Membrane contact sites between endolysosomes and the ER as novel hubs in Ca2+ signalling.

Lead Research Organisation: University College London
Department Name: Cell and Developmental Biology

Abstract

One critical feature which distinguishes our cells from those of bacteria is the presence of organelles - tiny membrane bound structures dedicated to performing specific tasks. But this division of labour comes at a price namely the need for the organelles to communicate in order for the cell to function properly as a whole. One emerging means by which organelles "chatter" is through membrane contact sites. These sites are regions where the organelles come together via bridges so as to allow exchange of small molecules such as lipids and calcium. Understanding the composition of these sites and how they are regulated is critical for understanding how cell function is coordinated.

In this application, we focus on contact sites between the endoplasmic reticulum (ER) where new cellular material is made and endolysosomes that take up, recycle and degrade material. Notably, both of these organelles are also stores of calcium. Calcium is perhaps most familiar as a mineral ion important for bones and teeth. This is certainly true. However, less familiar is its critical role in signalling. When cells are stimulated, calcium levels within the cell rapidly increase and this increase sets in train a series of events which change the behaviour of the cell in significant ways. This is critical for probably all cellular events from fertilisation through to the beating of the heart. In many cases this calcium comes from stores.

What is now clear is that release of calcium from endolysosomes "triggers" further calcium release from the ER - an excellent example of organelles working together. What is not so clear is how this occurs leading us to our hypothesis that this happens at contact sites between the two organelles. To this end, we have now succeeded in defining some of the proteins involved in connecting endolysosomes and the ER and shown that disrupting these contacts impairs calcium signalling between the two organelles. Surprisingly, we also find that contacts are regulated by calcium itself.

We will build on these findings by combining the expertise of the applicants in studying calcium (Patel) and contact sites (Futter). First, we will study a protein on the endolysosomes which we think channels the calcium to stabilize contacts. We will also interfere with the contacts and study how this impacts calcium changes when calcium release from endolysosomes is activated in different ways. In the second part of the program, we will study new proteins that we think are additional contact site components. Finally, we will examine the consequences of disrupting contacts on cell behaviour. Specifically, we will study how contacts affect the function of receptor proteins that are involved in receiving messages from outside of the cells. It is not easy to study contact sites because of their small size. So within the program we will strive to develop new methods to facilitate study. This will be of relevance to many in the scientific community.

The successful outcome of the project will provide us with key new insight into how cells control and use their calcium. Disturbances in calcium have already been linked to many disorders including Parkinson's disease and cancer. Could defective contact sites be an unrecognized culprit? Understanding how calcium regulates membrane contacts sites and vice versa could provide the basis for formulating drugs to make or break contact.

Technical Summary

Changes in cytosolic Ca2+ form the basis of a critical signalling pathway. Alongside the ER, it is now clear that the
endolysosomal system is also a key mobilizable Ca2+ store. Importantly, these two physically segregated Ca2+ sources are functionally coupled such that endolysosomal Ca2+ release is often amplified by Ca2+ release from the ER to regulate numerous Ca2+-dependent outputs. But the mechanistic and regulatory basis for this cross-talk is unclear.

Here, we consider a role for membrane contact sites in coupling Ca2+ stores. Contact sites are regions of close membrane apposition between organelles that are fast emerging as an important non-vesicular means of communication. This application builds on our new data which has i) revealed the composition of distinct contact sites between endolysosomes and the ER and ii) shown that these contacts are not only required for inter-organellar Ca2+ signalling but also regulated by Ca2+ itself.

Our aims are to bridge complementary expertise in Ca2+ signalling (Patel) and membrane traffic (Futter) to 1) define the role of endolysosome-ER membrane contact sites in integrating Ca2+ signals 2) identify novel endolysosome-ER membrane contact site regulators and 3) establish the physiological relevance of endolysosome-ER membrane contact sites in receptor-mediated signalling events. We will use a combination of molecular and imaging approaches and develop advanced methods to facilitate study of these challenging physiological junctions.

The successful outcome of this project will establish membrane contact sites between endolysosomes and the ER as novel hubs in the control of Ca2+-dependent function.

Planned Impact

We identify the following beneficiaries of this collaborative multi-disciplinary project:

1. International science base.
We will provide a broad range of scientific training through the combination of internationally-recognized expertise bought by the applicants within the infrastructure of a world-class university. This will be disseminated not only to the appointee but to the wider scientific community thus adding major value to this proposal.

2. General public.
This application is a basic science proposal addressing a ubiquitous signalling pathway (calcium) that underlies probably all cellular processes. The layman is likely only aware of the role of calcium as a mineral ion. This proposal will provide a better general understanding of the multiple roles that calcium plays physiologically.

3. Women SET.
Futter has been a champion of women in science, engineering and technology (SET). This proposal will provide an ideal vehicle to continue promoting female scientists as role models.

4. Clinicians, patients and the pharmaceutical industry.
The pathways we target are demonstrably relevant to disease. In particular, TPCs are emerging as potential therapeutic targets and our linking of these ubiquitous proteins to membrane contacts sites identifies this cellular locus as novel therapeutic target. The outcomes of this project are thus of potential major relevance to public health and Pharma.
 
Description We have found that physical junctions between two types of 'mini organs' inside our cells known as endosomes and the ER require calcium to form. We have identified TPC1 as the protein which releases calcium to do this. And we have found that breaking the junctions perturbs the ability of a growth factor to function which has important implications for cancer. We also found that other channel proteins related to TPC1 use the junctions to release calcium.
The key publications [1, 2] have attracted much attention evidenced by invitations to write reviews and commentaries and the formation of new collaborations.
Specifically, we have published two important reviews in the highly visible 'Current Opinions' series [3, 4] which summarise thinking on how these junctions work and the role of TPC proteins in regulating them. And another review on the more general role of TPCs proteins in disease [5]. Our thoughts were also sought on related work published by others [6].
Two new collaborations were formed as a result of this work, both with scientists in the USA. The first has come to fruition in a study showing that TPC proteins in Toxoplasma, the causative agent of Toxoplasmosis, similarly regulate junctions. This work [7] identifies TPC proteins as potential new drug targets. The other collaboration proposes to look at how junctions are affected by a protein called JPT2 which has emerged as a new partner for TPC proteins essential for their function [8]. This has led to funding from abroad through the National Institutes of Health, USA.
Our findings have also allowed us to address controversy surrounding the use of a compound called GPN which is used widely in the field [9]. Our data show that the compound likely works as originally proposed [10].
Overall, the key findings have had resulted in numerous impactful publications not only in our area of research but well beyond.
Key publications:
[1] B.S. Kilpatrick, E.R. Eden, A.H. Schapira, C.E. Futter, S. Patel, Direct mobilisation of lysosomal Ca2+ triggers complex Ca2+ signals, J. Cell Sci 126 (2013) 60-66.
[2] B.S. Kilpatrick, E.R. Eden, L.N. Hockey, E. Yates, C.E. Futter, S. Patel, An Endosomal NAADP-Sensitive Two-Pore Ca2+ Channel Regulates ER-Endosome Membrane Contact Sites to Control Growth Factor Signaling, Cell Rep 18(7) (2017) 1636-1645.
[3] T.P. Levine, S. Patel, Signalling at membrane contact sites: two membranes come together to handle second messengers, Curr. Opin. Cell Biol 39 (2016) 77-83.
[4] K. Vassileva, M. Marsh, S. Patel, Two-pore channels as master regulators of membrane trafficking and endocytic well-being, Current opinion in physiology (2020).
[5] S. Patel, B.S. Kilpatrick, Two-pore channels and disease, Biochimica et biophysica acta. Molecular cell research 1865(11 Pt B) (2018) 1678-1686.
[6] S. Patel, Getting close. Lysosome-ER contact sites tailor Ca(2+) signals, Cell Calcium 80 (2019) 194-196.
[7] Z.H. Li, T.P. King, L. Ayong, B. Asady, X. Cai, T. Rahman, S.A. Vella, I. Coppens, S. Patel, S.N.J. Moreno, A plastid two-pore channel essential for inter-organelle communication and growth of Toxoplasma gondii, Nature communications 12(1) (2021) 5802.
[8] G.S. Gunaratne, E. Brailoiu, S. He, E.M. Unterwald, S. Patel, J.T. Slama, T.F. Walseth, J.S. Marchant, Essential requirement for JPT2 in NAADP-evoked Ca(2+) signaling, Science signaling 14(675) (2021).
[9] A.J. Morgan, Y. Yuan, S. Patel, A. Galione, Does lysosomal rupture evoke Ca(2+) release? A question of pores and stores, Cell Calcium 86 (2020) 102139.
[10] Y. Yuan, B.S. Kilpatrick, S. Gerndt, F. Bracher, C. Grimm, A.H. Schapira, S. Patel, The lysosomotrope GPN mobilises Ca(2+) from acidic organelles, Journal of cell science 134(6) (2021).
Exploitation Route These findings will be of relevance to a large body of scientists who are studying junctions and cancer biologists who study growth factors. Our work might also be relevant to the treatment of Toxoplasmosis.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Successful scientific training and professional development of appointed PDRA. Further development of lay website. Educational outreach through hosting of summer placements.
First Year Of Impact 2017
Sector Education,Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic

 
Description Membrane contact sites between endolysosomes and the ER as novel hubs in Ca2+ signalling
Amount £430,122 (GBP)
Funding ID BB/N01524X/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 06/2016 
End 06/2019
 
Description Probing Ca2+ channel dysfunction in LRRK2-Parkinson's
Amount £49,924 (GBP)
Funding ID K-1802 
Organisation Parkinson's UK 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2018 
End 03/2020
 
Description Probing Ca2+ channel dysfunction in LRRK2-Parkinson's
Amount £121,748 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 08/2018 
End 09/2021
 
Description Probing Two-pore channel 2 in LRRK2-Parkinson's in vivo
Amount £282,374 (GBP)
Funding ID G-2006 
Organisation Parkinson's UK 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2020 
End 03/2023
 
Description Switching the ion selectivity of an ion channel on demand
Amount £492,120 (GBP)
Funding ID BB/T015853/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 06/2020 
End 07/2023
 
Description Collaboration with Jonathan Marchant on the regulation of two-pore channels by NAADP-binding proteins 
Organisation Medical College of Wisconsin
Country United States 
Sector Academic/University 
PI Contribution We helped identify NAADP-binding proteins in different organisms.
Collaborator Contribution The Marchant lab led all the experimental work.
Impact NIH grant in which we will characterise the effects of NAADP-binding proteins on membrane contact sites. Paper in Science Signalling: G.S. Gunaratne, E. Brailoiu, S. He, E.M. Unterwald, S. Patel, J.T. Slama, T.F. Walseth, J.S. Marchant, Essential requirement for JPT2 in NAADP-evoked Ca(2+) signaling, Science signaling 14(675) (2021).
Start Year 2018
 
Description Collaboration with Silvia Moreno on Two-pore channels in Toxoplasma 
Organisation University of Georgia
Country United States 
Sector Academic/University 
PI Contribution We helped characterise TPC proteins in Toxoplasma using in silico methods. And advising on generation of mutant channels.
Collaborator Contribution They lead the experimental work.
Impact A paper in Nature Communications: Z.H. Li, T.P. King, L. Ayong, B. Asady, X. Cai, T. Rahman, S.A. Vella, I. Coppens, S. Patel, S.N.J. Moreno, A plastid two-pore channel essential for inter-organelle communication and growth of Toxoplasma gondii, Nature communications 12(1) (2021) 5802
Start Year 2018