Adult-onset autosomal dominant spastic paraplegia linked to a GTPase-effector domain mutation of dynamin 2.

BACKGROUND: Hereditary Spastic Paraplegia (HSP) represents a large group of clinically and genetically heterogeneous disorders linked to over 70 different loci and more than 60 recognized disease-causing genes. A heightened vulnerability to disruption of various cellular processes inherent to the unique function and morphology of corticospinal neurons may account, at least in part, for the genetic heterogeneity. METHODS: Whole exome sequencing was utilized to identify candidate genetic variants in a four-generation Siberian kindred that includes nine individuals showing clinical features of HSP. Segregation of candidate variants within the family yielded a disease-associated mutation. Functional as well as in-silico structural analyses confirmed the selected candidate variant to be causative. RESULTS: Nine known patients had young-adult onset of bilateral slowly progressive lower-limb spasticity, weakness and hyperreflexia progressing over two-to-three decades to wheel-chair dependency. In the advanced stage of the disease, some patients also had distal wasting of lower leg muscles, pes cavus, mildly decreased vibratory sense in the ankles, and urinary urgency along with electrophysiological evidence of a mild distal motor/sensory axonopathy. Molecular analyses uncovered a missense c.2155C > T, p.R719W mutation in the highly conserved GTP-effector domain of dynamin 2. The mutant DNM2 co-segregated with HSP and affected endocytosis when expressed in HeLa cells. In-silico modeling indicated that this HSP-associated dynamin 2 mutation is located in a highly conserved bundle-signaling element of the protein while dynamin 2 mutations associated with other disorders are located in the stalk and PH domains; p.R719W potentially disrupts dynamin 2 assembly. CONCLUSION: This is the first report linking a mutation in dynamin 2 to a HSP phenotype. Dynamin 2 mutations have previously been associated with other phenotypes including two forms of Charcot-Marie-Tooth neuropathy and centronuclear myopathy. These strikingly different pathogenic effects may depend on structural relationships the mutations disrupt. Awareness of this distinct association between HSP and c.2155C > T, p.R719W mutation will facilitate ascertainment of additional DNM2 HSP families and will direct future research toward better understanding of cell biological processes involved in these partly overlapping clinical syndromes.

A hemi-fission intermediate links two mechanistically distinct stages of membrane fission.

Fusion and fission drive all vesicular transport. Although topologically opposite, these reactions pass through the same hemi-fusion/fission intermediate, characterized by a 'stalk' in which only the outer membrane monolayers of the two compartments have merged to form a localized non-bilayer connection. Formation of the hemi-fission intermediate requires energy input from proteins catalysing membrane remodelling; however, the relationship between protein conformational rearrangements and hemi-fusion/fission remains obscure. Here we analysed how the GTPase cycle of human dynamin 1, the prototypical membrane fission catalyst, is directly coupled to membrane remodelling. We used intramolecular chemical crosslinking to stabilize dynamin in its GDP·AlF4(-)-bound transition state. In the absence of GTP this conformer produced stable hemi-fission, but failed to progress to complete fission, even in the presence of GTP. Further analysis revealed that the pleckstrin homology domain (PHD) locked in its membrane-inserted state facilitated hemi-fission. A second mode of dynamin activity, fuelled by GTP hydrolysis, couples dynamin disassembly with cooperative diminishing of the PHD wedging, thus destabilizing the hemi-fission intermediate to complete fission. Molecular simulations corroborate the bimodal character of dynamin action and indicate radial and axial forces as dominant, although not independent, drivers of hemi-fission and fission transformations, respectively. Mirrored in the fusion reaction, the force bimodality might constitute a general paradigm for leakage-free membrane remodelling.

Dynamins and BAR Proteins-Safeguards against Cancer.

Dynamins and BAR proteins are crucial in a wide variety of cellular processes for their ability to mediate membrane remodeling, such as membrane curvature and membrane fission and fusion. In this review, we highlight dynamins and BAR proteins and the cellular mechanisms that are involved in the initiation and progression of cancer. We specifically discuss the roles of the seproteinsin endocytosis, endo-lysosomal trafficking, autophagy, and apoptosis as these processes are all tightly linked to membrane remodeling and cancer.

Regulating dynamin dynamics during endocytosis.

Dynamin is a large GTPase that mediates plasma membrane fission during clathrin-mediated endocytosis. Dynamin assembles into polymers on the necks of budding membranes in cells and has been shown to undergo GTP-dependent conformational changes that lead to membrane fission in vitro. Recent efforts have shed new light on the mechanisms of dynamin-mediated fission, yet exactly how dynamin performs this function in vivo is still not fully understood. Dynamin interacts with a number of proteins during the endocytic process. These interactions are mediated by the C-terminal proline-rich domain (PRD) of dynamin binding to SH3 domain-containing proteins. Three of these dynamin-binding partners (intersectin, amphiphysin and endophilin) have been shown to play important roles in the clathrin-mediated endocytosis process. They promote dynamin-mediated plasma membrane fission by regulating three important sequential steps in the process: recruitment of dynamin to sites of endocytosis; assembly of dynamin into a functional fission complex at the necks of clathrin-coated pits (CCPs); and regulation of dynamin-stimulated GTPase activity, a key requirement for fission.

A dynamin mutant defines a superconstricted prefission state.

Dynamin is a 100 kDa GTPase that organizes into helical assemblies at the base of nascent clathrin-coated vesicles. Formation of these oligomers stimulates the intrinsic GTPase activity of dynamin, which is necessary for efficient membrane fission during endocytosis. Recent evidence suggests that the transition state of dynamin's GTP hydrolysis reaction serves as a key determinant of productive fission. Here, we present the structure of a transition-state-defective dynamin mutant K44A trapped in a prefission state at 12.5 Å resolution. This structure constricts to 3.7 nm, reaching the theoretical limit required for spontaneous membrane fission. Computational docking indicates that the ground-state conformation of the dynamin polymer is sufficient to achieve this superconstricted prefission state and reveals how a two-start helical symmetry promotes the most efficient packing of dynamin tetramers around the membrane neck. These data suggest a model for the assembly and regulation of the minimal dynamin fission machine.

An endophilin-dynamin complex promotes budding of clathrin-coated vesicles during synaptic vesicle recycling.

Clathrin-mediated vesicle recycling in synapses is maintained by a unique set of endocytic proteins and interactions. We show that endophilin localizes in the vesicle pool at rest and in spirals at the necks of clathrin-coated pits (CCPs) during activity in lamprey synapses. Endophilin and dynamin colocalize at the base of the clathrin coat. Protein spirals composed of these proteins on lipid tubes in vitro have a pitch similar to the one observed at necks of CCPs in living synapses, and lipid tubules are thinner than those formed by dynamin alone. Tubulation efficiency and the amount of dynamin recruited to lipid tubes are dramatically increased in the presence of endophilin. Blocking the interactions of the endophilin SH3 domain in situ reduces dynamin accumulation at the neck and prevents the formation of elongated necks observed in the presence of GTPγS. Therefore, endophilin recruits dynamin to a restricted part of the CCP neck, forming a complex, which promotes budding of new synaptic vesicles.

Molecular basis for SH3 domain regulation of F-BAR-mediated membrane deformation.

Members of the Bin/amphiphysin/Rvs (BAR) domain protein superfamily are involved in membrane remodeling in various cellular pathways ranging from endocytic vesicle and T-tubule formation to cell migration and neuromorphogenesis. Membrane curvature induction and stabilization are encoded within the BAR or Fer-CIP4 homology-BAR (F-BAR) domains, alpha-helical coiled coils that dimerize into membrane-binding modules. BAR/F-BAR domain proteins often contain an SH3 domain, which recruits binding partners such as the oligomeric membrane-fissioning GTPase dynamin. How precisely BAR/F-BAR domain-mediated membrane deformation is regulated at the cellular level is unknown. Here we present the crystal structures of full-length syndapin 1 and its F-BAR domain. Our data show that syndapin 1 F-BAR-mediated membrane deformation is subject to autoinhibition by its SH3 domain. Release from the clamped conformation is driven by association of syndapin 1 SH3 with the proline-rich domain of dynamin 1, thereby unlocking its potent membrane-bending activity. We hypothesize that this mechanism might be commonly used to regulate BAR/F-BAR domain-induced membrane deformation and to potentially couple this process to dynamin-mediated fission. Our data thus suggest a structure-based model for SH3-mediated regulation of BAR/F-BAR domain function.

Regulation of synaptic vesicle recycling by complex formation between intersectin 1 and the clathrin adaptor complex AP2.

Clathrin-mediated synaptic vesicle (SV) recycling involves the spatiotemporally controlled assembly of clathrin coat components at phosphatidylinositiol (4, 5)-bisphosphate [PI(4,5)P(2)]-enriched membrane sites within the periactive zone. Such spatiotemporal control is needed to coordinate SV cargo sorting with clathrin/AP2 recruitment and to restrain membrane fission and synaptojanin-mediated uncoating until membrane deformation and clathrin coat assembly are completed. The molecular events underlying these control mechanisms are unknown. Here we show that the endocytic SH3 domain-containing accessory protein intersectin 1 scaffolds the endocytic process by directly associating with the clathrin adaptor AP2. Acute perturbation of the intersectin 1-AP2 interaction in lamprey synapses in situ inhibits the onset of SV recycling. Structurally, complex formation can be attributed to the direct association of hydrophobic peptides within the intersectin 1 SH3A-B linker region with the "side sites" of the AP2 alpha- and beta-appendage domains. AP2 appendage association of the SH3A-B linker region inhibits binding of the inositol phosphatase synaptojanin 1 to intersectin 1. These data identify the intersectin-AP2 complex as an important regulator of clathrin-mediated SV recycling in synapses.

Intersectin is a negative regulator of dynamin recruitment to the synaptic endocytic zone in the central synapse.

Intersectin is a multidomain dynamin-binding protein implicated in numerous functions in the nervous system, including synapse formation and endocytosis. Here, we demonstrate that during neurotransmitter release in the central synapse, intersectin, like its binding partner dynamin, is redistributed from the synaptic vesicle pool to the periactive zone. Acute perturbation of the intersectin-dynamin interaction by microinjection of either intersectin antibodies or Src homology 3 (SH3) domains inhibited endocytosis at the fission step. Although the morphological effects induced by the different reagents were similar, antibody injections resulted in a dramatic increase in dynamin immunoreactivity around coated pits and at constricted necks, whereas synapses microinjected with the GST (glutathione S-transferase)-SH3C domain displayed reduced amounts of dynamin in the neck region. Our data suggest that intersectin controls the amount of dynamin released from the synaptic vesicle cluster to the periactive zone and that it may regulate fission of clathrin-coated intermediates.

IL-18 accelerates atherosclerosis accompanied by elevation of IFN-gamma and CXCL16 expression independently of T cells.

OBJECTIVE: The proatherogenic effect of IL-18 is shown to be dependent on IFN-gamma production. It is believed that activated T cells play a proatherogenic role through secretion of IFN-gamma. However, recent studies in vitro have shown that macrophages, NK cells, and even vascular smooth muscle cells may also secrete IFN-gamma after stimulation by cytokines like IL-18. We therefore investigated whether cells other than activated T cells can play a proatherogenic role via IFN-gamma secretion under the stimulation of IL-18 in vivo. METHODS AND RESULTS: SCID/apoE knockout mice were injected intraperitoneally with either IL-18 or phosphate-buffered saline 3 times per week for 7 weeks. Our results show that administration of IL-18 leads to 3-fold larger lesions and 2-fold higher circulating IFN-gamma despite the absence of T cells. In addition, increased IFN-gamma, accompanied by elevation of the scavenger receptor/chemokine CXCL16, was observed in both lesions and spleens. Furthermore, our findings revealed that macrophages, NK cells, and vascular cells were the source of IFN-gamma under the stimulation of IL-18 in the absence of T cells in vivo. CONCLUSIONS: The current data suggest that the proatherogenic effect of IL-18 can occur in the absence of T cells and that IFN-gamma secreted by macrophages, NK cells, and vascular cells is sufficient for the disease progression.