The role of phosphatidylinositol 3-phosphate (PI3P) in the induction of autophagy

自噬是细胞对营养限制的一种重要反应，因为它允许细胞通过自身成分的自我消化来产生营养，从而在有限的时间内存活。自噬也是正常生长和发育期间质量控制机制的重要贡献者，因此，它被证明对维持适当的寿命至关重要。最后，已经发现几种癌症模型可以上调或下调自噬，这表明自噬也可能与肿瘤的进展有关(基因开发21：2861-73,2007)。自噬的诱导是一个关键的承诺步骤，受到严格的监管。在上游调控因子中，有营养感应的机制(目前尚不清楚)和参与PI-3-激酶信号传递的蛋白质，包括哺乳动物靶标雷帕霉素激酶。这些监管机构如何协调提供自噬信号，目前正在紧张调查中。最近的工作更清楚的是，该信号最终导致PI3P的形成，正是这种脂质允许自噬进行(自噬4：952-4,2008)。然而，PI3P在诱导过程中的确切功能直到最近才为人所知。通过跟踪活细胞中几种PI3P结合蛋白在氨基酸(AA)饥饿过程中的动态，我们为PI3P在自噬诱导中的功能提供了一些线索(J Cell Biol 182：685-701,2008)。我们发现，在AA饥饿后不久，PI3P开始在我们称为omegasome的新的膜室中积累。这些omegasome与内质网处于动态平衡状态，它们构成了自噬小体生物发生的场所。因此，我们最近的数据解释了PI3P在早期自噬中的作用。本次研究的目的将是进一步探讨PI3P在自噬诱导中的作用。大部分工作将涉及在饥饿反应的早期阶段对omegasome中的PI3P进行可视化的方法。我们建议与Innova Biosciences合作开展这项工作，Innova Biosciences是一家在生产荧光生物偶联物方面拥有广泛专业知识的公司。这些生物偶联物(以抗体或脂质结合域的形式)对于成功的结果将是必不可少的。在该项目的一个部分，学生将产生专门针对omegasome限制的PI3P池的荧光探针，以评估广泛的细胞系统和组织中的饥饿反应。为了实现这一点，我们将产生标记在GST上的ER-FYVE探针的重组版本，我们将使用Innova技术将它们连接到FITC或RITC等荧光记者上。在一种平行的方法中，我们将产生针对DFCP1的抗体，DFCP1是目前已知的唯一驻留在omegasome中的内源性蛋白，我们将再次将这些抗体与荧光记者结合，以产生有用的探针。一旦有了这些探针，我们将使用它们来检测饥饿期间细胞和(A)对照和饥饿动物的组织以及(B)正常和肿瘤组织样本中自噬特异性PI3P的积累。这种类型的实验将提供关于自噬对病理或生理状态的贡献的重要信息。第二个相关目标将是鉴定PI3P磷脂磷酸酶，该酶负责终止自噬过程中的诱导信号。在动物细胞中有14个肌管蛋白相关蛋白(MTMR，Trends Cell Biol 16：403-12,2006)，我们预计一旦PI3P完成其功能，将有一个或多个成员负责PI3P的去磷酸化。为了鉴定这些酶，我们将使用siRNA来对抗该家族的所有成员，并遵循omegasome动力学。我们预计，相关基因的敲除将导致omegasome中PI3P的增加，甚至可能足以增加基础自噬。这些结果的分析是在我的团队中建立的。

Autophagy is an important cellular response to nutrient limitation because it allows the cell to survive for a finite length of time by self digestion of its own components to generate nutrients. Autophagy is also an important contributor to quality control mechanisms during normal growth and development, and as such it has been shown to be critical for maintaining proper life span. Finally, several cancer models have been found to either up- or down-regulate autophagy, indicating that autophagy may also be involved in tumour progression (Genes Dev 21: 2861-73, 2007). The induction of autophagy represents a critical commitment step and is regulated tightly. Among the upstream regulators are mechanisms (as yet unknown) for nutrient sensing and proteins involved in PI 3-kinase signalling including the mammalian target of rapamycin kinase. How these regulators co-ordinate to provide the autophagy signal is currently under intense investigation. What is more clear from recent work is that the signal ultimately results in formation of PI3P, and it is this lipid that allows autophagy to proceed (Autophagy 4: 952-4, 2008). However, the exact function of PI3P in the induction step was unknown until recently. By following the dynamics of several PI3P-binding proteins during amino acid (AA) starvation in live cells we have provided some clues as to the function of PI3P in autophagy induction (J Cell Biol 182: 685-701, 2008). We found that PI3P starts to accumulate soon after AA starvation in novel membrane compartments that we termed omegasomes. These omegasomes are in dynamic equilibrium with the endoplasmic reticulum, and they constitute sites of autophagosome biogenesis. Therefore, our recent data provide an explanation of the role of PI3P in early autophagy. The aim of this studentship will be to further explore the role of PI3P in autophagy induction. The majority of the work will involve methods for the visualization of PI3P in omegasomes, during the earliest stages of the starvation response. We propose to do this work in collaboration with Innova Biosciences, a company that has broad expertise in generating fluorescent bio-conjugates. These bio-conjugates (in the form of antibodies or lipid-binding domains) will be essential for a successful outcome. In one part of the project the student will generate fluorescent probes specific for the omegasome-restricted pool of PI3P to evaluate starvation responses in a broad number of cell systems and in tissues. To achieve this we will generate recombinant versions of our ER-FYVE probes tagged to GST and we will use the Innova technology to conjugate them to fluorescent reporters such as FITC or RITC. In a parallel approach, we will generate antibodies against DFCP1, the only endogenous protein known so far to reside in omegasomes, and we will again conjugate these antibodies with fluorescent reporters in order to generate useful probes. Once these probes are at hand, we will use them to examine autophagy-specific PI3P accumulation in cells during starvation and in (a) tissues from control and starved animals and (b) from normal vs tumour tissue samples. This type of experiment will provide important information on the contribution of autophagy to pathological or physiological states. A second related aim will be the identification of the PI3P phospholipid phosphatase responsible for terminating the induction signal during autophagy. In animal cells there are 14 members of the myotubularin-related proteins (MTMRs, Trends Cell Biol 16: 403-12, 2006) and we expect that one or more will be responsible for de-phosphorylating PI3P once it has fulfilled its function. To identify these enzymes we will use siRNA against all members of the family and follow omegasome dynamics. We expect that knock-down of the relevant gene will lead to an increase in PI3P in omegasomes, and it may even be sufficient to increase basal autophagy. Assays for these outcomes are established in my group.