Mutations That Rescue the Paralysis of Caenorhabditis elegans ric-8 (Synembryn) Mutants Activate the Gαs Pathway and Define a Third Major Branch of the Synaptic Signaling Network

Schade, Michael A.; Reynolds, Nicole; Dollins, Claudia M.; Miller, Kenneth G.

doi:10.1534/genetics.104.032334

Cited by 119 publications

(152 citation statements)

References 81 publications

Supporting

Mentioning

141

Contrasting

Unclassified

Order By: Relevance

“…5A). Hyperactivity is characteristic of animals with increased activity of cAMP-dependent protein kinase A (PKA), such as mutants for the gene kin-2, which encodes for the regulatory subunit of PKA (40). To test the effect of red light, we grew wild-type and Punc-17:ilaC animals in the dark for a single generation.…”

Section: Resultsmentioning

confidence: 99%

Engineering adenylate cyclases regulated by near-infrared window light

Ryu

Kang

Nelson

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

108

114

View full text Add to dashboard Cite

Bacteriophytochromes sense light in the near-infrared window, the spectral region where absorption by mammalian tissues is minimal, and their chromophore, biliverdin IXα, is naturally present in animal cells. These properties make bacteriophytochromes particularly attractive for optogenetic applications. However, the lack of understanding of how light-induced conformational changes control output activities has hindered engineering of bacteriophytochromebased optogenetic tools. Many bacteriophytochromes function as homodimeric enzymes, in which light-induced conformational changes are transferred via α-helical linkers to the rigid output domains. We hypothesized that heterologous output domains requiring homodimerization can be fused to the photosensory modules of bacteriophytochromes to generate light-activated fusions. Here, we tested this hypothesis by engineering adenylate cyclases regulated by light in the near-infrared spectral window using the photosensory module of the Rhodobacter sphaeroides bacteriophytochrome BphG1 and the adenylate cyclase domain from Nostoc sp. CyaB1. We engineered several light-activated fusion proteins that differed from each other by approximately one or two α-helical turns, suggesting that positioning of the output domains in the same phase of the helix is important for light-dependent activity. Extensive mutagenesis of one of these fusions resulted in an adenylate cyclase with a sixfold photodynamic range. Additional mutagenesis produced an enzyme with a more stable photoactivated state. When expressed in cholinergic neurons in Caenorhabditis elegans, the engineered adenylate cyclase affected worm behavior in a light-dependent manner. The insights derived from this study can be applied to the engineering of other homodimeric bacteriophytochromes, which will further expand the optogenetic toolset.phytochrome | protein engineering | adenylyl cyclase | cAMP | locomotion

show abstract

Section: Resultsmentioning

confidence: 99%

Engineering adenylate cyclases regulated by near-infrared window light

Ryu

Kang

Nelson

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

108

114

View full text Add to dashboard Cite

show abstract

“…We constructed appropriate recipient strains for transformation containing the pha-1(e2123) mutation. Extrachromosomal arrays were integrated by gamma irradiation (Schade et al 2005).…”

Section: Methodsmentioning

confidence: 99%

Choline Transport and de novo Choline Synthesis Support Acetylcholine Biosynthesis in Caenorhabditis elegans Cholinergic Neurons

Mullen

Mathews

et al. 2007

Genetics

View full text Add to dashboard Cite

The cho-1 gene in Caenorhabditis elegans encodes a high-affinity plasma-membrane choline transporter believed to be rate limiting for acetylcholine (ACh) synthesis in cholinergic nerve terminals. We found that CHO-1 is expressed in most, but not all cholinergic neurons in C. elegans. cho-1 null mutants are viable and exhibit mild deficits in cholinergic behavior; they are slightly resistant to the acetylcholinesterase inhibitor aldicarb, and they exhibit reduced swimming rates in liquid. cho-1 mutants also fail to sustain swimming behavior; over a 33-min time course, cho-1 mutants slow down or stop swimming, whereas wildtype animals sustain the initial rate of swimming over the duration of the experiment. A functional CHO-1TGFP fusion protein rescues these cho-1 mutant phenotypes and is enriched at cholinergic synapses. Although cho-1 mutants clearly exhibit defects in cholinergic behaviors, the loss of cho-1 function has surprisingly mild effects on cholinergic neurotransmission. However, reducing endogenous choline synthesis strongly enhances the phenotype of cho-1 mutants, giving rise to a synthetic uncoordinated phenotype. Our results indicate that both choline transport and de novo synthesis provide choline for ACh synthesis in C. elegans cholinergic neurons.

show abstract

“…To determine this, we genetically tested a variety of candidate G␣ subunits for their ability to suppress aex-2 mutant phenotype. We built double mutants between an aex-2 loss-of-function mutant and a gsa-1 (G s ␣) gain-of-function mutant (20) and an egl-30 (G q ␣) gainof-function mutant (21)(22)(23). We also built an aex-2 and dgk-1 (which encodes a diacylglycerol kinase) double loss-of-function mutant, because dgk-1 normally antagonizes egl-30 signaling and its loss-of-function phenocopies egl-30 gain of function (21-23).…”

Section: Aex-2 Functions Through a Gs␣ Signaling Pathwaymentioning

confidence: 99%

Intestinal signaling to GABAergic neurons regulates a rhythmic behavior in Caenorhabditis elegans

Mahoney

Luo

Round

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

110

View full text Add to dashboard Cite

The Caenorhabditis elegans defecation motor program (DMP) is a highly coordinated rhythmic behavior that requires two GABAergic neurons that synapse onto the enteric muscles. One class of DMP mutants, called anterior body wall muscle contraction and expulsion defective (aex) mutants, exhibits similar defects to those caused by the loss of these two neurons. Here, we demonstrate that aex-2 encodes a G-protein-coupled receptor (GPCR) and aex-4 encodes an exocytic SNAP25 homologue. We found that aex-2 functions in the nervous system and activates a Gs␣ signaling pathway to regulate defecation. aex-4, on the other hand, functions in the intestinal epithelial cells. Furthermore, we show that aex-5, which encodes a pro-protein convertase, functions in the intestine to regulate the DMP and that its secretion from the intestine is impaired in aex-4 mutants. Activation of the Gs␣ GPCR pathway in GABAergic neurons can suppress the defecation defect of the intestinal mutants aex-4 and aex-5. Lastly, we demonstrate that activation of GABAergic neurons using the light-gated cation channel channelrhodopsin-2 is sufficient to suppress the behavioral defects of aex-2, aex-4, and aex-5. These results genetically place intestinal genes aex-4 and aex-5 upstream of GABAergic GPCR signaling. We propose a model whereby the intestinal genes aex-4 and aex-5 control the DMP by regulating the secretion of a signal, which activates the neuronal receptor aex-2. T he Caenorhabditis elegans defecation motor program (DMP)is a highly coordinated series of three muscle contractions that are executed every 45 sec [ Fig. 1A and supporting information (SI) Movie S1]. The cycle is initiated by a posterior body wall muscle contraction (pBoc), followed 2-3 sec later by an anterior body wall muscle contraction (aBoc). About 1 sec after the aBoc, enteric muscles contract, thus causing the expulsion (Exp) of intestinal contents. The process repeats itself Ϸ45 sec later with little variability in the timing of contractions (1). A genetic screen for mutants that displayed defects in the DMP isolated mutants defective in each of the three muscle contractions, known as pbo, abo, and exp (1). The screen also recovered mutants defective in the last two muscle contractions (aBoc and Exp [aex]) and mutants defective in the cycle periodicity (i.e., longer or shorter than normal DMP cycling times) (1). Molecular studies of these mutants have suggested that the behavior is orchestrated through the communication between the intestine, GABAergic neurons, and muscle.The periodicity of the DMP is regulated by the C. elegans intestine, a single-cell layer tube of polarized epithelial cells joined by gap junctions (2, 3). Intestinal Ca 2ϩ oscillations with Ϸ45-sec periodicity appear to play a central role in this timing. They consist of a posterior-to-anterior Ca 2ϩ wave whose levels peak in the posterior and anterior intestinal cells just before the pBoc and aBoc contractions, respectively (3-5). Mutations in genes involved in the maintenance of Ca 2ϩ oscillations or i...

show abstract

Mutations That Rescue the Paralysis of Caenorhabditis elegans ric-8 (Synembryn) Mutants Activate the Gαs Pathway and Define a Third Major Branch of the Synaptic Signaling Network

Cited by 119 publications

References 81 publications

Engineering adenylate cyclases regulated by near-infrared window light

Engineering adenylate cyclases regulated by near-infrared window light

Choline Transport and de novo Choline Synthesis Support Acetylcholine Biosynthesis in Caenorhabditis elegans Cholinergic Neurons

Intestinal signaling to GABAergic neurons regulates a rhythmic behavior in Caenorhabditis elegans

Contact Info

Product

Resources

About