A role for a novel animal endo-lysosomal Ca2+/H+ exchanger in Ca2+ signalling and chemotaxis.
Lead Research Organisation:
University College London
Department Name: Cell and Developmental Biology
Abstract
Calcium is familiar to perhaps all of us as a mineral that is important for healthy bones and teeth. But less familiar is its vital role within our cells where it controls nearly all processes essential for life. Calcium rises signal the very start of life (during fertilization) and are used, time and time again, during early development (in for example controlling the way in which cells migrate throughout the embryo). These rises drive our nerve impulses and our heart beats. Calcium changes are bought about by a delicately balanced network of proteins which act to deliver calcium at precisely defined times and at specific locations within our cells. Thus, even subtle defects in calcium levels can precipitate disease. Understanding exactly how calcium is controlled within cells is thus vital for understanding how cells work - a critical first step towards devising ways to correct faulty calcium levels in disease.
Lysosomes are small acid filled structures (organelles) present within our cells and which are traditionally thought of as the cell's recycling centre. But there is now much evidence stemming from early work in sea urchin eggs suggesting that they and a number of related structures are important sources of calcium. These so called "acidic calcium stores" regulate a number of cellular processes. Moreover, defective handling of calcium by these stores is also emerging as important in several diseases including Parkinson's disease. Work from our lab has helped to identify the proteins which open to allow calcium to be released from these stores. But how calcium is taken up is not known. This is a major gap in our knowledge that requires urgent filling.
Plant biologists have a much better understanding of how calcium is taken up in to acidic calcium stores in the cells they study. One way is by the actions of a family of proteins known as calcium hydrogen exchangers (CAXs). There is much evidence to suggest that these proteins also fill acidic calcium stores in animals but identification of the corresponding genes which encode them is only in its infancy.
In our initial experiments, we identified CAX genes from the sea urchin and frog - two commonly used laboratory animals. We showed that frog CAX localizes to acidic calcium stores and that it is involved in regulating calcium levels. We also showed that chemically interfering with acidic organelles prevents the normal migration of embryonic cells in response to a hormone-like substance which is known to drive changes in calcium. These data provide new insight in to the molecular identity and function of CAXs in animals.
In this application, we bring together a team of scientists with expertise in the study of acidic calcium stores, CAXs in plants and migration of cells. We will build on our preliminary findings to determine i) the precise location of CAX within cells, ii) details of the calcium "transporting" activity of CAX (including whether CAX can be used to reset defective calcium levels in diseased cells) and iii) to define the role of CAX in cell migration.
The successful outcome of this project will provide us with new information on how calcium is handled by acidic calcium stores and importantly provide us with new tools for further study. It will also provide new insight in to the role of these stores in migration of cells. This process is vital for proper development.
The collective expertise of the team means that we can apply a broad range of techniques to achieve our aims. It also means that the information we obtain will be useful to a number of scientists and not only those in our respective fields. These scientists include those studying i) calcium in other acid-filled organelles (such as stores of hormones), ii) established processes that acidic organelles fulfil (such as recycling materials), iii) related CAX proteins in other organisms (such as plants and yeast) and iv) specific diseases (such as cancer).
Lysosomes are small acid filled structures (organelles) present within our cells and which are traditionally thought of as the cell's recycling centre. But there is now much evidence stemming from early work in sea urchin eggs suggesting that they and a number of related structures are important sources of calcium. These so called "acidic calcium stores" regulate a number of cellular processes. Moreover, defective handling of calcium by these stores is also emerging as important in several diseases including Parkinson's disease. Work from our lab has helped to identify the proteins which open to allow calcium to be released from these stores. But how calcium is taken up is not known. This is a major gap in our knowledge that requires urgent filling.
Plant biologists have a much better understanding of how calcium is taken up in to acidic calcium stores in the cells they study. One way is by the actions of a family of proteins known as calcium hydrogen exchangers (CAXs). There is much evidence to suggest that these proteins also fill acidic calcium stores in animals but identification of the corresponding genes which encode them is only in its infancy.
In our initial experiments, we identified CAX genes from the sea urchin and frog - two commonly used laboratory animals. We showed that frog CAX localizes to acidic calcium stores and that it is involved in regulating calcium levels. We also showed that chemically interfering with acidic organelles prevents the normal migration of embryonic cells in response to a hormone-like substance which is known to drive changes in calcium. These data provide new insight in to the molecular identity and function of CAXs in animals.
In this application, we bring together a team of scientists with expertise in the study of acidic calcium stores, CAXs in plants and migration of cells. We will build on our preliminary findings to determine i) the precise location of CAX within cells, ii) details of the calcium "transporting" activity of CAX (including whether CAX can be used to reset defective calcium levels in diseased cells) and iii) to define the role of CAX in cell migration.
The successful outcome of this project will provide us with new information on how calcium is handled by acidic calcium stores and importantly provide us with new tools for further study. It will also provide new insight in to the role of these stores in migration of cells. This process is vital for proper development.
The collective expertise of the team means that we can apply a broad range of techniques to achieve our aims. It also means that the information we obtain will be useful to a number of scientists and not only those in our respective fields. These scientists include those studying i) calcium in other acid-filled organelles (such as stores of hormones), ii) established processes that acidic organelles fulfil (such as recycling materials), iii) related CAX proteins in other organisms (such as plants and yeast) and iv) specific diseases (such as cancer).
Technical Summary
The widespread physiological and patho-physiological role of Ca2+ signals demands a full understanding of the molecular mechanisms that drive them. Coordination of cellular Ca2+ signalling by acidic Ca2+ stores (such as lysosomes) is fast emerging as vital for homeostasis. But despite recent advances in defining the molecular basis for Ca2+ release from acidic organelles through the identification of endo-lysosomal two-pore channels, there is currently a paucity of information regarding the molecular basis for Ca2+ uptake into these stores in animals. In pilot studies, we have i) cloned animal orthologues of Ca2+/H+ exchangers (CAXs) which in plants and unicellular organisms are established in mediating Ca2+ uptake into acidic vacuolar Ca2+ stores ii) provided the first molecular evidence that Xenopus CAX is an endo-lysosomal protein which regulates cytosolic Ca2+ signals and iii) implicated endo-lysosomal Ca2+ signalling in chemotaxis of a highly motile embryonic cell type (neural crest). Building on these initial findings, the overall aim of this project is to combine expertise in endo-lysosomal Ca2+ signalling (Patel), vacuolar Ca2+ transporters (Pittman) and cell migration (Mayor) to test the central hypothesis that Xenopus CAX is an endo-lysosomal Ca2+ transporter required for chemotaxis. We will define 1) the subcellular location of CAX, 2) the functional role of CAX in Ca2+ signalling and 3) the physiological role of CAX in chemotaxis. We will use a combination of yeast, amphibian and mammalian cells and a range of molecular cell biology and imaging approaches. An essential feature of this proposal is the complementary skills brought by the team to provide an integrated molecular and functional view of Ca2+/H+ exchangers in animals. The successful outcome of this project will provide urgently needed molecular insight in to the mechanisms of endo-lysosomal Ca2+ signalling and extend the functional roles of acidic Ca2+ stores to a crucial developmental process.
Planned Impact
In this collaborative multi-disciplined project, we identify the international science base, the general public and the pharmaceutical industry as beneficiaries beyond the immediate academic community. Expert training of the appointee will contribute directly to the science base. The signalling pathway we target (calcium) is ubiquitous and demonstrably relevant to disease. Its understanding is therefore of major relevance to public health. That the molecular basis for handling of calcium by acidic organelles is only just emerging renders this pathway an attractive and novel therapeutic target, and thus of major potential interest to pharmaceutical companies. This is underscored by the identified link between these organelles and cell migration which is essential for proper development and also de-regulated in several diseases. To achieve maximal impact of the research, we will provide a broad range of scientific training through the combination of internationally recognized expertise bought by the applicants. In addition, professional training will be ensured through the infrastructure provided by the world-class universities in which the research will be performed. We will engage the public through generation of a website aimed at the lay person and promote of our findings through the general media, lay publications and outreach activities aimed at school children. The pharmaceutical industry will be engaged through our existing links and an identified collaborative scheme. Thus our research will provide major impact in several disparate areas.
Organisations
Publications
Abeti R
(2018)
Calcium Deregulation: Novel Insights to Understand Friedreich's Ataxia Pathophysiology.
in Frontiers in cellular neuroscience
Burgoyne T
(2015)
Calcium signaling at ER membrane contact sites.
in Biochimica et biophysica acta
Cai X
(2015)
Insights into the early evolution of animal calcium signaling machinery: a unicellular point of view.
in Cell calcium
Churamani D
(2013)
The N-terminal region of two-pore channel 1 regulates trafficking and activation by NAADP.
in The Biochemical journal
Fernández B
(2016)
Iron overload causes endolysosomal deficits modulated by NAADP-regulated 2-pore channels and RAB7A.
in Autophagy
Goodridge JP
(2019)
Remodeling of secretory lysosomes during education tunes functional potential in NK cells.
in Nature communications
Hockey LN
(2015)
Dysregulation of lysosomal morphology by pathogenic LRRK2 is corrected by TPC2 inhibition.
in Journal of cell science
Hooper R
(2015)
TPC1 Knockout Knocks Out TPC1.
in Molecular and cellular biology
Jha A
(2014)
Convergent regulation of the lysosomal two-pore channel-2 by Mg²?, NAADP, PI(3,5)P2 and multiple protein kinases.
in The EMBO journal
Kilpatrick B
(2017)
An Endosomal NAADP-Sensitive Two-Pore Ca 2+ Channel Regulates ER-Endosome Membrane Contact Sites to Control Growth Factor Signaling
in Cell Reports
Kilpatrick B
(2016)
Endo-lysosomal TRP mucolipin-1 triggers global ER Ca2+ release and Ca2+ influx
in Journal of Cell Science
Kilpatrick BS
(2015)
Methods for monitoring lysosomal morphology.
in Methods in cell biology
Kilpatrick BS
(2016)
Connecting Ca2+ and lysosomes to Parkinson disease.
in Messenger (Los Angeles, Calif. : Print)
Kilpatrick BS
(2016)
Endoplasmic reticulum and lysosomal Ca²? stores are remodelled in GBA1-linked Parkinson disease patient fibroblasts.
in Cell calcium
Kilpatrick BS
(2013)
Direct mobilisation of lysosomal Ca2+ triggers complex Ca2+ signals.
in Journal of cell science
Levine TP
(2016)
Signalling at membrane contact sites: two membranes come together to handle second messengers.
in Current opinion in cell biology
Lin-Moshier Y
(2014)
The Two-pore channel (TPC) interactome unmasks isoform-specific roles for TPCs in endolysosomal morphology and cell pigmentation.
in Proceedings of the National Academy of Sciences of the United States of America
Lloyd-Evans E
(2016)
On the move, lysosomal CAX drives Ca2+ transport and motility.
in The Journal of cell biology
Love NR
(2015)
NAD kinase controls animal NADP biosynthesis and is modulated via evolutionarily divergent calmodulin-dependent mechanisms.
in Proceedings of the National Academy of Sciences of the United States of America
Marchant J
(2013)
Questioning Regulation of Two-Pore Channels by NAADP
in Messenger
Marchant JS
(2015)
Two-pore channels at the intersection of endolysosomal membrane traffic.
in Biochemical Society transactions
Melchionda M
(2016)
Ca2+/H+ exchange by acidic organelles regulates cell migration in vivo.
in The Journal of cell biology
Melchionda M
(2016)
Ca 2+ /H + exchange by acidic organelles regulates cell migration in vivo
in The Journal of Experimental Medicine
Morgan AJ
(2020)
Does lysosomal rupture evoke Ca2+ release? A question of pores and stores.
in Cell calcium
Patel S
(2018)
Two-pore channels and disease.
in Biochimica et biophysica acta. Molecular cell research
Patel S
(2019)
Getting close. Lysosome-ER contact sites tailor Ca2+ signals.
in Cell calcium
Patel S
(2015)
Function and dysfunction of two-pore channels.
in Science signaling
Patel S
(2018)
Two-pore channels open up.
in Nature
Patel S
(2018)
Thinking through acidic Ca2+ stores.
in Science signaling
Patel S
(2016)
A Special Issue of Messenger 2016
in Messenger
Patel S
(2016)
Deviant lysosomal Ca2+ signalling in neurodegeneration. An introduction.
in Messenger (Los Angeles, Calif. : Print)
Patel S
(2015)
Evolution of acidic Ca²? stores and their resident Ca²?-permeable channels.
in Cell calcium
Patel S
(2017)
NAADP-evoked Ca2+ signals through two-pore channel-1 require arginine residues in the first S4-S5 linker.
in Cell calcium
Penny C
(2014)
A computational model of lysosome-ER Ca2+ microdomains
in Journal of Cell Science
Penny CJ
(2019)
Mining of Ebola virus entry inhibitors identifies approved drugs as two-pore channel pore blockers.
in Biochimica et biophysica acta. Molecular cell research
Penny CJ
(2016)
Isolated pores dissected from human two-pore channel 2 are functional.
in Scientific reports
Penny CJ
(2014)
A "mix-and-match" approach to designing Ca(2+) microdomains at membrane-contact sites.
in Communicative & integrative biology
Penny CJ
(2015)
Coupling acidic organelles with the ER through Ca²? microdomains at membrane contact sites.
in Cell calcium
Penny CJ
(2015)
Poring over two-pore channel pore mutants.
in Messenger (Los Angeles, Calif. : Print)
Pereira GJ
(2014)
NAADP-sensitive two-pore channels are present and functional in gastric smooth muscle cells.
in Cell calcium
Pittman JK
(2016)
CAX-ing a wide net: Cation/H(+) transporters in metal remediation and abiotic stress signalling.
in Plant biology (Stuttgart, Germany)
Rahman T
(2014)
Two-pore channels provide insight into the evolution of voltage-gated Ca2+ and Na+ channels.
in Science signaling
| Title | Additional file 4: Figure S2. of Phylogenetic analysis and protein structure modelling identifies distinct Ca2+/Cation antiporters and conservation of gene family structure within Arabidopsis and rice species |
| Description | Phylogenetic analysis of the CaCA superfamily. Trees derived from alignments of full-length sequences, conserved â core domainâ hydrophobic region sequences, and Îa2-repeat region sequences. (PNG 437 kb) |
| Type Of Art | Film/Video/Animation |
| Year Produced | 2016 |
| URL | https://springernature.figshare.com/articles/figure/Additional_file_4_Figure_S2_of_Phylogenetic_anal... |
| Title | Additional file 4: Figure S2. of Phylogenetic analysis and protein structure modelling identifies distinct Ca2+/Cation antiporters and conservation of gene family structure within Arabidopsis and rice species |
| Description | Phylogenetic analysis of the CaCA superfamily. Trees derived from alignments of full-length sequences, conserved â core domainâ hydrophobic region sequences, and Îa2-repeat region sequences. (PNG 437 kb) |
| Type Of Art | Film/Video/Animation |
| Year Produced | 2016 |
| URL | https://springernature.figshare.com/articles/figure/Additional_file_4_Figure_S2_of_Phylogenetic_anal... |
| Description | We have identified and characterized a new class of transporters known as the calcium hydrogen exchangers (CAXs) in animals. Our findings indicate that these proteins are responsible for filling acidic organelles such as lysosomes with calcium, and that this is important movement of cells in the embryo. This is a major breakthrough as we knew next to nothing about the molecular identity and function of CAXs in organisms other than plants and fungi. |
| Exploitation Route | Our work allows now affords the opportunity to specifically interfering with calcium uptake into acidic organelles to identify new functional roles and to correct defective calcium handling in disease. |
| Sectors | Pharmaceuticals and Medical Biotechnology |
| URL | http://jcb.rupress.org/content/212/7/755 |
| Description | Implicit to this work was to provide excellence in scientific training brought by the interdisciplinary collaboration. In this context, the appointed post-doctoral scientist was recruited to the prestigious Crick Institute as a Senior Laboratory Research Scientist upon completion of the project. As such, this is directly strengthening the UK science base to meet the future needs of the international economy. Stemming directly from the key publication associated with this work have been numerous invitations to speak at international meetings. This in turn has led to world-wide scientific partnerships with research groups in Spain, Norway and Germany, all of which have resulted in high impact publications in the area of Ca2+ signalling more broadly. This has significantly strengthened international cooperation. This publication also led to consultancy work for two major investment firms and a scientific advisory role in a company that runs science clubs for children. |
| First Year Of Impact | 2016 |
| Sector | Creative Economy |
| 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 | Mis-coordinated Ca2+ signalling in Parkinson's |
| Amount | £92,276 (GBP) |
| Organisation | Parkinson's UK |
| Sector | Charity/Non Profit |
| Country | United Kingdom |
| Start | 08/2013 |
| End | 09/2016 |
| 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 | Targeting two-pore channel 2 in LRRK2-dependent Parkinson's disease |
| Amount | £34,798 (GBP) |
| Funding ID | K-1412 |
| Organisation | Parkinson's UK |
| Sector | Charity/Non Profit |
| Country | United Kingdom |
| Start | 01/2015 |
| End | 09/2015 |
| Title | Additional file 2: Table S1. of Phylogenetic analysis and protein structure modelling identifies distinct Ca2+/Cation antiporters and conservation of gene family structure within Arabidopsis and rice species |
| Description | Reference number or accession number of the sequences used for the phylogenetic analysis. (XLSX 23 kb) |
| Type Of Material | Database/Collection of data |
| Year Produced | 2016 |
| Provided To Others? | Yes |
| URL | https://springernature.figshare.com/articles/dataset/Additional_file_2_Table_S1_of_Phylogenetic_anal... |
| Title | Additional file 2: Table S1. of Phylogenetic analysis and protein structure modelling identifies distinct Ca2+/Cation antiporters and conservation of gene family structure within Arabidopsis and rice species |
| Description | Reference number or accession number of the sequences used for the phylogenetic analysis. (XLSX 23 kb) |
| Type Of Material | Database/Collection of data |
| Year Produced | 2016 |
| Provided To Others? | Yes |
| URL | https://springernature.figshare.com/articles/dataset/Additional_file_2_Table_S1_of_Phylogenetic_anal... |