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

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.