hide them from neutralizing antibodies. The goal is to protect them from being cleared in the bloodstream before they reach tumor cells and exert their effect. Alternatively, aptamers were raised against the Fab fragment of anti-VSV polyclonal antibodies. These aptamers were then engineered as multivalent binders. The most effective constructs increased the viral infectivity by 70% in vitro. Another application in the field of immunology was described by Dr. Jean Gariépy : he designed a pegylated aptamer raised against a CD200R1 target that blocks inflammatory responses in mouse models. Importantly, this aptamer does not suppress cytotoxic T-lymphocyte induction in lymphocyte culture. Dr. H. Tom Soh  described real-time biosensor-integrating aptamers in a micro-fluidic device capable of tracking circulating molecules in living mice. He presented the results obtained for monitoring a cancer drug (doxorubicin) and an antibiotic (kanamycin). This device can also be used for feedback control of drug concentration.Saturday afternoon’s session chaired by Dr. Beatrix Suess  included five lectures on novel therapeutic applications of aptamers. The first speaker of the session, Dr. Paloma Giangrande , reported on cell-targeted RNA aptamers directed against vascular smooth muscle cells. Dr. Giangrande demonstrated that these aptamers have superior therapeutic effects when compared to the gold-standard drug-eluting stents ; the aptamers inhibit the pathologic proliferation of vascular smooth muscle cells without affecting the healing of the endothelial cell layer . Dr. Giangrande discussed how cell-targeting aptamers can be developed as therapeutics for many different diseases including cancer and diabetes, where it is beneficial to limit undesired toxic effects of the drug to normal cells. Dr. Ulrich Hahn  gave a nice overview of his aptamer program over the years and discussed his recent focus on the development of aptamers that inhibit cell adhesion. He provided examples of high-affinity DNA aptamers that target E- and P-selectin and demonstrated the inhibitory effects of these aptamers in cancer cells in culture. Dr. Hahn also touched on ongoing new projects aimed at the selection of aptamers specific for further components playing a central role in the promotion of tumor cell growth, invasion, and organotropic metastasis, such as integrins. Dr. Sullenger  reported on the therapeutic and diagnostic applications of rapidly reversible aptamers. He discussed the development and clinical evaluation of the factor IXa aptamer. This aptamer was recently evaluated in 2000 patients undergoing angioplasty. Although the aptamer-antidote pair failed due to safety issues with the formulation, it demonstrated that aptamers can rapidly and potently inhibit their target proteins in patients and that antidote molecules can rapidly reverse such activity in the minute time frame. Based on these initial promising results with the aptamer-antidote technology, Dr. Sullenger’s group is exploring the potential of using antidote-mediated control of aptamers for a variety of other therapeutic and diagnostic applications. One application includes the use of a rapidly controllable factor Xa anticoagulant aptamer to effectively control (reverse) blood coagulation during cardiopulmonary bypass surgery. A second application involves a rapidly reversible thrombin aptamer for imaging blood clots in real time. Finally, the group is also developing a rapidly reversible tumor-targeting aptamer to limit the side effects of chemotherapeutic agents. Dr. Brett Schrand  reported on the use of aptamers as an adjuvant to radiation therapy. He discussed the application of VEGF-targeted 4-1BB co-stimulation of T-cells to enhance radiation-induced immune control of tumor growth using a multimeric aptamer approach linking two aptamers (one to VEGF and the other to 4-1BB). Dr. Schrand reported efficacy in several animal models of cancer. For example, he showed that treatment of tumor-bearing mice with VEGF-4-1BB aptamer conjugates was effective against melanoma, breast cancer, fibrosarcoma and glioma. He went on to show that this effect was greatly enhanced with radiation, through the activation of VEGF following radiation treatment. Finally, he presented key findings that validated this approach in a model for abscopal responses and showed that VEGF-targeted 4-1BB costimulation was effective at inducing an immune response against a distant, non-irradiated tumor, comparable to that of CTLA-4 and/or PD-1, the gold standard in tumor immunotherapy, without the off-target effects associated with these therapies. Dr. Vittorio de Franciscis  reported on aptamer carriers for cancer stem cell–targeted delivery of therapeutic miRNAs for the treatment of glioblastiomas (GBM). GBM is the most common primary brain tumor and one of the most lethal kinds of cancer sustained by a small population of cancer stem cells . Recent interest has focused on the use of miRNAs and miRNA inhibitors (antimiRs) for the treatment of this disease. However, a major obstacle to the clinical translation of this new class of oligonucleotide drugs is the lack of a reliable targeted delivery method. Dr. de Franciscis reported on exciting work from his lab demonstrating the use of aptamers targeted to glioblastoma cells as potential delivery agents for therapeutic miRNAs and miR inhibitors. In addition, he presented evidence that when used in combination, these aptamer-miR conjugates result in enhanced therapeutic effects, leading to the suppression of tumor sphere formation. Results provide the rationale for the design of combined aptamer-miR–based gene therapies selectively targeting cancer stem cells.To highlight the remarkable work carried out by young investigators, two out of 69 posters were selected for awards. The first award was given to Nam Nguyen Quang, a PhD student supervised by Dr. Frédéric Ducongé , who presented PATTERNITY-seq, a new bioinformatics pipeline to analyze every SELEX round using next-generation sequencing. This method monitors the paternity relationship and the evolution of several patterns from primary sequences to predicted secondary structures. It can help to identify aptamer families and the best aptamers of a family. The second award was given to Marc Vogel, a PhD student in the laboratory of Dr. Beatrix Suess , who introduced a recently developed synthetic riboswitch which allows the control of alternative splicing in mammalian cells. Using the tetracycline-binding aptamer, a control device was developed which regulates the accessibility of the 3′ splice site, leading to exon skipping or the lack thereof. The functionality of this device was demonstrated in different host genes and cell lines including a suicide system that allows efficient, tetracycline-dependent induction of cell death via the controlled expression of a CD20 surface receptor.http://www.aptamers-in-bordeaux.com/) as a satellite of the annual meeting of the “Oligonucleotide Therapeutic Society” (24–27 September 2017).Following the success encountered by this symposium, a new edition will be organized in Bordeaux on 22–23 September 2017 (


For specific drug development and to study distinct arrhythmias, simple models are required to implement and analyze such mutations. Here, we introduced CPVT inducing mutations into the pharynx of Caenorhabditis elegans, which we previously established as an optogenetically paced heart model. By electrophysiology and video-microscopy, we characterized mutations in csq-1 (CASQ2 homologue) and unc-68 (RyR2 homologue). csq-1 deletion impaired pharynx function and caused missed pumps during 3.7 Hz pacing. Deletion mutants of unc-68, and in particular the point mutant UNC-68(R4743C), analogous to the established human CPVT mutant RyR2(R4497C), were unable to follow 3.7 Hz pacing, with progressive defects during long stimulus trains. The pharynx either locked in pumping at half the pacing frequency or stopped pumping altogether, possibly due to UNC-68 leakiness and/or malfunctional SR Ca2+ homeostasis. Last, we could reverse this ‘worm arrhythmia’ by the benzothiazepine S107, establishing the nematode pharynx for studying specific CPVT mutations and for drug screening.Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a condition of abnormal heart rhythm (arrhythmia), induced by physical activity or stress. Mutations in ryanodine receptor 2 (RyR2), a Ca If untreated, CPVT is highly lethal. The exact prevalence of CPVT is not known, but it has been estimated to be around 1:10,0001. The mean age of symptom onset is between 7 and 8 years2, but a wide range of ages has been observed. Most mutations that have been linked to CPVT are found in two genes, ryanodine receptor 2  and calsequestrin 2 , two proteins fundamentally involved in regulation of intracellular Ca2+ in cardiac myocytes4. The cardiac RyR2 is a Ca2+ release channel located in the SR. During CICR, Ca2+ is provided by the activated L-type voltage-gated Ca2+ channel  and binds to RyR2 thus triggering the opening of the channel, allowing fast Ca2+ efflux from the SR and subsequent muscle contraction. After reuptake into the SR lumen by the SERCA complex , Ca2+ is stored there at high concentrations. Calsequestrin is the major Ca2+ sequestering protein in the SR. It forms higher-order dynamic structures of monomers, dimers or long chains, depending on the Ca2+ concentration. Polymerization of calsequestrin occurs by back-to-back  and front-to-front  interactions6. In mammals calsequestrin is linked to the RyR through an interaction with triadin7 and junctin8.CPVT is an inherited disturbance of the heart rhythm (arrhythmia) induced by adrenergic stress. It derives from an alteration of intracellular CaC. elegans, CSQ-1, which shows ~40% sequence identity and 50–60% similarity to the human CASQ2. Cho et al.9 reported that UNC-68 appeared to affect the correct localization of CSQ-1 in vivo. Based on the genome sequence, there are no obvious homologs of junctin and triadin in C. elegans, therefore a direct interaction of the positively charged CSQ-1 C-terminus with the negatively charged luminal loops of UNC-68 is postulated10.For calsequestrin there is only one isoform present in C. elegans has a single RyR gene encoded by the unc-68 locus11. The UNC-68 protein of C. elegans shares 45% sequence identity and 63% homology with the human cardiac RyR2. A low-affinity Ca2+ binding site specific for RyR1 (aa 1872-1923) is not present in UNC-68, arguing that it is more related to RyR2. UNC-68 is expressed in the terminal bulb and posterior isthmus muscle cells of the pharynx11, but also in body wall muscles and neurons. unc-68 mutants are viable, but were identified as being defective in locomotion12 and to exhibit defects in pharyngeal pumping. In addition to visible defects in muscle function, unc-68 mutants grow more slowly and have fewer offspring than the wild type. In contrast, mutant mice lacking RyR2 die as embryos with morphological abnormalities in the heart tube13.In vertebrates three RyR isoforms are present, whereas 14 (http://triad.fsm.it/cardmoc/). These result in a Ca2+ leakage from the SR, which leads to cytosolic Ca2+ overload generating delayed afterdepolarizations (DADs) , triggered activity, and ventricular arrhythmias, in particular under adrenergic conditions15. Disease-causing mutations have also been identified in RyR1, and are linked to malignant hyperthermia or central core disease19. Recently, the significance of RyR function for aging was shown by single amino acid modifications, which conferred a reduction in lifespan and an accelerated decline in muscle integrity with age in C. elegans20.Up to now, many different (>170) CPVT mutations and polymorphisms are knownC. elegans pharynx, a rhythmically active muscular pump that we previously established as an optogenetically controlled arrhythmia test system21. We thus examined two available mutations of C. elegans calsequestrin. First, we analyzed possible defects of a deletion mutant. In addition, we tested a mutation at position P319 (mutated to S), a residue that corresponds to P308 in human CASQ2, and mutation of which is an established cause of CPVT. This mutation  caused decreased Ca2+-selectivity during polymerization of purified CASQ2-P308L protein23 as a structural disruptor for α-helices and as a turning point in β-sheets, thus it was suggested that a substitution of P308 causes a profoundly altered conformation of the CASQ2 protein, and that this could lead to reduced Ca2+ binding22. Therefore, we expected that any alteration of this proline in C. elegans CSQ-1 would also cause problems in folding or conformation, and consequently in Ca2+ binding capacity. Even though the worm allele introduces a polar residue instead of an aliphatic one, the structural consequences could be equally strong.In this study we aimed to model CPVT arrhythmias in the C. elegans UNC-68, identified in human patients with CPVT24, and demonstrated in mouse models to be causative to CPVT26. They represent two of the most investigated CPVT mutations in animal models or cell systems. Nevertheless, the mechanisms by which they induce arrhythmia are still not completely resolved. R2474S resides in the helical domain 1 segment 6b27. One study suggested that the CPVT-associated RyR2 mutation results in leaky RyR2 channels due to decreased binding of calstabin-2 (FKBP12.6), which stabilizes the closed state of the channel25. However, more recently it was found that the RyR2-FKBP12.6 interaction was not disrupted in R2474S/ + knock-in mice. These authors suggested that the R2474S mutation in the central domain of RyR2 induced a defective interaction between the central and the N-terminal domains, thus causing channel dysfunction28. Jiang et al.29 could also not observe effects of CPVT mutations on FKBP12.6 binding. The mutation R4497C is located in the S0 segment of the RyR channel domain27 affecting the channel gating and creating a SR Ca2+ leak30. A decreased binding of FKBP12.6 was also detected for this mutation25. However, in other studies a normal RyR2-FKBP12.6 interaction was shown in RyR2R4496C+/− mice31 or in coimmunoprecipitation studies from resting HL-1 cardiomyocytes (CM)32. The reasons for these discrepancies are unclear.In the case of RyR2, we inserted two mutations (R2474S and R4497C) in 37. Few RyR2 mutations have already been characterized in CM derived from induced pluripotent stem cells (iPSCs) and reflect basic aspects of CPVT that can be assessed well on the single-cell level45. Novak et al.46 found in iPSC-CMs with the D307H CPVT mutation of calsequestrin that the β-adrenergic agonist isoproterenol causes DADs, oscillatory arrhythmic prepotentials, after-contractions and a diastolic [Ca2+]i rise. However, iPSCs are an expensive and complicated experimental system, precluding large scale screening approaches, and, more importantly, they cannot reflect the complex disease as it is present in an intact organ. For this reason we assessed the pharynx, the feeding organ of C. elegans, which is a rhythmically active muscular pump47, as a heart model21. Beside the easily accessible genetics and its high throughput potential, there are observations suggesting that the pharynx is orthologous to the vertebrate heart48. Both the pharynx and the heart are tubes transporting material along their lumina. The pharynx utilizes homologues of most of the ion channels, pumps and transporters defining human CM physiology. Similar to the mammalian heart, the pharynx muscle cells are connected by gap junctions49. The C. elegans EGL-19 L-type voltage dependent Ca2+ channel α1-subunit maintains depolarization during the plateau phase and sustains muscle contraction similar to the cardiac action potential (AP). In fact, with a plateau phase of ~200 ms, the pharynx AP resembles the shape of the human cardiac AP better than that of the mouse. EGL-19 slowly inactivates, and membrane repolarization is executed by a voltage-gated K+-channel, encoded by exp-250 which, though homology is low, is functionally similar to the human ether-a-go-go-related gene (hERG) channel.To enable drug development for treatment of specific arrhythmias, but also to study distinct arrhythmia types, simple model systems are required, that allow implementing patient-specific mutations. Up to now, only a handful of mouse models for CPVT exist21. ChR2(H134R), a gain-of-function variant, was directed to the plasma membrane of the pharyngeal muscle cells by the specific promoter pmyo-2 and its expression allows stimulation of pumping. We assessed pharynx pumping by extracellular recordings , similar to electrocardiograms, of dissected (cut-head preparation) or intact worms, which allows accurate measurements of pump rate and duration, and distinguishing muscle activity of different parts of the pharynx, as well as activity of pharyngeal neurons52. Corpus and terminal bulb contraction/excitation, as well as their relaxation/repolarization, can be automatically analyzed53. In addition, we used a video-microscopy based method, which allows recording of multiple intact animals simultaneously21. Importantly, ChR2-paced pumping of the pharynx is not influenced by neuronal input21.Spontaneous pharynx pumping is too irregular to allow arrhythmia detection. To yield millisecond-precise and stable rhythmicity, we optically paced the pharynx using channelrhodopsin-2 (ChR2)csq-1 and unc-68 and examined their effect on optogenetically paced pumping, exhibiting clear arrhythmia phenotypes and progressive loss of the ability to lock into the pacing. Finally, we demonstrate ameliorating effects of the benzothiazepine S107, emphasizing that our model can be used to identify and/or test allele-specific drugs.Here, we extended our pharynx arrhythmia model to CPVT like arrhythmias. We characterized the role of RyR and calsequestrin in deletion mutants. Further we introduced different CPVT-related mutations of csq-1 deletion mutants  deletion mutant phenotypes in a swimming locomotion assay depending on normal BWM function  led to reduced pumping ability in the presence of bacterial food, and a complete rescue was observed by CSQ-1::CFP expression  deletion mutant was unable to follow the 3.7 Hz optogenetic pacing  did not show significant defects. Probably the polar serine residue is not affecting the Ca2+ binding and polymerization properties as much as the aliphatic leucine found in the human P308L mutation23 , we used fosmid recombineering. The fosmid we used contains a slightly truncated promoter, thus we first verified if this promoter fragment is expressed in pharyngeal muscles. This was the case, i.e. the punc-68 promoter fragment included in the fosmid, transcriptionally fused to CFP, was expressed in pharyngeal muscles and BWMs . The unc-68(r1162) mutant eliminates a large part of the channel pore domain, thus r1162 can be considered a molecular ‘null’ allele55.To modify the WMs Fig. . unc-68 unc-68 mutant animals suggest that intracellular Ca2+-release is not essential for excitation-contraction (EC) coupling in C. elegans, but may rather act to amplify a plasma membrane mediated calcium influx that itself is sufficient for contraction55. Thus, we observed a reduction of spontaneous pump rate on food of only 32% in the unc-68(r1162) mutant compared to wt , and a mild reduction of function mutant of the inositol trisphosphate (IP3)-receptor itr-1(sa73) exhibited a similarly reduced spontaneous pump rate (in the presence of food). A deletion of the presumptive FKBP12.6 homolog fkb-2 (fkb-2(ok3007); Supplementary Fig. unc-68 deletions. Interestingly, for spontaneous pumping, eliminating synaptic transmission in the unc-13(s69) mutant56 caused a more severe phenotype than unc-68 deletions  , a well-known activator of RyR. In EPG recordings, a 72% increase of the pump rate was observed in wt , and further suggesting an important contribution of the RyR to caffeine induced pumping.To demonstrate the conservation of function of the RyR between humans and unc-68 is a very large gene , we introduced mutations into a rescuing fosmid containing unc-68 by recombineering. We chose two mutations (described above) from human CPVT patients that were demonstrated to cause arrhythmia also in mice: The R4743C mutation, analogous to the CPVT mutation R4497C in humans , and R2729S analogous to the CPVT mutation R2474S in humans and mice . The engineered fosmids were used to generate transgenic lines containing extrachromosomal arrays, in the unc-68(r1162) mutant background. As a control, we also generated a rescuing transgene from the unmodified fosmid. Expression of mutant UNC-68 proteins, in the absence of the endogenous protein, resulted in complete  mutants with the wt fosmid rescue could follow the pace rhythm faithfully until the end of the stimulus train  deletion mutant, as well as the mutant rescued with UNC-68(R4743C) exhibited pumping defects. To analyze these in a quantitative manner, we counted the actual pumps for each 10 light pulses. Both the deletion mutant as well as the point mutation R4743C could briefly follow the 3.7 Hz pumping period in the beginning of the stimulation protocol, but then exhibited a decrease of the pump rate, that was particularly pronounced for the R4743C CPVT mutant specific drugs may be identified that could prevent cardiac arrhythmias. The 1,4-benzothiazepine derivative K201 (a.k.a. JTV519) is an anti-arrhythmic drug currently under clinical investigation. However, K201 is a non-specific blocker of sodium, potassium and calcium channels57. Thus, there are’mixed‘ results for K201, with side-effects outweighing benefits58. Treatment with K201 enhanced the affinity of FKBP12.6 for RyR2, which stabilized the closed state of RyR2 and prevented the Ca2+ leak triggering arrhythmias59. Furthermore, K201 was effective in reducing SR Ca2+ leak by specifically regulating RyR2 opening at diastolic [Ca2+]i in the absence of increased RyR2 phosphorylation at Ser2814, thus extending the potential use of K201 to conditions of acute cellular Ca2+ overload60. However, in another study on isolated myocytes, K201 treatment did not elicit a decrease in delayed after depolarizations and K201 failed to prevent induction of CPVT in RyR2 R4496C knock-in mice31.Up to now β-blockers are the first therapeutic option for patients with CPVT. Although β-blockers reduce the occurrence of ventricular tachycardia, 30% of patients still experience cardiac arrhythmias and eventually require cardioverter defibrillator implantation to prevent cardiac arrest25. Sasaki et al.43 could recently show in iPSCs for another C-terminal CPVT mutation (I4587V) that the development of DADs in the presence of isoproterenol was significantly suppressed by S107. But, to our knowledge, S107 was never tested on R4497C (R4743C in C. elegans). In fact we found after a 30 min incubation in S107 (50 µM) a complete rescue of the pump ability of R4743C mutation bearing nematodes  specific effect of S107, that was not elicited via other, unknown channels.Thus, we decided to test a novel RyR2-specific compound (S107), a more specific 1,4-benzothiazepine derivative, which in the rodent model enhanced the binding of FKBP12.6 to the mutant RyR2(R2474S) channel, inhibited the channel leak and prevented cardiac arrhythmiasdes Fig. . Whereasdes Fig.  indicati21 we established the C. elegans pharynx as a model for analyzing LQT8-like arrhythmogenic mutations affecting the Cav1.2 VGCC. Optogenetic stimulation of pharynx muscle via ChR2 enables pacing at about 4 Hz (and up to 6 Hz) for > 1 minute. Here, we extended this model for CPVT-related mutations and characterized the role of RyR2- and CASQ2-mutations in C. elegans pumping, including homologous mutations causing CPVT in humans. The UNC-68(R4743C) mutation, homologous to human RyR2(R4497C), causing channel leakiness, provoked a progressive drop in the ability to follow pacing. The experimental drug S107, a derivative of 1,4-benzothiazepine, caused a complete reversion of the phenotype, emphasizing that the C. elegans pharynx can indeed be used as a simple, genetically amenable model for cardiac arrhythmias, and possibly also for drug screening.In a former study2+ sequestering protein CSQ-1  weakly affected swimming locomotion (16% reduced swimming cycles), but had a more pronounced effect on spontaneous pharynx pump rate on food (38% reduction), which could be reversed by over-expression of CSQ-1::CFP. Inconsistently, in previous work, no significant effect on C. elegans locomotion speed could be observed, neither by RNAi against csq-1 nor in another deletion mutant csq-1(jh109)10. Interestingly, CASQ2-null mice are viable and display normal SR Ca2+ release and contractile function under basal conditions, although they exhibit striking increases in SR volume33. Nevertheless, these mice exhibit severe forms of CPVT, analogous to the phenotypes observed in humans lacking CASQ254.Eliminating the Cacsq-1 in the paced pharynx model, which was unable to follow the 3.7 Hz stimulation , loss of function was related to the observed loss of normal binding of CASQ2(D307H) to junctin and triadin61. The fact that the C. elegans genome apparently does not encode triadin and junctin10 could explain why we did not observe any strong effect of the P319S mutation.Here, we observed an arrhythmic phenotype for the deletion mutant of ion Fig.  and in aion Fig. . The poi62. The C. elegans RyR is required for normal locomotion and pumping. However, the unc-68 deletion mutant phenotype shows that UNC-68 is not essential for EC coupling or survival in the nematode. RyR could cooperate with the IP3 receptor for pumping at high rates, since a mild reduction of function mutant of itr-1(sa73)63 shows a similarly reduced pump rate on food as unc-68 deletions. Animals with strong itr-1 loss-of-function alleles are sterile64.Homozygous deletions of the RyR2 gene are embryonically lethal in miceC. elegans FKBP12.6 may be different than in vertebrates; this will have to be assessed in future work. A clear FKBP12.6 homolog is yet to be identified in C. elegans, which contains 8 members of the fkb gene class  to the same extent as in human RYR228. The C. elegans UNC-68 protein could have a slightly altered structure, and R2729S may thus not exhibit an arrhythmia phenotype. In the mouse, it was proposed that the mutation alters RyR2 structure such that the whole complex becomes more susceptible to protein kinase A (PKA)-mediated phosphorylation modulation, thus causing increased arrhythmia. This could occur either by increased propensity to ‘unzipping’ of the N-terminal and central domains, and thus result in a significant increase in the frequency of spontaneous Ca2+ transients as shown in R2474S/ + knock-in CM28. Alternatively, it could be effected by dissociation of FKBP12.6 from human RyR2 following PKA phosphorylation65. In C. elegans, no adrenergic signaling is present; however, serotonin and other neuromodulators affect pharynx function as a response to the presence or absence of food. Yet, food-stimulated pumping was unaffected in the R2729S mutant animal , but observed no effect on paced pump ability, only swimming locomotion was affected. Possibly, an interaction of UNC-68 with the mal Fig. . Last, wunc-68, caused a significant progressive decrease in the ability to follow paced pumping at high rate, both in EPG-recordings and video microscopy of intact animals, where the majority of animals were able to follow only every second light pulse or stopped pumping. This finding manifests a ‘worm arrhythmia’ phenotype of the CPVT-like mutation in our optogenetic model. The equivalent mutation R4496C in mice was suggested to play an essential role in RyR2 channel gating, and it was shown that the channel is more likely to open even at low Ca2+ concentration30. Thus, a faster depletion of Ca2+ could be underlying the inability of UNC-68(R4743C) pharynxes to follow 3.7 Hz pacing.The second CPVT-related mutation R4743C (R4497C in humans), as well as the deletion of C. elegans mutant UNC-68(R4743C) argues for our system as a valuable arrhythmia model that enables to study CPVT mutations. To assess whether this also enables drug testing, we analyzed effects of the benzothiazepine S107 on restoring the pump ability of the arrhythmic pharynx. Indeed this was possible. It is interesting that S107 was considered to stabilize the FKBP12.6-RyR2 interaction in humans, yet, here it also showed beneficial effects for R4743C (R4497C in humans). This strengthens the assumption that the respective mutation influences the FKBP12.6-RyR2 interaction65, as already shown by Lehnart et al.25 when using S107 treatment for the R2474S mutation. Our work demonstrates that potential anti-arrhythmogenic drugs can be studied in the C. elegans system, which may be used as a rapid, initial model for introducing patient-specific CPVT mutations, and then to screen potential drugs in a relatively straightforward manner. Of course, this may not always be possible if the affected residue in humans is not conserved in UNC-68. However, possibly the human RyR2 can be introduced into C. elegans to replace UNC-68, thus generating a ‘humanized’ model. Worms can be treated with drugs and then assessed in a high-throughput manner by video microscopy.The arrhythmic phenotype observed in the pharynx of the C. elegans as a model to characterize CPVT mutations, for two loci affected by this genetic condition, CASQ2 and RyR2 . The optogenetically paced pharynx model enables testing of drugs like S107 on specific mutations. The approach may enable characterizing new CPVT mutations found in patients, but possibly also mutations of RyR1, which are associated with malignant hyperthermia or central core disease.In conclusion, we have established the pharynx of C. elegans strains were cultivated at 20 °C on nematode growth medium (NGM) fed with E. coli strain OP-50-1. We used the following strains kindly provided by the Caenorhabditis Genetics Center (CGC): N2 (wild type), RB2019: csq-1(ok2672)X, VC40907:csq-1(gk876502)X; altering P319 to S, TR2171:unc-68(r1162)V, CB540:unc-68(e540)V, JT73: itr-1(sa73)IV and RB2222: fkb-2(ok3007)I. unc-13(s69)I was kindly provided by E. Jorgensen.ZX1534:csq-1(ok2672)X; zxEx754; zxIs20, ZX1581:csq-1(ok2672)X; zxEx754, ZX1651:unc-68(r1162)V; zxEx752; zxIs20 , ZX1654:unc-68(r1162)V; zxEx749; zxIs20, ZX1819:csq-1(gk876502)X; zxIs20, ZX1833:unc-68(r1162)V; zxEx748; zxIs20, ZX2121:unc-68(r1162)V; punc-68::unc-68 (exon1–4)::CFP (10 ng).Furthermore, we generated the following transgenic strains: pmyo-2::ChR2(H134R)::mCherry was generated as described in Schuler et al.21.Plasmid csq-1 gene was amplified by PCR from C. elegans genomic DNA  and inserted into pmyo-3::CFP (based on pPD115.46) via restriction sites BamHI and KpnI. All primer sequences are listed in Supplementary Table myo-3 promoter was exchanged to the endogenous csq-1 promoter, by obtaining a 2.4 kb DNA fragment upstream of the CSQ-1 encoding region by PCR from the genomic DNA  and insertion into the pmyo-3::csq-1::CFP plasmid via PstI and BsaBI restriction sites, resulting in pcsq-1::csq-1::CFP.The Punc-68::unc-68(exon1–4)::CFP, which was used for the verification of expression via punc-68, was generated using the In-Fusion cloning HD Kit . The pmyo-2::CFP plasmid was restricted by Acc65I and SalI and served as backbone. punc-68::unc-68 was amplified from fosmid WRM063aG12 by primer pair oEF150 and oEF151 containing homology arms annealing to the restricted pmyo-2::CFP plasmid and ligated.unc-68 gene was performed by a modified protocol of the heat induced recombination strategy in fosmids previously published by Tursun et al.66. Electro-competent E. coli SW105 which contain an arabinose-activatable FLP recombinase (not used in these experiments) and a heat-activatable λ red recombinase, were transfected with fosmid WRM063aG12 by electroporation  at 2000 V, 6 ms. Successful transfection was verified by isolation of fosmid DNA and test digestion with AgeI. For insertion of point mutations, a two-step recombination and selection strategy was used. For the first step, λ red recombinase was heat-activated and electro-competent SW105 containing fosmid WRM063aG12 were generated66 and transfected with a PCR product containing a galactokinase-K  encoding gene with corresponding em-7 promoter, serving as a selection marker and flanked by 50 bp unc-68-homology sequences including the respective point mutation .Insertion of point mutations in the unc-68 and GalK-unc-68 fosmids were observed and “onlyGalK” clones were separated by repeatedly streaking out on MacConkey agar plates67, containing the pH indicator neutral red for an indication of galactose degradation, and analyzed via colony PCR screening  . Mixed clones containing both WT-pmyo-3::mCherry (10 ng/µl) or pelt-2::mCherry (20 ng/µl) in the deletion mutant unc-68(r1162). Generation of a strain for verification of unc-68 expression in the pharynx occurred by microinjection of 10 ng/µl of punc-68::unc-68(exon1–4)::CFP in the deletion background unc-68(r1162)V. The CSQ-1 rescue construct was expressed in mutant csq-1(ok2672) after microinjection of 5ng/µl pcsq-1(2.4 kb)::csq-1::CFP with co-injection marker pmyo-3::mCherry (10 ng/µl).Transgenic animals expressing endogenous UNC-68 or UNC-68 with point mutations, respectively, were generated by microinjection of 1 ng/µl of the particular fosmid and the co-injection marker unc-68(r1162)V mutants were selected by locomotion phenotype, and were confirmed by PCR genotyping . Genotyping of csq-1(ok2672)X was likewise done by PCR . The mutation csq-1(P319S)68 was genotyped by PCR amplification , digestion with HinfI and analysis by electrophoresis on a 5% acrylamide gel to visualize the resulting small DNA fragments. The positively genotyped mutants were verified by sequencing.pcsq-1::csq-1::CFP and punc-68::unc-68(exon1–4)::CFP was analyzed on a Zeiss Axio Observer microscope, with an 40x/0.25 Zeiss ∞/- APlan oil objective and CFP filter set . Animals were transferred on 2% agarose pads in M9 buffer  and immobilized with 1 µL freshly prepared 50 mM sodium azide solution  in M9 buffer from a 1 M stock in water.Expression of One day before experiments, L4 larvae were picked on NGM dishes  seeded with 320 µl OP50 culture. Spontaneous pumping of animals on food was video recorded  and visually counted for 20 s. Mean values, standard error of the mean (SEM) and further statistics (1-way ANOVA with Bonferroni post-hoc test) were calculated with OriginPro .trans retinal . ATR (0.65 µl of a 100 mM stock in ethanol) was added to 650 µl of OP50 culture and spread onto 94 mm culture dishes  containing 25.2 ml of NGM. We performed EPG recordings on intact animals and on cut head preparations. For cut head preparations, animals were transferred into a recording chamber containing a Sylgard-coated cover slip (25 mm diameter) and filled with 1.5 ml of EmD50 buffer . The head of an animal was cut away from the body with a scalpel  directly posterior to the terminal bulb. Upon dissection, the body wall muscles contract and expose the posterior pharynx.One day before experiments young adult hermaphrodites were placed on fresh NGM plates with or without all-et al.21. The tip of the worm head was sucked into an EPG-suction electrode (~20 µm inner diameter tip) using a 10x objective. For optical pacing with a 470 nm LED  the pharynx was positioned below a 60x water immersion objective ; an EGFP-ET filter set  was used. Recording of EPGs and triggering of light pulses was synchronized by PatchMaster software .Electrophysiology was performed as described in Schuler 53 . Excel was used for calculation of means and SEM, 1-way ANOVA with Bonferroni post-hoc test was performed using OriginPro.Either spontaneous pumping (at least for 1 minute) was recorded or the pharynx was stimulated with 470 nm light pulses  over a period of 30 s or in a stress test . Review software  was used to translate PatchMaster files to ABF files. Pump rate and duration were analyzed by AutoEPGTransgenic L4 larvae cultivated with ATR were placed on fresh NGM dishes containing ATR one day prior to the assay. 1 µL of polystyrene microspheres  were added on pads composed of 10% agarose. About 10 animals were transferred into the beads and gently overlaid with a coverslip.Measurements were performed on a Zeiss Axio Observer, equipped with a 100 W HBO lamp, EGFP Filter  and an 10x/0.25 Zeiss ∞/- APlan objective. Animals were stimulated with blue light pulses  with a computer-controlled shutter  every 250 ms (4 Hz) over a period of 30 s. Recording was performed using an ORCA Flash 4.0 sCMOS camera  and µManager v1.4 software. For drug tests, animals were incubated 30 min in 50 µL of 50 µM S107  in M9 buffer containing 0.1% DMSO before the measurements.et al.21. Excel was used for calculation of means and SEM, 1-way ANOVA with Bonferroni post-hoc test was performed using OriginPro.Multi-kymographs of grinder movements were generated and analyzed as described in Schuler The data that support the findings of this study  are available from the corresponding authors upon reasonable request.Supplements


The dysregulation of monoamine clearance in the central nervous system occurs in various neuropsychiatric disorders, and the role of polyspecific monoamine transporters in monoamine clearance is increasingly highlighted in recent studies. However, no study to date has properly characterized polyspecific monoamine transporters in the mouse brain. In the present study, we examined the kinetic properties of three mouse polyspecific monoamine transporters  and compared the absolute mRNA expression levels of these transporters in various brain areas. First, we evaluated the affinities of each transporter for noradrenaline, dopamine, serotonin, and histamine, and found that mouse ortholog substrate affinities were similar to those of human orthologs. Next, we performed drug inhibition assays and identified interspecies differences in the pharmacological properties of polyspecific monoamine transporters; in particular, corticosterone and decynium‐22, which are widely recognized as typical inhibitors of human OCT3, enhanced the transport activity of mouse Oct3. Finally, we quantified absolute mRNA expression levels of each transporter in various regions of the mouse brain and found that while all three transporters were ubiquitously expressed, Pmat was the most highly expressed transporter. These results provide an important foundation for future translational research investigating the roles of polyspecific monoamine transporters in neurological and neuropsychiatric disease. CaMKII2+/calmodulin‐dependent protein kinase IICaOCTorganic cation transporterPMATplasma membrane monoamine transporterSNRIserotonin‐noradrenaline reuptake inhibitorSSRIserotonin‐selective reuptake inhibitorTEAtetraethylammoniumKm) value] and low‐capacity  transporters with high substrate specificity. Noradrenaline uptake through the noradrenaline transporter is one example of uptake1 transport. Synaptic reuptake through the uptake1 transport system plays a central role in limiting the extent of neurotransmission, and accordingly this system is accepted as a highly important system in the brain. To this end, multiple substrate‐specific transporter inhibitors such as serotonin‐selective reuptake inhibitors (SSRIs) and serotonin–noradrenaline reuptake inhibitors (SNRIs) have been developed and implemented for clinical use Km value and high Vmax value, respectively) transporters in a polyspecific manner; that is, these transporters interact with at least two different substrates Two distinct uptake systems are responsible for the transport of extracellular monoamine neurotransmitters in the brain. The uptake1 system facilitates the intracellular transport of extracellular neurotransmitters through high‐affinity ‐labeled substrate (1 μCi·mL−1) at 37 °C for 2–60 min (for time‐dependent uptake) or 5 min (for dose‐dependent uptake and Na+/Cl−/H+‐dependent uptake). [3H]‐labeled noradrenaline, dopamine, serotonin, and histamine were purchased from PerkinElmer  and [3H]‐1‐methyl‐4‐phenylpyridium acetate (MPP+) was purchased from American Radiolabelled Chemicals . Specific uptake rates in transfected cells were calculated by subtracting uptake in mock‐transfected CHO‐K1 cells.Uptake assays were performed as described previously + and/or Cl− was examined by comparing MPP+ transport in KRPH buffer, Na+‐free buffer, and Cl−‐free buffer The dependence of transporter activity on extracellular Na® qPCR (Life Technologies) for absolute quantification and β‐actin was used as an internal control. Vectors containing each cDNA were used for absolute quantification, and amplification efficiencies of qPCR were nearly 100%, with standard curve r2 values greater than 0.995. Differences between RT‐PCR reactions in the presence or absence of reverse transcriptase are shown as the results.mRNA expression levels of mOct2, mOct3, and mPmat were determined in total RNA isolated from 12 regions of the mouse brain; olfactory bulb, frontal cortex, cortex, striatum, posterior cortex, hippocampus, thalamus, hypothalamus, midbrain, pons, medulla, and cerebellum. Synthesized cDNA was amplified by TaqManKm, Vmax, and concentrations producing 50% inhibition (IC50) were performed using prism 6 software . Differences were identified using Student's t tests and considered to be statistically significant when P < 0.05.All data are expressed as the mean ± SE. Statistical analyses and calculations of Km, Vmax, and transport efficiency (Vmax/Km) values are shown in Tables We investigated time‐dependent transport of serotonin, dopamine, noradrenaline, and histamine for all three transporters. mOct2 transported serotonin and histamine in a time‐dependent manner, whereas dopamine and noradrenaline transport values were negligible Fig. A. In con+ or Cl− concentrations and pH are important factors that can modulate transporter activity + as a substrate, which is a stable and silent compound that retains a positive charge under a variety of physiological conditions + transport through mOct2, mOct3, and mPmat under physiological conditions are summarized in Fig. + or Cl−, the transport activity of mOct2 was decreased by 70% and 50%, respectively , mOct2 activity was significantly decreased, whereas mPmat activity tended to increase , completely abolished the potentiating effects of corticosterone and decynium‐22 on mOct3 transport activity (Figs We also investigated the effects of protein kinase inhibitors on corticosterone‐ and decynium‐22‐mediated enhancements in the transport activity of mOct3, since a previous study showed that structural analogs of decynium‐22 modulated the phosphorylation of rat Oct3 to enhance transport activity ity Figs  and 8.in vivo is partially related to their transport efficiency for various monoamines, functional expression of transporters in cellular membrane defines their impact on monoamine clearance. However, sensitive and specific antibodies against the transporters, which are required to reveal the protein expression localized to plasma membrane, are not available. Although mRNA levels do not always reflect protein levels, mRNA expression is one of the important factors to determine the amount of transporters. Therefore, the absolute expression levels of mOct2, mOct3, and mPmat were investigated using RT‐PCR. mOct2 was highly expressed in the olfactory bulb (Fig. Although the contributions of polyspecific monoamine transporters to monoamine clearance ulb Fig. A, whereaulb Fig. B, and mPulb Fig. C. In gen+ transport through mOct2 was dependent on extracellular Na+ and Cl−, and was enhanced in the presence of high extracellular pH. Drug inhibition assays revealed that cimetidine, decynium‐22, and imipramine decreased mOct2 transport activity at low concentrations.This study is the first to provide a detailed characterization of polyspecific monoamine transporters in the murine brain. We demonstrated that mOct2 only transported serotonin and histamine. Moreover, MPP50 values of human OCT2 for cimetidine and decynium‐22 were 171 and 13.8 μmm and 427 nm, respectively, indicating an interspecies difference in the potency of pharmacological reagents.Previous studies have shown that rat Oct2 and human OCT2 are selective for serotonin and histamine over dopamine and noradrenaline mOct2 gene expression was detected in various brain regions including the olfactory bulb and cerebral cortex, but was generally lower than that of mOct3 and mPmat. This gene expression pattern suggests that the contribution of mOct2 to monoamine regulation in the brain is minor with respect to mOct3 and mPmat; however, we did not investigate the protein or functional expression of mOct2 or other transporters to confirm this hypothesis. Moreover, a recent study demonstrated that mOct2 regulated the tone of serotonergic neurons and controlled stress vulnerability 50 value of 0.9 μm for human OCT3 m enhanced the transport activity of mOct3 in the present study. Isocyanine derivatives, which are structural analogs of decynium‐22, were also reported to increased rat Oct3‐mediated transport We found that while mOct3 transported all four monoamines assayed, higher transport efficiency values were noted for noradrenaline and histamine relative to serotonin and dopamine. Noradrenaline and histamine are also the favored substrates of human OCT3 The activities of OCTs can be regulated by phosphorylation, and accordingly putative phosphorylation sites have been reported for polyspecific transporters Corticosterone has high inhibitory potency and efficacy for human OCT3 et al. et al. In the present study, mOct3 expression was prominent in the striatum, thalamus, hypothalamus, midbrain and pons. Cui + in a manner independent of extracellular Na+ or Cl−+ and serotonin by mPmat was not dependent on extracellular Na+ or Cl−, although extracellular Na+ affected noradrenaline, dopamine, and histamine transport through mPmat. Decynium‐22 was previously reported to inhibit human PMAT activity with an IC50 value of 100 nmHuman PMAT has been reported to transport MPPet al. PMAT gene polymorphism has been reported in association with autism spectrum disorders and was posited to underlie dysfunction of the serotonergic system With regard to gene expression, mPmat was the mostly highly expressed transporter among the three polyspecific monoamine transporters investigated in this study. mPmat was most highly expressed in the medulla oblongata. Hosford In the present work, we characterized the monoamine transport activities of mOct2, mOct3, and mPmat, and demonstrated several similarities and differences with respect to the human orthologs OCT2, OCT3, and PMAT. Overall, these orthologs appeared to have similar functions for monoamine clearance. Accordingly, investigating the roles of these transporters in mouse models of human disease could lead to a better understanding of disease pathophysiology and lead to the identification of novel therapeutic targets. Importantly, specific and potent inhibitors are essential for studying the pathological roles of mOct2, mOct3, and mPmat in models of human disease; the development of novel pharmacological drugs for each mouse transporter is therefore necessary.To our knowledge, this is the first study to characterize the brain distribution and pharmacological kinetics of the mouse polyspecific monoamine transporters mOct2, mOct3, and mPmat. We hope that these results will lead to improvements in the understanding of the role of the monoaminergic system and specifically the uptake2 system in various neurological and neuropsychiatric disease states.TY and KY designed the project and wrote the paper. YM, TY, FN, TN, TI, and AK acquired the data. YM, TY, TM, AM, and RH analyzed and interpreted the data.


Neofusicoccum luteum species belongs to the Botryosphaeriaceae family and is involved in grapevine wood decay diseases. The present study reports the discovery and the molecular characterization of a novel mitovirus infecting this fungus. Double-stranded RNAs were purified from cultivated N. luteum and analysed by next generation sequencing. Using contigs showing BlastX homology with the RNA-dependent RNA polymerase (RdRp) gene of various members of the family Narnaviridae, a single contig of approximately 1.2 kb was constructed. The genomic sequence was completed and phylogenetic analyses indicated that this virus represents a new member of the genus Mitovirus, for which the name of “Neofusicoccum luteum mitovirus 1” is proposed. The genome is 2,389 nucleotides long and, based on the fungal mitochondrial genetic code, it encodes a putative protein of 710 amino acids, homologous to the RdRps of members of the Narnaviridae family. The neofusicoccum luteus mitovirus 1 (NLMV1) RdRp contains the six conserved motifs previously reported for mitoviral RdRps. Our findings represent the first evidence that a mycovirus can infect N. luteum, an important pathogenic fungus of grapevine.The online version of this article (doi:10.1007/s00705-017-3338-9) contains supplementary material, which is available to authorized users. Agaricus bisporus [Mitovirus have been proposed [Narnaviridae, which comprises the genera Mitovirus and Narnavirus. They are considered as the simplest mycoviruses, in that they contain a single linear molecule of positive-sense RNA, with a single open reading frame (ORF) encoding the viral RNA-dependent RNA polymerase (RdRp) in the mitochondrial genetic code. Mitoviruses do not form particles, but form lipid membrane-bound vesicles, and are confined to the mitochondria of their host cells, in contrast to narnaviruses, which replicate in the cytosol [Since the first discovery of viruses in bisporus , mycovirbisporus . With thproposed –6. Taxon cytosol . Several cytosol , 8 have  cytosol .Neofusicoccum luteum is involved in Botryosphaeria dieback, a worldwide grapevine trunk disease [N. luteum for which the name of “Neofusicoccum luteum mitovirus 1” is proposed. The complete genomic sequence of neofusicoccum luteum mitovirus 1 (NLMV1) was determined and consists of a 2,389 nucleotides long RNA molecule encoding a putative RdRp of 710 amino acids (aa). disease . In the N. luteum isolate used was NL-37 (GWEMA-UMR SAVE collection), sampled in a vineyard in 1996, on canes of Vitis vinifera. The fungus was grown as described previously [The eviously .Mitovirus. The 5’ and 3’ genome ends were determined using purified dsRNA as template and 5’ and 3’ Rapid Amplification of cDNA Ends (RACE) with internal primers designed from the contig, following the kit manufacturer’s instructions .Double-stranded RNAs were purified and analyzed on agarose prior to random amplification and Miseq sequencing in a multiplexed scheme as previously reported . After qMitovirus. The genetic organization of the genome is shown in Fig. G values of -35.7 kcal/mol and -52.3 kcal/mol, respectively. In contrast, no inverted complementarity between the two UTRs was detected, suggesting absence of a panhandle structure. This has been described in other mitoviruses, such as alternaria arborescens mitovirus 1 [Sclerotinia sclerotiorum mitovirus 18, with 38.4% of aa sequence identity. Considering the species demarcation criteria accepted for the genus Mitovirus , which is a typical size for a mitoviral genome . Its GC Rp gene, ), NLMV1 N. luteum involved in grapevine Botryosphaeria dieback. Further studies are needed to assess the virulence of NLMV1, and to explore the overall diversity of viruses infecting this pathogenic fungus.Some mitoviruses have been shown to affect the virulence of their hosts . This stSupplementary material 1 (TXT 2 kb)Below is the link to the electronic supplementary material.


Caenorhabditis elegans is constant. Mitochondrial density and transport are controlled both by the Kinesin heavy chain and the Dynein-Dynactin complex. However, unlike in other models, the presence of mitochondria in C. elegans TRNs depends on a Kinesin light chain as well. Mutants in the three C. elegans miro genes do not alter mitochondrial density in the TRNs. Mutants in the Kinesin-1 associated proteins, UNC-16/JIP3 and UNC-76/FEZ1, show increased mitochondrial density and also have elevated levels of both the Kinesin Heavy and Light Chains in neurons. Genetic analyses suggest that, the increased mitochondrial density at the distal end of the neuronal process in unc-16 and unc-76 depends partly on Dynein. We observe a net anterograde bias in the ratio of anterograde to retrograde mitochondrial flux in the neuronal processes of unc-16 and unc-76, likely due to both increased Kinesin-1 and decreased Dynein in the neuronal processes. Our study shows that UNC-16 and UNC-76 indirectly limit mitochondrial density in the neuronal process by maintaining a balance in anterograde and retrograde mitochondrial axonal transport.We investigate the role of axonal transport in regulating neuronal mitochondrial density. We show that the density of mitochondria in the touch receptor neuron (TRN) of adult  Additionally, neurons have defined mitochondrial density along neuronal processes that is altered in models of neurodegenerative disease4. Majority of the mitochondrial biogenesis in neurons is believed to occur in the cell body6. Therefore, transport is likely an important means to control both the presence and the density of mitochondria along the neuronal processes. Kinesin-1 and Dynein are the major anterograde and retrograde motors for mitochondrial transport10. Kinesin heavy chain in both Drosophila and vertebrates is recruited onto the mitochondrial surface via the adaptor Milton/TRAK, in addition, Miro, a Rho GTPase, is essential for mitochondrial transport14. Kinesin heavy chain (KHC), independent of Kinesin light chain (KLC), binds to Milton/TRAK14. Milton/TRAK in turn binds to Miro present on the mitochondrial surface and this binding facilitates mitochondrial transport14. Additionally, RIC-7, a C. elegans specific mitochondrial protein, has been shown to control the presence of mitochondria in the motor neuron process10, although its interaction with Kinesin-1 is not established.Mitochondria are present along the entire length of the neuron and at greater density in regions with high energy needs such as at the Nodes of Ranvier and at synapses20. UNC-16/JIP3/dSYD scaffolds the JNK pathway kinases and is a major Kinesin-1 adaptor21. JIP3/UNC-16 facilitates Kinesin-1 mediated transport of activated JNK Kinases22 and Dynein Light Intermediate Chain23. C. elegans JIP3/UNC-16 mutants also show an increase in levels of mitochondria, marked by TOMM20, in motor neuron axons24. JIP3/UNC-16 binds to both Kinesin heavy and light chains26 and increases motor velocity and run length25. Another protein, UNC-76/FEZ1 (fasciculation and elongation protein zeta 1), binds to the Kinesin heavy chain subunit and activates the motor29.Kinesin-1 has multiple interacting proteins, some of which are used to bind cargoC. elegans touch receptor neuron (TRN) as a model to study mitochondrial transport in vivo. The TRN allows us to track mitochondria in a single isolated neuron where its distribution and transport can be easily observed. In wild type animals, mitochondrial density is constant in the TRN. This density is regulated by the Kinesin heavy chain, Kinesin light chains and the Dynein-Dynactin complex. Our data suggest that the adaptors of Kinesin-1 limit the density of mitochondria in the TRNs through regulation of the balance of anterograde and retrograde mitochondrial transport.In this study, we use jsIs60930, a transgenic animal expressing matrix targeted GFP in the touch receptor neurons of C. elegans. The mitochondrial density in the anterior TRN  processes in 1-day adult animals is approximately 5 mitochondria/100 µm 32 and klc-2(km11)21 show significant reductions in their mitochondrial densities in the anterior TRN processes in both adult and L1 animals 33 has been shown to reduce mitochondrial density in motor neurons10 although we do not see a significant decrease in the anterior TRN 34 and klc-2(km28)21 show a complete absence of mitochondria along the neuronal process in just hatched L1 animals compared to similarly staged wild type L1 35 with a 1383 bp deletion in the gene was similar to that observed in wild type  and the two Kinesin Light Chain genes, als Fig. . unc-116TRN Fig. . The nul L1 Fig. . We coulype Fig. . Thus, b7. Thus, we assessed mitochondrial density in the mutants of the Dynein heavy chain (dhc-1), the Dynein light intermediate chain (dli-1) and the p150 Glued Dynactin subunit (dnc-1). We observed that a strong loss-of-function allele, dhc-1(js121)36, and dnc-1(or404ts)36 grown at the restrictive temperature, show a significant increase in overall mitochondrial density 36, show a significant increase in mitochondrial density at the distal quarter of the neuronal process , dhc-1(or195)37 and dnc-1(or404ts) all show a significant increase compared to wild type in the ratio of the density of mitochondria in the last quarter of the anterior TRN to the density in the first quarter , shows nearly a doubling of but not a statistically significant increase compared to wild type in the ratio of mitochondrial density in the distal TRN process to the proximal TRN process  is similar to that observed in the hypomorphic dhc-1(js319) allele  animals are sterile and are the progeny of dli-1/+ hermaphrodites36. The weaker phenotype observed in dli-1(js351) animals thus may arise from the known maternal perdurance of wild type DLI-138.Dynein is known to transport mitochondria in the retrograde directionity Fig. . All othype Fig. . Howeveress Fig. . This phr Figure . Despiter Figure , dli-1, miro-2(tm2933) and miro-3(tm3150) single mutants do not alter mitochondrial density in the anterior TRN process  show a significant increase in mitochondrial density along the anterior TRN process compared to wild type  show a significant but less pronounced increase in mitochondrial density than unc-16 when compared to wild type  alleles do not show a significant change in mitochondrial density compared to wild type in the TRN processes  and unc-76(n2397) animals  and the null allele klc-2(km28) result in an absence of mitochondria from the neuronal process identical to klc-2(km28) alone  null allele perhaps due to a combination of the lethality of f122 allele and that the two genes are about 0.2 map units apart. Using double mutants with hypomorphic alleles in the Kinesin-1 subunits, we observe that [unc-16 unc-116],  and  show significantly increased mitochondrial density compared to both wild type   Fig. , we concunc-116(e2310) and klc-2(km11) produce lower levels of full length UNC-116 and KLC-2 respectively48. One possible explanation to account for the elevated mitochondrial density in double mutants between the adaptors and hypomorphic alleles of Kinesin-1 is that, presence of even low levels of the Kinesin-1 motor maybe sufficient to cause increased mitochondrial density in unc-16 and unc-76 mutants.Both hypomorphic alleles unc-16 and unc-76 mutant animals. Therefore, we sought to determine if Kinesin-1 levels were altered in unc-16 and unc-76 mutants. Using transgenic UNC-116::GFP and KLC-2::GFP integrated lines, driven by their endogenous promoters, that rescue their respective null alleles48, we observe an increase in levels of UNC-116::GFP and KLC-2::GFP in neurons of both unc-16 and unc-76 animals  mutants  compared to the strong loss of function unc-76 alleles27   Fig. . The pheunc-16 and unc-76.Since, both UNC-16 and UNC-76 contribute to the anterograde transport of Dynein, we investigated the genetic interactions between mutants in both genes with a Dynein complex mutant. We examined whether a further decrease in Dynein function exacerbated the distal TRN mitochondria accumulation phenotypes of dhc-1; unc-16] and  do not show any significant increase in overall mitochondrial density compared respectively to unc-16 and unc-76 alone ; unc-16(tb109)],  and  all continue to show greater numbers of distal mitochondria compared to wild type ; unc-16(tb109)],  and  show a small but a statistically not significant increase in the distal mitochondrial density  compared to the distal mitochondria accumulation seen in the corresponding the unc-16 or unc-76 single mutant alleles in the same region  and klc-2(km11) have significantly reduced anterograde flux compared to wild type animals  decrease in anterograde transport compared to wild type  do not show a significant increase in anterograde transport compared to wild type , shows a significant reduction in the anterograde flux compared to wild type, contrary to the expectation that increased Kinesin-1 motors should lead to increased anterograde flux  perhaps arising from statistically not significant (p = 0.06) but a 40% elevation in anterograde motor levels seen in the double mutant compared to unc-116 alone  and the unc-16 single mutant (blue*) , dhc-1, klc-2 and unc-116 animals compared to wild type  and klc-2(km11) likely arises from both the fewer mitochondria in the neuronal process available for retrograde transport and the known role of these motors in transporting Dynein in the axon23. However neither allele of unc-16 nor unc-76(n2397) show significant changes in retrograde mitochondrial flux compared to wild type  and unc-16(e109) animals, there is an increase of > 30% compared to wild type  animals show a 10% increase and unc-76(rh116) animals show a 38% decrease in anterograde flux respectively compared to wild type. unc-16(tb109) and unc-16(e109) animals respectively show a 13% and 22% reduction in retrograde flux compared to wild type. unc-76(n2397) and unc-76(rh116) animals respectively show a 16% and 50% decrease in retrograde mitochondrial flux when compared to wild type. We thus examined whether the difference in anterograde and retrograde transport or its ratio might account for the observed overall mitochondrial density increase observed in adaptor mutants and adaptor; motor subunit double mutants. Anterograde and retrograde flux of mitochondria do not differ from each other in wild type but significantly differ from each other in both unc-16 mutants and all adaptor; Kinesin-1 subunit double mutant combinations examined , unc-16(e109), unc-76(n2397), unc-76(rh116) and dhc-1(js319) have higher anterograde/retrograde flux ratios respectively of 1.9, 2, 1.6, 1.5 and 2 compared to the wild type ratio of ~1. The double mutants [unc-116 unc-16],  and  have an anterograde to retrograde flux ratio of 3.5, 2.1 and 1.9 respectively. All genotypes that show an anterograde/retrograde flux ratio much greater than 1 also have significantly increased mitochondrial density in the neuron compared to wild type (flux ratio of ~1). These data suggest that, UNC-16 and UNC-76 may act to maintain the balance between anterograde and retrograde flux of mitochondria in the neuronal processes. This anterograde bias in the ratio of transport in unc-16 and unc-76 may contribute in part to the increased mitochondrial density in the TRN processes of these genotypes.The net mitochondrial density probably arises due to a balance in transport of anterograde and retrograde flux, thus we decided to examine the ratios of mitochondrial transport in the different mutants. Although there is little statistical change in the anterograde flux of ype Fig. . unc-76 The increase in Kinesin-1 levels in adaptor mutants. This increase coupled with the inability of the increased Kinesin-1 to recruit UNC-16 and UNC-76 dependent cargo may lead to more Kinesin available for mitochondrial anterograde transport. This is consistent with the striking observation where double mutants between Kinesin-1 and the adaptor mutants show higher levels of anterograde mitochondrial transport compared to motor mutants alone. (ii) In addition to the anterograde transport effects, we also see reduced retrograde transport. This may be due to reduced availability of dynein complex subunits at the distal end of the neuron as has been reportedin vivo show a defined density of mitochondria57. This mitochondrial density changes, both, during development57 and when neurons express human disease causing variants of TDP-43 and SOD156. We show that wild type C. elegans TRNs also have a defined density of mitochondria. Localization of mitochondria in specific regions in the neuronal process and their overall density in the axon are likely dependent on multiple factors. In our study, we find that changes in axonal transport act as one factor in regulating density of mitochondria. Specifically, our data suggest that cargo competition for the Kinesin-1 motor through the availability of adaptors may contribute to this organelle’s density. However, there are likely to be additional factors. This is illustrated by the vast reduction in anterograde transport in a weaker loss-of-function allele dhc-1 mutant, perhaps as a compensatory mechanism to maintain overall density of mitochondria in the neuronal process. Thus, we think that the relationship between motor levels, mitochondrial flux and average axonal mitochondrial density is likely to be complex.Cultured neurons from rats and zebrafish sensory neurons 58, mitochondrial density was assessed in worms grown without any contamination using 30 mM sodium azide or 10 mM sodium azide as an anesthetic. Each animal was imaged within 10 minutes of anesthetisation. Overlapping images were used to reconstruct the entire neuronal process. The neuronal length was measured using the background fluoresecence in the TRN and the total number of mitochondria were counted. Mitochondrial density was calculated as number of mitochondria/100 micron of the neuronal process using 20–25 animals. L3 animals were anaesthetized in 3 mM Levamisole solution and time lapse images were acquired at 2fps for 200s in the distal regions of the ALM . Kymographs were made using the ImageJ Kymograph plugin59 and anterograde and retrograde movements were determined using a displacement of 2 µm as the minimum cut-off. Each mitochondrion was counted only once. The strains used in this study have been mentioned in Table Worms were grown using standard protocolsL1 and L4 animals were anaesthetized with 30 mM Sodium azide and imaged using 60×/100× objective on a Olympus IX83 microscope with spinning disc  with a Hamamatsu  monochrome EMCCD camera. Images for a specific cell type were collected from all genotypes grown identically, at the same time, using identical imaging conditions. Imaging conditions varied for different cell types.2-1.5 mM; EDTA-0.1 mM; EGTA-0.5 mM; Sucrose-44 mM in water) with Protease Inhibitor Cocktail  was carried out from a growing plate. Western blot analysis was carried out as described in60. Polyclonal rabbit antibodies against UNC-116 and KLC-2 were raised by Bioklone, Chennai using HIS tagged proteins made from constructs as described earlier33. We validated the antibodies on Western blot using protein extracts from Ex[punc-116::unc-116::gfp]; unc-116(f122) and Ex[Pklc-2::klc-2::gfp]; klc-2(km28) worms (data not shown). Antibody dilutions used were Rabbit α-UNC-116 (1:40000), Rabbit α-KLC-2 (1:40000), mouse α-Actin (1:1500) (NeoLabMarkers)]. Secondary antibodies dilutions were anti-Rabbit-HRP (1:10000), anti-Mouse-HRP (1:6000) . Three or more independent biological repeats were carried out in duplicates.Protein extraction using Homogenization buffer  using random hexamers. qPCR was done with Kapa SYBR FAST universal qPCR mastermix on Roche LightCycler- LC96. For purposes of quantitation, the samples were normalized to wild type. At least three independent biological repeats were carried out with at least two technical repeats. No genomic DNA contamination was observed when using actin primers. Quantitation was carried out as described in62 and MyDomains63. The genomic DNA sequences were sourced from the NCBI Database.The domain structure has been generated using ScanPrositeAll data and reagents are available upon request.Supplementary material