| Phosphatidylinositol-3-phosphate (PI(3)P) is an intracellular signaling lipid which recruits lipid-binding proteins, tethering them to subcellular compartments where they function (Gaullier, Simonsen et al. 1998). PI(3)P is required for proper endocytosis and autophagy, two membrane trafficking processes pivotal to cellular homeostasis, and as such, its production, maintenance, and turnover must be tightly controlled (Funderburk, Wang et al. 2010). PI(3)P is produced by the class III member of the mammalian phosphoinositide-3-kinase (PI3K) family, Vps34 (vacuolar protein sorting 34) (Funderburk, Wang et al. 2010). Vps34-PI(3)P signaling is antagonized by PI(3)P lipid phosphatases, such as MTMR3 and MTMR14 (hJumpy), which function in autophagy (Vergne, Roberts et al. 2009; Taguchi-Atarashi, Hamasaki et al. 2010). Given the complexity of Vps34 function and the number of proteins essential for its function, we hypothesized that additional protein phosphatases exist to regulate Vps34-PI(3)P signaling.;To investigate this hypothesis, we performed a cell-based RNA interference screen to identify phosphatases whose loss of function alters cellular PI(3)P . We found that reduced expression of a receptor-like protein tyrosine phosphatase, PTPsigma, increased the abundance of PI(3)P-positive vesicles in cells. Intriguingly, the vesicles in these cells mimicked those observed in autophagic cells. Using a variety of cell biology approaches, we confirmed that PI(3)P signaling and autophagy are elevated in the absence of PTPsigma.;As a role for PTPsigma in PI(3)P signaling was unprecedented, much work was required, and still remains, to fully characterize its function in this process. We uncovered that PTPsigma resides on PI(3)P-positive vesicles during both basal and induced autophagy. Further, its internalization from the cell surface and presence on these vesicles is likely controlled through defined proteolytic processing. Finally, loss of PTPsigma in cells appears to promote Vps34 activity as measured in vitro and PTPsigma is capable of interacting with both Vps34 and at least one of its binding partners, Rubicon. We propose a working model where PTPsigma downregulates PI(3)P signaling through control of a phosphotyrosine substrate, yet to be identified, within or closely related to an endocytic Rubicon-containing Vps34 complex. These results are summarized in Chapter 2.;Although still elusive, the function of PTPsigma in PI(3)P signaling and autophagy may prove to have important consequences for cell fate. Autophagy is essential for cell survival during stress and accordingly, we would predict PTPsigma suppression to enhance autophagy-mediated survival (Meijer and Codogno 2004). The ability to increase autophagy could potentially provide therapeutic benefit in the treatment of several diseases, notably those involving neurodegeneration (Hara, Nakamura et al. 2006; Komatsu, Waguri et al. 2006). With this in mind, we utilized an in silico screening approach, outlined in Chapter 3, to identify small molecule inhibitors of PTPsigma. If selective, these compounds could prove to be useful molecular probes in the study of autophagy.;Finally, we aimed to capture a comprehensive understanding of autophagy dynamics through mathematical modeling. To this end, we utilized kinetic live-cell microscopy to develop an accurate and predictive model of autophagy vesicle dynamics, detailed in Chapter 4. This model, and its framework for a more large-scale autophagy network, can be used to generate novel hypotheses and be implemented as a tool to test experimentally-derived hypotheses, such as those presented for PTPsigma. |
