Structure And Function Of Dynamin, A 100kd GTPase Involved In Endocytosis

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

The dynamin family of proteins consists of unique GTPases involved in membrane fission and fusion events throughout the cell. The founding member, dynamin, is crucial for endocytosis, synaptic membrane recycling, membrane trafficking within the cell, and is associated with filamentous actin. Dynamin was first implicated in endocytosis when it was discovered to be the mammalian homologue of the shibire gene product in Drosophila. A temperature sensitive shibire allele causes a defect in clathrin-mediated endocytosis. Since then, overexpressing human dynamin mutants in mammalian cells was found to block clathrin-mediated endocytosis. Over the years, our cryo-electron microscopy (cryo-EM) structural work has played a leading role in dissecting the function of dynamin in membrane fission. We have shown that purified dynamin readily assembles into rings and spirals and forms similar structures on liposomes, generating dynamin-lipid tubes that constrict upon GTP hydrolysis. A potential mechanism for dynamin constriction was revealed when we solved the three-dimensional structure of dynamin in the non-constricted and constricted states by cryo-electron microscopy (cryo-EM). These results suggest dynamin wraps around the necks of budding vesicles as a helical polymer and upon GTP hydrolysis undergoes a significant constriction that ultimately leads to membrane fission. In 2018, we solved the first high-resolution cryo-EM structure of the membrane-associated helical polymer of human dynamin-1 in the GTP-bound state (3.75 Å; Kong et al, Nature, 2018). The dynamin helical structure allowed us to build an atomic model of the assembled dynamin polymer bound to lipid. Comparing soluble crystal structures to our new high-resolution cryo-EM structure revealed conformational changes that occur upon assembly and lipid binding. The structure defines the 1-start helical symmetry of the dynamin polymer and the positions of its oligomeric interfaces, which were validated by cell-based endocytosis assays in collaboration with Dr. Justin Taraska, NHLBI. The inner lumen of the dynamin-lipid tube is ~7 nm compared to ~20 nm observed in the apo state. In 2020, we examined the speed of dynamin constriction using a novel time-resolved machine at the New York Structural Biology Center. For this collaboration with Drs. Carragher and Potter, we sprayed dynamin tubes and GTP simultaneously onto grids and froze the sample in the milli-second range and found dynamin undergoes constriction and falls off the lipid within 150 ms (Dandey et al, Nat Methods, 2020). Compared to previous results that showed dynamin constricted within seconds using the traditional freezing devices, we now have a tighter timeline for dynamin-mediated constriction and fission during endocytosis. In the past few years, we solved two structures of a GTPase-defective dynamin mutant (K44A), full-length and delta-PRD, in the presence of GTP, to 3.6 Å resolution (Jimah, Kundu et al, Dev Cell, 2024), which provided evidence for a model of dynamin-mediated membrane constriction (Sundborger et al, Cell Rep, 2014). The high-resolution cryo-EM density indicates dynamin is in the GDP-bound state, further constricts the underlying lipid bilayer to achieve an inner lumen of 3.4 nm and assembles as a 2-start helix. Constriction of the membrane to 3.4 nm lumen is reaching the theoretical limit required for spontaneous membrane fission, supporting the model that dynamin alone can cause membrane fission. The K44A structure also reveals how a 2-start helical symmetry promotes the most efficient packing of dynamin tetramers around the membrane neck. In 2024, we contributed to two successful collaborations. For the first collaboration with Dr. Justin Taraska (NHLBI), funded by a Chan Zuckerberg grant, we developed a and characterized a high-resolution correlative cryo-electron tomography (cryo-ET) pipeline method to examine the plasma membrane (Sun, Michalak, Sochacki et al, Nat Commun, 2025). The pipeline provides fast, efficient, distributable, and low-cost sample preparation. With this method we localized dynamin helical structures on the plasma membrane in cells expressing the K44A mutant (Jimah et al, Dev Cell, 2024). Our pipeline also employs a genetically-encodable rapid chemically-induced electron microscopy visible tag for marking specific proteins within the complex cell environment. This tag allowed us to locate the clathrin associated proteins Hip1R and clathrin light chain. Overall, our method allows for plasma membrane-associated proteins such as ion channels, coats, receptors, signaling proteins, and cytoskeletal elements to be unambiguously identified for structural studies within their native biological context by cryo-ET. In the second collaboration with Dr. Ling-Gang Wu (NINDS), we demonstrate phosphatidylinositol-4-phosphate (PI4P) promotes dynamin-mediated constriction sufficient for fission/fusion pore closure, whereas phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] hinders dynamin from producing efficient constriction and thus inhibits fission/fusion pore closure (Guo, Wei, Kundu et al, under review). The control of pore closure by PI4P and PI(4,5)P2 regulates diverse endocytic modes in the synapse (ultrafast, fast, slow, overshoot, and bulk endocytosis) and every step of the fusion pathway (hemi-fusion, fusion pore opening, expansion, constriction, closure). These findings reveal how phospholipids initiate, mediate and control fission, endocytosis and fusion, and may have widespread impact on other fusion and fission pathways since phospholipids and dynamin family proteins are universally present in cells.