Clathrin-mediated endocytosis (CME) is the major endocytic pathway in mammalian cells. It is responsible for the uptake of transmembrane receptors and transporters, for remodeling plasma membrane composition in response to environmental changes, and for regulating cell surface signaling. CME occurs via the assembly and maturation of clathrin-coated pits that concentrate cargo as they invaginate and pinch off to form clathrin-coated vesicles. In addition to the major coat proteins, clathrin triskelia and adaptor protein complexes, CME requires a myriad of endocytic accessory proteins and phosphatidylinositol lipids. CME is regulated at multiple steps-initiation, cargo selection, maturation, and fission-and is monitored by an endocytic checkpoint that induces disassembly of defective pits. Regulation occurs via posttranslational modifications, allosteric conformational changes, and isoform and splice-variant differences among components of the CME machinery, including the GTPase dynamin. This review summarizes recent findings on the regulation of CME and the evolution of this complex process.

Clathrin-mediated endocytosis (CME) regulates signaling from the plasma membrane. Analysis of clathrin-coated pit (CCP) dynamics led us to propose the existence of a rate-limiting, regulatory step(s) that monitor the fidelity of early stages in CCP maturation. Here we show that nascent endocytic vesicles formed in mutant cells displaying rapid, dysregulated CME are defective in early endosomal trafficking, maturation and acidification, confirming the importance of this "checkpoint." Dysregulated CME also alters EGF receptor signaling and leads to constitutive activation of the protein kinase Akt. Dynamin-1, which was thought to be neuron specific, is activated by the Akt/GSK3b signaling cascade in nonneuronal cells to trigger rapid, dysregulated CME. Acute activation of dynamin-1 in RPE cells by inhibition of GSK3b accelerates CME, alters CCP dynamics and, unexpectedly, increases the rate of CCP initiation. CRISPR-Cas9n-mediated knockout and reconstitution studies establish that dynamin-1 is activated by Akt/GSK3b signaling in H1299 non-small lung cancer cells. These findings provide direct evidence for an isoform-specific role for dynamin in regulating CME and reveal a feed-forward pathway that could link signaling from cell surface receptors to the regulation of CME.

Protein aggregation is a hallmark of many diseases, including amyotrophic lateral sclerosis (ALS), where aggregation of Cu/Zn superoxide dismutase (SOD1) is implicated in causing neurodegeneration. Recent studies have suggested that destabilization and aggregation of the most immature form of SOD1, the disulfide-reduced, unmetallated (apo) protein is particularly important in causing ALS. We report herein in depth analyses of the effects of chemically and structurally diverse ALS-associated mutations on the stability and aggregation of reduced apo SOD1. In contrast with previous studies, we find that various reduced apo SOD1 mutants undergo highly reversible thermal denaturation with little aggregation, enabling quantitative thermodynamic stability analyses. In the absence of ALS-associated mutations, reduced apo SOD1 is marginally stable but predominantly folded. Mutations generally result in slight decreases to substantial increases in the fraction of unfolded protein. Calorimetry, ultracentrifugation, and light scattering show that all mutations enhance aggregation propensity, with the effects varying widely, from subtle increases in most cases, to pronounced formation of 40–100 nm soluble aggregates by A4V, a mutation that is associated with particularly short disease duration. Interestingly, although there is a correlation between observed aggregation and stability, there is minimal to no correlation between observed aggregation, predicted aggregation propensity, and disease characteristics. These findings suggest that reduced apo SOD1 does not play a dominant role in modulating disease. Rather, additional and/or multiple forms of SOD1 and additional biophysical and biological factors are needed to account for the toxicity of mutant SOD1 in ALS.

Membrane scission is a crucial step in all budding processes, from endocytosis to viral budding. Many proteins have been associated with scission, though the underlying molecular details of how scission is accomplished often remain unknown. Here, we investigate the process of M2-mediated membrane scission during the budding of influenza viruses. Residues 50–61 of the viral M2 protein bind membrane and form an amphipathic α-helix (AH). Membrane binding requires hydrophobic interactions with the lipid tails but not charged interactions with the lipid headgroups. Upon binding, the M2AH induces membrane curvature and lipid ordering, constricting and destabilizing the membrane neck, causing scission. We further show that AHs in the cellular proteins Arf1 and Epsin1 behave in a similar manner. Together, they represent a class of membrane-induced AH domains that alter membrane curvature and fluidity, mediating the scission of constricted membrane necks in multiple biological pathways.

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