In JoVE (1)

Other Publications (61)

Articles by Erik M. Jorgensen in JoVE

Other articles by Erik M. Jorgensen on PubMed

The Art and Design of Genetic Screens: Caenorhabditis Elegans

Nature Reviews. Genetics. May, 2002  |  Pubmed ID: 11988761

The nematode Caenorhabditis elegans was chosen as a model genetic organism because its attributes, chiefly its hermaphroditic lifestyle and rapid generation time, make it suitable for the isolation and characterization of genetic mutants. The most important challenge for the geneticist is to design a genetic screen that will identify mutations that specifically disrupt the biological process of interest. Since 1974, when Sydney Brenner published his pioneering genetic screen, researchers have developed increasingly powerful methods for identifying genes and genetic pathways in C. elegans.

Characterization of a Dominant Negative C. Elegans Twist Mutant Protein with Implications for Human Saethre-Chotzen Syndrome

Development (Cambridge, England). Jun, 2002  |  Pubmed ID: 12015302

Twist is a transcription factor that is required for mesodermal cell fates in all animals studied to date. Mutations of this locus in humans have been identified as the cause of the craniofacial disorder Saethre-Chotzen syndrome. The Caenorhabditis elegans Twist homolog is required for the development of a subset of the mesoderm. A semidominant allele of the gene that codes for CeTwist, hlh-8, has defects that occur earlier in the mesodermal lineage than a previously studied null allele of the gene. The semidominant allele has a charge change (E29K) in the basic DNA-binding domain of CeTwist. Surprisingly, the mutant protein retains DNA-binding activity as both a homodimer and a heterodimer with its partner E/Daughterless (CeE/DA). However, the mutant protein blocks the activation of the promoter of a target gene. Therefore, the mutant CeTwist may cause cellular defects as a dominant negative protein by binding to target promoters as a homo- or heterodimer and then blocking transcription. Similar phenotypes as those caused by the E29K mutation were observed when amino acid substitutions in the DNA-binding domain that are associated with the human Saethre-Chotzen syndrome were engineered into the C. elegans protein. These data suggest that Saethre-Chotzen syndrome may be caused, in some cases, by dominant negative proteins, rather than by haploinsufficiency of the locus.

Pharmacological Characterization of the Homomeric and Heteromeric UNC-49 GABA Receptors in C. Elegans

British Journal of Pharmacology. Mar, 2003  |  Pubmed ID: 12642390

(1) UNC-49B and UNC-49C are gamma-aminobutyric acid (GABA) receptor subunits encoded by the Caenorhabditis elegans unc-49 gene. UNC-49B forms a homomeric GABA receptor, or can co-assemble with UNC-49C to form a heteromeric receptor. The pharmacological properties of UNC-49B homomers and UNC-49B/C heteromers were investigated in Xenopus oocytes. (2) The UNC-49 subunits are most closely related to the bicuculline- and benzodiazepine-insensitive RDL GABA receptors of insects. Consistent with this classification, bicuculline (10 micro M) did not inhibit, nor did diazepam (10 micro M) enhance UNC-49B homomeric or UNC-49B/C heteromeric receptors. (3) The UNC-49C subunit strongly affects the pharmacology of UNC-49B/C heteromeric receptors. UNC-49B homomers were much more picrotoxin sensitive than UNC-49B/C heteromers (IC(50)=0.9+/-0.2 micro M and 166+/-42 micro M, respectively). Pentobarbitone enhancement was greater for UNC-49B homomers compared to UNC-49B/C heteromers. Propofol (50 micro M) slightly enhanced UNC-49B homomers but slightly inhibited UNC-49B/C heteromers. Penicillin G (10 mM) inhibited UNC-49B homomers less strongly than UNC-49B/C heteromers (30% compared to 53% inhibition, respectively). (4) Several aspects of UNC-49 pharmacology were unusual. Picrotoxin sensitivity strongly correlates with dieldrin sensitivity, yet UNC-49B homomers were highly dieldrin resistant. The enhancing neurosteroid pregnanolone (5beta-pregnan-3alpha-ol-20-one; 10 micro M) strongly inhibited both UNC-49 receptors. Alphaxalone (10 micro M), another enhancing neurosteroid, did not affect UNC-49B homomers, but slightly inhibited UNC-49B/C heteromers. (5) UNC-49 subunits and mammalian GABA(A) receptor alpha, beta, and gamma subunit classes all share roughly the same degree of sequence similarity. Thus, although they are most similar to other invertebrate GABA receptors, the UNC-49 receptors share significant structural and pharmacological overlap with mammalian GABA(A) receptors.

Controversies in Synaptic Vesicle Exocytosis

Journal of Cell Science. Sep, 2003  |  Pubmed ID: 12917353

Defects in Synaptic Vesicle Docking in Unc-18 Mutants

Nature Neuroscience. Oct, 2003  |  Pubmed ID: 12973353

Sec1-related proteins function in most, if not all, membrane trafficking pathways in eukaryotic cells. The Sec1-related protein required in neurons for synaptic vesicle exocytosis is UNC-18. Several models for UNC-18 function during vesicle exocytosis are under consideration. We have tested these models by characterizing unc-18 mutants of the nematode Caenorhabditis elegans. In the absence of UNC-18, the size of the readily releasable pool is severely reduced. Our results show that the near absence of fusion-competent vesicles is not caused by a reduction in syntaxin levels, by a mislocalization of syntaxin, by a defect in fusion or by a failure to open syntaxin during priming. Rather, we found a reduction of docked vesicles at the active zone in unc-18 mutants, suggesting that UNC-18 functions, directly or indirectly, as a facilitator of vesicle docking.

EXP-1 is an Excitatory GABA-gated Cation Channel

Nature Neuroscience. Nov, 2003  |  Pubmed ID: 14555952

Gamma-aminobutyric acid (GABA) mediates fast inhibitory neurotransmission by activating anion-selective ligand-gated ion channels. Although electrophysiological studies indicate that GABA may activate cation-selective ligand-gated ion channels in some cell types, such a channel has never been characterized at the molecular level. Here we show that GABA mediates enteric muscle contraction in the nematode Caenorhabditis elegans via the EXP-1 receptor, a cation-selective ligand-gated ion channel. The EXP-1 protein resembles ionotropic GABA receptor subunits in almost all domains. In the pore-forming domain of EXP-1, however, the residues that confer anion selectivity are exchanged for those that specify cation selectivity. When expressed in Xenopus laevis oocytes, EXP-1 forms a GABA receptor that is permeable to cations and not anions. We conclude that some of the excitatory functions assigned to GABA are mediated by cation channels rather than by anion channels.

Endophilin is Required for Synaptic Vesicle Endocytosis by Localizing Synaptojanin

Neuron. Nov, 2003  |  Pubmed ID: 14622579

Endophilin is a membrane-associated protein required for endocytosis of synaptic vesicles. Two models have been proposed for endophilin: that it alters lipid composition in order to shape membranes during endocytosis, or that it binds the polyphosphoinositide phosphatase synaptojanin and recruits this phosphatase to membranes. In this study, we demonstrate that the unc-57 gene encodes the Caenorhabditis elegans ortholog of endophilin A. We demonstrate that endophilin is required in C. elegans for synaptic vesicle recycling. Furthermore, the defects observed in endophilin mutants closely resemble those observed in synaptojanin mutants. The electrophysiological phenotype of endophilin and synaptojanin double mutants are virtually identical to the single mutants, demonstrating that endophilin and synaptojanin function in the same pathway. Finally, endophilin is required to stabilize expression of synaptojanin at the synapse. These data suggest that endophilin is an adaptor protein required to localize and stabilize synaptojanin at membranes during synaptic vesicle recycling.

Long Chain Polyunsaturated Fatty Acids Are Required for Efficient Neurotransmission in C. Elegans

Journal of Cell Science. Dec, 2003  |  Pubmed ID: 14625390

The complex lipid constituents of the eukaryotic plasma membrane are precisely controlled in a cell-type-specific manner, suggesting an important, but as yet, unknown cellular function. Neuronal membranes are enriched in long-chain polyunsaturated fatty acids (LC-PUFAs) and alterations in LC-PUFA metabolism cause debilitating neuronal pathologies. However, the physiological role of LC-PUFAs in neurons is unknown. We have characterized the neuronal phenotype of C. elegans mutants depleted of LC-PUFAs. The C. elegans genome encodes a single Delta6-desaturase gene (fat-3), an essential enzyme for LC-PUFA biosynthesis. Animals lacking fat-3 function do not synthesize LC-PUFAs and show movement and egg-laying abnormalities associated with neuronal impairment. Expression of functional fat-3 in neurons, or application of exogenous LC-PUFAs to adult animals rescues these defects. Pharmacological, ultrastructural and electrophysiological analyses demonstrate that fat-3 mutant animals are depleted of synaptic vesicles and release abnormally low levels of neurotransmitter at cholinergic and serotonergic neuromuscular junctions. These data indicate that LC-PUFAs are essential for efficient neurotransmission in C. elegans and may account for the clinical conditions associated with mis-regulation of LC-PUFAs in humans.

Preservation of Immunoreactivity and Fine Structure of Adult C. Elegans Tissues Using High-pressure Freezing

The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society. Jan, 2004  |  Pubmed ID: 14688212

The location of a protein labeled by immunogold techniques can be resolved under an electron beam to within nanometers of its epitope, a resolution that makes immunoelectron microscopy a valuable tool for studies of cell biology. However, tissues in the nematode Caenorhabditis elegans are difficult to preserve for immunoelectron microscopic studies. The animal's cuticle slows the diffusion of solutions into the animal and thus makes it difficult to preserve both immunoreactivity and cell morphology. Here we describe a protocol that circumvents these problems. Specifically, we instantly immobilized tissue in vitreous ice by freezing living adult animals under high pressure. Frozen specimens were then chemically fixed, dehydrated, and embedded at low temperatures. As a result, chemical diffusion across the cuticle could occur over an extended period without morphological deterioration. We show that this method is capable of preserving both cell morphology, including fine structures, and immunoreactivity. Therefore, it provides a means to characterize the localization of endogenous proteins and exogenous proteins, such as the green fluorescent protein (GFP), with respect to subcellular compartments in C. elegans tissues by using postembedding immunogold labeling.

Neuroscience. Vesicular Glutamate Transporter--shooting Blanks

Science (New York, N.Y.). Jun, 2004  |  Pubmed ID: 15205517

The GABA Nervous System in C. Elegans

Trends in Neurosciences. Jul, 2004  |  Pubmed ID: 15219740

GABA neurotransmission requires a specialized set of proteins to synthesize, transport or respond to GABA. This article reviews results from a genetic strategy in the nematode Caenorhabditis elegans designed to identify the genes responsible for these activities. These studies identified mutations in genes encoding five different proteins: the biosynthetic enzyme for GABA, the vesicular GABA transporter, a transcription factor that determines GABA neuron identity, a classic inhibitory GABA receptor and a novel excitatory GABA receptor. This review discusses the strategy employed to identify these genes as well as the conclusions about GABA transmission derived from study of the mutant phenotypes.

Dopamine: Should I Stay or Should I Go Now?

Nature Neuroscience. Oct, 2004  |  Pubmed ID: 15452567

A Conserved Metalloprotease Mediates Ecdysis in Caenorhabditis Elegans

Development (Cambridge, England). Dec, 2004  |  Pubmed ID: 15539494

Molting is required for progression between larval stages in the life cycle of nematodes. We have identified four mutant alleles of a Caenorhabditis elegans metalloprotease gene, nas-37, that cause incomplete ecdysis. At each molt the cuticle fails to open sufficiently at the anterior end and the partially shed cuticle is dragged behind the animal. The gene is expressed in hypodermal cells 4 hours before ecdysis during all larval stages. The NAS-37 protein accumulates in the anterior cuticle and is shed in the cuticle after ecdysis. This pattern of protein accumulation places NAS-37 in the right place and at the right time to degrade the cuticle to facilitate ecdysis. The nas-37 gene has orthologs in other nematode species, including parasitic nematodes, and they undergo a similar shedding process. For example, Haemonchus contortus molts by digesting a ring of cuticle at the tip of the nose. Incubating Haemonchus larvae in extracted exsheathing fluids causes a refractile ring of digested cuticle to form at the tip of the nose. When Haemonchus cuticles are incubated with purified NAS-37, a similar refractile ring forms. NAS-37 degradation of the Haemonchus cuticle suggests that the metalloproteases and the cuticle substrates involved in exsheathment of parasitic nematodes are conserved in free-living nematodes.

Characterization of Mos1-mediated Mutagenesis in Caenorhabditis Elegans: a Method for the Rapid Identification of Mutated Genes

Genetics. Mar, 2005  |  Pubmed ID: 15654093

Insertional mutagenesis with a heterologous transposon provides a method to rapidly determine the molecular identity of mutated genes. The Drosophila transposon Mos1 can be mobilized to cause mutations in Caenorhabditis elegans (Bessereau et al. 2001); however, the mutagenic rate was initially too low for use in most forward genetic screens. To increase the effectiveness of Mos1-mediated mutagenesis we examined the conditions influencing Mos1 transposition. First, optimal transposition occurs 24 hr after expression of the transposase and is unlikely to occur in differentiated sperm or oocytes. Second, transposition is limited to germ-cell nuclei that contain donor elements, but the transposase enzyme can diffuse throughout the gonad syncytium. Third, silencing of transposition is caused by changes in the donor array that occur over time. Finally, multiple transposition events occur in individual germ cells. By using screening techniques based on these results, Mos1 mutagenicity was increased to within an order of magnitude of chemical mutagens.

The Composition of the GABA Receptor at the Caenorhabditis Elegans Neuromuscular Junction

British Journal of Pharmacology. Feb, 2005  |  Pubmed ID: 15655525

1. The unc-49 gene of the nematode Caenorhabditis elegans encodes three gamma-aminobutyric acid type A (GABA(A)) receptor subunits. Two of these, UNC-49B and UNC-49C, are expressed at high abundance and co-localize at the neuromuscular junction. 2. The UNC-49B subunit is sufficient to form a GABA(A) receptor in vitro and in vivo. Furthermore, all loss-of-function unc-49 alleles lack functional UNC-49B. No mutations specifically inactivate UNC-49C. Thus, UNC-49C appears to be dispensable for receptor function; however, UNC-49C has been conserved among different nematode species, suggesting it plays a necessary role. 3. To ascertain whether UNC-49C is part of the GABA(A) receptor in vivo, we performed patch-clamp electrophysiology on C. elegans muscle cells. Sensitivity to GABA, and to the antagonists picrotoxin and pregnenolone sulfate, matched the UNC-49B/C heteromer rather than the UNC-49B homomer, for both exogenous and synaptically-released GABA. 4. The synaptic localization of UNC-49C requires the presence of UNC-49B, indicative of a physical association between the two subunits in vivo. Thus, the in vivo receptor is an UNC-49B/C heteromer. 5. UNC-49C plays a negative modulatory role. Using the rapid ligand-exchange technique in vitro, we determined that UNC-49C causes accelerated receptor desensitization. Previously, UNC-49C was shown to reduce single-channel conductance in UNC-49B/C heteromers. Thus, the function of UNC-49B is to provide GABA responsiveness and localization to synapses, while the function of UNC-49C is to negatively modulate receptor function and precisely shape inhibitory postsynaptic currents.

Heterozygous Insertions Alter Crossover Distribution but Allow Crossover Interference in Caenorhabditis Elegans

Genetics. Nov, 2005  |  Pubmed ID: 16118192

The normal distribution of crossover events on meiotic bivalents depends on homolog recognition, alignment, and interference. We developed a method for precisely locating all crossovers on Caenorhabditis elegans chromosomes and demonstrated that wild-type animals have essentially complete interference, with each bivalent receiving one and only one crossover. A physical break in one homolog has previously been shown to disrupt interference, suggesting that some aspect of bivalent structure is required for interference. We measured the distribution of crossovers in animals heterozygous for a large insertion to determine whether a break in sequence homology would have the same effect as a physical break. Insertions disrupt crossing over locally. However, every bivalent still experiences essentially one and only one crossover, suggesting that interference can act across a large gap in homology. Although insertions did not affect crossover number, they did have an effect on crossover distribution. Crossing over was consistently higher on the side of the chromosome bearing the homolog recognition region and lower on the other side of the chromosome. We suggest that nonhomologous sequences cause heterosynapsis, which disrupts crossovers along the distal chromosome, even when those regions contain sequences that could otherwise align. However, because crossovers are not completely eliminated distal to insertions, we propose that alignment can be reestablished after a megabase-scale gap in sequence homology.

Rapid Single Nucleotide Polymorphism Mapping in C. Elegans

BMC Genomics. 2005  |  Pubmed ID: 16156901

In C. elegans, single nucleotide polymorphisms (SNPs) can function as silent genetic markers, with applications ranging from classical two- and three-factor mapping to measuring recombination across whole chromosomes.


WormBook : the Online Review of C. Elegans Biology. 2005  |  Pubmed ID: 18050397

The most abundant synapses in the central nervous system of vertebrates are inhibitory synapses that use the neurotransmitter gamma-aminobutyric acid (GABA). GABA is also an important neurotransmitter in C. elegans; however, in contrast to vertebrates where GABA acts at synapses of the central nervous system, in nematodes GABA acts primarily at neuromuscular synapses. Specifically, GABA acts to relax the body muscles during locomotion and foraging and to contract the enteric muscles during defecation. The importance of this neurotransmitter for basic motor functions of the worm has facilitated the genetic analysis of proteins required for GABA function. Genetic screens have identified the GABA biosynthetic enzyme, the vesicular transporter, inhibitory and excitatory receptors, and a transcription factor required for the differentiation of GABA cell identity. The plasma membrane transporter and other GABA receptors have been identified by molecular criteria.

Residues in the First Transmembrane Domain of the Caenorhabditis Elegans GABA(A) Receptor Confer Sensitivity to the Neurosteroid Pregnenolone Sulfate

British Journal of Pharmacology. May, 2006  |  Pubmed ID: 16547524

The GABA(A) receptor is a target of endogenous and synthetic neurosteroids. Little is known about the residues required for neurosteroid action on GABA(A) receptors. We have investigated pregnenolone sulfate (PS) inhibition of the Caenorhabditis elegans UNC-49 GABA receptor, a close homolog of the mammalian GABA(A) receptor. The UNC-49 locus encodes two GABA receptor subunits, UNC-49B and UNC-49C. UNC-49C is sensitive to PS but UNC-49B is not sensitive. By analyzing chimeric receptors and receptors containing site-directed mutations, we identified two regions required for PS inhibition. Four residues in the first transmembrane domain are required for the majority of the sensitivity to PS, but a charged extracellular residue at the end of the M2 helix also plays a role. Strikingly, mutation of one additional M1 residue reverses the effect of PS from an inhibitor to an enhancer of receptor function. Mutating the M1 domain had little effect on sensitivity to the inhibitor picrotoxin, suggesting that these residues may mediate neurosteroid action specifically, and not allosteric regulation in general.

Synaptic Tetraspan Vesicle Membrane Proteins Are Conserved but Not Needed for Synaptogenesis and Neuronal Function in Caenorhabditis Elegans

Proceedings of the National Academy of Sciences of the United States of America. May, 2006  |  Pubmed ID: 16698939

Tetraspan vesicle membrane proteins (TVPs) comprise a major portion of synaptic vesicle proteins, yet their contribution to the synaptic vesicle cycle is poorly understood. TVPs are grouped in three mammalian gene families: physins, gyrins, and secretory carrier-associated membrane proteins (SCAMPs). In Caenorhabditis elegans, only a single member of each of these families exists. These three nematode TVPs colocalize to the same vesicular compartment when expressed in mammalian cells, suggesting that they could serve overlapping functions. To examine their function, C. elegans null mutants were isolated for each gene, and a triple mutant was generated. Surprisingly, these animals develop normally and exhibit normal neuronal architecture and synaptic contacts. In addition, functions of the motor and sensory systems are normal as determined by pharmacological, chemotaxis, and thermotaxis assays. Finally, direct electrophysiological analysis of the neuromuscular junction revealed no phenotype in the TVP mutants. We therefore conclude that TVPs are not needed for the basic neuronal machinery and instead may contribute to subtle higher order functions.

Induction and Repair of Zinc-finger Nuclease-targeted Double-strand Breaks in Caenorhabditis Elegans Somatic Cells

Proceedings of the National Academy of Sciences of the United States of America. Oct, 2006  |  Pubmed ID: 17060623

Zinc-finger nucleases are chimeric proteins consisting of engineered zinc-finger DNA-binding motifs attached to an endonuclease domain. These proteins can induce site-specific DNA double-strand breaks in genomic DNA, which are then substrates for cellular repair mechanisms. Here, we demonstrate that engineered zinc-finger nucleases function effectively in somatic cells of the nematode Caenorhabditis elegans. Although gene-conversion events were indistinguishable from uncut DNA in our assay, nonhomologous end joining resulted in mutations at the target site. A synthetic target on an extrachromosomal array was targeted with a previously characterized nuclease, and an endogenous genomic sequence was targeted with a pair of specifically designed nucleases. In both cases, approximately 20% of the target sites were mutated after induction of the corresponding nucleases. Alterations in the extrachromosomal targets were largely products of end-filling and blunt ligation. By contrast, alterations in the chromosomal target were mostly deletions. We interpret these differences to reflect the abundance of homologous templates present in the extrachromosomal arrays versus the paucity of such templates for repair of chromosomal breaks. In addition, we find evidence for the involvement of error-prone DNA synthesis in both homologous and nonhomologous pathways of repair. DNA ligase IV is required for efficient end joining, particularly of blunt ends. In its absence, a secondary end-joining pathway relies more heavily on microhomologies in producing deletions.

Axons Break in Animals Lacking Beta-spectrin

The Journal of Cell Biology. Jan, 2007  |  Pubmed ID: 17261846

Axons and dendrites can withstand acute mechanical strain despite their small diameter. In this study, we demonstrate that beta-spectrin is required for the physical integrity of neuronal processes in the nematode Caenorhabditis elegans. Axons in beta-spectrin mutants spontaneously break. Breakage is caused by acute strain generated by movement because breakage can be prevented by paralyzing the mutant animals. After breaking, the neuron attempts to regenerate by initiating a new growth cone; this second round of axon extension is error prone compared with initial outgrowth. Because spectrin is a major target of calpain proteolysis, it is possible that some neurodegenerative disorders may involve the cleavage of spectrin followed by the breakage of neural processes.

The Plasma Membrane Calcium ATPase MCA-3 is Required for Clathrin-mediated Endocytosis in Scavenger Cells of Caenorhabditis Elegans

Traffic (Copenhagen, Denmark). May, 2007  |  Pubmed ID: 17343680

Plasma membrane Ca2+ ATPases (PMCAs) maintain proper intracellular Ca2+ levels by extruding Ca2+ from the cytosol. PMCA genes and splice forms are expressed in tissue-specific patterns in vertebrates, suggesting that these isoforms may regulate specific biological processes. However, knockout mutants die as embryos or undergo cell death; thus, it is unclear whether other cell processes utilize PMCAs or whether these pumps are largely committed to the control of toxic levels of calcium. Here, we analyze the role of the PMCA gene, mca-3, in Caenorhabditis elegans. We report that partial loss-of-function mutations disrupt clathrin-mediated endocytosis in a class of scavenger cells called coelomocytes. Moreover, components of early endocytic machinery are mislocalized in mca-3 mutants, including phosphatidylinositol-4,5-bisphosphate, clathrin and the Eps15 homology (EH) domain protein RME-1. This defect in endocytosis in the coelomocytes can be reversed by lowering calcium. Together, these data support a function for PMCAs in the regulation of endocytosis in the C. elegans coelomocytes. In addition, they suggest that endocytosis can be blocked by high calcium levels.

PKC Defends Crown Against Munc13

Neuron. Apr, 2007  |  Pubmed ID: 17442237

Protein kinase C has long been thought to mediate DAG signaling at the synapse. Recently PKC has been supplanted by members of the Unc13 family as the predominant effectors of DAG signaling. Thanks to a study by Wierda and colleagues in this issue of Neuron, PKC returns to reclaim part of the kingdom: both pathways must be active to activate presynaptic potentiation.

UNC-31 (CAPS) is Required for Dense-core Vesicle but Not Synaptic Vesicle Exocytosis in Caenorhabditis Elegans

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Jun, 2007  |  Pubmed ID: 17553987

Previous studies indicated that CAPS (calcium-dependent activator protein for secretion) functions as an essential component for the Ca2+-dependent exocytosis of dense-core vesicles in neuroendocrine cells. However, recent mouse knock-out studies suggested an alternative role in the vesicular uptake or storage of catecholamines. To genetically assess the functional role of CAPS, we characterized the sole Caenorhabditis elegans CAPS ortholog UNC-31 (uncoordinated family member) and determined its role in dense-core vesicle-mediated peptide secretion and in synaptic vesicle recycling. Novel assays for dense-core vesicle exocytosis were developed by expressing a prepro-atrial natriuretic factor-green fluorescent protein fusion protein in C. elegans. unc-31 mutants exhibited reduced peptide release in vivo and lacked evoked peptide release in cultured neurons. In contrast, cultured neurons from unc-31 mutants exhibited normal stimulated synaptic vesicle recycling measured by FM4-64 [N-(3-triethylammoniumpropyl)-4-(6-(4-diethylamino)phenyl)hexatrienyl)pyridinium dibromide] dye uptake. Conversely, UNC-13, which exhibits sequence homology to CAPS/UNC-31, was found to be essential for synaptic vesicle but not dense-core vesicle exocytosis. These findings indicate that CAPS/UNC-31 function is not restricted to catecholaminergic vesicles but is generally required for and specific to dense-core vesicle exocytosis. Our results suggest that CAPS/UNC-31 and UNC-13 serve parallel and dedicated roles in dense-core vesicle and synaptic vesicle exocytosis, respectively, in the C. elegans nervous system.

UNC-46 is Required for Trafficking of the Vesicular GABA Transporter

Nature Neuroscience. Jul, 2007  |  Pubmed ID: 17558401

Mutations in unc-46 in Caenorhabditis elegans cause defects in all behaviors that are mediated by GABA. Here we show that UNC-46 is a sorting factor that localizes the vesicular GABA transporter to synaptic vesicles. The UNC-46 protein is related to the LAMP (lysosomal associated membrane protein) family of proteins and is localized at synapses. In unc-46 mutants, the vesicular transporter is not found specifically in synaptic vesicles but rather is diffusely spread along the axon. Mislocalization of the transporter severely reduces the frequency of miniature currents, but the remaining currents are normal in amplitude. Because the number of synaptic vesicles is not depleted, it is likely that only a fraction of vesicles harbor the transporter in unc-46 mutants. Our data indicate that the transporter and UNC-46 have mutual roles in sorting. The vesicular GABA transporter recruits UNC-46 to synaptic vesicle precursors in the cell body, and UNC-46 sorts the transporter at the cell body and during endocytosis at the synapse.

Open Syntaxin Docks Synaptic Vesicles

PLoS Biology. Aug, 2007  |  Pubmed ID: 17645391

Synaptic vesicles dock to the plasma membrane at synapses to facilitate rapid exocytosis. Docking was originally proposed to require the soluble N-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE) proteins; however, perturbation studies suggested that docking was independent of the SNARE proteins. We now find that the SNARE protein syntaxin is required for docking of all vesicles at synapses in the nematode Caenorhabditis elegans. The active zone protein UNC-13, which interacts with syntaxin, is also required for docking in the active zone. The docking defects in unc-13 mutants can be fully rescued by overexpressing a constitutively open form of syntaxin, but not by wild-type syntaxin. These experiments support a model for docking in which UNC-13 converts syntaxin from the closed to the open state, and open syntaxin acts directly in docking vesicles to the plasma membrane. These data provide a molecular basis for synaptic vesicle docking.

UNC-80 and the NCA Ion Channels Contribute to Endocytosis Defects in Synaptojanin Mutants

Current Biology : CB. Sep, 2007  |  Pubmed ID: 17825559

Synaptojanin is a lipid phosphatase required to degrade phosphatidylinositol 4,5 bisphosphate (PIP(2)) at cell membranes during synaptic vesicle recycling. Synaptojanin mutants in C. elegans are severely uncoordinated and are depleted of synaptic vesicles, possibly because of accumulation of PIP(2). To identify proteins that act downstream of PIP(2) during endocytosis, we screened for suppressors of synaptojanin mutants in the nematode C. elegans. A class of uncoordinated mutants called "fainters" partially suppress the locomotory, vesicle depletion, and electrophysiological defects in synaptojanin mutants. These suppressor loci include the genes for the NCA ion channels, which are homologs of the vertebrate cation leak channel NALCN, and a novel gene called unc-80. We demonstrate that unc-80 encodes a novel, but highly conserved, neuronal protein required for the proper localization of the NCA-1 and NCA-2 ion channel subunits. These data suggest that activation of the NCA ion channel in synaptojanin mutants leads to defects in recycling of synaptic vesicles.

A Calcium Wave Mediated by Gap Junctions Coordinates a Rhythmic Behavior in C. Elegans

Current Biology : CB. Sep, 2007  |  Pubmed ID: 17825560

Intercellular calcium waves can be observed in adult tissues, but whether they are instructive, permissive, or even required for behavior is predominantly unknown. In the nematode Caenorhabditis elegans, a periodic calcium spike in a pacemaker cell initiates a calcium wave in the intestine. The calcium wave is followed by three muscle contractions that comprise the defecation motor program. Normal wave propagation requires the pannexin gap-junction subunit INX-16 at the interfaces of the intestinal cells. In the absence of this gap-junction subunit, calcium waves are frequently absent. The remaining waves are slow, initiate at abnormal locations, or travel in the opposite direction. Abnormal waves are associated with parallel effects in the first step of the motor program: The contractions of the overlying muscles fail to propagate beyond the pacemaker cell, are slow, initiate in abnormal locations, or are reversed. Moreover, the last two motor steps are predominantly absent. Finally, the absence of this gap-junction subunit also affects the reliability of the pacemaker cell; cycle timing is often irregular. These data demonstrate that pannexin gap junctions propagate calcium waves in the C. elegans intestine. The calcium waves instruct the motor steps and regulate the pacemaker cell's authority and reliability.

Trio's Rho-specific GEF Domain is the Missing Galpha Q Effector in C. Elegans

Genes & Development. Nov, 2007  |  Pubmed ID: 17942708

The Galpha(q) pathway is essential for animal life and is a central pathway for driving locomotion, egg laying, and growth in Caenorhabditis elegans, where it exerts its effects through EGL-8 (phospholipase Cbeta [PLCbeta]) and at least one other effector. To find the missing effector, we performed forward genetic screens to suppress the slow growth and hyperactive behaviors of mutants with an overactive Galpha(q) pathway. Four suppressor mutations disrupted the Rho-specific guanine-nucleotide exchange factor (GEF) domain of UNC-73 (Trio). The mutations produce defects in neuronal function, but not neuronal development, that cause sluggish locomotion similar to animals lacking EGL-8 (PLCbeta). Strains containing null mutations in both EGL-8 (PLCbeta) and UNC-73 (Trio RhoGEF) have strong synthetic phenotypes that phenocopy the arrested growth and near-complete paralysis of Galpha(q)-null mutants. Using cell-based and biochemical assays, we show that activated C. elegans Galpha(q) synergizes with Trio RhoGEF to activate RhoA. Activated Galpha(q) and Trio RhoGEF appear to be part of a signaling complex, because they coimmunoprecipitate when expressed together in cells. Our results show that Trio's Rho-specific GEF domain is a major Galpha(q) effector that, together with PLCbeta, mediates the Galpha(q) signaling that drives the locomotion, egg laying, and growth of the animal.

The Sensory Circuitry for Sexual Attraction in C. Elegans Males

Current Biology : CB. Nov, 2007  |  Pubmed ID: 17964166

Why do males and females behave differently? Sexually dimorphic behaviors could arise from sex-specific neurons or by the modification of circuits present in both sexes. C. elegans males exhibit different behaviors than hermaphrodites. Although there is a single class of sex-specific sensory neurons in the head of males, most of their neurons are part of a core nervous system also present in hermaphrodites. Are the behavioral differences due to sex-specific or core neurons?

Molecular Basis of Synaptic Vesicle Cargo Recognition by the Endocytic Sorting Adaptor Stonin 2

The Journal of Cell Biology. Dec, 2007  |  Pubmed ID: 18166656

Synaptic transmission depends on clathrin-mediated recycling of synaptic vesicles (SVs). How select SV proteins are targeted for internalization has remained elusive. Stonins are evolutionarily conserved adaptors dedicated to endocytic sorting of the SV protein synaptotagmin. Our data identify the molecular determinants for recognition of synaptotagmin by stonin 2 or its Caenorhabditis elegans orthologue UNC-41B. The interaction involves the direct association of clusters of basic residues on the surface of the cytoplasmic domain of synaptotagmin 1 and a beta strand within the mu-homology domain of stonin 2. Mutation of K783, Y784, and E785 to alanine within this stonin 2 beta strand results in failure of the mutant stonin protein to associate with synaptotagmin, to accumulate at synapses, and to facilitate synaptotagmin internalization. Synaptotagmin-binding-defective UNC-41B is unable to rescue paralysis in C. elegans stonin mutant animals, suggesting that the mechanism of stonin-mediated SV cargo recognition is conserved from worms to mammals.

C. Elegans AP-2 and Retromer Control Wnt Signaling by Regulating Mig-14/Wntless

Developmental Cell. Jan, 2008  |  Pubmed ID: 18160346

While endocytosis can regulate morphogen distribution, its precise role in shaping these gradients is unclear. Even more enigmatic is the role of retromer, a complex that shuttles proteins between endosomes and the Golgi apparatus, in Wnt gradient formation. Here we report that DPY-23, the C. elegans mu subunit of the clathrin adaptor AP-2 that mediates the endocytosis of membrane proteins, regulates Wnt function. dpy-23 mutants display Wnt phenotypes, including defects in neuronal migration, neuronal polarity, and asymmetric cell division. DPY-23 acts in Wnt-expressing cells to promote these processes. MIG-14, the C. elegans homolog of the Wnt-secretion factor Wntless, also acts in these cells to control Wnt function. In dpy-23 mutants, MIG-14 accumulates at or near the plasma membrane. By contrast, MIG-14 accumulates in intracellular compartments in retromer mutants. Based on our observations, we propose that intracellular trafficking of MIG-14 by AP-2 and retromer plays an important role in Wnt secretion.

Protons Act As a Transmitter for Muscle Contraction in C. Elegans

Cell. Jan, 2008  |  Pubmed ID: 18191228

Muscle contraction is normally mediated by the release of neurotransmitters from motor neurons. Here we demonstrate that protons can act as a direct transmitter from intestinal cells to stimulate muscle contraction. During the C. elegans defecation motor program the posterior body muscles contract even in the absence of neuronal inputs or vesicular neurotransmission. In this study, we demonstrate that the space between the intestine and the muscle is acidified just prior to muscle contraction and that the release of caged protons is sufficient to induce muscle contraction. PBO-4 is a putative Na+/H+ ion exchanger expressed on the basolateral membrane of the intestine, juxtaposed to the posterior body muscles. In pbo-4 mutants the extracellular space is not acidified and the muscles fail to contract. The pbo-5 and pbo-6 genes encode subunits of a "cys-loop" proton-gated cation channel required for muscles to respond to acidification. In heterologous expression assays the PBO receptor is half-maximally activated at a pH of 6.8. The identification of the mechanisms for release and reception of proton signals establishes a highly unusual mechanism for intercellular communication.

CAPS and Syntaxin Dock Dense Core Vesicles to the Plasma Membrane in Neurons

The Journal of Cell Biology. Feb, 2008  |  Pubmed ID: 18250196

Docking to the plasma membrane prepares vesicles for rapid release. Here, we describe a mechanism for dense core vesicle docking in neurons. In Caenorhabditis elegans motor neurons, dense core vesicles dock at the plasma membrane but are excluded from active zones at synapses. We have found that the calcium-activated protein for secretion (CAPS) protein is required for dense core vesicle docking but not synaptic vesicle docking. In contrast, we see that UNC-13, a docking factor for synaptic vesicles, is not essential for dense core vesicle docking. Both the CAPS and UNC-13 docking pathways converge on syntaxin, a component of the SNARE (soluble N-ethyl-maleimide-sensitive fusion protein attachment receptor) complex. Overexpression of open syntaxin can bypass the requirement for CAPS in dense core vesicle docking. Thus, CAPS likely promotes the open state of syntaxin, which then docks dense core vesicles. CAPS function in dense core vesicle docking parallels UNC-13 in synaptic vesicle docking, which suggests that these related proteins act similarly to promote docking of independent vesicle populations.

Gene Activation Using FLP Recombinase in C. Elegans

PLoS Genetics. Mar, 2008  |  Pubmed ID: 18369447

The FLP enzyme catalyzes recombination between specific target sequences in DNA. Here we use FLP to temporally and spatially control gene expression in the nematode C. elegans. Transcription is blocked by the presence of an "off cassette" between the promoter and the coding region of the desired product. The "off cassette" is composed of a transcriptional terminator flanked by FLP recognition targets (FRT). This sequence can be excised by FLP recombinase to bring together the promoter and the coding region. We have introduced two fluorescent reporters into the system: a red reporter for promoter activity prior to FLP expression and a green reporter for expression of the gene of interest after FLP expression. The constructs are designed using the multisite Gateway system, so that promoters and coding regions can be quickly mixed and matched. We demonstrate that heat-shock-driven FLP recombinase adds temporal control on top of tissue specific expression provided by the transgene promoter. In addition, the temporal switch is permanent, rather than acute, as is usually the case for heat-shock driven transgenes. Finally, FLP expression can be driven by a tissue specific promoter to provide expression in a subset of cells that can only be addressed as the intersection of two available promoters. As a test of the system, we have driven the light chain of tetanus toxin, a protease that cleaves the synaptic vesicle protein synaptobrevin. We show that we can use this to inactivate synaptic transmission in all neurons or a subset of neurons in a FLP-dependent manner.

Gene Conversion and End-joining-repair Double-strand Breaks in the Caenorhabditis Elegans Germline

Genetics. Sep, 2008  |  Pubmed ID: 18757928

Excision of a Mos1 transposon in the germline of Caenorhabditis elegans generates a double-strand break in the chromosome. We demonstrate that breaks are most prominently repaired by gene conversion from the homolog, but also rarely by nonhomologous end-joining. In some cases, gene conversion events are resolved by crossing over. Surprisingly, expression of the transposase using an intestine-specific promoter can induce repair, raising the possibility that activation of transposase expression in somatic cells can lead to transposition of Mos1 in the germline.

Single-copy Insertion of Transgenes in Caenorhabditis Elegans

Nature Genetics. Nov, 2008  |  Pubmed ID: 18953339

At present, transgenes in Caenorhabditis elegans are generated by injecting DNA into the germline. The DNA assembles into a semistable extrachromosomal array composed of many copies of injected DNA. These transgenes are typically overexpressed in somatic cells and silenced in the germline. We have developed a method that inserts a single copy of a transgene into a defined site. Mobilization of a Mos1 transposon generates a double-strand break in noncoding DNA. The break is repaired by copying DNA from an extrachromosomal template into the chromosomal site. Homozygous single-copy insertions can be obtained in less than 2 weeks by injecting approximately 20 worms. We have successfully inserted transgenes as long as 9 kb and verified that single copies are inserted at the targeted site. Single-copy transgenes are expressed at endogenous levels and can be expressed in the female and male germlines.

Mu2 Adaptin Facilitates but is Not Essential for Synaptic Vesicle Recycling in Caenorhabditis Elegans

The Journal of Cell Biology. Dec, 2008  |  Pubmed ID: 19047463

Synaptic vesicles must be recycled to sustain neurotransmission, in large part via clathrin-mediated endocytosis. Clathrin is recruited to endocytic sites on the plasma membrane by the AP2 adaptor complex. The medium subunit (micro2) of AP2 binds to cargo proteins and phosphatidylinositol-4 ,5-bisphosphate on the cell surface. Here, we characterize the apm-2 gene (also called dpy-23), which encodes the only micro2 subunit in the nematode Caenorhabditis elegans. APM-2 is highly expressed in the nervous system and is localized to synapses; yet specific loss of APM-2 in neurons does not affect locomotion. In apm-2 mutants, clathrin is mislocalized at synapses, and synaptic vesicle numbers and evoked responses are reduced to 60 and 65%, respectively. Collectively, these data suggest AP2 micro2 facilitates but is not essential for synaptic vesicle recycling.

Differential Requirements for Clathrin in Receptor-mediated Endocytosis and Maintenance of Synaptic Vesicle Pools

Proceedings of the National Academy of Sciences of the United States of America. Jan, 2009  |  Pubmed ID: 19151157

Clathrin is a coat protein involved in vesicle budding from several membrane-bound compartments within the cell. Here we present an analysis of a temperature-sensitive (ts) mutant of clathrin heavy chain (CHC) in a multicellular animal. As expected Caenorhabditis elegans chc-1(b1025ts) mutant animals are defective in receptor-mediated endocytosis and arrest development soon after being shifted to the restrictive temperature. Steady-state clathrin levels in these mutants are reduced by more than 95% at all temperatures. Hub interactions and membrane associations are lost at the restrictive temperature. chc-1(b1025ts) animals become paralyzed within minutes of exposure to the restrictive temperature because of a defect in the nervous system. Surprisingly synaptic vesicle number is not reduced in chc-1(b1025ts) animals. Consistent with the normal number of vesicles, postsynaptic miniature currents occur at normal frequencies. Taken together, these results indicate that a high level of CHC activity is required for receptor-mediated endocytosis in nonneuronal cells but is largely dispensable for maintenance of synaptic vesicle pools.

Axon Regeneration Requires a Conserved MAP Kinase Pathway

Science (New York, N.Y.). Feb, 2009  |  Pubmed ID: 19164707

Regeneration of injured neurons can restore function, but most neurons regenerate poorly or not at all. The failure to regenerate in some cases is due to a lack of activation of cell-intrinsic regeneration pathways. These pathways might be targeted for the development of therapies that can restore neuron function after injury or disease. Here, we show that the DLK-1 mitogen-activated protein (MAP) kinase pathway is essential for regeneration in Caenorhabditis elegans motor neurons. Loss of this pathway eliminates regeneration, whereas activating it improves regeneration. Further, these proteins also regulate the later step of growth cone migration. We conclude that after axon injury, activation of this MAP kinase cascade is required to switch the mature neuron from an aplastic state to a state capable of growth.

Graded Synaptic Transmission at the Caenorhabditis Elegans Neuromuscular Junction

Proceedings of the National Academy of Sciences of the United States of America. Jun, 2009  |  Pubmed ID: 19528650

Most neurotransmission is mediated by action potentials, whereas sensory neurons propagate electrical signals passively and release neurotransmitter in a graded manner. Here, we demonstrate that Caenorhabditis elegans neuromuscular junctions release neurotransmitter in a graded fashion. When motor neurons were depolarized by light-activation of channelrhodopsin-2, the evoked postsynaptic current scaled with the strength of the stimulation. When motor neurons were hyperpolarized by light-activation of halorhodopsin, tonic release of synaptic vesicles was decreased. These data suggest that both evoked and tonic neurotransmitter release is graded in response to membrane potential. Acetylcholine synapses are depressed by high-frequency stimulation, in part due to desensitization of the nicotine-sensitve ACR-16 receptor. By contrast, GABA synapses facilitate before becoming depressed. Graded transmission and plasticity confer a broad dynamic range to these synapses. Graded release precisely transmits stimulation intensity, even hyperpolarizing inputs. Synaptic plasticity alters the balance of excitatory and inhibitory inputs into the muscle in a use-dependent manner.

Calcium: an Insignificant Thing

Nature Neuroscience. Oct, 2009  |  Pubmed ID: 19783977

A Neuronal Acetylcholine Receptor Regulates the Balance of Muscle Excitation and Inhibition in Caenorhabditis Elegans

PLoS Biology. Dec, 2009  |  Pubmed ID: 20027209

In the nematode Caenorhabditis elegans, cholinergic motor neurons stimulate muscle contraction as well as activate GABAergic motor neurons that inhibit contraction of the contralateral muscles. Here, we describe the composition of an ionotropic acetylcholine receptor that is required to maintain excitation of the cholinergic motor neurons. We identified a gain-of-function mutation that leads to spontaneous muscle convulsions. The mutation is in the pore domain of the ACR-2 acetylcholine receptor subunit and is identical to a hyperactivating mutation in the muscle receptor of patients with myasthenia gravis. Screens for suppressors of the convulsion phenotype led to the identification of other receptor subunits. Cell-specific rescue experiments indicate that these subunits function in the cholinergic motor neurons. Expression of these subunits in Xenopus oocytes demonstrates that the functional receptor is comprised of three alpha-subunits, UNC-38, UNC-63 and ACR-12, and two non-alpha-subunits, ACR-2 and ACR-3. Although this receptor exhibits a partially overlapping subunit composition with the C. elegans muscle acetylcholine receptor, it shows distinct pharmacology. Recordings from intact animals demonstrate that loss-of-function mutations in acr-2 reduce the excitability of the cholinergic motor neurons. By contrast, the acr-2(gf) mutation leads to a hyperactivation of cholinergic motor neurons and an inactivation of downstream GABAergic motor neurons in a calcium dependent manner. Presumably, this imbalance between excitatory and inhibitory input into muscles leads to convulsions. These data indicate that the ACR-2 receptor is important for the coordinated excitation and inhibition of body muscles underlying sinusoidal movement.

Targeted Gene Deletions in C. Elegans Using Transposon Excision

Nature Methods. Jun, 2010  |  Pubmed ID: 20418868

We developed a method, MosDEL, to generate targeted knockouts of genes in Caenorhabditis elegans by injection. We generated a double-strand break by mobilizing a Mos1 transposon adjacent to the region to be deleted; the double-stranded break is repaired using injected DNA as a template. Repair can delete up to 25 kb of DNA and simultaneously insert a positive selection marker.

Two Cyclin-dependent Kinase Pathways Are Essential for Polarized Trafficking of Presynaptic Components

Cell. May, 2010  |  Pubmed ID: 20510931

Polarized trafficking of synaptic proteins to axons and dendrites is crucial to neuronal function. Through forward genetic analysis in C. elegans, we identified a cyclin (CYY-1) and a cyclin-dependent Pctaire kinase (PCT-1) necessary for targeting presynaptic components to the axon. Another cyclin-dependent kinase, CDK-5, and its activator p35, act in parallel to and partially redundantly with the CYY-1/PCT-1 pathway. Synaptic vesicles and active zone proteins mostly mislocalize to dendrites in animals defective for both PCT-1 and CDK-5 pathways. Unlike the kinesin-3 motor, unc-104/Kif1a mutant, cyy-1 cdk-5 double mutants have no reduction in anterogradely moving synaptic vesicle precursors (SVPs) as observed by dynamic imaging. Instead, the number of retrogradely moving SVPs is dramatically increased. Furthermore, this mislocalization defect is suppressed by disrupting the retrograde motor, the cytoplasmic dynein complex. Thus, PCT-1 and CDK-5 pathways direct polarized trafficking of presynaptic components by inhibiting dynein-mediated retrograde transport and setting the balance between anterograde and retrograde motors.

Detection of Neuron Membranes in Electron Microscopy Images Using a Serial Neural Network Architecture

Medical Image Analysis. Dec, 2010  |  Pubmed ID: 20598935

Study of nervous systems via the connectome, the map of connectivities of all neurons in that system, is a challenging problem in neuroscience. Towards this goal, neurobiologists are acquiring large electron microscopy datasets. However, the shear volume of these datasets renders manual analysis infeasible. Hence, automated image analysis methods are required for reconstructing the connectome from these very large image collections. Segmentation of neurons in these images, an essential step of the reconstruction pipeline, is challenging because of noise, anisotropic shapes and brightness, and the presence of confounding structures. The method described in this paper uses a series of artificial neural networks (ANNs) in a framework combined with a feature vector that is composed of image intensities sampled over a stencil neighborhood. Several ANNs are applied in series allowing each ANN to use the classification context provided by the previous network to improve detection accuracy. We develop the method of serial ANNs and show that the learned context does improve detection over traditional ANNs. We also demonstrate advantages over previous membrane detection methods. The results are a significant step towards an automated system for the reconstruction of the connectome.

Syntaxin N-terminal Peptide Motif is an Initiation Factor for the Assembly of the SNARE-Sec1/Munc18 Membrane Fusion Complex

Proceedings of the National Academy of Sciences of the United States of America. Dec, 2010  |  Pubmed ID: 21139055

Intracellular membrane fusion is mediated by the concerted action of N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and Sec1/Munc18 (SM) proteins. During fusion, SM proteins bind the N-terminal peptide (N-peptide) motif of the SNARE subunit syntaxin, but the function of this interaction is unknown. Here, using FRET-based biochemical reconstitution and Caenorhabditis elegans genetics, we show that the N-peptide of syntaxin-1 recruits the SM protein Munc18-1/nSec1 to the SNARE bundle, facilitating their assembly into a fusion-competent complex. The recruitment is achieved through physical tethering rather than allosteric activation of Munc18-1. Consistent with the recruitment role, the N-peptide is not spatially constrained along syntaxin-1, and it is functional when translocated to another SNARE subunit SNAP-25 or even when simply anchored in the target membrane. The N-peptide function is restricted to an early initiation stage of the fusion reaction. After association, Munc18-1 and the SNARE bundle together drive membrane merging without further involving the N-peptide. Thus, the syntaxin N-peptide is an initiation factor for the assembly of the SNARE-SM membrane fusion complex.

Protein Localization in Electron Micrographs Using Fluorescence Nanoscopy

Nature Methods. Jan, 2011  |  Pubmed ID: 21102453

A complete portrait of a cell requires a detailed description of its molecular topography: proteins must be linked to particular organelles. Immunocytochemical electron microscopy can reveal locations of proteins with nanometer resolution but is limited by the quality of fixation, the paucity of antibodies and the inaccessibility of antigens. Here we describe correlative fluorescence electron microscopy for the nanoscopic localization of proteins in electron micrographs. We tagged proteins with the fluorescent proteins Citrine or tdEos and expressed them in Caenorhabditis elegans, fixed the worms and embedded them in plastic. We imaged the tagged proteins from ultrathin sections using stimulated emission depletion (STED) microscopy or photoactivated localization microscopy (PALM). Fluorescence correlated with organelles imaged in electron micrographs from the same sections. We used these methods to localize histones, a mitochondrial protein and a presynaptic dense projection protein in electron micrographs.

Complexin Maintains Vesicles in the Primed State in C. Elegans

Current Biology : CB. Jan, 2011  |  Pubmed ID: 21215631

Complexin binds the SNARE complex at synapses and regulates exocytosis, but genetic studies indicate contradictory roles: in flies it predominantly inhibits synaptic vesicle fusion, whereas in mice it promotes evoked responses.

CYY-1/cyclin Y and CDK-5 Differentially Regulate Synapse Elimination and Formation for Rewiring Neural Circuits

Neuron. May, 2011  |  Pubmed ID: 21609829

The assembly and maturation of neural circuits require a delicate balance between synapse formation and elimination. The cellular and molecular mechanisms that coordinate synaptogenesis and synapse elimination are poorly understood. In C. elegans, DD motoneurons respecify their synaptic connectivity during development by completely eliminating existing synapses and forming new synapses without changing cell morphology. Using loss- and gain-of-function genetic approaches, we demonstrate that CYY-1, a cyclin box-containing protein, drives synapse removal in this process. In addition, cyclin-dependent kinase-5 (CDK-5) facilitates new synapse formation by regulating the transport of synaptic vesicles to the sites of synaptogenesis. Furthermore, we show that coordinated activation of UNC-104/Kinesin3 and Dynein is required for patterning newly formed synapses. During the remodeling process, presynaptic components from eliminated synapses are recycled to new synapses, suggesting that signaling mechanisms and molecular motors link the deconstruction of existing synapses and the assembly of new synapses during structural synaptic plasticity.

UNC119 is Required for G Protein Trafficking in Sensory Neurons

Nature Neuroscience. Jul, 2011  |  Pubmed ID: 21642972

UNC119 is widely expressed among vertebrates and other phyla. We found that UNC119 recognized the acylated N terminus of the rod photoreceptor transducin α (Tα) subunit and Caenorhabditis elegans G proteins ODR-3 and GPA-13. The crystal structure of human UNC119 at 1.95-Å resolution revealed an immunoglobulin-like β-sandwich fold. Pulldowns and isothermal titration calorimetry revealed a tight interaction between UNC119 and acylated Gα peptides. The structure of co-crystals of UNC119 with an acylated Tα N-terminal peptide at 2.0 Å revealed that the lipid chain is buried deeply into UNC119's hydrophobic cavity. UNC119 bound Tα-GTP, inhibiting its GTPase activity, thereby providing a stable UNC119-Tα-GTP complex capable of diffusing from the inner segment back to the outer segment after light-induced translocation. UNC119 deletion in both mouse and C. elegans led to G protein mislocalization. Thus, UNC119 is a Gα subunit cofactor essential for G protein trafficking in sensory cilia.

Membrane Tension Regulates Motility by Controlling Lamellipodium Organization

Proceedings of the National Academy of Sciences of the United States of America. Jul, 2011  |  Pubmed ID: 21709265

Many cell movements proceed via a crawling mechanism, where polymerization of the cytoskeletal protein actin pushes out the leading edge membrane. In this model, membrane tension has been seen as an impediment to filament growth and cell motility. Here we use a simple model of cell motility, the Caenorhabditis elegans sperm cell, to test how membrane tension affects movement and cytoskeleton dynamics. To enable these analyses, we create transgenic worm strains carrying sperm with a fluorescently labeled cytoskeleton. Via osmotic shock and deoxycholate treatments, we relax or tense the cell membrane and quantify apparent membrane tension changes by the membrane tether technique. Surprisingly, we find that membrane tension reduction is correlated with a decrease in cell displacement speed, whereas an increase in membrane tension enhances motility. We further demonstrate that apparent polymerization rates follow the same trends. We observe that membrane tension reduction leads to an unorganized, rough lamellipodium, composed of short filaments angled away from the direction of movement. On the other hand, an increase in tension reduces lateral membrane protrusions in the lamellipodium, and filaments are longer and more oriented toward the direction of movement. Overall we propose that membrane tension optimizes motility by streamlining polymerization in the direction of movement, thus adding a layer of complexity to our current understanding of how membrane tension enters into the motility equation.

Transcriptional Profiling of C. Elegans DAF-19 Uncovers a Ciliary Base-associated Protein and a CDK/CCRK/LF2p-related Kinase Required for Intraflagellar Transport

Developmental Biology. Sep, 2011  |  Pubmed ID: 21740898

Cilia are ubiquitous cell surface projections that mediate various sensory- and motility-based processes and are implicated in a growing number of multi-organ genetic disorders termed ciliopathies. To identify new components required for cilium biogenesis and function, we sought to further define and validate the transcriptional targets of DAF-19, the ciliogenic C. elegans RFX transcription factor. Transcriptional profiling of daf-19 mutants (which do not form cilia) and wild-type animals was performed using embryos staged to when the cell types developing cilia in the worm, the ciliated sensory neurons (CSNs), still differentiate. Comparisons between the two populations revealed 881 differentially regulated genes with greater than a 1.5-fold increase or decrease in expression. A subset of these was confirmed by quantitative RT-PCR. Transgenic worms expressing transcriptional GFP fusions revealed CSN-specific expression patterns for 11 of 14 candidate genes. We show that two uncharacterized candidate genes, termed dyf-17 and dyf-18 because their corresponding mutants display dye-filling (Dyf) defects, are important for ciliogenesis. DYF-17 localizes at the base of cilia and is specifically required for building the distal segment of sensory cilia. DYF-18 is an evolutionarily conserved CDK7/CCRK/LF2p-related serine/threonine kinase that is necessary for the proper function of intraflagellar transport, a process critical for cilium biogenesis. Together, our microarray study identifies targets of the evolutionarily conserved RFX transcription factor, DAF-19, providing a rich dataset from which to uncover-in addition to DYF-17 and DYF-18-cellular components important for cilium formation and function.

Opposing Activities of LIT-1/NLK and DAF-6/patched-related Direct Sensory Compartment Morphogenesis in C. Elegans

PLoS Biology. Aug, 2011  |  Pubmed ID: 21857800

Glial cells surround neuronal endings to create enclosed compartments required for neuronal function. This architecture is seen at excitatory synapses and at sensory neuron receptive endings. Despite the prevalence and importance of these compartments, how they form is not known. We used the main sensory organ of C. elegans, the amphid, to investigate this issue. daf-6/Patched-related is a glia-expressed gene previously implicated in amphid sensory compartment morphogenesis. By comparing time series of electron-microscopy (EM) reconstructions of wild-type and daf-6 mutant embryos, we show that daf-6 acts to restrict compartment size. From a genetic screen, we found that mutations in the gene lit-1/Nemo-like kinase (NLK) suppress daf-6. EM and genetic studies demonstrate that lit-1 acts within glia, in counterbalance to daf-6, to promote sensory compartment expansion. Although LIT-1 has been shown to regulate Wnt signaling, our genetic studies demonstrate a novel, Wnt-independent role for LIT-1 in sensory compartment size control. The LIT-1 activator MOM-4/TAK1 is also important for compartment morphogenesis and both proteins line the glial sensory compartment. LIT-1 compartment localization is important for its function and requires neuronal signals. Furthermore, the conserved LIT-1 C-terminus is necessary and sufficient for this localization. Two-hybrid and co-immunoprecipitation studies demonstrate that the LIT-1 C-terminus binds both actin and the Wiskott-Aldrich syndrome protein (WASP), an actin regulator. We use fluorescence light microscopy and fluorescence EM methodology to show that actin is highly enriched around the amphid sensory compartment. Finally, our genetic studies demonstrate that WASP is important for compartment expansion and functions in the same pathway as LIT-1. The studies presented here uncover a novel, Wnt-independent role for the conserved Nemo-like kinase LIT-1 in controlling cell morphogenesis in conjunction with the actin cytoskeleton. Our results suggest that the opposing daf-6 and lit-1 glial pathways act together to control sensory compartment size.

Improved Mos1-mediated Transgenesis in C. Elegans

Nature Methods. Feb, 2012  |  Pubmed ID: 22290181

V-ATPase V1 Sector is Required for Corpse Clearance and Neurotransmission in Caenorhabditis Elegans

Genetics. Jun, 2012  |  Pubmed ID: 22426883

The vacuolar-type ATPase (V-ATPase) is a proton pump composed of two sectors, the cytoplasmic V(1) sector that catalyzes ATP hydrolysis and the transmembrane V(o) sector responsible for proton translocation. The transmembrane V(o) complex directs the complex to different membranes, but also has been proposed to have roles independent of the V(1) sector. However, the roles of the V(1) sector have not been well characterized. In the nematode Caenorhabditis elegans there are two V(1) B-subunit genes; one of them, vha-12, is on the X chromosome, whereas spe-5 is on an autosome. vha-12 is broadly expressed in adults, and homozygotes for a weak allele in vha-12 are viable but are uncoordinated due to decreased neurotransmission. Analysis of a null mutation demonstrates that vha-12 is not required for oogenesis or spermatogenesis in the adult germ line, but it is required maternally for early embryonic development. Zygotic expression begins during embryonic morphogenesis, and homozygous null mutants arrest at the twofold stage. These mutant embryos exhibit a defect in the clearance of apoptotic cell corpses in vha-12 null mutants. These observations indicate that the V(1) sector, in addition to the V(o) sector, is required in exocytic and endocytic pathways.

Semi-Automated Neuron Boundary Detection and Nonbranching Process Segmentation in Electron Microscopy Images

Neuroinformatics. May, 2012  |  Pubmed ID: 22644867

Neuroscientists are developing new imaging techniques and generating large volumes of data in an effort to understand the complex structure of the nervous system. The complexity and size of this data makes human interpretation a labor-intensive task. To aid in the analysis, new segmentation techniques for identifying neurons in these feature rich datasets are required. This paper presents a method for neuron boundary detection and nonbranching process segmentation in electron microscopy images and visualizing them in three dimensions. It combines both automated segmentation techniques with a graphical user interface for correction of mistakes in the automated process. The automated process first uses machine learning and image processing techniques to identify neuron membranes that deliniate the cells in each two-dimensional section. To segment nonbranching processes, the cell regions in each two-dimensional section are connected in 3D using correlation of regions between sections. The combination of this method with a graphical user interface specially designed for this purpose, enables users to quickly segment cellular processes in large volumes.

UNC-41/stonin Functions with AP2 to Recycle Synaptic Vesicles in Caenorhabditis Elegans

PloS One. 2012  |  Pubmed ID: 22808098

The recycling of synaptic vesicles requires the recovery of vesicle proteins and membrane. Members of the stonin protein family (Drosophila Stoned B, mammalian stonin 2) have been shown to link the synaptic vesicle protein synaptotagmin to the endocytic machinery. Here we characterize the unc-41 gene, which encodes the stonin ortholog in the nematode Caenorhabditis elegans. Transgenic expression of Drosophila stonedB rescues unc-41 mutant phenotypes, demonstrating that UNC-41 is a bona fide member of the stonin family. In unc-41 mutants, synaptotagmin is present in axons, but is mislocalized and diffuse. In contrast, UNC-41 is localized normally in synaptotagmin mutants, demonstrating a unidirectional relationship for localization. The phenotype of snt-1 unc-41 double mutants is stronger than snt-1 mutants, suggesting that UNC-41 may have additional, synaptotagmin-independent functions. We also show that unc-41 mutants have defects in synaptic vesicle membrane endocytosis, including a ∼50% reduction of vesicles in both acetylcholine and GABA motor neurons. These endocytic defects are similar to those observed in apm-2 mutants, which lack the µ2 subunit of the AP2 adaptor complex. However, no further reduction in synaptic vesicles was observed in unc-41 apm-2 double mutants, suggesting that UNC-41 acts in the same endocytic pathway as µ2 adaptin.

Visualizing Proteins in Electron Micrographs at Nanometer Resolution

Methods in Cell Biology. 2012  |  Pubmed ID: 22857934

To understand protein function, we need a detailed description of the molecular topography of the cell. The subcellular localization of proteins can be revealed using genetically encoded fluorescent proteins or immunofluorescence. However, the precise localization of proteins cannot be resolved due to the diffraction limit of light. Recently, the diffraction barrier has been overcome by employing several microscopy techniques. Using super-resolution fluorescence microscopy, one can pinpoint the location of proteins at a resolution of 20 nm or even less. However, the cellular context is often absent in these images. Recently, we developed a method for visualizing the subcellular structures in super-resolution images. Here we describe the method with two technical improvements. First, we optimize the method to preserve more fluorescence without compromising the morphology. Second, we implement ground-state depletion and single-molecule return (GSDIM) imaging, which does not rely on photoactivatable fluorescent proteins. These improvements extend the utility of nano-resolution fluorescence electron microscopy (nano-fEM).

Sensation in a Single Neuron Pair Represses Male Behavior in Hermaphrodites

Neuron. Aug, 2012  |  Pubmed ID: 22920252

Pheromones elicit innate sex-specific mating behaviors in many species. We demonstrate that in C. elegans, male-specific sexual attraction behavior is programmed in both sexes but repressed in hermaphrodites. Repression requires a single sensory neuron pair, the ASIs. To repress attraction in adults, the ASIs must be present, active, and capable of sensing the environment during development. The ASIs release TGF-β, and ASI function can be bypassed by experimental activation of TGF-β signaling. Sexual attraction in derepressed hermaphrodites requires the same sensory neurons as in males. The sexual identity of both these sensory neurons and a distinct subset of interneurons must be male to relieve repression and release attraction. TGF-β may therefore act to change connections between sensory neurons and interneurons during development to engage repression. Thus, sensation in a single sensory neuron pair during development reprograms a common neural circuit from male to female behavior.

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