Award details

Functions of TRPM channels in the control of an ultradian rhythm in C. elegans

Reference	BB/C507661/1
Principal Investigator / Supervisor	Dr Howard Baylis
Co-Investigators / Co-Supervisors
Institution	University of Cambridge
Department	Zoology
Funding type	Research
Value (£)	252,024
Status	Completed
Type	Research Grant
Start date	01/12/2004
End date	30/04/2008
Duration	41 months

Abstract

Ultradian rhythms are important components of an animal¿s ability to maintain homeostasis and to interact with its environment. Changes in the levels of ions such as calcium are likely to be an important part of the mechanism of many such rhythms. The regulation of defecation in C. elegans offers an excellent system in which to develop a multilevel and coherent model that combines the actions of genes, changes in levels and distribution of ions, the network of components that allow not only the oscillator to function but also its inputs and outputs to be determined, intercellular communication between like (intestinal) and different tissue types and the function of the whole animal. It has been shown that calcium signalling plays a central role in the defecation oscillator. We have recently shown that members of the relatively recently discovered and poorly understood family of TRPM channels play a key role in determining the variability of the defecation rhythm in C. elegans. This proposal aims to establish an integrated model of the function of TRPM channels in determining the variability of the defecation oscillator of C. elegans. By so doing we aim to contribute to our understanding of, both the mechanisms that power and regulate ultradian rhythms and the function of TRPM channels. Our first aim is to identify interactions between the TRPM channel genes gtl-1 and gon-2 and other genes that regulate the defecation process in C. elegans thus dissecting the mechanism underlying the behaviour of the oscillator. To do this we will combine: a) Dissecting the interactions of gtl-1, gon-2 with genes that are known to be involved in the defecation oscillator. This will be achieved by combining gene knock-down by RNAi and genetic analysis. These interactions will be defined with respect to their effect (or not) on both the mean period and the variability of the process by measuring defecation behaviour of whole animals under the stereomicroscope. b) Identifying specific modifiers of gtl-1 action using suppressor screens to identify mutations (induced using EMS) that can suppress the phenotypes of gtl-1 mutants. Such mutants will be characterised with respect to their interaction with gtl-1 and gon-2 and the defecation rhythm. The information from these experiments will be used to create interaction maps centred on gtl-1 and gon-2 and a functional classification of the genes involved in defecation. We will also determine the effects of disrupting gtl-1, gon-2 and other interacting genes on the calcium signals in the intestine and on the membrane properties of the intestinal cells. We will do this by using calcium sensitive dyes (e.g. fluo-4) or calcium sensitive GFP indicator proteins in isolated intestines or whole animals respectively. The calcium conductances of isolated intestinal cells will be analysed using patch clamp. This will enable us to correlate physiological changes to perturbations in the network of interacting genes and to the behaviour of the whole animal. To complement these studies we will also determine the sub-cellular localisation and extent or co-localisation of GON-2, GTL-1 and interacting genes such as flr-1 by making reporter constructs in which the gene of interest is fused to fluorescent markers (CFP, GFP, YFP and dsRED) and introduced into C. elegans. We will also test the tissue specificity of gon-2 and gtl-1 action using cell specific RNAi. We will establish tissue specific transgenes that express hairpin loops in order to produce dsRNA of gon-2 and ftl-1 in mutants of C. elegans that exhibit non-systemic RNAi (e.g. sld-1).

Summary

unavailable

Committee	Closed Committee - Animal Sciences (AS)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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