Microautophagy is a poorly-characterized autophagic pathway defined by direct invagination of vacuolar (fungi) or lysosomal (higher eukaryotes) membrane for engulfment and degradation of organelles and cytosolic components. Microautophagy is required for cell survival under conditions of stress, such as nutrient limitation, and for resumption of cell growth on recovery from stress. Strikingly, microautophagy is also a key regulator of TOR signaling, which plays well-established roles in growth, survival and lifespan control. The molecular mechanism of microautophagy and how it regulates TOR signaling is not understood. Our long-term goal is to understand the mechanism of microautophagy and how microautophagy is harnessed to regulate signaling cascades required for growth and development. Our preliminary data demonstrates that the membrane remodeling required for microautophagy depends on the dynamin-related protein (DRP) Vps1 and the ESCRTIII component Snf7. DRPs and ESCRTs are fascinating membrane remodeling machines that are vital for several fundamental cellular processes including membrane trafficking, mitochondrial dynamics, cytokinesis and viral budding. DRPs and ESCRTs both couple membrane deformation to self-assembly. Deficiencies in DRPs and ESCRTs are associated with numerous pathologies due to their central roles in homeostasis, including neurodegenerative disorders. Our central hypothesis is that the functional interaction between DRPs and ESCRTs forms the molecular basis for membrane invagination in microautophagy. Furthermore, we propose that nutrient and stress signaling pathways converge on Vps1 and ESCRT to regulate their novel function in microautophagy to, ultimately, control TOR signaling and cell growth. Using genetic, proteomic, cytological and structural approaches, we will characterize the mechanism of recruitment of DRP and ESCRT to sites of microautophagy, as well as the regulatory determinants of their function in TOR signaling. Completion of this work will provide an in-depth understanding of the machinery required for microautophagy. It will shed mechanistic light on novel aspects of DRP and ESCRT function that may have broad implications for membrane remodeling processes in general. Finally, this work will provide important insight into the mechanisms whereby microautophagy regulates TOR signaling. Dysregulation of TOR signaling is associated with several human cancers. Hence, the machinery of microautophagy, as a regulator of TOR signaling, represents a novel target for development of anticancer and antifungal drugs.
The work described in this proposal is directly relevant to human health as it studies how autophagy, an adaptive process required for cell survival upon stress, is regulated by membrane remodeling machineries. Both autophagy and membrane remodeling are processes of fundamental importance for cell growth and survival under normal, stressful and pathological conditions and dysregulation of autophagy and membrane remodeling are linked to a wide range of human diseases including cancers and neurodegenerative disorders. This proposed work is therefore relevant to the part of the NIH mission that supports and promotes basic discoveries in cell biology that will lead to the development of novel therapeutics and treatment strategies for a wide range of human diseases.
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| Varlakhanova, Natalia V; Mihalevic, Michael J; Bernstein, Kara A et al. (2017) Pib2 and the EGO complex are both required for activation of TORC1. J Cell Sci 130:3878-3890 |
