Jane E. Mendel

Participation of the protein Go in multiple aspects of behavior in C. elegans

The goa-1 gene encoding the alpha subunit of the heterotrimeric guanosine triphosphate-binding protein (G protein) Go from Caenorhabditis elegans is expressed in most neurons, and in the muscles involved in egg laying and male mating. Reduction-of-function mutations in goa-1 caused a variety of behavioral defects including hyperactive movement, premature egg laying, and male impotence. Expression of the activated Go alpha subunit (G alpha o) in transgenic nematodes resulted in lethargic movement, delayed egg laying, and reduced mating efficiency. Induced expression of activated G alpha o in adults was sufficient to cause these phenotypes, indicating that G alpha o mediates behavior through its role in neuronal function and the functioning of specialized muscles.

Mutations in a C. elegans Gqα Gene Disrupt Movement, Egg Laying, and Viability

We find that C. elegans egl-30 encodes a heterotrimeric G protein α subunit more than 80% identical to mammalian Gqα family proteins, and which can function as a Gqα subunit in COS-7 cells. We have identified new egl-30 alleles in a selection for genes involved in the C. elegans acetylcholine response. Two egl-30 alleles specify premature termination of Gqα and are essentially lethal in homozygotes. Animals homozygous for six other egl-30 alleles are viable and fertile, but exhibit delayed egg laying and leave flattened tracks. Overexpression of the wild-type egl-30 gene produces the opposite behavior. Analysis of these mutants suggest that their phenotypes reflect defects in the muscle or neuromuscular junction.

An automated system for measuring parameters of nematode sinusoidal movement

BackgroundNematode sinusoidal movement has been used as a phenotype in many studies of C. elegans development, behavior and physiology. A thorough understanding of the ways in which genes control these aspects of biology depends, in part, on the accuracy of phenotypic analysis. While worms that move poorly are relatively easy to describe, description of hyperactive movement and movement modulation presents more of a challenge. An enhanced capability to analyze all the complexities of nematode movement will thus help our understanding of how genes control behavior.ResultsWe have developed a user-friendly system to analyze nematode movement in an automated and quantitative manner. In this system nematodes are automatically recognized and a computer-controlled microscope stage ensures that the nematode is kept within the camera field of view while video images from the camera are stored on videotape. In a second step, the images from the videotapes are processed to recognize the worm and to extract its changing position and posture over time. From this information, a variety of movement parameters are calculated. These parameters include the velocity of the worms centroid, the velocity of the worm along its track, the extent and frequency of body bending, the amplitude and wavelength of the sinusoidal movement, and the propagation of the contraction wave along the body. The length of the worm is also determined and used to normalize the amplitude and wavelength measurements.To demonstrate the utility of this system, we report here a comparison of movement parameters for a small set of mutants affecting the Go/Gq mediated signaling network that controls acetylcholine release at the neuromuscular junction. The system allows comparison of distinct genotypes that affect movement similarly (activation of Gq-alpha versus loss of Go-alpha function), as well as of different mutant alleles at a single locus (null and dominant negative alleles of the goa-1 gene, which encodes Go-alpha). We also demonstrate the use of this system for analyzing the effects of toxic agents. Concentration-response curves for the toxicants arsenite and aldicarb, both of which affect motility, were determined for wild-type and several mutant strains, identifying P-glycoprotein mutants as not significantly more sensitive to either compound, while cat-4 mutants are more sensitive to arsenite but not aldicarb.ConclusionsAutomated analysis of nematode movement facilitates a broad spectrum of experiments. Detailed genetic analysis of multiple alleles and of distinct genes in a regulatory network is now possible. These studies will facilitate quantitative modeling of C. elegans movement, as well as a comparison of gene function. Concentration-response curves will allow rigorous analysis of toxic agents as well as of pharmacological agents. This type of system thus represents a powerful analytical tool that can be readily coupled with the molecular genetics of nematodes.

Go Directly (or Indirectly) to Gq

1999). However, egl-30 G q ␣ probably has additional ef-(or Indirectly) to Gq fectors, since a presumptive loss-of-function allele of egl-8 PLC␤ causes less severe phenotypes than strong reduction-of-function alleles of egl-30 G q ␣ (Miller et al., 1999). A fundamental mechanism for regulating synaptic trans-Since diacylglycerol (DAG) is a major end product mission is the control of neurotransmitter secretion by of the G q ␣–PLC␤ pathway in vertebrates, both groups presynaptic neurons. Recent papers, including two in asked whether DAG can positively regulate ACh release this issue of Neuron (Lackner et al., 1999; Miller et al., in C. elegans. Treatment of worms with phorbol esters, 1999), indicate that a hierarchical network of G proteins analogs of DAG, increased sensitivity to aldicarb, indi-controls, among other things, release of the neurotrans-cating that DAG positively regulates synaptic transmis-mitter acetylcholine (ACh) at the neuromuscular junction sion. Lackner et al. (1999) additionally show that treat-in C. elegans. In this organism, ventral cord motor neu-ment with phorbol esters restores aldicarb sensitivity to rons use ACh to stimulate contraction of body wall mus-egl-30 G q ␣ and egl-8 PLC␤ mutants, as is expected if cles. This stimulation, alternating with a coordinated these genes normally function to produce DAG. This relaxation program, results in the typical sinusoidal move-group also demonstrates that one target of DAG is UNC-ment of nematodes (Herman, 1993; Rand and Nonet, 13, a presynaptic DAG-binding protein, although there 1997). is evidence that UNC-13 is not the only target responsi-Mutations in two G protein ␣ subunit genes, goa-1 ble for the stimulation of ACh release by the egl-30 G q ␣ G o ␣ and egl-30 G q ␣, cause opposite effects on nematode pathway. The figure diagrams the relationship among movement. Loss-of-function mutations in goa-1 cause these molecules. Muscarinic agonists such as arecoline hyperactive movement, while reduction-of-function al-activate egl-30 G q ␣, which in turn activates egl-8 PLC␤. leles of egl-30 result in lethargy. Overexpression of wild-Activated egl-8 PLC␤ cleaves phosphatidylinositol 4,5-type or constitutively activated transgenes for each G␣ bisphosphate (PIP 2) into DAG and inositol 1,4,5-tris-subunit also results in opposite phenotypes: too much phosphate (IP 3 ; not shown). DAG stimulates ACh release G o ␣ activity makes worms lethargic, whereas too much through UNC-13 and possibly other molecules. EGL-30 G q ␣ activity makes them hyperactive (Mendel et al. The papers presented here provide insight into the The G protein pathway involving …