A new pH-sensitive red fluorescent protein called pHuji, in combination with green fluorescent superecliptic pHluorin, allows two-color detection of endocytic events in live cells.

Endocytosis in neuronal dendrites is known to play a critical role in synaptic transmission and plasticity such as long-term depression (LTD). However, the inability to detect endocytosis directly in living neurons has hampered studies of its dynamics and regulation. Here, we visualized the formation of individual endocytic vesicles containing pHluorin-tagged receptors with high temporal resolution in the dendrites of cultured hippocampal neurons. We show that transferrin receptors (TfRs) are constitutively internalized at optically static clathrin-coated structures. These structures are slightly enriched near synapses that represent preferential sites for the endocytosis of postsynaptic AMPA-type receptors (AMPARs), but not for non-synaptic TfRs. Moreover, the frequency of AMPAR endocytosis events increases after the induction of NMDAR-dependent chemical LTD, but the activity of perisynaptic endocytic zones is not differentially regulated. We conclude that endocytosis is a highly dynamic and stereotyped process that internalizes receptors in precisely localized endocytic zones.

Phosphoinositide (PtdIns) homeostasis requires a tight spatial and temporal regulation during the endocytic process [1]. Indeed, PtdIns(4,5)P2 plays a crucial role in endocytosis by controlling clathrin-coated pit formation, whereas its conversion into PtdIns4P right after scission of clathrin-coated vesicles (CCVs) is essential for successful uncoating and cargo sorting [1-6]. In non-neuronal cells, endosomal PtdIns(4,5)P2 hydrolysis critically relies on the lipid phosphatase OCRL [7-9], the inactivation of which causes the Oculo-Cerebro-Renal syndrome of Lowe [10, 11]. To understand the coupling between PtdIns(4,5)P2 hydrolysis and endosome formation, a key issue is thus to unravel the mechanism by which OCRL is recruited on CCVs precisely after their scission from the plasma membrane. Here we found that the Rab35 GTPase, which plays a fundamental but poorly understood role in endosomal trafficking after cargo internalization [12-21], directly recruits the OCRL phosphatase immediately after scission of the CCVs. Consistent with Rab35 and OCRL acting together, depletion of either Rab35 or OCRL leads to retention of internalized receptors such as the endogenous cation-independent mannose-6-phosphate receptor (CI-MPR) in peripheral clathrin-positive endosomes that display abnormal association with PtdIns(4,5)P2- and actin-binding proteins. Remarkably, Rab35 loading on CCVs rapidly follows the recruitment of the AP2-binding Rab35 GEF/activator DENND1A (connecdenn 1) and the disappearance of the Rab35 GAP/inhibitor EPI64B. We propose that the precise spatial and temporal activation of Rab35 acts as a major switch for OCRL recruitment on newborn endosomes, post-scission PtdIns(4,5)P2 hydrolysis, and subsequent endosomal trafficking.