Autophagy is a fundamental cellular process whose classical function is the clearance of long-lived proteins and the recycling of cellular components for resources. During this dynamic process, a double membrane isolation structure, termed the autophagosome, surrounds portions of the cytoplasm;the contents of this new organelle are subsequently delivered to the lysosome for degradation. In short, ATG proteins, with Beclin-1 and the class III PI3K, VPS34, initiate the formation of the isolation membrane or autophagophore. The elongation and ultimate closure of the autophagophore around the cytoplasmic material is regulated by two ubiquitylation- like, protein conjugation systems: the ATG12 and LC3-PE conjugation pathways. The resulting ATG5-12-16 complex is necessary for the expansion and/or curvature of the autophagosome, as well as the generation of the lipidated version of LC3, LC3-II. Both the ATG5-12-16 complex and LC3-II decorate the membranes of the autophagosome;however, it is only LC3-II that remains during its fusion with the lysosome. It is therefore believed that LC3-II is critical for the targeting of autophagosomes to lysosomal organelles. It has recently been demonstrated that TLR signaling during phagocytosis recruits LC3 to the phagosome, in a manner dependent on ATG5 and ATG7 and involving Beclin-1 and VPS34 association. Despite the use of classical autophagy machinery, this process, herein referred to as LC3-associated phagocytosis (LAP), lacks the double membrane structure reminiscent of the autophagosome. Taken together, this data suggest a convergence of the phagocytic and autophagic pathways, where classical autophagy machinery is annexed to the phagosome resulting in accelerated phagosome maturation and increased ability to kill ingested pathogens. A recent report has described T and B cell hyperactivity, leading to dysregulated lymphocyte activation and autoimmunity, in mice deficient for Tim4, a phosphatidylserine receptor on phagocytes that facilitates the clearance of apoptotic cells. The role of LAP in the expedited clearance of phagocytosed material indicates that perhaps efficient clearance of dying cells could require crosstalk with the autophagic machinery. The overall goal of this proposal is to elucidate the role and mechanisms of LC3-associated phagocytosis in terms of uptake of dying cells, as well as characterize its divergence from traditional autophagy. Firstly, we will investigate the ability of dying cells, both apoptotic and necrotic, to induce LAP. Furthermore, we will use synthetic inhibitors, siRNA, and primary cells from deficient animals to investigate which molecules downstream of the initiating signal are required for LAP. These techniques will aid to further define LAP, in terms of its divergence from autophagy. These studies will include examination of the ULK1-ATG13-FIP200 complex, mTOR, and the Beclin1-VPS34- ATG14L-UVRAG complex, all important mediators of the autophagic response. In addition, we will An important approach to studying LAP will be the use of live cell imaging in conjunction with fluorescently-labeled molecules to establish the associations required for LAP. This study will elucidate the molecules required for LAP specification to the phagosome. Emergent data indicates that autophagy is an important aspect of cancer;this role, however, seems paradoxical, as autophagy has been reported to have both cytoprotective and anti- tumor effects. Indeed, it will be critical to further understand the mechanisms by which autophagy, or in this case, LAP, can be triggered, thus providing researchers with novel therapeutic targets.
Our understanding of the role of autophagy in carcinogenesis is in its infancy, with data supporting both its promotion and the suppression of tumor growth. Analysis of clinical samples and cancer cell lines demonstrates that several components of autophagy are deregulated during tumorigenesis. Further understanding the mechanisms by which autophagy, or in this case, LAP, can be triggered will provide researchers with novel therapeutic targets to the treatment of cancer.