Probing Endocytosis during the cell cycle with minimal experimental perturbation

Endocytosis mediates the cellular uptake of nutrients, modulates signaling by regulating levels of cell surface receptors, and is usurped by pathogens during infection. Endocytosis activity is known to vary during the cell cycle, in particular during mitosis. Importantly, different experimental conditions can lead to opposite results and conclusions, thereby emphasizing the need for a careful design of protocols. For example, experiments using serum-starvation, ice-cold steps or using mitotic arrest produced by chemicals widely used to synchronize cells (nocodazole, RO-3306, or S-trityl-L-cysteine) induce a blockage of clathrin-mediated endocytosis during mitosis not observed in unperturbed, dividing cells. In addition, perturbations produced by mRNA interference or dominant-negative mutant overexpression affect endocytosis long before cells are being assayed. Here, we describe simple experimental procedures to assay endocytosis along the cell cycle with minimal perturbations

Endophilin marks and controls a clathrin-independent endocytic pathway

Endocytosis is required for internalization of micronutrients and turnover of membrane components. Endophilin has been assigned as a component of clathrin-mediated endocytosis. Here we show in mammalian cells that endophilin marks and controls a fast-acting tubulovesicular endocytic pathway that is independent of AP2 and clathrin, activated upon ligand binding to cargo receptors, inhibited by inhibitors of dynamin, Rac, phosphatidylinositol-3-OH kinase, PAK1 and actin polymerization, and activated upon Cdc42 inhibition. This pathway is prominent at the leading edges of cells where phosphatidylinositol-3,4-bisphosphate—produced by the dephosphorylation of phosphatidylinositol-3,4,5-triphosphate by SHIP1 and SHIP2—recruits lamellipodin, which in turn engages endophilin. This pathway mediates the ligand-triggered uptake of several G-protein-coupled receptors such as α2a- and β1-adrenergic, dopaminergic D3 and D4 receptors and muscarinic acetylcholine receptor 4, the receptor tyrosine kinases EGFR, HGFR, VEGFR, PDGFR, NGFR and IGF1R, as well as interleukin-2 receptor. We call this new endocytic route fast endophilin-mediated endocytosis (FEME)