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The class III PI3K Vacuolar protein sorting 34 (Vps34) plays a role in both canonical and noncanonical autophagy, key processes that control the presentation of antigens by dendritic cells (DCs) to naive T lymphocytes. We generated DC-specific -deficient mice to assess the contribution of Vps34 to DC functions. We found that DCs from these animals have a partially activated phenotype, spontaneously produce cytokines, and exhibit enhanced activity of the classic MHC class I and class II antigen-presentation pathways. Surprisingly, these animals displayed a defect in the homeostatic maintenance of splenic CD8α DCs and in the capacity of these cells to cross-present cell corpse-associated antigens to MHC class I-restricted T cells, a property that was associated with defective expression of the T-cell Ig mucin (TIM)-4 receptor. Importantly, mice deficient in the Vps34-associated protein Rubicon, which is critical for a noncanonical form of autophagy called "Light-chain 3 (LC3)-associated phagocytosis" (LAP), lacked such defects. Finally, consistent with their defect in the cross-presentation of apoptotic cells, DC-specific -deficient animals developed increased metastases in response to challenge with B16 melanoma cells. Collectively, our studies have revealed a critical role of Vps34 in the regulation of CD8α DC homeostasis and in the capacity of these cells to process and present antigens associated with apoptotic cells to MHC class I-restricted T cells. Our findings also have important implications for the development of small-molecule inhibitors of Vps34 for therapeutic purposes.
Autophagy plays a critical role in multiple aspects of the immune system, including the development and function of T lymphocytes. In mammalian cells, the class III PI3K vacuolar protein sorting (Vps)34 is thought to play a critical role in autophagy. However, recent studies have cast doubt on the role of Vps34 in autophagy, at least in certain cell types. To study the effects of Vps34 on autophagy in T lymphocytes, we generated mice that selectively lack Vps34 in the T cell lineage. Vps34 ablation in T cells caused profound defects in autophagic flux, resulting in accumulation of cellular organelles and apoptosis. These animals exhibited normal intrathymic development of conventional T cells, but they were profoundly impaired in the intrathymic development of invariant NKT cells. In peripheral organs, T cell-specific ablation of Vps34 had a profound impact on T cell homeostasis and function. Furthermore, aged animals developed an inflammatory wasting syndrome characterized by weight loss, intestinal inflammation, and anemia. Consistent with this phenotype, Vps34 was required for the peripheral maintenance and function of CD4(+)Foxp3(+) regulatory T cells. Collectively, our study reveals a critical role for Vps34 in autophagy and for the peripheral homeostasis and function of T lymphocytes.
Recent studies have suggested that autophagy is a key mechanism in maintaining the integrity of podocytes. The mammalian homologue of yeast vacuolar protein sorting defective 34 (mVps34) has been implicated in the regulation of autophagy, but its role in podocytes is unknown. We generated a line of podocyte-specific mVps34-knockout (mVps34(pdKO)) mice, which were born at Mendelian ratios. These mice appeared grossly normal at 2 weeks of age but exhibited growth retardation and were significantly smaller than control mice by 6 weeks of age, with no difference in ratios of kidney to body weight. mVps34(pdKO) mice developed significant proteinuria by 3 weeks of age, developed severe kidney lesions by 5-6 weeks of age, and died before 9 weeks of age. There was striking podocyte vacuolization and proteinaceous casts, with marked glomerulosclerosis and interstitial fibrosis by 6 weeks of age. Electron microscopy revealed numerous enlarged vacuoles and increased autophagosomes in the podocytes, with complete foot process effacement and irregular and thickened glomerular basement membranes. Immunoblotting of isolated glomerular lysates revealed markedly elevated markers specific for lysosomes (LAMP1 and LAMP2) and autophagosomes (LC3-II/I). Immunofluorescence staining confirmed that the enlarged vacuoles originated from lysosomes. In conclusion, these results demonstrate an indispensable role for mVps34 in the trafficking of intracellular vesicles to protect the normal cellular metabolism, structure, and function of podocytes.
Although phosphatidylinositol 5-phosphate (PtdIns5P) is present in many cell types and its biogenesis is increased by diverse stimuli, its precise cellular function remains elusive. Here we show that PtdIns5P levels increase when cells are stimulated to move and we find PtdIns5P to promote cell migration in tissue culture and in a Drosophila in vivo model. First, class III phosphatidylinositol 3-kinase, which produces PtdIns3P, was shown to be involved in migration of fibroblasts. In a cell migration screen for proteins containing PtdIns3P-binding motifs, we identified the phosphoinositide 5-kinase PIKfyve and the phosphoinositide 3-phosphatase MTMR3, which together constitute a phosphoinositide loop that produces PtdIns5P via PtdIns(3,5)P(2). The ability of PtdIns5P to stimulate cell migration was demonstrated directly with exogenous PtdIns5P and a PtdIns5P-producing bacterial enzyme. Thus, the identified phosphoinositide loop defines a new role for PtdIns5P in cell migration.