Functional dynamics of endogenous phosphatidylinositol-5-phosphate 4-kinases

多磷肌醇脂是构成哺乳动物细胞膜的磷脂的少量成分（约1%），但它们在生理和病理上的重要性与其数量不成比例。它们一共有7个，每一个都至少有一种（大多数都不止一种）生理作用来调节细胞的功能，而这些作用几乎发生在细胞内每一个可以想象到的位置。当这些过程没有得到适当的调节时，就可能发生病理：例如，多磷酸肌醇脂与癌症、炎症、神经系统疾病和心血管疾病有关。本应用的主题是最近发现的多磷酸肌醇脂质，磷脂酰肌醇5-磷酸（PI5P），它存在于细胞的许多位置（包括细胞核内，这是本项目的关键焦点之一）。大量的间接证据表明，PI5P与细胞对压力（紫外线、热量、氧化自由基）的反应方式有关，这些反应显然必须作为它们抵御环境诱发疾病的一部分。PI5P的水平（以及它的功能，因为它的作用主要是由在给定位置存在的多少来调节的）由三种酶家族调节，这是本研究的重点：pi5p4激酶（PI5P5Ks）。我们发现我们的（哺乳动物）PI5P4Ks在不同的组织中以不同的数量存在，并且在细胞内的不同位置。此外，它们具有显著不同的酶活性（PI5P4K α是迄今为止最活跃的），我们最近的发现是，出乎意料的是，它们彼此配对产生各种混合物（例如，α和β: α / α， α / β和β / β）。因此，我们发现非常活跃的PI5P4K α通过与其他PI5P4ks结合而“靶向”细胞中的不同位置（具体来说，PI5P4K β将PI5P4K α带入细胞核，可能PI5P4K γ将其带入细胞质中的囊泡）。到目前为止，这是一个静态的，没有功能的图片，这个项目的主要目的是解决这个缺点。我们计划将荧光“标签”附着在PI5P4Ks上，然后使用绝对尖端的荧光成像技术来获得前所未有的洞察力，了解它们的确切去向，它们从一个位置移动到另一个位置的速度，它们彼此之间的行为方式，以及这一切对细胞对压力的反应方式意味着什么。细胞模型,我们计划使用的这些研究是非常强大的DT40细胞系基因,但这些细胞的起源和鸡没有基因PI5P4Kγ(虽然这是有益的,它使得研究α和β容易!),我们还计划利用人类细胞系目前成为细胞系模型同样强大的DT40s, Nalm-6细胞,以类似的方式探索PI5P4Kγ。最后，爱丁堡的科学家培育了一只缺乏PI5P4K γ基因的小鼠，由于PI5P4K γ在肾小管系统的特定部位被发现水平异常高，我们将与爱丁堡的同事合作，详细研究这些小鼠的肾脏生理学。

Polyphophoinositol lipids are minor components (about 1%) of the phospholipids that make up mammalian cell membranes, but they have a physiological and pathological importance way out of proportion to their quantity. There are seven of them in all, each with at least one (most with more than one) physiological action to regulate how cells function - and these actions take place in almost every conceivable location within a cell. When these processes are not appropriately regulated, pathologies can occur: polyphosphoinositol lipids are implicated in, for example, cancer, inflammation, neurological disorders and cardiovascular disease. The subject of this application is the most recently discovered polyphosphoinositol lipid, phosphatidylinositol 5-phophate (PI5P), which is present in many locations of the cell (including within the nucleus, one of the key foci of this project). PI5P has, by a large body of circumstantial evidence, been implicated in the way in which cells respond to stress (uv light, heat, oxidizing radicals), responses which self-evidently must act as part of their defense against environmentally-induced disease. The levels of PI5P (and thus its functions, because its actions are regulated primarily by how much is present at a given location) are regulated by a family of three enzymes, which are the focus of this study: the PI5P 4-kinases (PI5P5Ks). We have found that our (mammalian) PI5P4Ks are present at different amounts in different tissues, and at very different locations within a cell. Moreover, they have remarkably different enzymic activities (PI5P4K alpha being by far the most active), and our most recent discovery is that, unexpectedly, they associate with one another as pairs to generate a variety of mixtures (e.g. for alpha and beta: alpha/alpha, alpha/beta and beta/beta). As a result of this, we find that the very active PI5P4K alpha is 'targeted' to different places in the cell by association with the other PI5P4ks (specifically, the PI5P4K beta takes PI5P4K alpha into the nucleus, and probably PI5P4K gamma takes it to vesicles in the cytoplasm).So far this is a static and rather functionless picture we have, and the main thrust of this project it to address that shortcoming. We plan to attach fluorescent 'tags' to the PI5P4Ks, and then use the absolute cutting edge of fluorescence imaging techniques to gain unprecedented insight into exactly where they go, how quickly they move from location to location, how they behave with respect to each other, and what this all means for the way in which cells react to stress. The cellular model that we plan to use for most of these studies is the genetically very powerful DT40 cell line, but these cells are of chicken origin and do not have a gene for PI5P4K gamma (though that is helpful in that it makes studying alpha and beta easier!), so we also plan to use a human cell line which is currently emerging as a model cell line equally powerful to DT40s, Nalm-6 cells, to explore PI5P4K gamma in similar fashion. Finally, scientists in Edinburgh have generated a mouse lacking the PI5P4K gamma gene, and because PI5P4K gamma is found at exceptionally high levels in specific parts of the kidney tubular system we will contribute to a collaboration with our colleagues in Edinburgh to study the kidney physiology of these mice in great detail.