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

我们将研究信息如何从细胞外环境传递到控制细胞活动的细胞内蛋白质。人体中有超过1000亿个细胞，其中大多数都是高度特化的。每个细胞都必须找到到达目的地的路，并与其他细胞沟通，才能有效地履行其特殊功能。这两个特征在这个方案中都得到了解决，它们都要求细胞检测周围环境中的特定刺激，并通过包围每个细胞的屏障——膜——传递这些信息。通过这种方式，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个。细胞投入大量的能量将钙挤出细胞膜，细胞膜围绕着细胞和细胞器。但是这些膜包括可以按需打开的孔，允许钙快速下坡流入细胞。这就会产生钙浓度的短暂增加，从而调节许多细胞活动。IP3受体是本研究的重点，是这些通过膜调节的钙渗透途径中最重要的。所有动物细胞都表达IP3受体，并且大多数发生在最广泛的胞内细胞器内质网的膜内，内质网是侵入细胞每个角落的网状网络。大量证据表明，细胞外刺激和IP3受体之间，以及由此产生的钙信号和细胞内靶标之间的通信是有组织的，允许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，它允许相当少的细胞内信使选择性地调节许多不同的事物。然而，一个问题是，细胞器本身也在不断地运动。这就像在暴风雨中颠簸的小船之间有选择地传递邮袋一样。我们关心的是了解细胞器是如何移动的，以及钙信号通路中信息转移的重新配置的后果。我们最近的工作意外地揭示了IP3R在动物细胞中表达的三种形式中的一种（IP3R2）的行为与其他形式不同。迄今为止，人们还不清楚为什么细胞会如此不遗余力地控制它们所表达的IP3Rs的混合物。我们已经证明IP3R1和IP3R3在网状内质网中表达，而IP3R2在一种未知但非常可移动的囊泡结构中表达，这与内质网明显不同。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，而这些钙信号似乎是允许转向特定刺激所必需的。成纤维细胞是这一建议的重点。它们是修复组织所必需的，它们被首先对组织损伤作出反应的血细胞释放的PDGF吸引到损伤部位。本提案应用多种先进方法来解决与含ip3r2囊泡相关的三个重要问题：表达IP3R2的细胞器是什么？ IP3R2上的地址标签是什么？这些囊泡对钙信号有什么贡献？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?