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Title: Characterisation of the Ca2+ signaling pathway involved in the control of temperature dependent locomotion by Neuronal Calcium Sensor-1 in Caenorhabditis elegans
Author: Todd, Paul Anthony Christopher
ISNI:       0000 0004 6422 5743
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2017
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Calcium (Ca2+) signaling is critical in regulating a number of neuronal functions including synaptic transmission, axonal growth, development and neurotransmitter release/recycling. The EF-hand containing Ca2+ binding protein Neuronal Ca2+ Sensor 1 (NCS-1) has been shown to be important in a number of these processes. NCS-1 is a member of the NCS family of proteins encoded in mammals by 14 genes. Ca2+ bound NCS-1 exposes a hydrophobic binding domain through an altered conformation allowing regulation of downstream processes, dependent on a diverse range of target proteins. Many interacting partners have been identified for NCS-1, some of which are common to Calmodulin (CaM) with others unique to NCS-1. Mammalian NCS-1 has been shown to regulate PQ-type voltage-gated Ca2+ channels (VGCCs), although evidence of a direct interaction between the two proteins has been lacking. The initial part of this study investigated interactions between NCS-1 and PQ-type VGCCs (CaV2.1). Within the CaV2.1 C-terminal tail, there are two Ca2+-sensor binding regions; the CBD (CaM binding domain) and the IM/IQ domain. Through cell transfection and fluorescence based experiments, an interaction between these two proteins was established. Co-expression of NCS-1 and a PQ channel C-terminal domain led to the appearance of hallmark aggregated cellular structures. Furthermore, the kinetic behaviour of NCS-1 and PQ in these cells was significantly different to those observed when each protein was expressed in isolation. The second part of this study used C. elegans as a model organism to study in vivo interactions of NCS-1. In C. elegans NCS-1 is primarily found in the nervous system, with highest expression in sensory neurons. Previous studies have identified a defect in isothermal tracking in an ncs-1 defective C. elegans animal. When tested in a locomotion assay, characterised in this thesis, the locomotion rate of wild-type C. elegans decreased at an elevated temperature of 28oC whilst the ncs-1 null C. elegans showed a slight yet significant increase in locomotion at the elevated temperature. During this project a reversible paralysis phenotype was identified in the temperature dependent locomotion assay (TDLA). In the second part of the project this assay was exploited to quantify the paralysis phenotype in various mutant C. elegans strains. Based on the mammalian NCS-1 interactome, a sub-set of protein orthologues were identified within the C. elegans nervous system as potential interacting proteins to be studied. It was established that potential targets were TRP1 & 2, ARF-1.1 and PI4K of which various double mutants were generated through C. elegans genetic crosses. The double mutants identified a genetic interaction between ARF-1.1 and NCS-1 in the TDLA suggestive of a possible functional interaction. Additional studies using a chemical inhibitor of phosphatidylinositol 4kinaseIIIb suggests a potential network comprising PI4K-1, NCS-1 and ARF-1.1 regulates this behaviour. These findings allowed a partial elucidation of the interactions and functional relationships between these proteins in a physiological context with an identifiable behavioural output.
Supervisor: Burgoyne, R. D. ; Barclay, J. W. ; Haynes, L. P. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral