Phosphorylation of clathrin light chains (CLCs) regulates GPCR uptake but is dispensable for transferrin internalization. Maib et al. show that CLCb phosphorylation is required for efficient auxilin-mediated clathrin exchange to promote coated pit invagination in a cargo-specific manner.

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Polarized trafficking is necessary for the development of eukaryotes and is regulated by a conserved molecular machinery. Late steps of cargo delivery are mediated by the exocyst complex, which integrates lipid and protein components to tether vesicles for plasma membrane fusion. However, the molecular mechanisms of this process are poorly defined. Here, we reconstitute functional octameric human exocyst, demonstrating the basis for holocomplex coalescence and biochemically stable subcomplexes. We determine that each subcomplex independently binds to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), which is minimally sufficient for membrane tethering. Through reconstitution and epithelial cell biology experiments, we show that Arf6-mediated recruitment of the lipid kinase PIP5K1C rapidly converts phosphatidylinositol 4-phosphate (PI(4)P) to PI(4,5)P2, driving exocyst recruitment and membrane tethering. These results provide a molecular mechanism of exocyst-mediated tethering and a unique functional requirement for phosphoinositide signaling on late-stage vesicles in the vicinity of the plasma membrane.

Clathrin-mediated endocytosis (CME) is a fundamental process in cell biology and has been extensively investigated over the past several decades. Every cell biologist learns about it at some point during his or her education, and the beauty of this process has led many of us to go deeper and make it the topic of our research. Great progress has been made toward elucidating the mechanisms of CME, and the field is becoming increasingly complex, with several hundred new publications every year. This makes it easy to get lost in the vast amount of literature and forget about the fundamentals of the field, which are based on the careful interpretation of simple observations made >40 years ago, as exemplified by a study performed by Anderson, Brown, and Goldstein in 1977. We examine how this seminal study was pivotal to our understanding of CME and its progression into ever-increasing complexity over the past four decades.

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Phosphoinositide signaling lipids (PIPs) are key regulators of membrane identity and trafficking. Of these, PI(3,5)P2 is one of the least well-understood, despite key roles in many endocytic pathways including phagocytosis and macropinocytosis. PI(3,5)P2 is generated by the phosphoinositide 5-kinase PIKfyve, which is critical for phagosomal digestion and antimicrobial activity. However PI(3,5)P2 dynamics and regulation remain unclear due to lack of reliable reporters. Using the amoeba Dictyostelium discoideum, we identify SnxA as a highly selective PI(3,5)P2-binding protein and characterize its use as a reporter for PI(3,5)P2 in both Dictyostelium and mammalian cells. Using GFP-SnxA, we demonstrate that Dictyostelium phagosomes and macropinosomes accumulate PI(3,5)P2 3 min after engulfment but are then retained differently, indicating pathway-specific regulation. We further find that PIKfyve recruitment and activity are separable and that PIKfyve activation stimulates its own dissociation. SnxA is therefore a new tool for reporting PI(3,5)P2 in live cells that reveals key mechanistic details of the role and regulation of PIKfyve/PI(3,5)P2.

Phosphoinositide signalling lipids (PIPs) are key regulators of membrane identity and vesicle trafficking. Of these, PI(3,5)P2 is one of the least understood, despite key roles in many endocytic pathways including phagocytosis and macropinocytosis. PI(3,5)P2 is predominantly generated by the phosphoinositide 5-kinase PIKfyve, and is critical for phagosomal digestion and the killing of engulfed microbes. However, the regulation of PIKfyve activity, PI(3,5)P2 dynamics, and how they control phagosome maturation remains unclear. Using the model professional phagocyte Dictyostelium discoideum we have identified a highly selective PI(3,5)P2-binding protein that allows faithful observation of PI(3,5)P2 dynamics in live cells. Using this probe we demonstrate that PIKfyve recruitment and activity are separable, and that PIKfyve activation stimulates its own dissociation from membranes. We show that PI(3,5)P2 accumulates on phagosomes 2-3 minutes after engulfment drives fusion of a specific population of PI(3,5)P2 and Rab7-positive macropinosomes. We find PIKfyve is required for the fusion between macropinosomes and phagosomes, which enables phagosomes to efficiently accumulate Rab7 and other components of the lysosomal machinery. These findings uncover key mechanistic details of the role and regulation of PIKfyve/PI(3,5)P2 likely to have general relevance across endocytic pathways.

Epidermal growth factor receptor (EGFR) signalling results in a variety of cell behaviours, including cell proliferation, migration and apoptosis, which depend on cell context. Here we have explored how the Rab5GEF, Rme-6, regulates EGFR signalling by modulating endocytic flux. We demonstrate that Rme-6, which acts early in the endocytic pathway, regulates EGFR trafficking through an endocytic compartment that is competent for ERK1/2 signalling. While overexpression of Rme-6 results in enhanced ERK1/2 nuclear localisation and c-Fos activation, loss of Rme-6 results in aberrant ERK1/2 signalling with increased cytoplasmic ERK1/2 phosphorylation (Thr202/Tyr204) but decreased ERK1/2 nuclear translocation and c-Fos activation, the latter leading to decreased cell proliferation. Phosphorylation of ERK1/2 by protein kinase 2 (CK2) is required for its nuclear translocation and our data support a model whereby Rme-6 provides a scaffold for a population of CK2 which is required for efficient nuclear translocation of ERK1/2. Rme-6 is itself a substrate for CK2 on Thr642 and Ser996 and phosphorylation on these sites can activate its Rab5GEF activity and endocytic trafficking of EGFR. Together our results indicate that that Rme-6 co-ordinates EGFR trafficking and signalling to regulate the assembly and disassembly of an ERK1/2 signalosome.