Rapid constriction of lipid bilayers by the mechanochemical enzyme dynamin

Dynamin, a large GTPase, is located at the necks of clathrin-coated pits where it facilitates the release of coated vesicles from the plasma membrane upon GTP binding, and hydrolysis. Previously, we have shown by negative stain electron microscopy that wild-type dynamin and a dynamin mutant lacking the C-terminal proline-rich domain, DeltaPRD, form protein-lipid tubes that constrict and vesiculate upon addition of GTP. Here, we show by time-resolved cryo-electron microscopy (cryo-EM) that DeltaPRD dynamin in the presence of GTP rapidly constricts the underlying lipid bilayer, and then gradually disassembles from the lipid. In agreement with the negative stain results, the dynamin tubes constrict from 50 to 40 nm, and their helical pitch decreases from approximately 13 to 9.4 nm. However, in contrast to the previous results, examination by cryo-EM shows that the lipid bilayer remains intact and small vesicles or fragments do not form upon GTP binding and hydrolysis. Therefore, the vesicle formation seen by negative stain may be due to the lack of mobility of the dynamin tubes on the grid during the GTP-induced conformational changes. Our results confirm that dynamin is a mechanochemical enzyme and suggest that during endocytosis dynamin is directly responsible for membrane constriction. In the cell, other proteins may enhance the activity of dynamin or the constraints induced by the surrounding coated pit and plasma membrane during constriction may cause the final membrane fission event.     

The stalk region of dynamin drives the constriction of dynamin tubes

The GTPase dynamin is essential for numerous vesiculation events including clathrin-mediated endocytosis. Upon GTP hydrolysis, dynamin constricts a lipid bilayer. Previously, a three-dimensional structure of mutant dynamin in the constricted state was determined by helical reconstruction methods. We solved the nonconstricted state by a single-particle approach and show that the stalk region of dynamin undergoes a large conformational change that drives tube constriction.     

The Mechanoenzymatic Core of Dynamin-related Protein 1 Comprises the Minimal Machinery Required for Membrane Constriction

Mitochondria are dynamic organelles that continually undergo cycles of fission and fusion. Dynamin-related protein 1 (Drp1), a large GTPase of the dynamin superfamily, is the main mediator of mitochondrial fission. Like prototypical dynamin, Drp1 is composed of a mechanochemical core consisting of the GTPase, middle, and GTPase effector domain regions. In place of the pleckstrin homology domain in dynamin, however, Drp1 contains an unstructured variable domain, whose function is not yet fully resolved. Here, using time-resolved EM and rigorous statistical analyses, we establish the ability of full-length Drp1 to constrict lipid bilayers through a GTP hydrolysis-dependent mechanism. We also show the variable domain limits premature Drp1 assembly in solution and promotes membrane curvature. Furthermore, the mechanochemical core of Drp1, absent of the variable domain, is sufficient to mediate GTP hydrolysis-dependent membrane constriction.     

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.     

Physical and functional connection between auxilin and dynamin during endocytosis

During clathrin-mediated endocytosis, the GTPase dynamin promotes formation of clathrin-coated vesicles, but its mode of action is unresolved. We provide evidence that a switch in three functional states of dynamin (dimers, tetramers, rings/spirals) coordinates its GTPase cycle. Dimers exhibit negative cooperativity whereas tetramers exhibit positive cooperativity with respect to GTP. Our study identifies tetramers as the kinetically most stable GTP-bound conformation of dynamin, which is required to promote further assembly into higher order structures such as rings or spirals. In addition, using fluorescence lifetime imaging microscopy, we show that interactions between dynamin and auxilin in cells are GTP-, endocytosis- and tetramer-dependent. Furthermore, we show that the cochaperone activity of auxilin is required for constriction of clathrin-coated pits, the same early step in endocytosis known to be regulated by the lifetime of dynamin:GTP. Together, our findings support the model that the GTP-bound conformation of dynamin tetramers stimulates formation of constricted coated pits at the plasma membrane by regulating the chaperone activity of hsc70/auxilin.     

Domain structure and intramolecular regulation of dynamin GTPase.

Dynamin is a 100 kDa GTPase required for receptor-mediated endocytosis, functioning as the key regulator of the late stages of clathrin-coated vesicle budding. It is specifically targeted to clathrin-coated pits where it self-assembles into 'collars' required for detachment of coated vesicles from the plasma membrane. Self-assembly stimulates dynamin GTPase activity. Thus, dynamin-dynamin interactions are critical in regulating its cellular function. We show by crosslinking and analytical ultracentrifugation that dynamin is a tetramer. Using limited proteolysis, we have defined structural domains of dynamin and evaluated the domain interactions and requirements for self-assembly and GTP binding and hydrolysis. We show that dynamin's C-terminal proline- and arginine-rich domain (PRD) and dynamin's pleckstrin homology (PH) domain are, respectively, positive and negative regulators of self-assembly and GTP hydrolysis. Importantly, we have discovered that the alpha-helical domain interposed between the PH domain and the PRD interacts with the N-terminal GTPase domain to stimulate GTP hydrolysis. We term this region the GTPase effector domain (GED) of dynamin.     

Domain structure and function of dynamin probed by limited proteolysis

Dynamin is a 100-kDa GTPase with multiple domains. Some of these have known functions, namely, the N-terminal GTPase domain, the PH domain that binds phosphatidylinositol lipids, and the C-terminal proline-arginine-rich domain (PRD) that binds to several SH3 domain-containing dynamin partners. Others, for example, the "middle" located between the GTPase domain and the PH domain and a predicted alpha-helical domain located between the PH domain and PRD, have unknown functions. Dynamin exists as a homotetramer in solution and self-assembles into higher-order structures resembling rings and helical stacks of rings. Dynamin self-assembly stimulates its GTPase activity. We used limited proteolysis to dissect dynamin's domain structure and to gain insight into intradomain interactions that regulate dynamin self-assembly and stimulate GTPase activity. We found that the PH domain functions as a negative regulator of dynamin self-assembly and stimulates GTPase activity and that the alpha-helical domain, termed GED for GTPase effector domain, is required for stimulated GTPase activity.     

Dynamin architecture--from monomer to polymer

Dynamins form a family of eukaryotic and prokaryotic proteins involved in membrane fission, fusion and restructuring. They have complex mechanisms of self-assembly, which are coupled to the tubulation and destabilization of lipid bilayers. Recent structural data has revolutionized our understanding and is now yielding detailed insights into dynamin structure, from monomer through to polymer. Traditional division of the dynamin subunit into GTPase domain, middle domain and GTPase effector domain based on sequence alignments and biochemistry is not supported by recent structural data. A unified model of dynamin architecture is presented here, based on observation that the basic dynamin fold is conserved across evolutionary kingdoms.     

Dynamin is membrane-active: lipid insertion is induced by phosphoinositides and phosphatidic acid

Dynamin is a large GTPase involved in the regulation of membrane constriction and fission during receptor-mediated endocytosis. Dynamin contains a pleckstrin-homology domain which is essential for endocytosis and which binds to anionic phospholipids. Here, we show for the first time that dynamin is a membrane-active molecule capable of penetrating into the acyl chain region of membrane lipids. Lipid penetration is strongly stimulated by phosphatidic acid (PA), phosphatidylinositol 4-phosphate, and phosphatidylinositol 4, 5-bisphosphate. Though binding is more efficient in the presence of the phosphoinositides, a much larger part of the dynamin molecule penetrates into PA-containing mixed-lipid systems. Thus, local lipid metabolism will dramatically influence dynamin-lipid interactions, and dynamin-lipid interactions are likely to play an important role in dynamin-dependent endocytosis. Our data suggest that dynamin is directly involved in membrane destabilization, a prerequisite to membrane fission.     

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.     

SNX9 activities are regulated by multiple phosphoinositides through both PX and BAR domains

Sorting nexin 9 (SNX9) functions at the interface between membrane remodeling and the actin cytoskeleton. In particular, SNX9 links membrane binding to potentiation of N-WASP and dynamin GTPase activities. SNX9 is one of a growing number of proteins that contain two lipid-binding domains, a phox homology (PX) and a Bin1/Amphiphysin/RVS167 (BAR) domain, and localizes to diverse membranes that are enriched in different phosphoinositides. Here, we investigate the mechanism by which SNX9 functions at these varied membrane environments. We show that SNX9 has low-lipid-binding affinity and harnesses a broad range of phosphoinositides to synergistically enhance both dynamin and N-WASP activities. We introduced point mutations in either the PX domain, BAR domain or both that are predicted to disrupt their functions and examined their respective roles in lipid-binding, and dynamin and N-WASP activation. We show that the broad lipid specificity of SNX9 is not because of independent and additive contributions by individual domains. Rather, the two domains appear to function in concert to confer lipid-binding and SNX9's membrane active properties. We also demonstrate that the two domains are differentially required for full SNX9 activity in N-WASP and dynamin regulation, and for localization of SNX9 to clathrin-coated pits and dorsal ruffles. In total, our results suggest that SNX9 can integrate signals from varied lipids through two domains to direct membrane remodeling events at multiple cellular locations.     

Specific Interaction with Cardiolipin Triggers Functional Activation of Dynamin-Related Protein 1

Dynamin-Related Protein 1 (Drp1), a large GTPase of the dynamin superfamily, is required for mitochondrial fission in healthy and apoptotic cells. Drp1 activation is a complex process that involves translocation from the cytosol to the mitochondrial outer membrane (MOM) and assembly into rings/spirals at the MOM, leading to membrane constriction/division. Similar to dynamins, Drp1 contains GTPase (G), bundle signaling element (BSE) and stalk domains. However, instead of the lipid–interacting Pleckstrin Homology (PH) domain present in the dynamins, Drp1 contains the so-called B insert or variable domain that has been suggested to play an important role in Drp1 regulation. Different proteins have been implicated in Drp1 recruitment to the MOM, although how MOM-localized Drp1 acquires its fully functional status remains poorly understood. We found that Drp1 can interact with pure lipid bilayers enriched in the mitochondrion-specific phospholipid cardiolipin (CL). Building on our previous study, we now explore the specificity and functional consequences of this interaction. We show that a four lysine module located within the B insert of Drp1 interacts preferentially with CL over other anionic lipids. This interaction dramatically enhances Drp1 oligomerization and assembly-stimulated GTP hydrolysis. Our results add significantly to a growing body of evidence indicating that CL is an important regulator of many essential mitochondrial functions.     

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.     

SH3 Domains Differentially Stimulate Distinct Dynamin I Assembly Modes and G Domain Activity

Dynamin I is a highly regulated GTPase enzyme enriched in nerve terminals which mediates vesicle fission during synaptic vesicle endocytosis. One regulatory mechanism involves its interactions with proteins containing Src homology 3 (SH3) domains. At least 30 SH3 domain-containing proteins bind dynamin at its proline-rich domain (PRD). Those that stimulate dynamin activity act by promoting its oligomerisation. We undertook a systematic parallel screening of 13 glutathione-S-transferase (GST)-tagged endocytosis-related SH3 domains on dynamin binding, GTPase activity and oligomerisation. No correlation was found between dynamin binding and their potency to stimulate GTPase activity. There was limited correlation between the extent of their ability to stimulate dynamin activity and the level of oligomerisation, indicating an as yet uncharacterised allosteric coupling of the PRD and G domain. We examined the two variants, dynamin Iab and Ibb, which differ in the alternately splice middle domain α2 helix. They responded differently to the panel of SH3s, with the extent of stimulation between the splice variants varying greatly between the SH3s. This study reveals that SH3 binding can act as a heterotropic allosteric regulator of the G domain via the middle domain α2 helix, suggesting an involvement of this helix in communicating the PRD-mediated allostery. This indicates that SH3 binding both stabilises multiple conformations of the tetrameric building block of dynamin, and promotes assembly of dynamin-SH3 complexes with distinct rates of GTP hydrolysis.     

Dynamin 2 Mutants Linked to Centronuclear Myopathies Form Abnormally Stable Polymers

Mutations in the dynamin 2 gene have been identified in patients with autosomal dominant forms of centronuclear myopathy (CNM). Dynamin 2 is a ubiquitously expressed ∼100-kDa GTPase that assembles around the necks of vesiculating membranes and promotes their constriction and scission. It has also been implicated in regulation of the actin and microtubule cytoskeletons. At present, the cellular functions of dynamin 2 that are affected by CNM-linked mutations are not well defined, and the effects of these mutations on the physical and enzymatic properties of dynamin have been not examined. Here, we report the expression, purification, and characterization of four CNM-associated dynamin mutants. All four mutants display higher than wild-type GTPase activities, and more importantly, the mutants form high order oligomers that are significantly more resistant than wild-type dynamin 2 to disassembly by guanine nucleotides or high ionic strength. These observations suggest that the corresponding wild-type residues serve to prevent excessive or prolonged dynamin assembly on cellular membranes or inappropriate self-assembly in the cytoplasm. To our knowledge, this report contains the first identification of point mutations that enhance the stability of dynamin polymers without impairing their ability to bind and/or hydrolyze GTP. We envision that the formation of abnormally large and stable complexes of these dynamin mutants in vivo contributes to their role in CNM pathogenesis.     

An Intramolecular Signaling Element that Modulates Dynamin Function In Vitro and In Vivo

Dynamin exhibits a high basal rate of GTP hydrolysis that is enhanced by self-assembly on a lipid template. Dynamin''s GTPase effector domain (GED) is required for this stimulation, though its mechanism of action is poorly understood. Recent structural work has suggested that GED may physically dock with the GTPase domain to exert its stimulatory effects. To examine how these interactions activate dynamin, we engineered a minimal GTPase-GED fusion protein (GG) that reconstitutes dynamin''s basal GTPase activity and utilized it to define the structural framework that mediates GED''s association with the GTPase domain. Chemical cross-linking of GG and mutagenesis of full-length dynamin establishes that the GTPase-GED interface is comprised of the N- and C-terminal helices of the GTPase domain and the C-terminus of GED. We further show that this interface is essential for structural stability in full-length dynamin. Finally, we identify mutations in this interface that disrupt assembly-stimulated GTP hydrolysis and dynamin-catalyzed membrane fission in vitro and impair the late stages of clathrin-mediated endocytosis in vivo. These data suggest that the components of the GTPase-GED interface act as an intramolecular signaling module, which we term the bundle signaling element, that can modulate dynamin function in vitro and in vivo.     

Mitochondria-specific function of the dynamin family protein DLP1 is mediated by its C-terminal domains

The dynamin superfamily of large GTPases has been implicated in a variety of distinct intracellular membrane remodeling events. One of these family members, DLP1/Drp1, is similar to conventional dynamins as it contains an N-terminal GTPase domain followed by a middle region (MID), an unconserved region (UC), and a coiled-coil (CC) domain. DLP1 has been shown to function in membrane-based processes distinct from conventional dynamin, most notably mitochondrial fission. In this study, we tested whether the functional specificities of DLP1 and dynamin stems from differences in the individual domains of these proteins by generating dynamin/DLP1 chimeras in which correlate domains had been interchanged. Here we report that three consecutive C-terminal domains of DLP1 (MID-UC-CC) contain information necessary for DLP1-specific function and removing any one of these domains results in a loss of DLP1 function. Importantly, the coiled-coil (CC) domain of DLP1 alone targets specifically and exclusively to mitochondria, implicating its involvement in localizing DLP1 to this organelle in vivo. The mitochondrial targeting information within the DLP1 CC domain is not sufficient to retarget dynamin to mitochondria but is still able to adequately function as an assembly domain in a dynamin background. These data suggest that whereas the GTPase domain of DLP1 provides an enzymatic function, other domains contain information for intermolecular assembly and mitochondrial targeting.     

Recruitment of endophilin to clathrin-coated pit necks is required for efficient vesicle uncoating after fission

Endophilin is a membrane-binding protein with curvature-generating and -sensing properties that participates in clathrin-dependent endocytosis of synaptic vesicle membranes. Endophilin also binds the GTPase dynamin and the phosphoinositide phosphatase synaptojanin and is thought to coordinate constriction of coated pits with membrane fission (via dynamin) and subsequent uncoating (via synaptojanin). We show that although synaptojanin is recruited by endophilin at bud necks before fission, the knockout of all three mouse endophilins results in the accumulation of clathrin-coated vesicles, but not of clathrin-coated pits, at synapses. The absence of endophilin impairs but does not abolish synaptic transmission and results in perinatal lethality, whereas partial endophilin absence causes severe neurological defects, including epilepsy and neurodegeneration. Our data support a model in which endophilin recruitment to coated pit necks, because of its curvature-sensing properties, primes vesicle buds for subsequent uncoating after membrane fission, without being critically required for the fission reaction itself.     

Auxilin-dynamin interactions link the uncoating ATPase chaperone machinery with vesicle formation

The large GTPase dynamin is required for budding of clathrin-coated vesicles from the plasma membrane, after which the clathrin coat is removed by the chaperone Hsc70 and its cochaperone auxilin. Recent evidence suggests that the GTP-bound form of dynamin may recruit factors that execute the fission reaction. Here, we show that dynamin:GTP binds to Hsc70 and auxilin. We mapped two domains within auxilin that interact with dynamin, and these domains inhibit endocytosis when overexpressed in HeLa cells or when added in a permeable cell assay. The inhibition is not due to impairment of clathrin uncoating or to altered clathrin distribution in cells. Thus, in addition to its requirement for clathrin uncoating, our results show that auxilin also acts during the early steps of clathrin-coated vesicle formation. The data suggest that dynamin regulates the action of molecular chaperones in vesicle budding during endocytosis.     

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.