Functional properties of a mobile organelle expressing type 2 inositol 1,4,5-trisphosphate receptors

We will examine how information passes from the extracellular environment to the intracellular proteins that control cellular activity.

There are more than 10 million million cells in a human body, and most are highly specialized. Each cell must both find its way to its destination and communicate with other cells if it is to fulfil its specialized functions effectively. Both features are addressed in this proposal, and both require that cells detect specific stimuli in their surroundings and transmit that information across the barrier - the membrane - that surrounds every cell. In this way extracellular signals regulate activities within a cell by generating intracellular messengers. Calcium is one of the most important of these messengers. Cells invest considerable energy extruding calcium across the membranes that surround both the cell and the organelles that reside within it. But these membranes include pores that can be opened on-demand to allow calcium to flow rapidly downhill into the cell. This then generates the transient increase in calcium concentration that regulates many cellular activities. IP3 receptors, the focus of this proposal, are the most important of these regulated calcium-permeable routes through membranes.

All animal cells express IP3 receptors, and most occur within the membranes of the most extensive of the intracellular organelles, the ER, a reticular network that invades every corner of the cell. Considerable evidence suggests that communication between extracellular stimuli and IP3 receptors, and between the resulting calcium signals and their intracellular targets is organized to allow local delivery of signals specifically to closely associated proteins. This spatial organization is thought to be important in allowing rather few intracellular messengers to nevertheless selectively regulate many different things. A problem, however, is that the organelles are themselves constantly moving. It is as if mail bags were being passed selectively between small boats tossed in a stormy sea. We are concerned with understanding how the organelles move and the consequences for reconfiguring transfer of information within calcium signalling pathways.

Our recent work has unexpectedly revealed that one of the three forms of IP3R expressed in animal cells (IP3R2) behaves differently to the others. It has hitherto been unclear why cells go to such considerable lengths to control which mixture of IP3Rs they express. We have shown that whereas IP3R1 and IP3R3 are expressed in reticular ER, IP3R2 is expressed in an unidentified but very mobile vesicular structure that is clearly distinct from ER. Furthermore, we have evidence that these structures move when cells migrate, and we speculate that their movement is required to allow migrating cells to generate the local calcium signals that seem to be required to allow turning towards specific stimuli. Fibroblasts are the focus of much of this proposal. They are required to repair tissue, and they are drawn to sites of injury by PDGF released by the blood cells that first respond to tissue damage.

This proposal applies a variety of advanced methods to address three important questions related to the IP3R2-containing vesicles:

1. What are the organelles in which IP3R2 are expressed, and what is the address label on IP3R2 that gets them there?

2. What contribution do these vesicles make to calcium signals?

3. What role do these vesicles play in controlling migration of fibroblasts towards chemoattractants?

Our preliminary analyses have established that one of the three subtypes of mammalian IP3R, IP3R2, is uniquely expressed in a mobile, vesicular organelle (R2V) that is clearly distinct from the reticular ER in which IP3R1 and IP3R3 are expressed. The finding is significant because IP3R2 are the most sensitive IP3R subtype, they specifically associate with adenylyl cyclase, and both published work and our preliminary analyses demonstrate that IP3R2 specifically contribute to chemotaxis of migrating cells. Furthermore, we have shown that R2V rapidly move to the leading edge of migrating cells. We suggest R2V are mobile organelles that allow regulated movement of IP3R and thereby rapid reconfiguring of intracellular calcium signalling pathways. Our hypothesis is that directed intracellular movement of R2V contributes to formation of the leading edge and its turning towards chemoattractant. We suggest that R2V provide the small focal sources of Ca2+ that generate Ca2+ flickers at the leading edge and which are proposed to initiate turning. We will assess this hypothesis in fibroblasts migrating towards PDGF. We address three broad questions:

1. What are R2V and how are IP3R2 targeted to them?
2. What contributions do R2V make to Ca2+ signalling?
3. How are R2V moved to and within the leading edge and what is their role in chemotaxis?

Training and skills
Staff are encouraged to develop the skills and experience required for independence. They engage fully with every aspect of the project from developing proposals, managing budgets, reviewing and developing research programmes, to preparing publications and presenting work. All staff apply a range of state-of-the art techniques directly or via collaboration. Staff are expected to gain experience of teaching by supervising project/PhD students, teaching practical classes and in a lecture on advanced techniques to PhD/final year students. All staff contribute fully to weekly lab meetings, where they present work and critically evaluate work. In my absence, lab meetings are chaired by postdocs. A major impact is the proven ability of my lab to train staff well-equipped to meet future needs of industry, the public sector and academia.

International and interdisciplinary interactions
BBSRC has international collaboration as a priority. International interactions are a strength of my lab. The work described in this proposal takes us into a new area (cell migration) and combines that with our existing expertise in Ca2+ signalling. Our existing work is sustained by many international/cross-disciplinary collaborations and enhanced by international visitors. We have had preliminary discussions with Falcke (Berlin) on future opportunities for stochastic modelling of relationships between Ca2+ signals and chemotaxis. We expect to develop additional interactions as our work on migration develops. Many impacts arise from these interactions. They encourage interactions at boundaries between disciplines and by fostering extensive international interactions, they ensure that the UK remains fully engaged with a world-wide science community.

Public understanding and schools
My lab provides a Pharmacology Masterclass for year-12 students in which intensive practicals expose students about to finalise their post-school options with insight into science research and its economic impact. We intend to continue with this activity and to assimilate our growing experience of cell migration into it.
We provide at least one placement for an undergraduate student to gain research experience before they decide on postgraduate options.
Staff contribute to the Cambridge Science Festival. Typically, the contribution involves a hands-on demonstration of the actions of common drugs on waterfleas. We provide occasional visits to local schools providing practical experience of, for example, insect biology and microscopy.
Cell migration is an important and visually appealing phenomenon that we will exploit as a means of informing general audiences of how cells respond to changes in their environment and of how these questions are addressed.
We work with press offices to maximize the impact of our work. This as an important opportunity to bring the significance of fundamental research to the attention of a wide audience.
The impacts of these activities are to encourage interest in science from students who have not yet finalised their careers, and to facilitate widespread appreciation of the importance of addressing fundamental questions in biology.

Health and wealth
Cell migration is essential for wound-healing, the spread of cancers, embryological development and for effective use of stem cells to replace defective tissues. Ca2+ signalling pathways also contribute to disease and they provide effective drug targets. Our work attempting to establish the relationships between IP3R2, Ca2+ and chemotaxis will, therefore, feed into a deeper understanding of the basic biology of these clinically important phenomena. The impacts for clinical medicine are impossible to predict, but we will actively engage with clinicians and the pharmaceutical industry to ensure that our findings are presented at an early stage to communities with direct interests in clinical development.