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

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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?