The dynamin family of proteins contains unique GTPases involved in membrane fission and fusion events throughout the cell. Our goal is to understand the dynamic structural properties of these proteins and correlate them with their diverse cellular functions. The founding member, dynamin, is crucial for endocytosis, synaptic membrane recycling, and membrane trafficking within the cell, and is associated with filamentous actin. Mutations in dynamin have been shown to cause the peripheral neuropathy, Charcot-Marie-Tooth disease (CMT), and centronuclear myopathy (CNM). Over the years, structural studies have 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 and twist upon GTP hydrolysis. The ability of dynamin to constrict and generate a force on the underlying lipid bilayer makes it unique among GTPases as a mechanochemical enzyme. Docking crystal structures of dynamin domains into our 3-dimensional maps of dynamin has revealed a conformational change induced by GTP hydrolysis, driven by a large swing in the BSE (GTPase/GED interaction domain) that suggests a mechanism for dynamin constriction. Therefore, the combination of the tension created by dynamin constricting the necks of coated pits followed by its release from the lipid bilayer may be sufficient for spontaneous membrane fission in the cell.
Additional dynamin family members have been implicated in a variety of fundamental cellular processes, including mitochondrial fission and fusion, antiviral activity, plant cell plate formation, and chloroplast biogenesis. Among these proteins, self-assembly and oligomerization into ordered structures is a common characteristic and, for the majority, is essential for their function. Although there is a wealth of information regarding dynamin, less is known about the structural properties of dynamin-related proteins. In recent years, we have examined two dynamin family members, one involved in mitochondria fission (Dnm1) and the other involved in mitochondrial fusion (Opa1). In the cell, healthy mitochondria are maintained by multiple cycles of fusion and fission. A dynamin-related protein (Drp1 in mammals or Dnm1 in yeast) is essential for mitochondrial fission and localizes to sites of mitochondrial division. We solved the structure of Dnm1-lipid tubes by cryo-electron microscopy and in comparison to dynamin, the structure of Dnm1 is significantly larger in diameter and has a minimal attachment to the underlying lipid bilayer. However, similar to dynamin, the Dnm1-lipid tubes constrict from ~120 nm to ~60 nm in diameter upon GTP hydrolysis, followed by a rapid dissociation from the lipid bilayer. These results imply that Dnm1 has the ability to impart a large contractile force at the site of membrane fission during mitochondrial division. The dynamin-related protein OPA1 is involved in the fusion of inner-mitochondrial membranes. Mutations within this protein are a major cause of dominant optic atrophy (DOA) due to the neuropathy of retinal ganglion cells. There exists a short and long form of OPA1 and both are necessary for mitochondrial fusion. We have characterized several mutants of OPA1 associated with DOA and showed that the short form self-assembles around liposomes forming protein-lipid tubes. The OPA1 mutants reveal defects in lipid binding, GTP hydrolysis, and membrane tubulation.
We are just beginning to understand the mechanism of action for dynamin in membrane fission; however, how this protein is regulated in the cell is not clearly understood. In addition, it remains a mystery how dynamin family members are also involved in membrane fusion.