α-actinin is required for the proper assembly of Z-disk/focal-adhesion-like structures and for efficient locomotion in Caenorhabditis elegans.

The actin binding protein α-actinin is a major component of focal adhesions found in vertebrate cells and of focal-adhesion-like structures found in the body wall muscle of the nematode Caenorhabditis elegans. To study its in vivo function in this genetic model system, we isolated a strain carrying a deletion of the single C. elegans α-actinin gene. We assessed the cytological organization of other C. elegans focal adhesion proteins and the ultrastructure of the mutant. The mutant does not have normal dense bodies, as observed by electron microscopy; however, these dense-body-like structures still contain the focal adhesion proteins integrin, talin, and vinculin, as observed by immunofluorescence microscopy. Actin is found in normal-appearing I-bands, but with abnormal accumulations near muscle cell membranes. Although swimming in water appeared grossly normal, use of automated methods for tracking the locomotion of individual worms revealed a defect in bending. We propose that the reduced motility of α-actinin null is due to abnormal dense bodies that are less able to transmit the forces generated by actin/myosin interactions.

Whole-genome profiling of mutagenesis in Caenorhabditis elegans.

Deep sequencing offers an unprecedented view of an organism's genome. We describe the spectrum of mutations induced by three commonly used mutagens: ethyl methanesulfonate (EMS), N-ethyl-N-nitrosourea (ENU), and ultraviolet trimethylpsoralen (UV/TMP) in the nematode Caenorhabditis elegans. Our analysis confirms the strong GC to AT transition bias of EMS. We found that ENU mainly produces A to T and T to A transversions, but also all possible transitions. We found no bias for any specific transition or transversion in the spectrum of UV/TMP-induced mutations. In 10 mutagenized strains we identified 2723 variants, of which 508 are expected to alter or disrupt gene function, including 21 nonsense mutations and 10 mutations predicted to affect mRNA splicing. This translates to an average of 50 informative mutations per strain. We also present evidence of genetic drift among laboratory wild-type strains derived from the Bristol N2 strain. We make several suggestions for best practice using massively parallel short read sequencing to ensure mutation detection.

Towards a mutation in every gene in Caenorhabditis elegans.

The combined efforts of the Caenorhabditis elegans Knockout Consortium and individuals within the worm community are moving us closer to the goal of identifying mutations in every gene in the nematode C. elegans. At present, we count about 7000 deletion alleles that fall within 5500 genes. The principal method used to detect deletion mutations in the nematode utilizes polymerase chain reaction (PCR). More recently, the Moerman group has incorporated array comparative genome hybridization (aCGH) to detect deletions across the entire coding genome. Other methods used to detect mutant alleles in C. elegans include targeting induced local lesion in genomes (TILLING), transposon tagging, using either Tc1 or Mos1 and resequencing. These combined strategies have improved the overall throughput of the gene-knockout labs, and have broadened the types of mutations that we, and others, can identify. In this review, we will discuss these different approaches.

An intracellular serpin regulates necrosis by inhibiting the induction and sequelae of lysosomal injury.

Extracellular serpins such as antithrombin and alpha1-antitrypsin are the quintessential regulators of proteolytic pathways. In contrast, the biological functions of the intracellular serpins remain obscure. We now report that the C. elegans intracellular serpin, SRP-6, exhibits a prosurvival function by blocking necrosis. Minutes after hypotonic shock, srp-6 null animals underwent a catastrophic series of events culminating in lysosomal disruption, cytoplasmic proteolysis, and death. This newly defined hypo-osmotic stress lethal (Osl) phenotype was dependent upon calpains and lysosomal cysteine peptidases, two in vitro targets of SRP-6. By protecting against both the induction of and the lethal effects from lysosomal injury, SRP-6 also blocked death induced by heat shock, oxidative stress, hypoxia, and cation channel hyperactivity. These findings suggest that multiple noxious stimuli converge upon a peptidase-driven, core stress response pathway that, in the absence of serpin regulation, triggers a lysosomal-dependent necrotic cell death routine.

C. elegans deletion mutant screening.

The methods used by the Caenorhabditis elegans Gene Knockout Consortium are conceptually simple. One does a chemical mutagenesis of wild-type C. elegans, and then screens the progeny of the mutagenized animals, in small mixed groups, using polymerase chain reaction (PCR) to identify populations with animals where a portion of DNA bounded by the PCR primers has been deleted. Animals from such populations are then selected and grown clonally to recover a pure genetic strain. We categorize the steps needed to do this as follows: (1) mutagenesis and DNA template preparation, (2) PCR detection of deletions, (3) sibling selection, and (4) deletion stabilization. These are discussed in detail in this chapter.

The Caenorhabditis elegans snf-11 gene encodes a sodium-dependent GABA transporter required for clearance of synaptic GABA.

Sodium-dependent neurotransmitter transporters participate in the clearance and/or recycling of neurotransmitters from synaptic clefts. The snf-11 gene in Caenorhabditis elegans encodes a protein of high similarity to mammalian GABA transporters (GATs). We show here that snf-11 encodes a functional GABA transporter; SNF-11-mediated GABA transport is Na+ and Cl- dependent, has an EC50 value of 168 microM, and is blocked by the GAT1 inhibitor SKF89976A. The SNF-11 protein is expressed in seven GABAergic neurons, several additional neurons in the head and retrovesicular ganglion, and three groups of muscle cells. Therefore, all GABAergic synapses are associated with either presynaptic or postsynaptic (or both) expression of SNF-11. Although a snf-11 null mutation has no obvious effects on GABAergic behaviors, it leads to resistance to inhibitors of acetylcholinesterase. In vivo, a snf-11 null mutation blocks GABA uptake in at least a subset of GABAergic cells; in a cell culture system, all GABA uptake is abolished by the snf-11 mutation. We conclude that GABA transport activity is not essential for normal GABAergic function in C. elegans and that the localization of SNF-11 is consistent with a GABA clearance function rather than recycling.

The stn-1 syntrophin gene of C.elegans is functionally related to dystrophin and dystrobrevin.

Syntrophins are a family of PDZ domain-containing adaptor proteins required for receptor localization. Syntrophins are also associated with the dystrophin complex in muscles. We report here the molecular and functional characterization of the Caenorhabditis elegans gene stn-1 (F30A10.8), which encodes a syntrophin with homology to vertebrate alpha and beta-syntrophins. stn-1 is expressed in neurons and in muscles of C.elegans. stn-1 mutants resemble dystrophin (dys-1) and dystrobrevin (dyb-1) mutants: they are hyperactive, bend their heads when they move forward, tend to hypercontract, and are hypersensitive to the acetylcholinesterase inhibitor aldicarb. These phenotypes are suppressed when stn-1 is expressed under the control of a muscular promoter, indicating that they are caused by the absence of stn-1 in muscles. These results suggest that the role of syntrophin is linked to dystrophin function in C.elegans.

Towards understanding the polycystins.

Autosomal dominant polycystic kidney disease (ADPKD) is a very common inherited disease caused by mutations in PKD1 or PKD2 genes characterized by progressive enlargement of fluid-filled cysts and loss of renal function [1]. Previous studies proposed a role for human polycystin-1 in renal morphogenesis acting as a matrix receptor in focal adhesions and for polycystin-2 as a putative calcium channel [2, 3]. The genome of Caenorhabditis elegans contains 2 new members of the polycystin family: lov-1, the homolog for PKD1; and pkd-2, the homolog for PKD2 [4; this paper]. Mutation analysis in C. elegans showed similarly compromised male mating behaviors in all single and double lov-1 and pkd-2 mutants, indicating their participation in a single genetic pathway. Expression analysis localized LOV-1 and PKD-2 to the ends of sensory neurons in male tails and to the tips of CEM neurons in the head, consistent with functions as chemo- or mechanosensors. Human and C. elegans PKD1 and PKD2 homologs, transfected into mammalian renal epithelial cells, co-localized with paxillin in focal adhesions suggesting function in a single biological pathway. Based on the role of polycystins in C. elegans sensory neuron function and the conservation of PKD pathways we suggest that polycystins act as sensors of the extracellular environment, initiating, via focal adhesion assembly, intracellular transduction events in neuronal or morphogenetic processes.

Combinatorial expression of TRPV channel proteins defines their sensory functions and subcellular localization in C. elegans neurons.

C. elegans OSM-9 is a TRPV channel protein involved in sensory transduction and adaptation. Here, we show that distinct sensory functions arise from different combinations of OSM-9 and related OCR TRPV proteins. Both OSM-9 and OCR-2 are essential for several forms of sensory transduction, including olfaction, osmosensation, mechanosensation, and chemosensation. In neurons that express both OSM-9 and OCR-2, tagged OCR-2 and OSM-9 proteins reside in sensory cilia and promote each other's localization to cilia. In neurons that express only OSM-9, tagged OSM-9 protein resides in the cell body and acts in sensory adaptation rather than sensory transduction. Thus, alternative combinations of TRPV proteins may direct different functions in distinct subcellular locations. Animals expressing the mammalian TRPV1 (VR1) channel in ASH nociceptor neurons avoid the TRPV1 ligand capsaicin, allowing selective, drug-inducible activation of a specific behavior.

A conserved RNA-binding protein controls germline stem cells in Caenorhabditis elegans.

Germline stem cells are defined by their unique ability to generate more of themselves as well as differentiated gametes. The molecular mechanisms controlling the decision between self-renewal and differentiation are central unsolved problems in developmental biology with potentially broad medical implications. In Caenorhabditis elegans, germline stem cells are controlled by the somatic distal tip cell. FBF-1 and FBF-2, two nearly identical proteins, which together are called FBF ('fem-3 mRNA binding factor'), were originally discovered as regulators of germline sex determination. Here we report that FBF also controls germline stem cells: in an fbf-1 fbf-2 double mutant, germline proliferation is initially normal, but stem cells are not maintained. We suggest that FBF controls germline stem cells, at least in part, by repressing gld-1, which itself promotes commitment to the meiotic cell cycle. FBF belongs to the PUF family ('Pumilio and FBF') of RNA-binding proteins. Pumilio controls germline stem cells in Drosophila females, and, in lower eukaryotes, PUF proteins promote continued mitoses. We suggest that regulation by PUF proteins may be an ancient and widespread mechanism for control of stem cells.

The MEP-1 zinc-finger protein acts with MOG DEAH box proteins to control gene expression via the fem-3 3' untranslated region in Caenorhabditis elegans.

Cell fates in the Caenorhabditis elegans germline are regulated, at least in part, at the posttranscriptional level. For example, the switch from spermatogenesis to oogenesis in the hermaphrodite relies on posttranscriptional repression of the fem-3 mRNA via its 3' untranslated region (UTR). Previous studies identified three DEAH box proteins, MOG-1, MOG-4, and MOG-5, that are critical for the fem-3 3' UTR control. Here we describe MEP-1, a zinc-finger protein that binds specifically to each of these three MOG proteins and that is required for repression by the fem-3 3' UTR in vivo. To investigate its in vivo function, we generated a mep-1 deletion mutant. The mep-1 null phenotype suggests a broad role for MEP-1 in C. elegans development, as it is associated with early larval arrest. In addition, mep-1 mutants can be defective in gonadogenesis and oocyte production when derived from a heterozygous mother. We suggest that MEP-1 acts together with the MOG proteins to repress fem-3 mRNA and that it also functions in other pathways to control development more broadly.

Improved detection of small deletions in complex pools of DNA.

About 40% of the genes in the nematode Caenorhabditis elegans have homologs in humans. Based on the history of this model system, it is clear that the application of genetic methods to the study of this set of genes would provide important clues to their function in humans. To facilitate such genetic studies, we are engaged in a project to derive deletion alleles in every gene in this set. Our standard methods make use of nested PCR to hunt for animals in mutagenized populations that carry deletions at a given locus. The deletion bearing animals exist initially in mixed populations where the majority of the animals are wild type at the target. Therefore, the production of the PCR fragment representing the deletion allele competes with the production of the wild type fragment. The size of the deletion fragment relative to wild type determines whether it can compete to a level where it can be detected above the background. Using our standard conditions, we have found that when the deletion is <600 bp, the deletion fragment does not compete effectively with the production of the wild type fragment in PCR. Therefore, although our standard methods work well to detect mutants with deletions >600 bp, they do not work well to detect mutants with smaller deletions. Here we report a new strategy to detect small deletion alleles in complex DNA pools. Our new strategy is a modification of our standard PCR based screens. In the first round of the nested PCR, we include a third PCR primer between the two external primers. The presence of this third primer leads to the production of three fragments from wild type DNA. We configure the system so that two of these three fragments cannot serve as a template in the second round of the nested PCR. The addition of this third primer, therefore, handicaps the amplification from wild type template. On the other hand, the amplification of mutant fragments where the binding site for the third primer is deleted is unabated. Overall, we see at least a 500-fold increase in the sensitivity for small deletion fragments using our new method. Using this new method, we report the recovery of new deletion alleles within 12 C.elegans genes.