Dynamin-Catalyzed Membrane Fission Requires Coordinated GTP Hydrolysis

Dynamin is the most-studied membrane fission machinery and has served as a paradigm for studies of other fission GTPases; however, several critical questions regarding its function remain unresolved. In particular, because most dynamin GTPase domain mutants studied to date equally impair both basal and assembly-stimulated GTPase activities, it has been difficult to distinguish their respective roles in clathrin-mediated endocytosis (CME) or in dynamin catalyzed membrane fission. Here we compared a new dynamin mutant, Q40E, which is selectively impaired in assembly-stimulated GTPase activity with S45N, a GTP-binding mutant equally defective in both basal and assembly-stimulated GTPase activities. Both mutants potently inhibit CME and effectively recruit other endocytic accessory proteins to stalled coated pits. However, the Q40E mutant blocks at a later step than S45N, providing additional evidence that GTP binding and/or basal GTPase activities of dynamin are required throughout clathrin coated pit maturation. Importantly, using in vitro assays for assembly-stimulated GTPase activity and membrane fission, we find that the latter is much more potently inhibited by both dominant-negative mutants than the former. These studies establish that efficient fission from supported bilayers with excess membrane reservoir (SUPER) templates requires coordinated GTP hydrolysis across two rungs of an assembled dynamin collar.     

Regulated interactions between dynamin and the actin-binding protein cortactin modulate cell shape

The dynamin family of large GTPases has been implicated in the formation of nascent vesicles in both the endocytic and secretory pathways. It is believed that dynamin interacts with a variety of cellular proteins to constrict membranes. The actin cytoskeleton has also been implicated in altering membrane shape and form during cell migration, endocytosis, and secretion and has been postulated to work synergistically with dynamin and coat proteins in several of these important processes. We have observed that the cytoplasmic distribution of dynamin changes dramatically in fibroblasts that have been stimulated to undergo migration with a motagen/hormone. In quiescent cells, dynamin 2 (Dyn 2) associates predominantly with clathrin-coated vesicles at the plasma membrane and the Golgi apparatus. Upon treatment with PDGF to induce cell migration, dynamin becomes markedly associated with membrane ruffles and lamellipodia. Biochemical and morphological studies using antibodies and GFP-tagged dynamin demonstrate an interaction with cortactin. Cortactin is an actin-binding protein that contains a well defined SH3 domain. Using a variety of biochemical methods we demonstrate that the cortactin-SH3 domain associates with the proline-rich domain (PRD) of dynamin. Functional studies that express wild-type and mutant forms of dynamin and/or cortactin in living cells support these in vitro observations and demonstrate that an increased expression of cortactin leads to a significant recruitment of endogenous or expressed dynamin into the cell ruffle. Further, expression of a cortactin protein lacking the interactive SH3 domain (CortDeltaSH3) significantly reduces dynamin localization to the ruffle. Accordingly, transfected cells expressing Dyn 2 lacking the PRD (Dyn 2(aa)DeltaPRD) sequester little of this protein to the cortactin-rich ruffle. Interestingly, these mutant cells are viable, but display dramatic alterations in morphology. This change in shape appears to be due, in part, to a striking increase in the number of actin stress fibers. These findings provide the first demonstration that dynamin can interact with the actin cytoskeleton to regulate actin reorganization and subsequently cell shape.     

Defective membrane remodeling in neuromuscular diseases: insights from animal models

Proteins involved in membrane remodeling play an essential role in a plethora of cell functions including endocytosis and intracellular transport. Defects in several of them lead to human diseases. Myotubularins, amphiphysins, and dynamins are all proteins implicated in membrane trafficking and/or remodeling. Mutations in myotubularin, amphiphysin 2 (BIN1), and dynamin 2 lead to different forms of centronuclear myopathy, while mutations in myotubularin-related proteins cause Charcot-Marie-Tooth neuropathies. In addition to centronuclear myopathy, dynamin 2 is also mutated in a dominant form of Charcot-Marie-Tooth neuropathy. While several proteins from these different families are implicated in similar diseases, mutations in close homologues or in the same protein in the case of dynamin 2 lead to diseases affecting different tissues. This suggests (1) a common molecular pathway underlying these different neuromuscular diseases, and (2) tissue-specific regulation of these proteins. This review discusses the pathophysiology of the related neuromuscular diseases on the basis of animal models developed for proteins of the myotubularin, amphiphysin, and dynamin families. A better understanding of the common mechanisms between these neuromuscular disorders will lead to more specific health care and therapeutic approaches.     

Direct interaction between endothelial nitric-oxide synthase and dynamin-2. Implications for nitric-oxide synthase function

Endothelial nitric-oxide synthase (eNOS) is regulated in part through specific protein interactions. Dynamin-2 is a large GTPase residing within similar membrane compartments as eNOS. Here we show that dynamin-2 binds directly with eNOS thereby augmenting eNOS activity. Double label confocal immunofluorescence demonstrates colocalization of eNOS and dynamin in both Clone 9 cells cotransfected with green fluorescent protein-dynamin and eNOS, as well as in bovine aortic endothelial cells (BAEC) expressing both proteins endogenously, predominantly in a Golgi membrane distribution. Immunoprecipitation of eNOS from BAEC lysate coprecipitates dynamin and, conversely, immunoprecipitation of dynamin coprecipitates eNOS. Additionally, the calcium ionophore, a reagent that promotes nitric oxide release, enhances coprecipitation of dynamin with eNOS in BAEC, suggesting the interaction between the proteins can be regulated by intracellular signals. In vitro studies demonstrate that glutathione S-transferase (GST)-dynamin-2 quantitatively precipitates both purified recombinant eNOS protein as well as in vitro transcribed (35)S-labeled eNOS from solution indicating a direct interaction between the proteins in vitro. Scatchard analysis of binding studies demonstrates an equilibrium dissociation constant (K(d)) of 27.6 nm. Incubation of purified recombinant eNOS protein with GST-dynamin-2 significantly increases eNOS activity as does overexpression of dynamin-2 in ECV 304 cells stably transfected with eNOS-green fluorescent protein. These studies demonstrate a direct protein-protein interaction between eNOS and dynamin-2, thereby identifying a new NOS-associated protein and providing a novel function for dynamin. These events may have relevance for eNOS regulation and trafficking within vascular endothelium.     

Dynamin GTPase regulation is altered by PH domain mutations found in centronuclear myopathy patients

The large GTPase dynamin has an important membrane scission function in receptor‐mediated endocytosis and other cellular processes. Self‐assembly on phosphoinositide‐containing membranes stimulates dynamin GTPase activity, which is crucial for its function. Although the pleckstrin‐homology (PH) domain is known to mediate phosphoinositide binding by dynamin, it remains unclear how this promotes activation. Here, we describe studies of dynamin PH domain mutations found in centronuclear myopathy (CNM) that increase dynamin's GTPase activity without altering phosphoinositide binding. CNM mutations in the PH domain C‐terminal α‐helix appear to cause conformational changes in dynamin that alter control of the GTP hydrolysis cycle. These mutations either ‘sensitize’ dynamin to lipid stimulation or elevate basal GTPase rates by promoting self‐assembly and thus rendering dynamin no longer lipid responsive. We also describe a low‐resolution structure of dimeric dynamin from small‐angle X‐ray scattering that reveals conformational changes induced by CNM mutations, and defines requirements for domain rearrangement upon dynamin self‐assembly at membrane surfaces. Our data suggest that changes in the PH domain may couple lipid binding to dynamin GTPase activation at sites of vesicle invagination.     

Dynamin II regulates hormone secretion in neuroendocrine cells

The dynamin family of GTP-binding proteins has been implicated as playing an important role in endocytosis. In Drosophila shibire, mutations of the single dynamin gene cause blockade of endocytosis and neurotransmitter release, manifest as temperature-sensitive neuromuscular paralysis. Mammals express three dynamin genes: the neural specific dynamin I, ubiquitous dynamin II, and predominantly testicular dynamin III. Mutations of dynamin I result in a blockade of synaptic vesicle recycling and receptor-mediated endocytosis. Here, we show that dynamin II plays a key role in controlling constitutive and regulated hormone secretion from mouse pituitary corticotrope (AtT20) cells. Dynamin II is preferentially localized to the Golgi apparatus where it interacts with G-protein betagamma subunit and regulates secretory vesicle release. The presence of dynamin II at the Golgi apparatus and its interaction with the betagamma subunit are mediated by the pleckstrin homology domain of the GTPase. Overexpression of the pleckstrin homology domain, or a dynamin II mutant lacking the C-terminal SH3-binding domain, induces translocation of endogenous dynamin II from the Golgi apparatus to the plasma membrane and transformation of dynamin II from activity in the secretory pathway to receptor-mediated endocytosis. Thus, dynamin II regulates secretory vesicle formation from the Golgi apparatus and hormone release from mammalian neuroendocrine cells.     

Contribution of the GTPase Domain to the Subcellular Localization of Dynamin in the Nematode Caenorhabditis elegans

Caenorhabditis elegans dynamin is expressed at high levels in neurons and at lower levels in other cell types, consistent with the important role that dynamin plays in the recycling of synaptic vesicles. Indirect immunofluorescence showed that dynamin is concentrated along the dorsal and ventral nerve cords and in the synapse-rich nerve ring. Green fluorescent protein (GFP) fused to the N terminus of dynamin is localized to synapse-rich regions. Furthermore, this chimera was detected along the apical membrane of intestinal cells, in spermathecae, and in coelomocytes. Dynamin localization was not affected by disrupting axonal transport of synaptic vesicles in the unc-104 (kinesin) mutant. To investigate the alternative mechanisms that dynamin might use for translocation to the synapse, we systematically tested the localization of different protein domains by fusion to GFP. Localization of each chimera was measured in one specific neuron, the ALM. The GTPase, a middle domain, and the putative coiled coil each contribute to synaptic localization. Surprisingly, the pleckstrin homology domain and the proline-rich domain, which are known to bind to coated-pit constituents, did not contribute to synaptic localization. The GFP-GTPase chimera was most strongly localized, although the GTPase domain has no known interactions with proteins other than with dynamin itself. Our results suggest that different dynamin domains contribute to axonal transport and the sequestration of a pool of dynamin molecules in synaptic cytosol.     

Dynamin-mediated Internalization of Caveolae

The dynamins comprise an expanding family of ubiquitously expressed 100-kD GTPases that have been implicated in severing clathrin-coated pits during receptor-mediated endocytosis. Currently, it is unclear whether the different dynamin isoforms perform redundant functions or participate in distinct endocytic processes. To define the function of dynamin II in mammalian epithelial cells, we have generated and characterized peptide-specific antibodies to domains that either are unique to this isoform or conserved within the dynamin family. When microinjected into cultured hepatocytes these affinity-purified antibodies inhibited clathrin-mediated endocytosis and induced the formation of long plasmalemmal invaginations with attached clathrin-coated pits. In addition, clusters of distinct, nonclathrin-coated, flask-shaped invaginations resembling caveolae accumulated at the plasma membrane of antibody-injected cells. In support of this, caveola-mediated endocytosis of labeled cholera toxin B was inhibited in antibody-injected hepatocytes. Using immunoisolation techniques an anti-dynamin antibody isolated caveolar membranes directly from a hepatocyte postnuclear membrane fraction. Finally, double label immunofluorescence microscopy revealed a striking colocalization between dynamin and the caveolar coat protein caveolin. Thus, functional in vivo studies as well as ultrastructural and biochemical analyses indicate that dynamin mediates both clathrin-dependent endocytosis and the internalization of caveolae in mammalian cells.     

Identification and function of conformational dynamics in the multidomain GTPase dynamin

Vesicle release upon endocytosis requires membrane fission, catalyzed by the large GTPase dynamin. Dynamin contains five domains that together orchestrate its mechanochemical activity. Hydrogen–deuterium exchange coupled with mass spectrometry revealed global nucleotide‐ and membrane‐binding‐dependent conformational changes, as well as the existence of an allosteric relay element in the α2^S helix of the dynamin stalk domain. As predicted from structural studies, FRET analyses detect large movements of the pleckstrin homology domain (PHD) from a ‘closed’ conformation docked near the stalk to an ‘open’ conformation able to interact with membranes. We engineered dynamin constructs locked in either the closed or open state by chemical cross‐linking or deletion mutagenesis and showed that PHD movements function as a conformational switch to regulate dynamin self‐assembly, membrane binding, and fission. This PHD conformational switch is impaired by a centronuclear myopathy‐causing disease mutation, S619L, highlighting the physiological significance of its role in regulating dynamin function. Together, these data provide new insight into coordinated conformational changes that regulate dynamin function and couple membrane binding, oligomerization, and GTPase activity during dynamin‐catalyzed membrane fission.     

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.     

The proline/arginine-rich domain is a major determinant of dynamin self-activation

Dynamins induce membrane vesiculation during endocytosis and Golgi budding in a process that requires assembly-dependent GTPase activation. Brain-specific dynamin 1 has a weaker propensity to self-assemble and self-activate than ubiquitously expressed dynamin 2. Here we show that dynamin 3, which has important functions in neuronal synapses, shares the self-assembly and GTPase activation characteristics of dynamin 2. Analysis of dynamin hybrids and of dynamin 1-dynamin 2 and dynamin 1-dynamin 3 heteropolymers reveals that concentration-dependent GTPase activation is suppressed by the C-terminal proline/arginine-rich domain of dynamin 1. Dynamin proline/arginine-rich domains also mediate interactions with SH3 domain-containing proteins and thus regulate both self-association and heteroassociation of dynamins.     

Fission of biological membranes: interplay between dynamin and lipids

Membrane budding and fission are the key stages of ubiquitous processes of formation of intracellular transport vesicles. We present a theoretical consideration of one of the most important types of fission machinery, which is mediated by GTPase dynamin and controlled by lipid composition of the membrane. We suggest a mechanism for collapse of a membrane neck driven by interplay between the dynamin collar and the bending elastic energy of the neck membrane. The collar plays a role of a rigid external skeleton, which imposes mechanical constraints on the neck. We show that in certain conditions the membrane of the neck loses its stability and collapses. Collapse can result from: (i) shifting of the spontaneous curvature of the neck membrane towards negative values, (ii) stretching of the dynamin collar, (iii) tightening of the dynamin collar. The three factors can act separately or concertedly. The suggested model accounts for the major experimental knowledge on membrane fission mediated by dynamin. It includes the elements of all previous models of dynamin action based on different sets of experimental results [Sever et al., Traffic 2000; 1: 385-392]. It reconciles, at least partially, the apparent contradictions between the existing alternative views on biomembrane fission machinery.     

Real-time detection reveals that effectors couple dynamin's GTP-dependent conformational changes to the membrane

The GTPase dynamin is a mechanochemical enzyme involved in membrane fission, but the molecular nature of its membrane interactions and their regulation by guanine nucleotides and protein effectors remain poorly characterized. Using site-directed fluorescence labeling and several independent fluorescence spectroscopic techniques, we have developed robust assays for the detection and real-time monitoring of dynamin-membrane and dynamin-dynamin interactions. We show that dynamin interacts preferentially with highly curved, PIP2-dense membranes and inserts partially into the lipid bilayer. Our kinetic measurements further reveal that cycles of GTP binding and hydrolysis elicit major conformational rearrangements in self-assembled dynamin that favor dynamin-membrane association and dissociation, respectively. Sorting nexin 9, an abundant dynamin partner, transiently stabilizes dynamin on the membrane at the onset of stimulated GTP hydrolysis and may function to couple dynamin's mechanochemical conformational changes to membrane destabilization. Amphiphysin I has the opposite effect. Thus, dynamin's mechanochemical properties on a membrane surface are dynamically regulated by its GTPase cycle and major binding partners.     

Dynamin Regulates Specific Membrane Fusion Events Necessary for Acrosomal Exocytosis in Mouse Spermatozoa

Mammalian spermatozoa must complete an acrosome reaction prior to fertilizing an oocyte. The acrosome reaction is a unique exocytotic event involving a series of prolonged membrane fusions that ultimately result in the production of membrane vesicles and release of the acrosomal contents. This event requires the concerted action of a large number of fusion-competent signaling and scaffolding proteins. Here we show that two different members of the dynamin GTPase family localize to the developing acrosome of maturing mouse germ cells. Both dynamin 1 and 2 also remain within the periacrosomal region of mature mouse spermatozoa and are thus well positioned to regulate the acrosome reaction. Two pharmacological inhibitors of dynamin, dynasore and Dyngo-4a, blocked the in vitro induction of acrosomal exocytosis by progesterone, but not by the calcium ionophore A23187, and elicited a concomitant reduction of in vitro fertilization. In vivo treatment with these inhibitors also resulted in spermatozoa displaying reduced acrosome reaction potential. Dynamin 1 and 2 phosphorylation increased on progesterone treatment, and this was also selectively blocked by dynasore. On the basis of our collective data, we propose that dynamin could regulate specific membrane fusion events necessary for acrosomal exocytosis in mouse spermatozoa.     

Multiple distinct coiled-coils are involved in dynamin self-assembly

Dynamin, a 100-kDa GTPase, has been implicated to be involved in synaptic vesicle recycling, receptor-mediated endocytosis, and other membrane sorting processes. Dynamin self-assembles into helical collars around the necks of coated pits and other membrane invaginations and mediates membrane scission. In vitro, dynamin has been reported to exist as dimers, tetramers, ring-shaped oligomers, and helical polymers. In this study we sought to define self-assembly regions in dynamin. Deletion of two closely spaced sequences near the dynamin-1 C terminus abolished self-association as assayed by co-immunoprecipitation and the yeast interaction trap, and reduced the sedimentation coefficient from 7.5 to 4.5 S. Circular dichroism spectroscopy and equilibrium ultracentrifugation of synthetic peptides revealed coiled-coil formation within the C-terminal assembly domain and at a third, centrally located site. Two of the peptides formed tetramers, supporting a role for each in the monomer-tetramer transition and providing novel insight into the organization of the tetramer. Partial deletions of the C-terminal assembly domain reversed the dominant inhibition of endocytosis by dynamin-1 GTPase mutants. Self-association was also observed between different dynamin isoforms. Taken altogether, our results reveal two distinct coiled-coil-containing assembly domains that can recognize other dynamin isoforms and mediate endocytic inhibition. In addition, our data strongly suggests a parallel model for dynamin subunit self-association.     

Disruption of a Dynamin Homologue Affects Endocytosis, Organelle Morphology, and Cytokinesis in Dictyostelium discoideum

The identification and functional characterization of Dictyostelium discoideum dynamin A, a protein composed of 853 amino acids that shares up to 44% sequence identity with other dynamin-related proteins, is described. Dynamin A is present during all stages of D. discoideum development and is found predominantly in the cytosolic fraction and in association with endosomal and postlysosomal vacuoles. Overexpression of the protein has no adverse effect on the cells, whereas depletion of dynamin A by gene-targeting techniques leads to multiple and complex phenotypic changes. Cells lacking a functional copy of dymA show alterations of mitochondrial, nuclear, and endosomal morphology and a defect in fluid-phase uptake. They also become multinucleated due to a failure to complete normal cytokinesis. These pleiotropic effects of dynamin A depletion can be rescued by complementation with the cloned gene. Morphological studies using cells producing green fluorescent protein-dynamin A revealed that dynamin A associates with punctate cytoplasmic vesicles. Double labeling with vacuolin, a marker of a postlysosomal compartment in D. discoideum, showed an almost complete colocalization of vacuolin and dynamin A. Our results suggest that that dynamin A is likely to function in membrane trafficking processes along the endo-lysosomal pathway of D. discoideum but not at the plasma membrane.     

A pseudoatomic model of the dynamin polymer identifies a hydrolysis-dependent powerstroke

The GTPase dynamin catalyzes membrane fission by forming a collar around the necks of clathrin-coated pits, but the specific structural interactions and conformational changes that drive this process remain a mystery. We present the GMPPCP-bound structures of the truncated human dynamin 1 helical polymer at 12.2 ? and a fusion protein, GG, linking human dynamin 1's catalytic G domain to its GTPase effector domain (GED) at 2.2 ?. The structures reveal the position and connectivity of dynamin fragments in the assembled structure, showing that G domain dimers only form between tetramers in sequential rungs of the dynamin helix. Using chemical crosslinking, we demonstrate that dynamin tetramers are made of two dimers, in which the G domain of one molecule interacts in trans with the GED of another. Structural comparison of GG(GMPPCP) to the GG transition-state complex identifies a hydrolysis-dependent powerstroke that may play a role in membrane-remodeling events necessary for fission.     

Dynamin: possible mechanism of "Pinchase" action.

Dynamin is a GTPase playing an essential role in ubiquitous intra cellular processes involving separation of vesicles from plasma membranes and membranes of cellular compartments. Recent experimental progress (. Cell. 93:1021-1029;. Cell. 94:131-141) has made it possible to attempt to understand the action of dynamin in physical terms. Dynamin molecules are shown to bind to a lipid membrane, to self-assemble into a helicoidal structure constricting the membrane into a tubule, and, as a result of GTP hydrolysis, to mediate fission of this tubule (). In a similar way, dynamin is supposed to mediate fission of a neck connecting an endocytic bud and the plasma membrane, i.e., to complete endocytosis. We suggest a mechanism of this "pinchase" action of dynamin. We propose that, as a result of GTP hydrolysis, dynamin undergoes a conformational change manifested in growth of the pitch of the dynamin helix. We show that this gives rise to a dramatic change of shape of the tubular membrane constricted inside the helix, resulting in a local tightening of the tubule, which is supposed to promote its fission. We treat this model in terms of competing elasticities of the dynamin helix and the tubular membrane and discuss the predictions of the model in relation to the previous views on the mechanism of dynamin action.     

Endocytosis: How dynamin sets vesicles PHree!

The dynamin GTPase is required for clathrin-dependent, receptor-mediated endocytosis. Exciting new studies have shown that dynamin's pleckstrin homology domain binds to phosphatidylinositol 4, 5-bisphosphate in vivo, thus localising dynamin directly at the plasma membrane and ultimately enabling vesiculation.     

Caveolin-1 interacts directly with dynamin-2

Caveolin is the principal component of caveolae in vivo. In addition to a structural role, it is believed to play a scaffolding function to organize and inactivate signaling molecules that are concentrated on the cytoplasmic surface of caveolar membranes. The large GTPase dynamin has been shown to mediate the scission of caveolae from the plasma membrane, although it is unclear if dynamin interacts directly with caveolin or via accessory proteins. Therefore, the goal of this study was to test whether dynamin associates with caveolae via a direct binding to the caveolin 1 (Cav1) protein. Immunoelectron microscopy of lung endothelium or a cultured hepatocyte cell line stained with antibodies for Dyn2 and Cav1 shows that these proteins co-localize to caveolae. To further define this interaction biochemically, in vitro experiments were performed using glutathione-S-transferase (GST)-Dyn2 and GST-Cav1 fusion proteins, which demonstrated a direct interaction between these proteins. This interaction appears to be mediated by the proline-arginine-rich domain (PRD) of Dyn2, as a GST-PRD fragment binds Cav1 while GST-Dyn2DeltaPRD does not. Further, in vitro binding studies using two Dyn2 spliced forms and Cav1 peptides immobilized on paper identify specific domains of Cav1 that bind Dyn2. Interestingly, these Cav1-binding domains differ markedly between two spliced variant forms of Dyn2. In support of these distinctive physical interactions, we find that the different Dyn2 forms, when expressed as GTPase-defective mutants, exert markedly different inhibitory effects on caveolae internalization, as assayed by cholera toxin uptake. These studies provide the first evidence for a direct interaction between dynamin and the caveolin coat, and demonstrate a selectivity of one Dyn2 form toward the caveolae-mediated endocytosis.