Abstract

Formation and fission of tubules from lysosomal organelles, such as autolysosomes, endolysosomes or phagolysosomes, are required for lysosome reformation. However, the mechanisms governing these processes in the different forms of lysosomal organelles are poorly understood. For instance, the role of phosphatidylinositol-4-phosphate (PI(4)P) is unclear as it was shown to promote the formation of tubules from phagolysosomes but was proposed to inhibit tubule formation on autolysosomes because the loss of PI4KIII{beta} causes extensive lysosomal tubulations. Using super-resolution live-cell imaging, we show that Arf1-PI4KIII{beta} positive vesicles are recruited to tubule fission sites from autolysosomes, endolysosomes and phagolysosomes. Moreover, we show that PI(4)P is required to form autolysosomal tubules and that increased lysosomal tubulation caused by loss of PI4KIII{beta} represents impaired tubule fission. At the site of fission, we propose that Arf1-PI4KIII{beta} positive vesicles mediate a PI(3)P signal on lysosomes in a process requiring the lipid transfer protein SEC14L2. Our findings indicate that Arf1-PI4KIII{beta} positive vesicles and their regulation of PI(3)P are critical components of the lysosomal tubule fission machinery.