Inactivation of V-ATPase induces autophagy and ectopic Notch activation to promote intestinal stem cell differentiation in adult <i>drosophila</i> midgut

The self-renewal and differentiation of stem cells are tightly controlled to maintain tissue homeostasis. Failure in stem cell maintenance results in stem cell depletion and precocious aging. However, how stem cells are maintained still remains not fully understood. Here, through a large-scale RNAi screen for maintenance and proliferation of adult Drosophila intestinal stem cells (ISCs), we identify several subunits of V-ATPase including Vha68-2/ATP6V1A, required for ISC proliferation, differentiation and tissue regeneration. Inactivation of Vha68-2 results in accumulation of plasma membrane (PM)-derived structures in autophagosomes and lysosomes through Atg16 and Rab5. Furthermore, Vha68-2-defective ISCs undergo direct differentiation due to ectopic Notch activation. Mechanistically, Vha68-2 facilitates Notch receptor internalization and subsequent degradation in autolysosomes to quench ectopic Notch activation, thereby maintaining ISC fate. Truncated Notch product devoid of its extracellular domain is accumulated in autolysosomes upon Vha68-2 deficiency. The Notch products accumulated in autolysosomes still require further cleavage to drive ISC differentiation. The functions of Vha68-2 in macroautophagy/autophagy and ISC maintenance are evolutionarily conserved. Our results provide new insights into the underlying mechanism of how autophagy is involved in stem cell maintenance under physiological conditions.