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

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.

发动蛋白三磷酸鸟苷水解酶（GTPases）因其在网格蛋白介导的内吞作用（CME）的终末膜剪切过程中的功能而被广泛研究，但现有研究也表明其可调控CME的早期阶段。尽管发动蛋白-1（Dyn1）在神经元中富集度极高，但实际上它与Dyn2广泛共表达，不过在非神经元细胞中会被糖原合成激酶3β（GSK3β）磷酸化而失活。本研究聚焦于Dyn1与Dyn2作为CME调控因子的亚型特异性差异功能。我们在两种Dyn1表达水平迥异的细胞系中，对内源表达的Dyn1与Dyn2分别或共同进行荧光标记。通过定量活细胞双通道及三通道全内反射荧光显微镜成像，我们发现Dyn2相较于Dyn1可更高效地被招募至网格蛋白包被小窝（CCPs）；且仅Dyn2而非Dyn1会呈现显著的组装爆发现象，推测其组装为超分子环状结构，进而驱动膜剪切与网格蛋白包被囊泡（CCV）的形成。通过急性抑制GSK3β激活Dyn1，可加快转铁蛋白受体的内吞速率、提升CCP的起始频率并缩短CCP的存活时间，但不会显著影响Dyn1向CCPs的招募程度。因此，即使相较于Dyn2处于低水平的亚化学计量比状态，激活后的Dyn1仍可调控剪切事件上游的CME早期阶段。在生理条件下，Dyn1可通过表皮生长因子受体（EGFR）信号通路的下游途径被激活，进而改变CCP的动态变化过程。我们鉴定出分选连接蛋白9（SNX9）是激活态Dyn1的优选结合伴侣，且该蛋白部分介导了Dyn1对CCP成熟早期阶段的调控作用。综上，本研究解耦了发动蛋白的调控功能与剪切功能，并揭示了Dyn1在CME中发挥不依赖于剪切的亚型特异性调控功能。