Flower initiates a positive feedback loop upon PIP2 enrichment at periactive zones to control bulk endocytosis

Synaptic vesicle (SV) endocytosis is coupled to exocytosis to maintain SV pool size and thus neurotransmitter release. Intense stimulation induces activity-dependent bulk endocytosis (ADBE) to recapture large quantities of SV constituents in large endosomes from which SVs reform. How these consecutive processes are spatiotemporally coordinated remains unknown. Here, we show that the Flower Ca2+ channel-dependent phosphatidylinositol 4,5-bisphosphate (PIP2) compartmentalization governs such control. Strong stimuli trigger PIP2 microdomain formation at periactive zones. Upon exocytosis Flower translocates from SVs to periactive zones, where it increases PIP2 levels via Ca2+ influxes. Remarkably, PIP2 directly enhances Flower channel activity, thereby establishing a positive feedback loop for PIP2 microdomain compartmentalization. The PIP2 microdomains drive ADBE and SV reformation from bulk endosomes. PIP2 further sorts Flower to bulk endosomes, thereby terminating endocytosis. Hence, we propose that the interplay between Flower and PIP2 is the crucial spatiotemporal cue that couples exocytosis to ADBE and subsequent SV reformation.