Characterisation of the PI3P-dependent signalling network responsible for nutrient sensing and autophagy

Lead Research Organisation: Babraham Institute
Department Name: Signalling

Abstract

One of the most important requirements for healthy cellular growth is the ability to sense and respond to extracellular nutrients. When nutrient supply is plentiful, cells need only to fine tune their anabolic and catabolic rates so that their energy requirements match the available resources. When nutrients become limiting or are completely unavailable, a much stronger course of action is required to allow the cells to survive. During such times of nutrient withdrawal, the cells respond by initiating autophagy, a degradative pathway that allows the breakdown of intracellular proteins into amino acids that can be used subsequently for new protein synthesis or energy generation. An important question in autophagy concerns the mechanisms by which cells sense their extracellular nutrient content. Recent work from many groups has indicated that a small lipid molecule termed phosphatidylinositol 3 phosphate (PI3P) is an important signal for nutrient sensing. Our own work also indicates that autophagy is induced partially as a result of the formation of PI3P in specialised membrane compartments called omegasomes. Therefore, the mechanisms and signals that generate PI3P early during autophagy are likely to provide important information on the control of autophagy by nutrient sensing. The aim of this grant is to identify all human genes that are involved in this pathway. We plan to systematically silence all known human genes and then look at the effect that this will have in formation of PI3P during autophagy. By identifying all such genes we will be able to construct a wiring diagram of the cellular pathways that are implicated in nutrient sensing and respond during nutrient limitation by the induction of autophagy.

Technical Summary

During nutrient withdrawal, cells respond by initiating autophagy, a degradative pathway that allows the breakdown of intracellular proteins into amino acids that can be used subsequently for new protein synthesis or ATP generation. In addition to this role in nutrient sensing, autophagy is important for elimination of unneeded organelles, for the defence against pathogens, for the pathogenesis of various diseases and for healthy ageing. Our recent work has suggested that at least some autophagosomes (double membrane vesicles mediating autophagy) are formed in novel membrane specializations termed omegasomes which are connected to the endoplasmic reticulum and are enriched in a phosphatidylinositol 3-phosphate (PI3P). This is consistent with earlier work showing that formation of PI3P is essential for the induction of autophagy. However, the signalling pathways leading from nutrient sensing to PI3P formation and autophagy induction are still unknown. The aim of this grant is to identify the main gene products whose activity is important for the PI3P-dependent induction of autophagy. We will use an siRNA screen of all known human genes, to identify those whose down-regulation (a) leads to omegasome formation in normal nutrient conditions or (b) inhibits amino acid starvation-induced formation of omegasomes. Based on our preliminary data we expect the following classes of molecules to be identified: (1) Those involved in amino acid sensing [AA sensor(s)] (2) Those involved in signalling to calcium following amino acid sensing (3) Those involved in signalling from calcium to Vps34 (4) Those involved in AA-dependent mTOR activation (5) Those involved in omegasome formation, expansion and collapse (5) Those involved in termination of PI3P signal (6) Those involved in feedback inhibition of autophagosome biogenesis. The ultimate aim of this grant is to construct a signalling diagram leading from nutrient sensing to PI3P formation and autophagy induction.

Publications

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Burman C (2010) Autophagosome formation in mammalian cells. in Seminars in immunopathology

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Galluzzi L (2017) Molecular definitions of autophagy and related processes. in The EMBO journal

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Karanasios E (2014) Imaging autophagy. in Current protocols in cytometry

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Ktistakis NT (2013) PIPing on lysosome tubes. in The EMBO journal

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Ktistakis NT (2016) Digesting the Expanding Mechanisms of Autophagy. in Trends in cell biology

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Ktistakis NT (2015) Monitoring the Localization of MAP1LC3B by Indirect Immunofluorescence. in Cold Spring Harbor protocols

 
Description Several genes affecting autophagy responses
Exploitation Route Understand how autophagy is induced
Sectors Chemicals,Healthcare

 
Description Many scientists are using our reporter cell lines to follow autophagy
First Year Of Impact 2011
Sector Chemicals,Healthcare
 
Description Colaborration with Japanese 
Organisation University of Tokyo
Country Japan 
Sector Academic/University 
PI Contribution Paper together with Prof Noboru Mizushima
Collaborator Contribution Paper together with Prof Noboru Mizushima
Impact paper together visit of scientist
Start Year 2011
 
Description Roger Williams 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom 
Sector Public 
PI Contribution Exchange of reagents and cell lines
Collaborator Contribution Exchange of reagents and cell lines
Impact Publications and grants
Start Year 2012
 
Description Tamotsu 
Organisation Osaka University
Country Japan 
Sector Academic/University 
PI Contribution Papers together
Collaborator Contribution Papers together
Impact Published 2 papers together
Start Year 2009
 
Title DFCP1 cell line 
Description Reporter cell line for autophagy induction 
IP Reference  
Protection Protection not required
Year Protection Granted 2010
Licensed Yes
Impact WE have distributed this cell line to over 40 laboratories in UK, Europe, USA, China, Japan
 
Description Cambridge Enterprise & Technology Club 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Talk on autophagy and degenerative diseases
Year(s) Of Engagement Activity 2016
URL http://www.cetc.info/degenerative-diseases/
 
Description Cambridge Hellenic Learned Society 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact President of Society of Greek and Cypriot academics in Cambridge, charged with organizing events with well-known speakers in the fields of Science of Humanities and addressed to a lay/educated audience. So far we have organized 7 such events with average attendance of 90 people.
Year(s) Of Engagement Activity 2014,2015,2016
URL https://camlearnhelsoc.wordpress.com/
 
Description Presentation at Instutute Ageing Conference 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Invited talk at Ageing conference, Babraham Institute
Year(s) Of Engagement Activity 2017