Subcellular compartmentalization established by mobilization of intracellular calcium stores in RIA interneurons provides a means of self-motion monitoring and a cellular basis for integrating sensory and motor signals in nematodes’ brains. Most neurons in the small brains of roundworms such as Caenorhabditis elegans rely on a single axon both to receive inputs from and to send outputs to a number of other cells. Now, Yun Zhang and colleagues reveal that neural function in the RIA interneurons, which are crucial for complex sensorimotor tasks such as isothermal tracking and olfactory learning, is compartmentalized. Subcellular axonal activity dependent on acetylcholine release from head motor neurons is simultaneously present but independent of glutamate-dependent activity evoked by sensory inputs. Such subcellular computation prevents sensory and motor signals from getting mixed across the relatively small neuronal circuitry of nematodes' brains. The confinement of neuronal activity to specific subcellular regions is a mechanism for expanding the computational properties of neurons. Although the circuit organization underlying compartmentalized activity has been studied in several systems1,2,3,4, its cellular basis is still unknown. Here we characterize compartmentalized activity in Caenorhabditis elegans RIA interneurons, which have multiple reciprocal connections to head motor neurons and receive input from sensory pathways. We show that RIA spatially encodes head movement on a subcellular scale through axonal compartmentalization. This subcellular axonal activity is dependent on acetylcholine release from head motor neurons and is simultaneously present and additive with glutamate-dependent globally synchronized activity evoked by sensory inputs. Postsynaptically, the muscarinic acetylcholine receptor GAR-3 acts in RIA to compartmentalize axonal activity through the mobilization of intracellular calcium stores. The compartmentalized activity functions independently of the synchronized activity to modulate locomotory behaviour.

/pdf/compartmentalized-calcium-dynamics-in-a-c-elegans-14y6we6371.pdf

Compartmentalized calcium dynamics in a C. elegans interneuron encode head movement.

Neuropeptidergic signaling in the nematode Caenorhabditis elegans

Biogenic amine receptors in parasitic nematodes: what can be learned from Caenorhabditis elegans?

https://littlelab.bio.ed.ac.uk/resources/Research/Garbutt-et-al_2012_InsectBiochemistryMolecularBiology.pdf

Induction of RNA interference genes by double-stranded RNA; implications for susceptibility to RNA interference

It was demonstrated previously that polar and non-polar surface extracts of the brown alga Fucus vesiculosus collected during winter from the Kiel Bight (Germany) inhibited bacterial attachment at natural concentrations. The present study describes the bioassay-guided identification of the active metabolites from the polar fraction. Chromatographic separation on a size-exclusion liquid chromatography column and bioassays identified an active fraction that was further investigated using nuclear magnetic resonance spectroscopy and mass spectrometry. This fraction contained the metabolites dimethylsulphopropionate (DMSP), proline and alanine. DMSP and proline caused the anti-attachment activity. The metabolites were further quantified on the algal surface together with its associated boundary layer. DMSP and proline were detected in the range 0.12–1.08 ng cm−2 and 0.09–0.59 ng cm−2, respectively. These metabolites were tested in the concentration range from 0.1 to 1000 ng cm−2 against the attachment of five bacterial strains isolated from algae and sediment co-occurring with F. vesiculosus. The surface concentrations for 50% inhibition of attachment of these strains were always <0.38 ng cm−2 for DMSP and in four cases <0.1 ng cm−2 for proline, while one strain required 1.66 ng cm−2 of proline for 50% inhibition. Two further bacterial strains that had been directly isolated from F. vesiculosus were also tested, but proved to be the least sensitive. This study shows that DMSP and proline have an ecologically relevant role as surface inhibitors against bacterial attachment on F. vesiculosus.

Dimethylsulphopropionate (DMSP) and proline from the surface of the brown alga Fucus vesiculosus inhibit bacterial attachment

Alternative splicing of the muscarinic acetylcholine receptor GAR-3 in Caenorhabditis elegans☆

A Gram-negative, aerobic, rod-shaped, motile, marine bacterium, strain AR11T, was isolated from Arctic marine sediment. Strain AR11T grew with 0.5–7 % NaCl and at 7–37 °C and pH 5.5–9.0. It utilized propionate, 3-hydroxybenzoate, l-proline, acetate, d- and l-lactate, l-alanine, malate and phenylacetic acid. Alkaline phosphatase, esterase lipase (C8), leucine arylamidase and acid phosphatase activity tests were positive. Acid was produced from 5-ketogluconate and aesculin. Strain AR11T possessed C16 : 0 (22.0 %), summed feature 4 (C16 : 1
ω7c and/or iso-C15 : 0 2-OH; 28.1 %) and summed feature 7 (one or more of C18 : 1
ω7c, ω9t and ω12t; 34.0 %) as the major cellular fatty acids. The major ubiquinone was Q-8. Comparative 16S rRNA gene sequence studies showed that strain AR11T belonged to the Gammaproteobacteria and was most closely related to Marinobacterium stanieri DSM 7027T, Marinobacterium halophilum mano11T and Marinobacterium georgiense KW-40T (97.8, 97.0 and 96.7 % similarity, respectively). The G+C content of the genomic DNA of strain AR11T was 57.9 mol%. DNA–DNA relatedness data indicated that strain AR11T represented a distinct species that was separated from M. stanieri DSM 7027T, M. halophilum KCTC 12240T and M. georgiense JCM 21667T. On the basis of evidence from this polyphasic study, it is proposed that strain AR11T (=KCTC 22254T=JCM 15134T) represents the type strain of a novel species, Marinobacterium maritimum sp. nov.

Marinobacterium maritimum sp. nov., a marine bacterium isolated from Arctic sediment.

Marine sediment was collected from the Yellow Sea, Korea, and enriched using 1 mM thiosulfate under aerobic conditions. After 2 weeks, a facultative sulfur-oxidizing strain, BR13, was isolated. Phylogenetic analysis based on the 16S rRNA gene sequence showed that this strain belonged to the genus Marinobacter in the gamma-proteobacteria and was most closely related to Marinobacter pelagius H225T (99.5% 16S rRNA gene sequence similarity) and Marinobacter koreensis DD-M3T (97.7% 16S rRNA gene sequence similarity). The isolate was a sulfur-oxidizer, Gram-negative, aerobic, and slightly halophilic bacterium and grew in 0.5–9% NaCl at 7–37oC. Strain BR13 shared some physiological and biochemical properties with Marinobacter pelagius H225T and Marinobacter koreensis DD-M3T. The G+C content of the genomic DNA of strain BR13 was 58.4 mol%. Strain BR13 possessed C16:0, summed feature 4 (C16:1
 ω7c and/or C15:1iso 2OH), C16:0 N alcohol, and C18:1
 ω9c as the major cellular fatty acids. Our results showed that strain BR13 (=KCTC 22253 =JCM 15135) is a novel facultative sulfur-oxidizing strain in the genus of Marinobacter.

Characterization of facultative sulfur-oxidizing Marinobacter sp. BR13 isolated from marine sediment of Yellow Sea, Korea

Double-stranded RNA (dsRNA) induces gene silencing in a sequence-specific manner by a process known as RNA interference (RNAi). The RNA-induced silencing complex (RISC) is a multi-subunit ribonucleoprotein complex that plays a key role in RNAi. VIG (Vasa intronic gene) has been identified as a component of Drosophila RISC; however, the role VIG plays in regulating RNAi is poorly understood. Here, we examined the spatial and temporal expression patterns of VIG-1, the C. elegans ortholog of Drosophila VIG, using a vig-1::gfp fusion construct. This construct contains the 908-bp region immediately upstream of vig-1 gene translation initiation site. Analysis by confocal microscopy demonstrated GFP-VIG-1 expression in a number of tissues including the pharynx, body wall muscle, hypodermis, intestine, reproductive system, and nervous system at the larval and adult stages. Furthermore, western blot analysis showed that VIG-1 is present in each developmental stage examined. To investigate regulatory sequences for vig-1 gene expression, we generated constructs containing deletions in the upstream region. It was determined that the GFP expression pattern of a deletion construct (delta-908 to -597) was generally similar to that of the non-deletion construct. In contrast, removal of a larger segment (delta-908 to -191) resulted in the loss of GFP expression in most cell types. Collectively, these results indicate that the 406-bp upstream region (-596 to -191) contains essential regulatory sequences required for VIG-1 expression.

Analysis of C. elegans VIG-1 expression.

Many membrane-bound neurotransmitter receptors are known to be internalized by exposure to agonist. This agonist-induced receptor internalization is considered to play important roles in receptor-mediated signaling. Here we investigated the internalization of GAR-3, a Caenorhabditis elegans muscarinic acetylcholine receptor, using cultured mammalian cells. When Chinese hamster ovary cells stably expressing GAR-3 were treated with carbachol, GAR-3 was internalized in a dose- and time-dependent manner. Approximately 60% of the cell surface receptor was internalized by exposure to 1 mM carbachol for 1 h. Carbachol-induced GAR-3 internalization was suppressed by treatment with hypertonic sucrose, which blocks the formation of clathrin-coated pits. Overexpression of a dominant-negative dynamin mutant (DynK44A), but not of a dominant-negative β-arrestin mutant (Arr319–418), substantially inhibited carbachol-induced internalization of GAR-3. Thus, these data suggest that GAR-3 undergoes agonist-induced internalization via a clathrin- and dynamin-dependent but β-arrestin-independent pathway. Depletion of Ca2+ by simultaneous treatment of the cells with BAPTA/AM (Ca2+ mobilization blocker) and EGTA (Ca2+ influx blocker) almost completely blocked agonist-induced GAR-3 internalization. Moreover, treatment of the cells with the Ca2+ ionophore A23187 led to GAR-3 internalization in the absence of agonist. These results indicate that Ca2+ plays a critical role in GAR-3 internalization. We tested whether the third intracellular (i3) loop of GAR-3 is involved in agonist-stimulated receptor internalization. A GAR-3 deletion mutant lacking a large central portion of the i3 loop exhibited an internalization pattern comparable to that of the wild type, suggesting that the central i3 loop is not required for the internalization of GAR-3.

Boram Choi

Papers

Alternative splicing of the muscarinic acetylcholine receptor GAR-3 in Caenorhabditis elegans☆

Marinobacterium maritimum sp. nov., a marine bacterium isolated from Arctic sediment.

Characterization of facultative sulfur-oxidizing Marinobacter sp. BR13 isolated from marine sediment of Yellow Sea, Korea

Analysis of C. elegans VIG-1 expression.

Agonist-induced Internalization of the Caenorhabditis elegans Muscarinic Acetylcholine Receptor GAR-3 in Chinese Hamster Ovary Cells