A role for a novel animal endo-lysosomal Ca2+/H+ exchanger in Ca2+ signalling and chemotaxis.

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).

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.

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.