A noncanonical role for dynamin-1 in regulating early stages of clathrin-mediated endocytosis in non-neuronal cells

摘要: Dynamin Guanosine Triphosphate hydrolases (GTPases) are best studied for their role in the terminal membrane fission process of clathrin-mediated endocytosis (CME), but they have also been proposed to regulate earlier stages CME. Although highly enriched neurons, dynamin-1 (Dyn1) is, fact, widely expressed along with Dyn2 inactivated non-neuronal cells via phosphorylation by glycogen synthase kinase-3 beta (GSK3β) kinase. Here, we study differential, isoform-specific functions Dyn1 and as regulators Endogenously were fluorescently tagged either separately or together two cell lines contrasting expression levels. By quantitative live dual- triple-channel total internal reflection fluorescence microscopy, find that is more efficiently recruited clathrin-coated pits (CCPs) than Dyn1, not exhibits a pronounced burst assembly, presumably into supramolecular collar-like structures drive scission vesicle (CCV) formation. Activation acute inhibition GSK3β results rapid transferrin receptors, increased rates CCP initiation, decreased lifetimes did significantly affect extent recruitment CCPs. Thus, activated can early CME occur well upstream fission, even when present at low, substoichiometric levels relative Dyn2. Under physiological conditions, downstream epidermal growth factor receptor (EGFR) signaling alter dynamics. We identify sorting nexin 9 (SNX9) preferred binding partner partially required Dyn1-dependent effects on maturation. Together, decouple regulatory dynamins report scission-independent,