Control of Cell Signalling and Membrane Trafficking Pathways by Phosphoinositide Regulatory Networks.

Lead Research Organisation: University College London

Abstract

Our cells are divided into distinct membrane-bound compartments some of which form essential pathways that transport cargo into and out of cells. These membrane trafficking pathways control key cellular processes including nutrient uptake, clearance of activated growth factor receptors, and the secretion of hormones and neurotransmitters. However our understanding of membrane trafficking systems remains incomplete. We are studying the control of membrane trafficking pathways by specialized signalling lipids named phosphoinositides. Phosphoinositides (PI) are essential lipids in cellular membranes that have important roles in cell growth and development. Defects in PI metabolism have been implicated in many human diseases including cancer, diabetes, and neurodegenerative disorders. It is vital that we understand how cells maintain and use these signalling molecules. Our research integrates cell biology, biochemistry, and genetic strategies to uncover roles for PI signalling in membrane trafficking and cell signalling pathways to understand how cells respond to cues in their environment, such as growth factors, nutrients, and stress. These studies are especially relevant to the biology of neurons, as defects in PI signalling result in neurological and neurodegenerative disorders including Down’s syndrome, Alzheimer’s disease, and Parkinson’s disease. Our long-term goal is to develop new therapeutics for the treatment of diseases caused by PI signalling and membrane trafficking defects.

Technical Summary

The transmission of signals across the plasma membrane allows cells to sense their surroundings and mount responses to external cues. Accordingly, the plasma membrane is highly organized and undergoes dynamic remodelling by the delivery and removal of material via the secretory and endocytic pathways. Our research focuses on the control of cell signalling and membrane trafficking pathways by phosphoinositide (PI) kinase regulatory networks. PI kinase signalling networks impact on several key cellular processes including cell growth and survival, cell proliferation and differentiation, cell polarity and migration, and membrane trafficking pathways. Consequently, defects in PI kinase regulatory networks have been implicated in numerous human diseases including cancer, diabetes, and neurodegenerative disorders. It is vital that we understand how cells maintain and use these essential signalling molecules. We are currently undertaking multidisciplinary approaches–including cell biological assays, high-resolution microscopy, system-wide functional genomics and proteomics, and biochemical approaches–to make new discoveries into the regulation of PI kinase signalling and membrane trafficking networks. We expect that these investigations will reveal new insights into the complex regulatory processes that ensure the temporal and spatial specificity of membrane trafficking systems, including regulated exocytosis and endocytosis during neurotransmission. As such, we expect these studies will be particularly relevant to neuronal physiology, as defects in PI metabolism have been implicated in several neurological and neurodegenerative disorders including Down’s syndrome, Alzheimer’s disease, and Parkinson’s disease.

Specifically, we have uncovered a pathway for crosstalk between the plasma membrane (PM) and endoplasmic reticulum (ER) involving PI kinase signalling at ER-PM junctions. Our findings indicate that ER-PM crosstalk allows the ER to mount essential responses (calcium dynamics, protein and lipid biogenesis) to ensure the integrity of the PM even under membrane stress conditions. Another striking result is our finding that ER–PM crosstalk is essential for normal ER morphology and function. Loss of ER-PM junctions causes dramatic changes in ER organization and architecture, resulting in activation of ER stress response pathways. We are currently investigating the roles for ER-PM crosstalk in maintaining ER homeostasis, as well as PM domain organization. Recent findings have placed ER stress as a key component of neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and amyotrophic lateral sclerosis (ALS). We expect that ER-PM crosstalk may serve critical functions at nerve terminals, including membrane lipid and calcium dynamics that drive synapse biogenesis and function. We are investigating the impact that PM-ER membrane contacts have on ER homeostasis, as well as the membrane lipid dynamics that modulate exocytosis and endocytosis during neurotransmission.

Publications

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Henne WM (2015) Mdm1/Snx13 is a novel ER-endolysosomal interorganelle tethering protein. in The Journal of cell biology

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Malia PC (2018) Control of vacuole membrane homeostasis by a resident PI-3,5-kinase inhibitor. in Proceedings of the National Academy of Sciences of the United States of America

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Omnus DJ (2016) Phosphoinositide kinase signaling controls ER-PM cross-talk. in Molecular biology of the cell

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Stefan CJ (2018) Building ER-PM contacts: keeping calm and ready on alarm. in Current opinion in cell biology

 
Description Organizer and Chair, Workshop on "Quantitative Imaging of Cellular Lipids", ASBMB Annual Meeting, San Diego, CA, April 2014.
Geographic Reach Multiple continents/international 
Policy Influence Type Influenced training of practitioners or researchers
Impact Impact on technological developments for quantitative imaging in cell biological research.
 
Description Japan Society for the Promotion of Science
Amount £60,000 (GBP)
Organisation Japan Society for the Promotion of Science (JSPS) 
Sector Public
Country Japan
Start 01/2016 
End 12/2017
 
Description Therapeutic Innovation Fund
Amount £22,500 (GBP)
Organisation National Institute for Health Research 
Department NIHR Biomedical Research Centre
Sector Public
Country United Kingdom
Start 08/2014 
End 07/2015
 
Description Therapeutic Innovation Fund
Amount £15,000 (GBP)
Organisation National Institute for Health Research 
Department NIHR Biomedical Research Centre
Sector Public
Country United Kingdom
Start 08/2014 
End 07/2015
 
Description Therapeutic Innovation Fund
Amount £22,500 (GBP)
Organisation Wellcome Trust 
Department Wellcome Trust Bloomsbury Centre
Sector Charity/Non Profit
Country United Kingdom
Start 08/2014 
End 07/2015
 
Description WTISSF 3: Institutional Strategic Support Fund (ISSF) Third Tranche
Amount £20,000 (GBP)
Funding ID 163090 
Organisation Wellcome Trust 
Department Wellcome Trust Institutional Strategic Support Fund
Sector Charity/Non Profit
Country United Kingdom
Start 03/2014 
End 05/2015
 
Description Wenner-Gren Foundation Fellowship
Amount £65,000 (GBP)
Organisation The Wenner-Gren Institute 
Sector Academic/University
Country Sweden
Start 01/2015 
End 12/2016
 
Title High content screens for modulators of motor neuron disease 
Description Amyotrophic Lateral Sclerosis (ALS) is inexorably fatal because no treatment halting or preventing the disease is available. Mechanisms implicated in the disease process are poorly defined and highly complex. To identify chemical entities that target the underlying toxicity, it is essential to embrace unbiased and innovative approaches in which the search for effective therapeutics is accompanied and supported by an increased understanding of disease mechanisms. Dr. Stefan is a scientist with a longstanding interest in the biology of VAPB, the ALS8 causative gene. Dr. Stefan and his coworkers have conducted an HCS in a neuronal cellular model of ALS8 recapitulating major hallmarks of the human disease. Because inclusion formation is an easy assayable phenotype and represents a fundamental pathological feature, a screen of small molecule compound libraries (with more than 45,000 compounds currently available) was conducted to identify compounds that disrupt inclusion formation. Subsequently, compounds were tested in a series of phenotypic and functional readouts including changes in neurite extension, quantitative evaluation of ER stress, alterations in lipid metabolism, intracellular translocation of the disease protein and neuronal cell survival. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2017 
Provided To Others? No  
Impact Candidate compounds have been identified and further tested in a series of phenotypic and functional readouts including changes in neurite extension, quantitative evaluation of ER stress, alterations in lipid metabolism, intracellular translocation of the disease protein and neuronal cell survival. Candidate compounds are now in tests to validate their effectiveness in other forms of ALS. 
 
Title High throughput assays for regulators of PI kinases and phosphatases 
Description High throughput assays for small molecule regulators of PI kinases and phosphatases 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
Impact Phosphoinositide (PI) kinase signalling networks control several key cellular processes including cell growth and survival, cell proliferation and differentiation, cell polarity and migration, and membrane trafficking pathways. Defects in PI kinase regulatory networks have been implicated in numerous human diseases including cancer, diabetes, and neurodegenerative disorders. Our assays are designed to (1) validate PI kinases and phosphatases as potential drug targets, and (2) to identify small molecule compounds (chemical) that directly target and regulate these enzymes. We expect these studies will be particularly relevant to neuronal physiology, as defects in PI metabolism have been implicated in several neurological and neurodegenerative disorders including Down's syndrome, Alzheimer's disease, Parkinson's disease, bipolar disorders, and autism. 
 
Title YPA-Yeast Protein Atlas 
Description At UCL, the Stefan lab is undertaking system-wide approaches to make new discoveries in the cell biology. As part of this research program, Dr. Stefan's group is carrying out high-throughput genomic screens using yeast cells as a model system. Yeast cells are widely used in system-wide genomic studies, such as yeast genetic approaches (YGA), epistatic mini-array profiling (E-MAP), and chemical-genetic profiling techniques. To apply functional genomics approaches to cell biological problems, this new lab will combine existing yeast genomic approaches (YGA) with high-content quantitative imaging for system-wide analysis of changes in protein localization and abundance (YPA) under a variety of genetic, environmental, and chemical stresses using rapid image acquisition and quantitative analysis. Combined YGA-YPA approaches will provide new ways to monitor dynamic changes in cellular architecture. 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? Yes  
Impact With the establishment of a new high-throughput yeast cell biology facility at the LMCB, UCL will become one of only a few research institutions in the world capable of coupling functional genomics with high-content protein localization screens in yeast. The yeast protein atlas (YPA) database and technology will increase the resolution of existing yeast genetic approaches (YGA), enabling the detailed investigation of numerous biological processes. As such, this new YPA database and technology will benefit many yeast cell biology groups across the UK. 
 
Description Control of lysosome membrane homeostasis 
Organisation University of Osnabrück
Department School of Biology/Chemistry Osnabrück
Country Germany 
Sector Academic/University 
PI Contribution Lysosomes have an important role in cellular protein and organelle quality control, metabolism and signaling. On the surface of lysosomes, the PIKfyve/Fab1 complex generates phosphatidylinositol 3,5-bisphosphate, or PI(3,5)P2, which is critical for lysosomal membrane homeostasis and for lysosomal signaling during acute cellular stress. However, it is still unknown how he PIKfyve/Fab1 complex senses changes in the lysosomal membrane and is regulated during cellular stress. We are elucidating regulation of the PIKfyve/Fab1 complex with a direct influence on PI(3,5)P2 levels and vacuole homeostasis.
Collaborator Contribution The Ungermann lab studies lysosomal membrane dynamics and cross talk with PIKfyve/Fab1-mediated PI(3,5)P2 synthesis to counterbalance membrane stress.
Impact Malia P, Numrich J, Nishimura T, Gonzalez Montoro A, Stefan C, and C Ungermann (2018). Control of vacuole membrane homeostasis by a resident PI3P 5-kinase inhibitor. Proc Nat Acad Sci. 115(18):4684-4689. PMID: 29674454 PMCID: PMC5939101 DOI: 10.1073/pnas.1722517115
Start Year 2017
 
Description Cross talk between MAPK and ER stress signalling pathways 
Organisation University at Buffalo
Country United States 
Sector Academic/University 
PI Contribution Signaling mucins are evolutionarily conserved regulators of signal transduction pathways. The signaling mucin Msb2p regulates the Cdc42p-dependent mitogen-activated protein kinase (MAPK) pathway that controls filamentous growth in yeast. The cleavage and release of the glycosylated inhibitory domain of Msb2p is required for MAPK activation. We show here that proteolytic processing of Msb2p was induced by underglycosylation of its extracellular domain. Cleavage of underglycosylated Msb2p required the unfolded protein response (UPR), a quality control (QC) pathway that operates in the endoplasmic reticulum (ER). The UPR regulator Ire1p, which detects misfolded/underglycosylated proteins in the ER, controlled Msb2p cleavage by regulating transcriptional induction of Yps1p, the major protease that processes Msb2p. Accordingly, the UPR was required for differentiation to the filamentous cell type. Cleavage of Msb2p occurred in conditional trafficking mutants that trap secretory cargo in the endomembrane system. Processed Msb2p was delivered to the plasma membrane, and its turnover by the ubiquitin ligase Rsp5p and ESCRT attenuated the filamentous-growth pathway. We speculate that the QC pathways broadly regulate signaling glycoproteins and their cognate pathways by recognizing altered glycosylation patterns that can occur in response to extrinsic cues.
Collaborator Contribution We provided expertise in analysis of PI kinase and ER stress signalling pathways for this study.
Impact Adhikari H, Vadaie N, Chow J, Caccamise L, Chavel C, Li B, Bowithch A, Stefan CJ and Cullen P (2015). Role of the unfolded protein response in regulating the mucin-dependent filamentous growth MAPK pathway. Mol Cell Biol. 35: 1414-32.
Start Year 2014
 
Description ER-endosomal membrane dynamics 
Organisation University of Texas Southwestern Medical Center
Country United States 
Sector Academic/University 
PI Contribution Although endolysosomal trafficking is well defined, how it is regulated and coordinates with cellular metabolism is unclear. To identify genes governing endolysosomal dynamics, we conducted a global fluorescence-based screen to reveal endomembrane effector genes. Screening implicated Phox (PX) domain-containing protein Mdm1 in endomembrane dynamics. Surprisingly, we demonstrate that Mdm1 is a novel interorganelle tethering protein that localizes to endoplasmic reticulum (ER)-vacuole/lysosome membrane contact sites (MCSs). We show that Mdm1 is ER anchored and contacts the vacuole surface in trans via its lipid-binding PX domain. Strikingly, overexpression of Mdm1 induced ER-vacuole hypertethering, underscoring its role as an interorganelle tether. We also show that Mdm1 and its paralogue Ydr179w-a (named Nvj3 in this study) localize to ER-vacuole MCSs independently of established tether Nvj1. Finally, we find that Mdm1 truncations analogous to neurological disease-associated SNX14 alleles fail to tether the ER and vacuole and perturb sphingolipid metabolism. Our work suggests that human Mdm1 homologues may play previously unappreciated roles in interorganelle communication and lipid metabolism.
Collaborator Contribution Although endolysosomal trafficking is well defined, how it is regulated and coordinates with cellular metabolism is unclear. To identify genes governing endolysosomal dynamics, we conducted a global fluorescence-based screen to reveal endomembrane effector genes. Screening implicated Phox (PX) domain-containing protein Mdm1 in endomembrane dynamics. Surprisingly, we demonstrate that Mdm1 is a novel interorganelle tethering protein that localizes to endoplasmic reticulum (ER)-vacuole/lysosome membrane contact sites (MCSs). We show that Mdm1 is ER anchored and contacts the vacuole surface in trans via its lipid-binding PX domain. Strikingly, overexpression of Mdm1 induced ER-vacuole hypertethering, underscoring its role as an interorganelle tether. We also show that Mdm1 and its paralogue Ydr179w-a (named Nvj3 in this study) localize to ER-vacuole MCSs independently of established tether Nvj1. Finally, we find that Mdm1 truncations analogous to neurological disease-associated SNX14 alleles fail to tether the ER and vacuole and perturb sphingolipid metabolism. Our work suggests that human Mdm1 homologues may play previously unappreciated roles in interorganelle communication and lipid metabolism.
Impact Henne WM, Balogi Z, Zhu L, Stefan CJ, Pleiss J and Emr S (2015). Mdm1/Snx13 is a novel ER-endolysosomal inter-organelle tethering protein. J Cell Biol. 210: 541-51.
Start Year 2013
 
Description Role of Calcium and Membrane Lipid Dynamics in Immune Cell Responses 
Organisation Cornell University
Department Department of Chemistry and Chemical Biology
Country United States 
Sector Academic/University 
PI Contribution Ca(2+) mobilization in response to cross-linking of IgE bound to its high affinity receptor, FceRI, on mast cells is central to immune allergic responses. Stimulated tyrosine phosphorylation caused by this cross-linking activates store-operated Ca(2+)entry that results in sustained Ca(2+)oscillations dependent on Rho family GTPases and phosphoinositide synthesis. Coupling of the endoplasmic reticulum (ER) Ca(2+)sensor, stromal interaction molecule 1 (STIM1), to the Ca(2+)-selective channel, Orai1, is regulated by these elements and depends on membrane organization, both at the plasma membrane and at the ER. Mitochondria also contribute to the regulation of Ca(2+)mobilization, and we describe recent evidence that the ER membrane protein vesicle-associated membrane protein-associated protein (VAP) plays a significant role in the coupling between ER and mitochondria in this process. In addition to granule exocytosis, Ca(2+)mobilization in these cells also contributes to stimulated outward trafficking of recycling endosomes and to antigen-stimulated chemotaxis, and it is pathologically regulated by protozoan parasitic invasion.
Collaborator Contribution This work is discovering regulatory mechanisms for mast cell responses by calcium and phosphoinositide signaling networks.
Impact To date, one publication has resulted from this collaboration. Roles for Ca2+ mobilization and its regulation in mast cell functions: recent progress David Holowka, Marcus Wilkes, Christopher Stefan, Barbara Baird Biochemical Society Transactions Apr 11, 2016, 44 (2) 505-509; DOI: 10.1042/BST20150273
Start Year 2014
 
Description Targeting Membrane Homestasis in Motor Neuron Disease 
Organisation University of Edinburgh
Department Euan Macdonald Centre for Motor Neurone Disease Research
Country United Kingdom 
Sector Academic/University 
PI Contribution Amyotrophic Lateral Sclerosis (ALS) is inexorably fatal because no treatment halting or preventing the disease is available. Mechanisms implicated in the disease process are poorly defined and highly complex. To identify chemical entities that target the underlying toxicity, it is essential to embrace unbiased and innovative approaches in which the search for effective therapeutics is accompanied and supported by an increased understanding of disease mechanisms. Drosophila is emerging as a promising animal model for testing therapeutic options mainly because of excellent in vivo readouts of pathology, numerous genetic resources available and high degree of conservation of genetic pathways between flies and humans. Mammalian models have the advantage of being much more similar to humans but the length of the time and the cost required to perform experiments comparable to those possible in flies, can be prohibitive. Cell-based models can be used for high-throughput drug screens but they may not recapitulate the response of the entire organism. Dr. Stefan is a scientist with a longstanding interest in the biology of VAPB, the ALS8 causative gene. Dr. Stefan and his coworkers have conducted an high-content screen (HCS) in a neuronal cellular model of ALS8 recapitulating major hallmarks of the human disease. Because inclusion formation is an easy assayable phenotype and represents a fundamental pathological feature, a screen of small molecule compound libraries was conducted to identify compounds that disrupt inclusion formation. Subsequently, compounds were tested in a series of phenotypic and functional readouts including changes in neurite extension, quantitative evaluation of ER stress, alterations in lipid metabolism, intracellular translocation of the disease protein and neuronal cell survival.
Collaborator Contribution Dr. Pennetta's lab generated a Drosophila model of ALS8 in which the expression of the pathogenic transgene in the eye photoreceptors or in all neurons, induces neurotoxicity that can be monitored by measuring the reduction in eye size, progressive motor abnormalities and early organismal death. These phenotypic readouts are easy-to-score and can be used for quantitative and sensitive analyses of compound-mediated effects. The phenotypic analysis could also be extended to include aggregate formation, alterations in synaptic structure and function and muscle defects. A genome-wide screen aimed at identifying genetic modifiers of disease phenotypes in a fly model for ALS8 identified genes and pathways important for ALS pathogenesis, indicating potential targets for therapeutic intervention. Moreover, these pathways largely overlap with those identified by a high-content screen (HCS) of small molecule compounds performed in an ALS8 cell-based model by the lab of Dr. C. Stefan at University College London, UK. In a collaborative effort with Dr. Stefan's lab, a multisystem, cross-species approach in which Drosophila is be used for: 1) exploring the possibility of targeting the Hippo pathway for therapeutic intervention; 2) testing and mechanistically validate compounds identified by the cell-based screen with particular emphasis on those affecting proteins and pathways shown to be important for ALS pathogenesis by the genetic modifier screen.
Impact In progress
Start Year 2017
 
Description Highlight article on Organelle Crosstalk for The Biochemist newsletter 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Feature highlight piece on "Organelle Crosstalk in Membrane Dynamics and Cell Signaling", a Biochemical Society sponsored Focus meeting. This report has sparked further interest in holding future conferences in this area of cell biology.
Year(s) Of Engagement Activity 2016
URL http://www.biochemistry.org/381/TheBiochemist/index.html
 
Description Interview for national television program 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Appeared on TV broadcast of "How to lose weight well" as cell membrane expert and provided a lab demonstration.
Year(s) Of Engagement Activity 2018
 
Description School visit (Radboud University Honours Academy Day at the MRC LMCB ) 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact 2nd year students (from biology, chemistry, physics, and mathematics) selected for the Radboud University Honours Academy, a special program for gifted undergraduate students, attended a visit to the MRC LMCB.

We put together a program which sparked questions and discussion afterwards to increase interest in cross discipline studies in cell biology, biochemistry, and physics and to discuss opportunities as post-graduate students.
Year(s) Of Engagement Activity 2016