A dynamin GTPase mutation causes a rapid and reversible temperature-inducible locomotion defect in C. elegans.
ABSTRACT: Drosophila shibire and its mammalian homologue dynamin regulate an early step in endocytosis. We identified a Caenorhabditis elegans dynamin gene, dyn-1, based upon hybridization to the Drosophila gene. The dyn-1 RNA transcripts are trans-spliced to the spliced leader 1 and undergo alternative splicing to code for either an 830- or 838-amino acid protein. These dyn-1 proteins are highly similar in amino acid sequence, structure, and size to the Drosophila and mammalian dynamins: they contain an N-terminal GTPase, a pleckstrin homology domain, and a C-terminal proline-rich domain. We isolated a recessive temperature-sensitive dyn-1 mutant containing an alteration within the GTPase domain that becomes uncoordinated when shifted to high temperature and that recovers when returned to lower temperatures, similar to D. shibire mutants. When maintained at higher temperatures, dyn-1 mutants become constipated, egg-laying defective, and produce progeny that die during embryogenesis. Using a dyn-1::lacZ gene fusion, a high level of dynamin expression was observed in motor neurons, intestine, and pharyngeal muscle. Our results suggest that dyn-1 function is required during development and for normal locomotion.
Project description:shibire(ts1), a temperature-sensitive mutation of the Drosophila gene encoding a Dynamin orthologue, blocks vesicle endocytosis and thus synaptic transmission, at elevated, or restrictive temperatures. By targeted Gal4 expression, UAS-shibire(ts1) has been used to dissect neuronal circuits. We investigated the effects of UAS-shibire(ts1) overexpression in Drosophila photoreceptors at permissive (19 degrees C) and restrictive (31 degrees C) temperatures. At 19 degrees C, overexpression of UAS-shi(ts1) causes decelerated phototransduction and reduced neurotransmitter release. This phenotype is exacerbated with dark adaptation, age and in white mutants. Photoreceptors overexpressing UAS-shibire(ts1) contain terminals with widespread vacuolated mitochondria, reduced numbers of vesicles and bundled microtubules. Immuno-electron microscopy reveals that the latter are dynamin coated. Further, the microtubule phenotype is not restricted to photoreceptors, as UAS-shibire(ts1) overexpression in lamina cells also bundles microtubules. We conclude that dynamin has multiple functions that are interrupted by UAS-shibire(ts1) overexpression in Drosophila photoreceptors, destabilizing their neural communication irreversibly at previously reported permissive temperatures.
Project description: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.
Project description: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.
Project description:Dynamins form a family of multidomain GTPases involved in endocytosis, vesicle trafficking and maintenance of mitochondrial morphology. In contrast to the classical switch GTPases, a force-generating function has been suggested for dynamins. Here we report the 2.3 A crystal structure of the nucleotide-free and GDP-bound GTPase domain of Dictyostelium discoideum dynamin A. The GTPase domain is the most highly conserved region among dynamins. The globular structure contains the G-protein core fold, which is extended from a six-stranded beta-sheet to an eight-stranded one by a 55 amino acid insertion. This topologically unique insertion distinguishes dynamins from other subfamilies of GTP-binding proteins. An additional N-terminal helix interacts with the C-terminal helix of the GTPase domain, forming a hydrophobic groove, which could be occupied by C-terminal parts of dynamin not present in our construct. The lack of major conformational changes between the nucleotide-free and the GDP-bound state suggests that mechanochemical rearrangements in dynamin occur during GTP binding, GTP hydrolysis or phosphate release and are not linked to loss of GDP.
Project description:Dynamin, the product of the shibire gene of Drosophila, is a GTPase critically required for endocytosis. Some studies have suggested a functional link between dynamin and the actin cytoskeleton. This link is of special interest, because there is evidence implicating actin dynamics in endocytosis. Here we show that endogenous dynamin 2, as well as green fluorescence protein fusion proteins of both dynamin 1 and 2, is present in actin comets generated by Listeria or by type I PIP kinase (PIPK) overexpression. In PIPK-induced tails, dynamin is further enriched at the interface between the tails and the moving organelles. Dynamin mutants harboring mutations in the GTPase domain inhibited nucleation of actin tails induced by PIPK and moderately reduced their speed. Although dynamin localization to the tails required its proline-rich domain, expression of a dynamin mutant lacking this domain also diminished tail formation. In addition, this mutant disrupted a membrane-associated actin scaffold (podosome rosette) previously shown to include dynamin. These findings suggest that dynamin is part of a protein network that controls nucleation of actin from membranes. At endocytic sites, dynamin may couple the fission reaction to the polymerization of an actin pool that functions in the separation of the endocytic vesicles from the plasma membrane.
Project description:The dynamins represent a superfamily of proteins that have been shown to function in a wide range of membrane fusion and fission events. An increasing number of mutations in the human classical dynamins, Dyn-1 and Dyn-2 has been reported, with diseases caused by these changes ranging from Charcot-Marie-Tooth disorder to epileptic encephalopathies. The budding yeast, <i>Saccharomyces cerevisiae</i> expresses a single dynamin-related protein that functions in membrane trafficking, and is considered to play a similar role to Dyn-1 and Dyn-2 during scission of endocytic vesicles at the plasma membrane. Large parts of the dynamin protein are highly conserved across species and this has enabled us in this study to select a number of disease causing mutations and to generate equivalent mutations in Vps1. We have then studied these mutants using both cellular and biochemical assays to ascertain functions of the protein that have been affected by the changes. Specifically, we demonstrate that the Vps1-G397R mutation (Dyn-2 G358R) disrupts protein oligomerization, Vps1-A447T (Dyn-1 A408T) affects the scission stage of endocytosis, while Vps1-R298L (Dyn-1 R256L) affects lipid binding specificity and possibly an early stage in endocytosis. Overall, we consider that the yeast model will potentially provide an avenue for rapid analysis of new dynamin mutations in order to understand the underlying mechanisms that they disrupt.
Project description:Drosophila temperature-sensitive rolling blackout (rbo(ts) ) mutants display a total block of endocytosis in non-neuronal cells and a weaker, partial defect at neuronal synapses. RBO is an integral plasma membrane protein and is predicted to be a serine esterase. To determine if lipase activity is required for RBO function, we mutated the catalytic serine 358 to alanine in the G-X-S-X-G active site, and assayed genomic rescue of rbo mutant non-neuronal and neuronal phenotypes. The rbo(S358A) mutant is unable to rescue rbo null 100% embryonic lethality, indicating that the lipase domain is critical for RBO essential function. Likewise, the rbo(S358A) mutant cannot provide any rescue of endocytic blockade in rbo(ts) Garland cells, showing that the lipase domain is indispensable for non-neuronal endocytosis. In contrast, rbo(ts) conditional paralysis, synaptic transmission block and synapse endocytic defects are all fully rescued by the rbo(S358A) mutant, showing that the RBO lipase domain is dispensable in neuronal contexts. We identified a synthetic lethal interaction between rbo(ts) and the well-characterized dynamin GTPase conditional shibire (shi(ts1)) mutant. In both non-neuronal cells and neuronal synapses, shi(ts1); rbo(ts) phenocopies shi(ts1) endocytic defects, indicating that dynamin and RBO act in the same pathway, with dynamin functioning upstream of RBO. We conclude that RBO possesses both lipase domain-dependent and scaffolding functions with differential requirements in non-neuronal versus neuronal endocytosis mechanisms downstream of dynamin GTPase activity.
Project description:Mitochondrial fission is mediated by a dynamin-related GTPase that assembles at constricted sites on the organelle. The mechanism of action of this GTPase in fission is related to that of classical dynamin, which severs the necks of clathrin-coated pits at the plasma membrane. The scale of these membrane remodeling events differs by an order of magnitude, however, and structural studies have revealed variations in the assembly properties of classical and mitochondrial dynamins that accommodate these differences. Despite this progress, structural and mechanistic models have not yet incorporated a growing number of adaptor proteins that are required for the membrane recruitment and function of mitochondrial dynamins. Here, we review the structure and assembly properties of the yeast and mammalian mitochondrial dynamins and discuss what is known about the activities of their adaptor proteins.
Project description: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.
Project description:Cells can expand their plasma membrane laterally by unfolding membrane undulations and by exocytosis. Here, we describe a third mechanism involving invaginations held shut by the membrane adapter, dynamin. Compartments open when Ca activates the lipid scramblase, TMEM16F, anionic phospholipids escape from the cytoplasmic monolayer in exchange for neutral lipids, and dynamins relax. Deletion of TMEM16F or dynamins blocks expansion, with loss of dynamin expression generating a maximally expanded basal plasma membrane state. Re-expression of dynamin2 or its GTPase-inactivated mutant, but not a lipid binding mutant, regenerates reserve compartments and rescues expansion. Dynamin2-GFP fusion proteins form punctae that rapidly dissipate from these compartments during TMEM16F activation. Newly exposed compartments extend deeply into the cytoplasm, lack numerous organellar markers, and remain closure-competent for many seconds. Without Ca, compartments open slowly when dynamins are sequestered by cytoplasmic dynamin antibodies or when scrambling is mimicked by neutralizing anionic phospholipids and supplementing neutral lipids. Activation of Ca-permeable mechanosensitive channels via cell swelling or channel agonists opens the compartments in parallel with phospholipid scrambling. Thus, dynamins and TMEM16F control large plasma membrane reserves that open in response to lateral membrane stress and Ca influx.