ASSESSING MAMMALIAN AUTOPHAGY BY WIPI-1/ATG18 PUNCTA FORMATION

Macroautophagy (autophagy) is an evolutionarily highly conserved self-digestive mechanism that secures eukaryotic cellular homeostasis. Importantly, this process of intracellular bulk degradation is tightly regulated to prevent pathological consequences of disbalanced autophagic activity such as tumor development, neurodegeneration, myopathies and heart disease. A hallmark of the process of autophagy is the generation of autophagosomes, unique vesicles with double membranes of unknown origin and composition. Required for autophagosome formation is the delivery of phospholipids, such as the phosphoinositide phsophatidylinositol-3-phosphate (PI(3)P) to phagophore assembly sites (PAS). We identified the human WIPI family of 7-bladed beta-propeller proteins and found that WIPI-1 (Atg18) functions as a novel PI(3)P scaffold at the onset of autophagy. Upon binding to PI(3)P, the WIPI-1 protein accumulation at the phagophore (WIPI-1 puncta formation) can be visualized by fluorescence microscopy. Quantification of WIPI-1 puncta formation is suitable to analyze basal and induced or inhibited levels of phagophore formation, thereby assessing mammalian autophagy. Here we present an experimental step-to-step guide for assaying WIPI-1 puncta formation in human cells by confocal microscopy, live-cell imaging, and phosphoinositide binding.