Three-dimensional reconstruction of dynamin in the constricted state.

Members of the dynamin family of GTPases have unique structural properties that might reveal a general mechanochemical basis for membrane constriction. Receptor-mediated endocytosis, caveolae internalization and certain trafficking events in the Golgi all require dynamin for vesiculation. The dynamin-related protein Drp1 (Dlp1) has been implicated in mitochondria fission and a plant dynamin-like protein phragmoplastin is involved in the vesicular events leading to cell wall formation. A common theme among these proteins is their ability to self-assemble into spirals and their localization to areas of membrane fission. Here we present the first three-dimensional structure of dynamin at a resolution of approximately 20 A, determined from cryo-electron micrographs of tubular crystals in the constricted state. The map reveals a T-shaped dimer consisting of three prominent densities: leg, stalk and head. The structure suggests that the dense stalk and head regions rearrange when GTP is added, a rearrangement that generates a force on the underlying lipid bilayer and thereby leads to membrane constriction. These results indicate that dynamin is a force-generating 'contrictase'.

Dynamin family of mechanoenzymes.

The dynamin family of proteins is continually growing, and in recent years members have been localized to areas of mitochondrial fission, plant phragmoplasts and chloroplasts, and viral ribonucleoprotein complexes. All the dynamin-like proteins examined to-date appear to assemble into oligomers, such as rings or spirals; however, it remains to be determined if a global mechanism of action exists. Even the role of dynamin in vesicle formation remains controversial as to whether it behaves as a molecular switch or as a mechanochemical enzyme.

Dynamin and its role in membrane fission.

Dynamin, a 100-kDa GTPase, is an essential component of vesicle formation in receptor-mediated endocytosis, synaptic vesicle recycling, caveolae internalization, and possibly vesicle trafficking in and out of the Golgi. In addition to the GTPase domain, dynamin also contains a pleckstrin homology domain (PH) implicated in membrane binding, a GTPase effector domain (GED) shown to be essential for self-assembly and stimulated GTPase activity, and a C-terminal proline-rich domain (PRD), which contains several SH3-binding sites. Dynamin partners bind to the PRD and may either stimulate dynamin's GTPase activity or target dynamin to the plasma membrane. Purified dynamin readily self-assembles into rings or spirals. This striking structural property supports the hypothesis that dynamin wraps around the necks of budding vesicles where it plays a key role in membrane fission. The focus of this review is on the relationship between the GTPase and self-assembly properties of dynamin and its cellular function.

Dynamin spirals.

Dynamin is an important component of membrane recycling at the plasma membrane and, potentially, within the cell. The role of dynamin in clathrin-mediated endocytosis has been based on numerous endocytosis assays, as well as on the discovery and gross characterization of the assembled spiral structure of dynamin. Recently, it has been shown that dynamin can also bind to liposomes and form helical tubes that constrict and vesiculate upon GTP addition. This suggests that dynamin is capable of and may be responsible for the pinching off of clathrin-coated vesicles from the plasma membrane during clathrin-mediated endocytosis.

Dynamin undergoes a GTP-dependent conformational change causing vesiculation.

The dynamin family of GTPases is essential for receptor-mediated endocytosis and synaptic vesicle recycling, and it has recently been shown to play a role in vesicle formation from the trans-Golgi network. Dynamin is believed to assemble around the necks of clathrin-coated pits and assist in pinching vesicles from the plasma membrane. This role would make dynamin unique among GTPases in its ability to act as a mechanochemical enzyme. Data presented here demonstrate that purified recombinant dynamin binds to a lipid bilayer in a regular pattern to form helical tubes that constrict and vesiculate upon GTP addition. This suggests that dynamin alone is sufficient for the formation of constricted necks of coated pits and supports the hypothesis that dynamin is the force-generating molecule responsible for membrane fission.

Dynamin assembles into spirals under physiological salt conditions upon the addition of GDP and gamma-phosphate analogues.

Dynamin is a 100-kDa GTPase that is believed to be involved in the constriction of clathrin-coated pits and the fission of clathrin-coated vesicles during receptor-mediated endocytosis and during membrane retrieval in nerve termini. It has been shown that purified dynamin incubated under low salt conditions forms rings and spirals that, in dimension and appearance, resemble the dense material occasionally observed at the necks of coated pits. In this report we show that purified dynamin forms spirals under physiological salt conditions when incubated with GDP and gamma-phosphate analogues (beryllium and aluminum fluoride) or when dialyzed into guanosine 5'-3-O-(thio)triphosphate. Moreover, spirals still form when dynamin is proteolyzed to either a predominant approximately 90-kDa species, lacking the C terminus, or to two smaller fragments, a approximately 55-kDa species originating from the N-terminal half of the protein and a approximately 30-kDa species lacking both the N and C termini. This work indicates that the addition of GDP and gamma-phosphate analogues arrests dynamin in a GTP or transition state that markedly stabilizes the spiral conformation under physiological ionic strength conditions and thereby suggests that dynamin in the absence of a receptor is capable of assembly into spirals at the necks of coated pits prior to vesicle fission.

Dynamin self-assembly stimulates its GTPase activity.

GTP hydrolysis by dynamin is required to drive coated vesicle budding at the plasma membrane. A diverse set of molecules including microtubules, grb2, and acidic phospholipids stimulate dynamin GTPase activity in vitro, although the physiological relevance of these effectors remains to be determined. Dynamin has been shown to assemble around microtubules, the most potent stimulatory molecule, into structures indistinguishable by electron microscopy from collars captured in vivo at the necks of endocytic coated pits. Under low ionic strength conditions purified dynamin self-assembles into rings and helical stacks of rings. Here we show that dynamin self-assembly stimulates its GTPase activity as much as 10-fold. Thus, we identify dynamin, itself, as the first effector of dynamin GTPase activity known to be physiologically relevant. Assembled dynamin's stimulated GTPase activity is not dependent on the direct interaction of high affinity GTP binding sites since a mutant defective in GTP binding and hydrolysis can coassemble with and stimulate GTP hydrolysis by wild-type dynamin. Finally, we find that GTP destabilizes assembled dynamin structures, suggesting that the activated rates of GTP hydrolysis reflect a continuing cycle of assembly, GTP hydrolysis, and disassembly.

Dynamin self-assembles into rings suggesting a mechanism for coated vesicle budding.

DYNAMIN, a 100K member of the GTPase superfamily, is the mammalian homologue of the Drosophila shibire gene product. Mutations in shibire cause a defect in endocytosis leading to accumulation of coated pits and deep invaginations at the plasma membrane of all tissues examined. Similarly, invaginated coated pits accumulate in mammalian cells overexpressing dominant-negative mutants of dynamin, establishing that dynamin is required for the formation of 'constricted' coated pits and for coated vesicle budding. Whether dynamin functions in the classic GTPase mode as a molecular switch to regulate events leading to coated vesicle budding or instead actively participates as a mechanochemical enzyme driving coated vesicle formation is unclear. Here we show that dynamin spontaneously self-assembles into rings and stacks of interconnected rings, comparable in dimension to the 'collars' observed at the necks of invaginated coated pits that accumulate at synaptic terminals in shibire flies. We propose that invaginated coated pits become constricted by the assembly of dynamin into rings around their necks. A concerted conformational change would then close the rings and pinch off the budding coated vesicles.

Architecture of the nuclear pore complex and its involvement in nucleocytoplasmic transport.

Recent structural analyses of the nuclear pore complex (NPC) have described in some detail the numerous sub-domains which make up this supramolecular assembly. Three dimensional image analysis of detergent-extracted NPCs reveals that the NPC framework is made up of spoke units, each containing four major domains, arranged with 822 symmetry. As shown by freeze-drying/metal shadowing techniques, attached to this framework are several peripheral components including particles and fibers on the cytoplasmic face and a cage-like structure on the nucleoplasmic face. While it is known that traffic between cytoplasm and nucleus occurs exclusively through the nuclear pore, the specifics of NPC involvement in such traffic remain unknown at present. Proteins destined for the nucleus contain nuclear localization sequences (NLSs). Several proteins have been identified which bind to these NLSs and may act to direct these proteins to the NPC, either releasing them prior to or remaining attached during translocation through the NPC. These NLS-binding proteins have been localized to the cytoplasm, nucleus, nuclear envelope and nucleolus, suggesting some of these proteins transverse the NPC bound or unbound to NLS-containing proteins.

Mapping the lateral distribution of photosystem II and the cytochrome b6/f complex by direct immune labeling of the thylakoid membrane.

By direct immunolabeling we have mapped the distribution of photosystem II (PS II) and cytochrome b6/f on the surfaces of photosynthetic membranes isolated from spinach. Photosynthetic membranes were attached to a support and gently disrupted to expose the occluded outer stacked surface, prior to labeling. Polyclonal antibodies against PS II intensely labeled the outer stacked surfaces while the outer nonstacked surface had minimal labeling. This confirms previous fractionation and immunolocalization studies which demonstrated that PS II is largely restricted to the stacked regions of the membrane. Inside-out membranes were also heavily labeled with PS II antibodies. Antibodies against cytochrome f were evenly distributed between the stacked and nonstacked outer surfaces and were found clumped together on the membrane outer surface. Previous fractionation and immunolocalization studies have indicated that cytochrome b6/f is located in both the stacked and nonstacked regions, but this is the first report to provide direct evidence that the complex may be clustered in the membrane. The clustering of antibodies to cytochrome b6/f supports the idea that this electron transport component exists as a multimeric complex within the membranes and that such complexes are found in both stacked and nonstacked regions of the photosynthetic membrane. No evidence was seen of any special differentiation of the marginal regions of the membrane, which link stacked and nonstacked regions.

Programs for visualization in three-dimensional microscopy.

Three-dimensional data representing biological structures can be derived using several methods, including serial section reconstruction, optical sectioning, and tomography. The investigation, comprehension, and communication of structural relationships to others is greatly facilitated by computer-based visualization procedures. We describe SYNU, a suite of programs developed for interactive investigation of three-dimensional structure and for the production of high-quality three-dimensional images and animations. We illustrate the capabilities of SYNU in applications to biological data obtained by confocal light microscopy, serial section, and high-resolution electron microscopy from investigations at the cellular, subcellular, and molecular levels.

Architecture and design of the nuclear pore complex.

A three-dimensional analysis of the nuclear pore complex reveals the underlying, highly symmetric framework of this supramolecular assembly, how it is anchored in the nuclear membrane, and how it is built from many distinct, interconnected subunits. The arrangement of the subunits within the membrane pore creates a large central channel, through which active nucleocytoplasmic transport is known to occur, and eight smaller peripheral channels that are probable routes for passive diffusion of ions and small molecules.

Localization of light-harvesting complex II to the occluded surfaces of photosynthetic membranes.

The photosynthetic membranes of green plants are organized into stacked regions interconnected by nonstacked regions that have been shown to be biochemically and structurally distinct. Because the stacking process occludes the surfaces of appressed membranes, it has been impossible to conduct structural or biochemical studies of the outer surfaces of the photosynthetic membrane in regions of membrane stacking. Although stacking is mediated at this surface, it has not been possible to determine whether membrane components implicated in the stacking process, including a major light-harvesting complex (LHC-II), are in fact exposed at the membrane surface. We have been able to expose this surface for study in the electron microscope and directly label it with antibodies to determine protein exposure. The appearance of the newly exposed outer stacked surface highlights the extreme lateral heterogeneity of the photosynthetic membrane. The surface is smooth in contrast to the neighboring nonstacked surface that is covered with distinct particles. Although some investigators have suggested the existence of a cytochrome b6/f-rich boundary region between stacked and nonstacked membranes, our results provide no structural support for this concept. To explore the biochemical nature of the occluded membrane surface, we have used an mAb against the amino terminal region of the LHC-II. This mAb clearly labels the newly exposed outer stacked surface but does not label the inner surface or the outer nonstacked surface. These experimental results confirm the presence of the amino terminal region of this complex at the outer surface of the membrane in stacked regions, and also show that this complex is largely absent from nonstacked membranes.

Chylomicron metabolism by normal and injured isolated rat lungs.

Since the lung is the first highly vascularized organ to which chylomicrons are exposed, we sought to determine whether the lung vasculature is capable of metabolizing triglyceride contained in circulating, native chylomicrons. In addition, since acute lung injury can depress other endothelial cell associated metabolic functions, we determined whether acute injury due to alpha-naphthylthiourea (ANTU) changed chylomicron triglyceride metabolism by lungs. We compared the hydrolysis of radiolabelled chylomicrons from rat mesenteric lymph by perfused lungs isolated from rats pretreated with ANTU; with the vehicle, Tween 80, alone; or untreated control rats. In all groups of lungs, we found that perfusate content and concentration of triglyceride decreased over 30 minutes of perfusion, while that of free fatty acid increased, indicating that isolated lungs are able to hydrolyze chylomicron triglyceride. Despite enhancement of hydrolysis by perfusates containing 6 gm/100 ml of bovine serum albumin, there were no differences among the groups of lungs in the extent or rate of triglyceride metabolism. The [1-14C]-oleate from chylomicron triglyceride was taken up into lung tissue during 30 minutes of perfusion and incorporated into neutral lipid, phosphatidylcholine, and phosphatidylethanolamine to a similar degree by ANTU-injured and control lungs. Lipoprotein lipase activity in homogenates of lungs from ANTU and Tween treated rats did not differ. We conclude that lungs are capable of hydrolysis of triglyceride contained in chylomicrons and that this endothelial cell associated metabolic function is not altered by acute lung injury caused by ANTU.