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To sustain neurotransmission, synaptic vesicles and their associated proteins must be recycled locally at synapses. Synaptic vesicles are thought to be regenerated approximately 20 s after fusion by the assembly of clathrin scaffolds or in approximately 1 s by the reversal of fusion pores via 'kiss-and-run' endocytosis. Here we use optogenetics to stimulate cultured hippocampal neurons with a single stimulus, rapidly freeze them after fixed intervals and examine the ultrastructure using electron microscopy--'flash-and-freeze' electron microscopy. Docked vesicles fuse and collapse into the membrane within 30 ms of the stimulus. Compensatory endocytosis occurs within 50 to 100 ms at sites flanking the active zone. Invagination is blocked by inhibition of actin polymerization, and scission is blocked by inhibiting dynamin. Because intact synaptic vesicles are not recovered, this form of recycling is not compatible with kiss-and-run endocytosis; moreover, it is 200-fold faster thanclathrin-mediated endocytosis. It is likely that 'ultrafast endocytosis' is specialized to restore the surface area of the membrane rapidly., Recycling of synaptic vesicle membrane and proteins is required to keep membrane surface area constant and ensure efficient neurotransmission during sustained synaptic activity. Classical ultrastructural analysis of frog neuromuscular junctions [...]

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 ([micro]2) 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 [micro]2 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 [micro]2 facilitates but is not essential for synaptic vesicle recycling.

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.

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.

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.

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.

Author(s): Jean-Louis Bessereau [1]; Ashley Wright; Daniel C. Williams; Kim Schuske; M. Wayne Davis; Erik M. Jorgensen (corresponding author) Transposons have been enormously useful for genetic analysis in both Drosophila [...]

Author(s): Janet E. Richmond [1]; Robby M. Weimer [1]; Erik M. Jorgensen (corresponding author) The priming step of synaptic vesicle exocytosis is thought to require the formation of the SNARE [...]

The inositol triphosphate receptor, which regulates a 50-second behavioral rhythm in Caenorhabditis elegans, a nematode, is discussed. A study has shown that the defecation cycle, which is characterized by contraction of three distinct sets of muscles at 50 second intervals, is regulated by periodic calcium release mediated by the inositol trisphosphate receptor. The receptor is an essential timekeeper component for the cycle and represents a novel mechanism for control of behavioral rhythms.

The gene unc-47 has been implicated in the release of gamma-aminobutryic acid (GABA), in studies of the nematode Caenorhabditis elegans. Further research shows that the sequence of unc-47 predicts a 10 transmembrane domain protein, the gene is expressed by GABA neurons and that the protein colocalizes with synaptic vesicles. This vesicular GABA transport (VGAT) is compared with a vesicular transporter for monoamines, and indicates differences in bioenergetic transport dependence.

The synaptotagmin mutants of the nematode Caenorhabditis elegans has almost no synaptic vesicles at the synaptic terminals. The absence of synaptotagmin impairs both endocytosis and exocytosis which are involved in the recycling of synaptic vescicles. Therefore, synaptotagmin participates both in exocytosis and endocytosis of synaptic vesicles. Defective accumulation of synaptic vesicles from the plasma membrane or defective vesicular recycling causes such reduction.

Abstract Background 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. Results Here, we describe a set of 48 primer pairs that flank SNPs evenly spaced across the C. elegans genome and that work under identical PCR conditions. Each SNP in this set alters a DraI site, enabling rapid and parallel scoring. We describe a procedure using these reagents to quickly and reliably map mutations. We show that these techniques correctly map a known gene, dpy-5. We then use these techniques to map mutations in an uncharacterized strain, and show that its behavioral phenotype can be simultaneously mapped to three loci. Conclusion Together, the reagents and methods described represent a significant advance in the accurate, rapid and inexpensive mapping of genes in C. elegans.

A study providing the first insights into the relative importance of the RhoGEF and PLC[beta] G[[alpha].sub.q] effector pathways in the context of a living animal is presented. Rho-specific GEF domain is an important G[[alpha].sub.q] effector that along with PLC[beta], mediates the G[[alpha].sub.q] signaling.