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

我们将研究信息是如何从细胞外环境传递到控制细胞活动的细胞内蛋白质的。人体内有超过1000万个细胞，其中大多数是高度专业化的。每个细胞都必须找到通往目的地的道路，并与其他细胞沟通，才能有效地履行其专门职能。这两个特征都在这个提议中得到了解决，这两个特征都需要细胞检测周围环境中的特定刺激，并将信息传递到围绕每个细胞的屏障-膜。这样，细胞外信号通过产生细胞内信使来调节细胞内的活动。钙是这些信使中最重要的一种。细胞消耗大量的能量将钙挤出细胞膜和细胞器，但这些细胞膜上有小孔，可以按需打开，使钙快速向下流入细胞。然后，这产生钙浓度的瞬时增加，调节许多细胞活动。所有动物细胞都表达IP 3受体，并且大多数存在于细胞内最广泛的细胞器--内质网的膜上，内质网是一个网状网络，可以侵入细胞的每个角落。大量证据表明，细胞外刺激与IP 3受体之间以及产生的钙信号与其细胞内靶点之间的通讯是有组织的，以允许将信号特异性地局部传递到密切相关的蛋白质。这种空间组织被认为是重要的，允许相当少的细胞内信使，但选择性地调节许多不同的东西。然而，一个问题是细胞器本身也在不断地移动。这就好像邮袋被选择性地在暴风雨中颠簸的小船之间传递。我们关心的是了解细胞器如何移动和钙信号转导通路内的信息重新配置的后果。我们最近的工作出乎意料地揭示了在动物细胞中表达的三种形式的IP 3R之一（IP 3R 2）的行为与其他不同。到目前为止，人们还不清楚为什么细胞要花这么大的力气来控制它们表达的IP 3R的混合物。我们已经表明，而IP 3 R1和IP 3 R3表达在网状ER，IP 3 R2表达在一个身份不明，但非常移动的囊泡结构，这是明显不同于ER。此外，我们有证据表明，当细胞迁移时，这些结构会移动，我们推测，它们的移动是允许迁移细胞产生局部钙信号所必需的，这些信号似乎是允许转向特定刺激所必需的。成纤维细胞是这项提案的重点。它们需要修复组织，并且它们被首先对组织损伤做出反应的血细胞释放的PDGF吸引到损伤部位。该提案应用各种先进的方法来解决与含IP 3R 2的囊泡相关的三个重要问题：1。表达IP 3R 2的细胞器是什么？IP 3R 2上的地址标签是什么？2.这些囊泡对钙信号有什么贡献？3.这些囊泡在控制成纤维细胞向化学引诱物迁移中起什么作用？

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?