Dynamin Rings: Not Just for Fission

The GTPase dynamin has captivated researchers for over two decades, even managing to establish its own research field. Dynamin's allure is partly due to its unusual biochemical properties as well as its essential role in multiple cellular processes, which include the regulation of clathrin‐mediated endocytosis and of actin cytoskeleton. On the basis of the classic model, dynamin oligomerization into higher order oligomers such as rings and helices directly executes the final fission reaction in endocytosis, which results in the generation of clathrin‐coated vesicles. Dynamin's role in the regulation of actin cytoskeleton is mostly explained by its interactions with a number of actin‐binding and ‐regulating proteins; however, the molecular mechanism of dynamin's action continues to elude us. Recent insights into the mechanism and role of dynamin oligomerization in the regulation of actin polymerization point to a novel role for dynamin oligomerization in the cell .      

Regulation of Dynamin Oligomerization in Cells: The Role of Dynamin–Actin Interactions and Its GTPase Activity

Dynamin is a 96‐kDa protein that has multiple oligomerization states that influence its GTPase activity. A number of different dynamin effectors, including lipids, actin filaments, and SH3‐domain‐containing proteins, have been implicated in the regulation of dynamin oligomerization, though their roles in influencing dynamin oligomerization have been studied predominantly in vitro using recombinant proteins. Here, we identify higher order dynamin oligomers such as rings and helices in vitro and in live cells using fluorescence lifetime imaging microscopy (FLIM). FLIM detected GTP‐ and actin‐dependent dynamin oligomerization at distinct cellular sites, including the cell membrane and transition zones where cortical actin transitions into stress fibers. Our study identifies a major role for direct dynamin–actin interactions and dynamin's GTPase activity in the regulation of dynamin oligomerization in cells.      

A Dynamin Homolog Promotes the Transition from Hemifusion to Content Mixing in Intracellular Membrane Fusion

The convergence of the antagonistic reactions of membrane fusion and fission at the hemifusion/hemifission intermediate has generated a captivating enigma of whether Soluble N‐ethylmaleimide sensitive factor Attachment Protein Receptor (SNAREs) and dynamin have unusual counter‐functions in fission and fusion, respectively. SNARE‐mediated fusion and dynamin‐driven fission are fundamental membrane flux reactions known to occur during ubiquitous cellular communication events such as exocytosis, endocytosis and vesicle transport. Here we demonstrate the influence of the dynamin homolog Vps1 (Vacuolar protein sorting 1) on lipid mixing and content mixing properties of yeast vacuoles, and on the incorporation of SNAREs into fusogenic complexes. We propose a novel concept that Vps1, through its oligomerization and SNARE domain binding, promotes the hemifusion‐content mixing transition in yeast vacuole fusion by increasing the number of trans‐SNAREs .      

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: 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.     

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.     

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.     

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.     

Live-Cell Imaging in Caenorhabditis elegans Reveals the Distinct Roles of Dynamin Self-Assembly and Guanosine Triphosphate Hydrolysis in the Removal of Apoptotic Cells

Dynamins are large GTPases that oligomerize along membranes. Dynamin''s membrane fission activity is believed to underlie many of its physiological functions in membrane trafficking. Previously, we reported that DYN-1 (Caenorhabditis elegans dynamin) drove the engulfment and degradation of apoptotic cells through promoting the recruitment and fusion of intracellular vesicles to phagocytic cups and phagosomes, an activity distinct from dynamin''s well-known membrane fission activity. Here, we have detected the oligomerization of DYN-1 in living C. elegans embryos and identified DYN-1 mutations that abolish DYN-1''s oligomerization or GTPase activities. Specifically, abolishing self-assembly destroys DYN-1''s association with the surfaces of extending pseudopods and maturing phagosomes, whereas inactivating guanosine triphosphate (GTP) binding blocks the dissociation of DYN-1 from these membranes. Abolishing the self-assembly or GTPase activities of DYN-1 leads to common as well as differential phagosomal maturation defects. Whereas both types of mutations cause delays in the transient enrichment of the RAB-5 GTPase to phagosomal surfaces, only the self-assembly mutation but not GTP binding mutation causes failure in recruiting the RAB-7 GTPase to phagosomal surfaces. We propose that during cell corpse removal, dynamin''s self-assembly and GTP hydrolysis activities establish a precise dynamic control of DYN-1''s transient association to its target membranes and that this control mechanism underlies the dynamic recruitment of downstream effectors to target membranes.     

Dynamin‐2 in nervous system disorders

Dynamin‐2 is a pleiotropic GTPase whose best‐known function is related to membrane scission during vesicle budding from the plasma or Golgi membranes. In the nervous system, dynamin‐2 participates in synaptic vesicle recycling, post‐synaptic receptor internalization, neurosecretion, and neuronal process extension. Some of these functions are shared with the other two dynamin isoforms. However, the involvement of dynamin‐2 in neurological illnesses points to a critical function of this isoform in the nervous system. In this regard, mutations in the dynamin‐2 gene results in two congenital neuromuscular disorders. One of them, Charcot‐Marie‐Tooth disease, affects myelination and peripheral nerve conduction, whereas the other, Centronuclear Myopathy, is characterized by a progressive and generalized atrophy of skeletal muscles, yet it is also associated with abnormalities in the nervous system. Furthermore, single nucleotide polymorphisms located in the dynamin‐2 gene have been associated with sporadic Alzheimer's disease. In the present review, we discuss the pathogenic mechanisms implicated in these neurological disorders.

     

To mdivi‐1 or not to mdivi‐1: Is that the question?

The fission/division and fusion of mitochondria are fundamental aspects of mitochondrial biology. The balance of fission and fusion sets the length of mitochondria in cells to serve their physiological requirements. The fission of mitochondria is markedly induced in many disease states and in response to cellular injury, resulting in the fragmentation of mitochondria into dysfunctional units. The mechanism that drives fission is dependent on the dynamin related protein 1 (Drp1) GTPase. mdivi‐1 is a quinazolinone originally described as a selective inhibitor of Drp1, over other dynamin family members, and reported to inhibit mitochondrial fission. A recent study has challenged the activity of mdivi‐1 as an inhibitor of Drp1. This study raises serious issues regarding the interpretation of data addressing the effects of mdivi‐1 as reflective of the inhibition of Drp1 and thus fission. This commentary considers the evidence for and against mdivi‐1 as an inhibitor of Drp1 and presents the following considerations; (1) the activity of mdivi‐1 toward Drp1 GTPase activity requires further biochemical investigation, (2) as there is a large body of literature using mdivi‐1 in vitro with effects as predicted for inhibition of Drp1 and mitochondrial fission, reviewed herein, the evidence is in favor of mdivi‐1's originally described bioactivity, and (3) until the issue is resolved, experimental interpretations for the effects of mdivi‐1 on inhibition of fission in cell and tissue experiments warrants stringent positive controls directly addressing the effects of mdivi‐1 on fission. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1260–1268, 2017     

Mitochondrial dynamics modulate the expression of pro‐inflammatory mediators in microglial cells

Over‐activation of microglia cells in the brain contributes to neurodegenerative processes promoted by the production of various neurotoxic factors including pro‐inflammatory cytokines and nitric oxide. Recently, accumulating evidence has suggested that mitochondrial dynamics are an important constituent of cellular quality control and function. However, the role of mitochondrial dynamics in microglial activation is still largely unknown. In this study, we determined whether mitochondrial dynamics are associated with the production of pro‐inflammatory mediators in lipopolysaccharide (LPS)‐stimulated immortalization of murine microglial cells (BV‐2) by a v‐raf/v‐myc carrying retrovirus (J2). Excessive mitochondrial fission was observed in lentivirus‐transfected BV‐2 cells stably expressing DsRed2‐mito following LPS stimulation. Furthermore, mitochondrial localization of dynamin‐related protein 1 (Drp1) (a key regulator of mitochondrial fission) was increased and accompanied by de‐phosphorylation of Ser637 in Drp1. Interestingly, inhibition of LPS‐induced mitochondrial fission and reactive oxygen species (ROS) generation by Mdivi‐1 and Drp1 knock‐down attenuated the production of pro‐inflammatory mediators via reduced nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) and mitogen‐activated protein kinase (MAPK) signaling. Our results demonstrated for the first time that mitochondrial fission regulates mitochondrial ROS production in activated microglial cells and influences the expression of pro‐inflammatory mediators through the activation of NF‐κB and MAPK. We therefore suggest that mitochondrial dynamics may be essential for understanding pro‐inflammatory mediator expression in activated microglial cells. This could represent a new therapeutic approach for preventing neurodegenerative diseases.

     

Nucleotide-dependent conformational changes in dynamin: evidence for a mechanochemical molecular spring

The GTPase dynamin plays an essential part in endocytosis by catalysing the fission of nascent clathrin-coated vesicles from the plasma membrane. Using preformed phosphatidylinositol-4,5-bisphosphate-containing lipid nanotubes as a membrane template for dynamin self-assembly, we investigate the conformational changes that arise during GTP hydrolysis by dynamin. Electron microscopy reveals that, in the GTP-bound state, dynamin rings appear to be tightly packed together. After GTP hydrolysis, the spacing between rings increases nearly twofold. When bound to the nanotubes, dynamin's GTPase activity is cooperative and is increased by three orders of magnitude compared with the activity of unbound dynamin. An increase in the Kcat (but not the K(m) of GTP hydrolysis accounts for the pronounced cooperativity. These data indicate that a novel, lengthwise ('spring-like') conformational change in a dynamin helix may participate in vesicle fission.     

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.     

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.     

Distinct complexes of yeast Snx4 family SNX‐BARs mediate retrograde trafficking of Snc1 and Atg27

The yeast SNX4 sub‐family of sorting nexin containing a Bin‐Amphiphysin‐Rvs domain (SNX‐BAR) proteins, Snx4/Atg24, Snx41 and Atg20/Snx42, are required for endocytic recycling and selective autophagy. Here, we show that Snx4 forms 2 functionally distinct heterodimers: Snx4‐Atg20 and Snx4‐Snx41. Each heterodimer coats an endosome‐derived tubule that mediates retrograde sorting of distinct cargo; the v‐SNARE, Snc1, is a cargo of the Snx4‐Atg20 pathway, and Snx4‐Snx41 mediates retrograde sorting of Atg27, an integral membrane protein implicated in selective autophagy. Live cell imaging of individual endosomes shows that Snx4 and the Vps5‐Vps17 retromer SNX‐BAR heterodimer operate concurrently on a maturing endosome. Consistent with this, the yeast dynamin family protein, Vps1, which was previously shown to promote fission of retromer‐coated tubules, promotes fission of Snx4‐Atg20 coated tubules. The results indicate that the yeast SNX‐BAR proteins coat 3 distinct types of endosome‐derived carriers that mediate endosome‐to‐Golgi retrograde trafficking.      

Dynamin at the Neck of Caveolae Mediates Their Budding to Form Transport Vesicles by GTP-driven Fission from the Plasma Membrane of Endothelium

The molecular mechanisms mediating cell surface trafficking of caveolae are unknown. Caveolae bud from plasma membranes to form free carrier vesicles through a “pinching off” or fission process requiring cytosol and driven by GTP hydrolysis (Schnitzer, J.E., P. Oh, and D.P. McIntosh. 1996. Science. 274:239–242). Here, we use several independent techniques and functional assays ranging from cell-free to intact cell systems to establish a function for dynamin in the formation of transport vesicles from the endothelial cell plasma membrane by mediating fission at the neck of caveolae. This caveolar fission requires interaction with cytosolic dynamin as well as its hydrolysis of GTP. Expression of dynamin in cytosol as well as purified recombinant dynamin alone supports GTP-induced caveolar fission in a cell-free assay whereas its removal from cytosol or the addition to the cytosol of specific antibodies for dynamin inhibits this fission. Overexpression of mutant dynamin lacking normal GTPase activity not only inhibits GTP-induced fission and budding of caveolae but also prevents caveolae-mediated internalization of cholera toxin B chain in intact and permeabilized endothelial cells. Analysis of endothelium in vivo by subcellular fractionation and immunomicroscopy shows that dynamin is concentrated on caveolae, primarily at the expected site of action, their necks. Thus, through its ability to oligomerize, dynamin appears to form a structural collar around the neck of caveolae that hydrolyzes GTP to mediate internalization via the fission of caveolae from the plasma membrane to form free transport vesicles.     

Phenothiazine‐Derived Antipsychotic Drugs Inhibit Dynamin and Clathrin‐Mediated Endocytosis

Chlorpromazine is a phenothiazine‐derived antipsychotic drug (APD) that inhibits clathrin‐mediated endocytosis (CME) in cells by an unknown mechanism. We examined whether its action and that of other APDs might be mediated by the GTPase activity of dynamin. Eight of eight phenothiazine‐derived APDs inhibited dynamin I (dynI) in the 2–12 µm range, the most potent being trifluoperazine (IC₅₀ 2.6 ± 0.7 µm ). They also inhibited dynamin II (dynII) at similar concentrations. Typical and atypical APDs not based on the phenothiazine scaffold were 8‐ to 10‐fold less potent (haloperidol and clozapine) or were inactive (droperidol, olanzapine and risperidone). Kinetic analysis showed that phenothiazine‐derived APDs were lipid competitive, while haloperidol was uncompetitive with lipid. Accordingly, phenothiazine‐derived APDs inhibited dynI GTPase activity stimulated by lipids but not by various SH3 domains. All dynamin‐active APDs also inhibited transferrin (Tfn) CME in cells at related potencies. Structure–activity relationships (SAR) revealed dynamin inhibition to be conferred by a substituent group containing a terminal tertiary amino group at the N2 position. Chlorpromazine was previously proposed to target AP‐2 recruitment in the formation of clathrin‐coated vesicles (CCV). However, neither chlorpromazine nor thioridazine affected AP‐2 interaction with amphiphysin or clathrin. Super‐resolution microscopy revealed that chlorpromazine blocks neither clathrin recruitment by AP‐2, nor AP‐2 recruitment, showing that CME inhibition occurs downstream of CCV formation. Overall, potent dynamin inhibition is a shared characteristic of phenothiazine‐derived APDs, but not other typical or atypical APDs, and the data indicate that dynamin is their likely in‐cell target in endocytosis.      

Structural basis for conformational switching and GTP loading of the large G protein atlastin

Atlastin, a member of the dynamin superfamily, is known to catalyse homotypic membrane fusion in the smooth endoplasmic reticulum (ER). Recent studies of atlastin have elucidated key features about its structure and function; however, several mechanistic details, including the catalytic mechanism and GTP hydrolysis‐driven conformational changes, are yet to be determined. Here, we present the crystal structures of atlastin‐1 bound to GDP·AlF₄^? and GppNHp, uncovering an intramolecular arginine finger that stimulates GTP hydrolysis when correctly oriented through rearrangements within the G domain. Utilizing Förster Resonance Energy Transfer, we describe nucleotide binding and hydrolysis‐driven conformational changes in atlastin and their sequence. Furthermore, we discovered a nucleotide exchange mechanism that is intrinsic to atlastin's N‐terminal domains. Our results indicate that the cytoplasmic domain of atlastin acts as a tether and homotypic interactions are timed by GTP binding and hydrolysis. Perturbation of these mechanisms may be implicated in a group of atlastin‐associated hereditary neurodegenerative diseases.