Quantitative membrane contact site biology in autophagy

/Abstract Macroautophagy (hereafter ‘autophagy’) is an intracellular degradation process essential for cellular homeostasis and health conserved from yeast to humans. Defects in autophagy are linked to many diseases including neurodegeneration, metabolic disease and cancer. Thus, understanding the mechanisms and physiological functions of autophagy has important and broad implications for human disease and health. Autophagy is characterized by formation of transient double-membrane organelles, termed autophagosomes. Autophagosomes form ‘de novo’ upon fusion of vesicles to nucleate a small single- membrane cisterna called ‘phagophore’ (or isolation membrane). The membrane of the nucleated phagophore then rapidly expands in form of a large cup-shaped structure to enclose cytoplasmic substrates. Upon closure, the phagophore membrane divides into the outer and inner membrane of the double-membrane autophagosome. The outer membrane fuses with the vacuole or lysosome exposing the inner membrane and enclosed substrates to resident hydrolases for degradation and metabolite recycling. The particular mechanisms of autophagosome biogenesis endow cells with an unprecedented capacity for turnover of an unparalleled scope of substrates in size and nature. Our goals in this research proposal are to gain mechanistic and quantitative understanding of the committed regulatory steps in autophagosome biogenesis that control the key parameters of number, size, duration and substrate scope of autophagosomes in response to diverse intracellular and environmental challenges. First, we will analyze the mechanisms underlying autophagosome biogenesis during non- selective autophagy with the goal to develop a general, quantitative and predictive model of autophagosome biogenesis. In this context, we are particularly interested in understanding the mechanisms of phospholipid transfer across specific membrane contact sites. Specifically, we discovered that three conserved bridge-like phospholipid transfer proteins cooperate at the membrane contact site formed between the phagophore and the endoplasmic reticulum to drive the membrane assembly of forming autophagosomes. Second, we aim at understanding how cells modify the mechanisms of autophagosome biogenesis in order to target selective substrates according to specific signal inputs. For this, we will examine the specific mechanisms of autophagosome biogenesis during a homeostatic form of mitophagy, which targets mitochondria specifically for organelle size regulation. Third, we are interested in understanding how cells maintain the membrane composition of mitochondria and how a specific form of mitophagy targets large mitochondria-derived vesicles formed from excess outer mitochondrial membrane. We want to understand how inter- and intraorganellar membrane contact sites are tuned to balance the membrane distribution within mitochondria, drive the biogenesis of outer mitochondrial membrane-derived vesicles and generate autophagosomes for their specific turnover. Our long-term goal is to understand how cells convert complex metabolic, functional and size parameters into finetuned autophagy responses at mechanistic, cellular and physiological level. Deep mechanistic understanding of autophagosome biogenesis has the potential to enable modifying autophagy in a targeted and rational manner to maximize the benefits for a broad spectrum of pathophysiological outcomes. Project Number: 1R35GM161597-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Martin Graef | Institution: CORNELL UNIVERSITY, ITHACA, NY | Award Amount: $404,864 | Activity Code: R35 | Study Section: Special Emphasis Panel[ZRG1 CDB-N (55)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11260405