Analysis of Ca2+ Signaling Motifs That Regulate Proton Signaling through the Na+/H+ Exchanger NHX-7 during a Rhythmic Behavior in Caenorhabditis elegans

Membrane proton transporters contribute to pH homeostasis but have also been shown to transmit information between cells in close proximity through regulated proton secretion. For example, the nematode intestinal Na⁺/H⁺ exchanger NHX-7 causes adjacent muscle cells to contract by transiently acidifying the extracellular space between the intestine and muscle. NHX-7 operates during a Ca²⁺-dependent rhythmic behavior and contains several conserved motifs for regulation by Ca²⁺ input, including motifs for calmodulin and phosphatidylinositol 4,5-bisphosphate binding, protein kinase C- and calmodulin-dependent protein kinase type II phosphorylation, and a binding site for calcineurin homologous protein. Here, we tested the idea that Ca²⁺ input differentiates proton signaling from pH housekeeping activity. Each of these motifs was mutated, and their contribution to NHX-7 function was assessed. These functions included pH recovery from acidification in cells in culture expressing recombinant NHX-7, extracellular acidification measured during behavior in live moving worms, and muscle contraction strength as a result of this acidification. Our data suggest that multiple levels of Ca²⁺ input regulate NHX-7, whose transport capacity normally exceeds the minimum necessary to cause muscle contraction. Furthermore, extracellular acidification limits NHX-7 proton transport through feedback inhibition, likely to prevent metabolic acidosis from occurring. Our findings are consistent with an integrated network whereby both Ca²⁺ and pH contribute to proton signaling. Finally, our results obtained by expressing rat NHE1 in Caenorhabditis elegans suggest that a conserved mechanism of regulation may contribute to cell-cell communication or proton signaling by Na⁺/H⁺ exchangers in mammals.     

Small Collagenous Proteins Present during the Molt in Caenorhabditis elegans

Immunoblotting experiments using antibodies directed against the large collagenous cuticle proteins of Caenorhabditis elegans revealed a class of small collagenous proteins (CP) of apparent molecular weight 38,000-52,000 present during the L4 to adult molt. These CP are smaller than most vertebrate collagens characterized to date and share many characteristics with the small collagenous products translated in vitro from RNA isolated at this molt. C. elegans collagen genes, collagen-coding mRNA, and collagenous in vitro products that have been characterized are also small. Detection of small CP in vivo in C. elegans thus lends further support to the hypothesis that such small collagenous proteins are the primary gene product precursors to the larger collagenous proteins isolated from the C. elegans cuticle.     

Sex Determination in Caenorhabditis elegans

In Caenorhabditis elegans, the decision to develop as a hermaphrodite or male is controlled by a cascade of regulatory genes. These genes and other tissue-specific regulatory genes also control sexual fate in the hermaphrodite germline, which makes sperm first and then oocytes. In this review, we summarize the genetic and molecular characterization of these genes and speculate how they mutually interact to specify sexual fate.     

Genomic organization, expression, and analysis of the troponin C gene pat-10 of Caenorhabditis elegans.

We have cloned and characterized the troponin C gene, pat-10 of the nematode Caenorhabditis elegans. At the amino acid level nematode troponin C is most similar to troponin C of Drosophila (45% identity) and cardiac troponin C of vertebrates. Expression studies demonstrate that this troponin is expressed in body wall muscle throughout the life of the animal. Later, vulval muscles and anal muscles also express this troponin C isoform. The structural gene for this troponin is pat-10 and mutations in this gene lead to animals that arrest as twofold paralyzed embryos late in development. We have sequenced two of the mutations in pat-10 and both had identical two mutations in the gene; one changes D64 to N and the other changes W153 to a termination site. The missense alteration affects a calcium-binding site and eliminates calcium binding, whereas the second mutation eliminates binding to troponin I. These combined biochemical and in vivo studies of mutant animals demonstrate that this troponin is essential for proper muscle function during development.     

Crossover Heterogeneity in the Absence of Hotspots in Caenorhabditis elegans

Crossovers play mechanical roles in meiotic chromosome segregation, generate genetic diversity by producing new allelic combinations, and facilitate evolution by decoupling linked alleles. In almost every species studied to date, crossover distributions are dramatically nonuniform, differing among sexes and across genomes, with spatial variation in crossover rates on scales from whole chromosomes to subkilobase hotspots. To understand the regulatory forces dictating these heterogeneous distributions a crucial first step is the fine-scale characterization of crossover distributions. Here we define the wild-type distribution of crossovers along a region of the C. elegans chromosome II at unprecedented resolution, using recombinant chromosomes of 243 hermaphrodites and 226 males. We find that well-characterized large-scale domains, with little fine-scale rate heterogeneity, dominate this region’s crossover landscape. Using the Gini coefficient as a summary statistic, we find that this region of the C. elegans genome has the least heterogeneous fine-scale crossover distribution yet observed among model organisms, and we show by simulation that the data are incompatible with a mammalian-type hotspot-rich landscape. The large-scale structural domains—the low-recombination center and the high-recombination arm—have a discrete boundary that we localize to a small region. This boundary coincides with the arm-center boundary defined both by nuclear-envelope attachment of DNA in somatic cells and GC content, consistent with proposals that these features of chromosome organization may be mechanical causes and evolutionary consequences of crossover recombination.     

Ca2+-dependent Muscle Dysfunction Caused by Mutation of the Caenorhabditis elegans Troponin T-1 Gene

We have investigated the functions of troponin T (CeTnT-1) in Caenorhabditis elegans embryonic body wall muscle. TnT tethers troponin I (TnI) and troponin C (TnC) to the thin filament via tropomyosin (Tm), and TnT/Tm regulates the activation and inhibition of myosin-actin interaction in response to changes in intracellular [Ca²⁺]. Loss of CeTnT-1 function causes aberrant muscle trembling and tearing of muscle cells from their exoskeletal attachment sites (Myers, C.D., P.-Y. Goh, T. StC. Allen, E.A. Bucher, and T. Bogaert. 1996. J. Cell Biol. 132:1061–1077). We hypothesized that muscle tearing is a consequence of excessive force generation resulting from defective tethering of Tn complex proteins. Biochemical studies suggest that such defective tethering would result in either (a) Ca²⁺-independent activation, due to lack of Tn complex binding and consequent lack of inhibition, or (b) delayed reestablishment of TnI/TnC binding to the thin filament after Ca²⁺ activation and consequent abnormal duration of force. Analyses of animals doubly mutant for CeTnT-1 and for genes required for Ca²⁺ signaling support that CeTnT-1 phenotypes are dependent on Ca²⁺ signaling, thus supporting the second model and providing new in vivo evidence that full inhibition of thin filaments in low [Ca²⁺] does not require TnT.     

Illuminating neural circuits and behaviour in Caenorhabditis elegans with optogenetics

The development of optogenetics, a family of methods for using light to control neural activity via light-sensitive proteins, has provided a powerful new set of tools for neurobiology. These techniques have been particularly fruitful for dissecting neural circuits and behaviour in the compact and transparent roundworm Caenorhabditis elegans. Researchers have used optogenetic reagents to manipulate numerous excitable cell types in the worm, from sensory neurons, to interneurons, to motor neurons and muscles. Here, we show how optogenetics applied to this transparent roundworm has contributed to our understanding of neural circuits.     

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.     

CRISPR-Based Methods for Caenorhabditis elegans Genome Engineering

The advent of genome editing techniques based on the clustered regularly interspersed short palindromic repeats (CRISPR)–Cas9 system has revolutionized research in the biological sciences. CRISPR is quickly becoming an indispensible experimental tool for researchers using genetic model organisms, including the nematode Caenorhabditis elegans. Here, we provide an overview of CRISPR-based strategies for genome editing in C. elegans. We focus on practical considerations for successful genome editing, including a discussion of which strategies are best suited to producing different kinds of targeted genome modifications.     

A High-Throughput Method for the Analysis of Larval Developmental Phenotypes in Caenorhabditis elegans

Caenorhabditis elegans postembryonic development consists of four discrete larval stages separated by molts. Typically, the speed of progression through these larval stages is investigated by visual inspection of the molting process. Here, we describe an automated method to monitor the timing of these discrete phases of C. elegans maturation, from the first larval stage through adulthood, using bioluminescence. The method was validated with a lin-42 mutant strain that shows delayed development relative to wild-type animals and with a daf-2 mutant that shows an extended second larval stage. This new method is inherently high-throughput and will finally allow dissecting the molecular machinery governing the speed of the developmental clock, which has so far been hampered by the lack of a method suitable for genetic screens.     

Gap junctions synchronize action potentials and Ca2+ transients in Caenorhabditis elegans body wall muscle

The sinusoidal locomotion of Caenorhabditis elegans requires synchronous activities of neighboring body wall muscle cells. However, it is unknown whether the synchrony results from muscle electrical coupling or neural inputs. We analyzed the effects of mutating gap junction proteins and blocking neuromuscular transmission on the synchrony of action potentials (APs) and Ca2+ transients among neighboring body wall muscle cells. In wild-type worms, the percentage of synchronous APs between two neighboring cells varied depending on the anatomical relationship and junctional conductance (Gj) between them, and Ca2+ transients were synchronous among neighboring muscle cells. Compared with the wild type, knock-out of the gap junction gene unc-9 resulted in greatly reduced coupling coefficient and asynchronous APs and Ca2+ transients. Inhibition of unc-9 expression specifically in muscle by RNAi also reduced the synchrony of APs and Ca2+ transients, whereas expression of wild-type UNC-9 specifically in muscle rescued the synchrony defect. Loss of the stomatin-like protein UNC-1, which is a regulator of UNC-9-based gap junctions, similarly impaired muscle synchrony as unc-9 mutant did. The blockade of muscle ionotropic acetylcholine receptors by (+)-tubocurarine decreased the frequencies of APs and Ca2+ transients, whereas blockade of muscle GABAA receptors by gabazine had opposite effects. However, both APs and Ca2+ transients remained synchronous after the application of (+)-tubocurarine and/or gabazine. These observations suggest that gap junctions in C. elegans body wall muscle cells are responsible for synchronizing muscle APs and Ca2+ transients.     

Transgene-Free Genome Editing in Caenorhabditis elegans Using CRISPR-Cas

CRISPR-Cas is an efficient method for genome editing in organisms from bacteria to human cells. We describe a transgene-free method for CRISPR-Cas-mediated cleavage in nematodes, enabling RNA-homology-targeted deletions that cause loss of gene function; analysis of whole-genome sequencing indicates that the nuclease activity is highly specific.     

The topology of the C-terminal sections of the NCX1 Na+/Ca2+ exchanger and the NCKX2 Na+/Ca2+-K+ exchanger

Mammalian Na⁺/Ca²⁺ (NCX) and Na⁺/Ca²⁺-K⁺ exchangers (NCKX) are polytopic membrane proteins that play critical roles in calcium homeostasis in many cells. Although hydropathy plots for NCX and NCKX are very similar, reported topological models for NCX1 and NCKX2 differ in the orientation of the three C-terminal transmembrane segments (TMS). NCX1 is thought to have 9 TMS and a re-entrant loop, whereas NCKX2 is thought to have 10 TMS. The current topological model of NCKX2 is very similar to the 10 membrane spanning helices seen in the recently reported crystal structure of NCX_MJ, a distantly related archaebacterial Na⁺/Ca²⁺ exchanger. Here we reinvestigate the orientation of the three C-terminal TMS of NCX1 and NCKX2 using mass-tagging experiments of substituted cysteine residues. Our results suggest that NCX1, NCKX2 and NCX_MJ all share the same 10 TMS topology.     

CRISPR/Cas9-Targeted Mutagenesis in Caenorhabditis elegans

The generation of genetic mutants in Caenorhabditis elegans has long relied on the selection of mutations in large-scale screens. Directed mutagenesis of specific loci in the genome would greatly speed up analysis of gene function. Here, we adapt the CRISPR/Cas9 system to generate mutations at specific sites in the C. elegans genome.     

Ecdysteroids in Axenically Propagated Caenorhabditis elegans and Culture Medium

Ecdysteroids (insect molting hormones) from Caenorhabditis elegans were chromatographically purified and quantified by radioimmunoassay. Nematodes from semidefined medium contained the immunoreactive equivalent of 460 pg ecdysone per gram dry weight. Culture medium, however, contained the immunoreactive equivalent of 68 times the quantity within the nematodes. In a defined medium lacking immunoreactivity, C. elegans contained 520 pg ecdysone equivalents per gram dry weight but reproduced slowly. Reproduction of C. elegans in defined medium was enhanced by formulation in agar. Propagation of C. elegans in either agar-based or aqueous defined medium supplemented with [¹⁴C]cholesterol of high specific activity failed to result in production of radiolabeled free ecdysteroids or polar or apolar ecdysteroid conjugates. Failure to demonstrate ecdysteroid biosynthesis in C. elegans raises questions about the ecdysteroids identified previously in nematodes being products of endogenous biosynthesis, a necessary condition for these compounds to be nematode hormones.     

The Genetic Basis of Natural Variation in Caenorhabditis elegans Telomere Length

Telomeres are involved in the maintenance of chromosomes and the prevention of genome instability. Despite this central importance, significant variation in telomere length has been observed in a variety of organisms. The genetic determinants of telomere-length variation and their effects on organismal fitness are largely unexplored. Here, we describe natural variation in telomere length across the Caenorhabditis elegans species. We identify a large-effect variant that contributes to differences in telomere length. The variant alters the conserved oligonucleotide/oligosaccharide-binding fold of protection of telomeres 2 (POT-2), a homolog of a human telomere-capping shelterin complex subunit. Mutations within this domain likely reduce the ability of POT-2 to bind telomeric DNA, thereby increasing telomere length. We find that telomere-length variation does not correlate with offspring production or longevity in C. elegans wild isolates, suggesting that naturally long telomeres play a limited role in modifying fitness phenotypes in C. elegans.     

Plasmalemmal Na+/Ca2+ exchanger modulates Ca2+-dependent exocytotic release of glutamate from rat cortical astrocytes

Astroglial excitability operates through increases in Ca²⁺_cyt (cytosolic Ca²⁺), which can lead to glutamatergic gliotransmission. In parallel fluctuations in astrocytic Na⁺_cyt (cytosolic Na⁺) control metabolic neuronal-glial signalling, most notably through stimulation of lactate production, which on release from astrocytes can be taken up and utilized by nearby neurons, a process referred to as lactate shuttle. Both gliotransmission and lactate shuttle play a role in modulation of synaptic transmission and plasticity. Consequently, we studied the role of the PMCA (plasma membrane Ca²⁺-ATPase), NCX (plasma membrane Na⁺/Ca²⁺ exchanger) and NKA (Na⁺/K⁺-ATPase) in complex and coordinated regulation of Ca²⁺_cyt and Na⁺_cyt in astrocytes at rest and upon mechanical stimulation. Our data support the notion that NKA and PMCA are the major Na⁺ and Ca²⁺ extruders in resting astrocytes. Surprisingly, the blockade of NKA or PMCA appeared less important during times of Ca²⁺ and Na⁺ cytosolic loads caused by mechanical stimulation. Unexpectedly, NCX in reverse mode appeared as a major contributor to overall Ca²⁺ and Na⁺ homoeostasis in astrocytes both at rest and when these glial cells were mechanically stimulated. In addition, NCX facilitated mechanically induced Ca²⁺-dependent exocytotic release of glutamate from astrocytes. These findings help better understanding of astrocyte-neuron bidirectional signalling at the tripartite synapse and/or microvasculature. We propose that NCX operating in reverse mode could be involved in fast and spatially localized Ca²⁺-dependent gliotransmission, that would operate in parallel to a slower and more widely distributed gliotransmission pathway that requires metabotropically controlled Ca²⁺ release from the ER (endoplasmic reticulum).     

Targeted Heritable Mutation and Gene Conversion by Cas9-CRISPR in Caenorhabditis elegans

We have achieved targeted heritable genome modification in Caenorhabditis elegans by injecting mRNA of the nuclease Cas9 and Cas9 guide RNAs. This system rapidly creates precise genomic changes, including knockouts and transgene-instructed gene conversion.