To map the organisation of nutrient signalling pathways

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We propose to begin to characterize the responses of freshly-prepared, collagenase-liberated isolated mouse hepatocytes to a range of specific nutrient challenges, that cannot be applied in vivo, in terms of their effect on the class I & III PI3K pathways to mTor, mTor activity, the intiation of autophagy and ROS production. So that in the second half of the project we can look at aged and diet-restricted animals. We aim to publish our first major study indicating that amino acids controlling mTor activity are taken-up by macro-pinocytosis and to have advanced our work addressing the nature of the Ca2+ signals controlling mTor at lysosomes near to a point of submission. We will have tested several amino acid analogues for their effect on mTORC1 activation and then image their distribution within cells. A project following-up the confirmed hits from our genome-wide screens for suppressors and activators of vps34 complexes in autophagy. This work will focus on specific hypotheses arising from that work using a combination of molecular, cell biology and systems approaches to assemble subsets of hits into meaningful regulatory networks. Within that project we aim to have isolated the subset of hits that appear to affect mTORC1 localization and activity and begun to generate a signalling diagram leading from starvation to autophagy. We will have begun to analyse the products of DAz-2 derivatised cell lysates to reveal the complexity of the samples we will be attempting to analyse. Following on from our new data we will define the activation status of ERK1/2, p38, MNK1/2 and eIF4E during nutrient starvation and during acute and chronic mTOR inhibition in primary human fibroblasts and assess their activation in young and aged fibroblasts. This will be a pre-requisite to gene expression studies and analysis of young and aged samples.

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