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Zusammenfassung:
The process of autophagy is initiated by phosphatidylinositol 3-phosphate (PtdIns3P) production and dynamic membrane rearrangements that lead to the formation of autophagosomes. WDrepeat protein interacting with phosphoinositides (WIPI) members are essential autophagy-related (ATG) proteins considered to function as PtdIns3P effectors at the nascent autophagosome. Here, we characterized the PtdIns3P-dependent membrane accumulation of WIPI1 (referred to as puncta) at the onset of autophagy. We demonstrate that the induction of autophagy imposed a rapid formation of fluorescent WIPI1 puncta within the first 5 min of stimulation. Delayed in time, WIPI1 puncta formation was accompanied by the formation of large, often perinuclear WIPI1 structures. Both, WIPI1 puncta and large perinuclear WIPI1 structures colocalized with the endoplasmic reticulum (ER) and ATGs considered to function upstream (ATG14L, ATG2A) and downstream of WIPI1 (ATG12, ATG16L, LC3). By quantitative live-cell imaging (>1000 individual cells), we provide evidence that in up to 10% of WIPI1 puncta-positive cells, large perinuclear WIPI1 structures dynamically rearrange into WIPI1/p62-positive ER-arrays that we characterized further. We discuss possible interpretations for the appearance of such WIPI1-positive ER-arrays during autophagy initiation.
