Dynamin1 long- and short-tail isoforms exploit distinct recruitment and spatial patterns to form endocytic nanoclusters.

Endocytosis requires a coordinated framework of molecular interactions that ultimately lead to the fission of nascent endocytic structures. How cytosolic proteins such as dynamin concentrate at discrete sites that are sparsely distributed across the plasma membrane remains poorly understood. Two dynamin-1 major splice variants differ by the length of their C-terminal proline-rich region (short-tail and long-tail). Using sptPALM in PC12 cells, neurons and MEF cells, we demonstrate that short-tail dynamin-1 isoforms ab and bb display an activity-dependent recruitment to the membrane, promptly followed by their concentration into nanoclusters. These nanoclusters are sensitive to both Calcineurin and dynamin GTPase inhibitors, and are larger, denser, and more numerous than that of long-tail isoform aa. Spatiotemporal modelling confirms that dynamin-1 isoforms perform distinct search patterns and undergo dimensional reduction to generate endocytic nanoclusters, with short-tail isoforms more robustly exploiting lateral trapping in the generation of nanoclusters compared to the long-tail isoform.

Mechanistic analysis of a novel membrane-interacting variable loop in the pleckstrin-homology domain critical for dynamin function.

Classical dynamins are best understood for their ability to generate vesicles by membrane fission. During clathrin-mediated endocytosis (CME), dynamin is recruited to the membrane through multivalent protein and lipid interactions between its proline-rich domain (PRD) with SRC Homology 3 (SH3) domains in endocytic proteins and its pleckstrin-homology domain (PHD) with membrane lipids. Variable loops (VL) in the PHD bind lipids and partially insert into the membrane thereby anchoring the PHD to the membrane. Recent molecular dynamics (MD) simulations reveal a novel VL4 that interacts with the membrane. Importantly, a missense mutation that reduces VL4 hydrophobicity is linked to an autosomal dominant form of Charcot-Marie-Tooth (CMT) neuropathy. We analyzed the orientation and function of the VL4 to mechanistically link data from simulations with the CMT neuropathy. Structural modeling of PHDs in the cryo-electron microscopy (cryo-EM) cryoEM map of the membrane-bound dynamin polymer confirms VL4 as a membrane-interacting loop. In assays that rely solely on lipid-based membrane recruitment, VL4 mutants with reduced hydrophobicity showed an acute membrane curvature-dependent binding and a catalytic defect in fission. Remarkably, in assays that mimic a physiological multivalent lipid- and protein-based recruitment, VL4 mutants were completely defective in fission across a range of membrane curvatures. Importantly, expression of these mutants in cells inhibited CME, consistent with the autosomal dominant phenotype associated with the CMT neuropathy. Together, our results emphasize the significance of finely tuned lipid and protein interactions for efficient dynamin function.

Wiskostatin and other carbazole scaffolds as off target inhibitors of dynamin I GTPase activity and endocytosis.

Wiskostatin (1-(3,6-dibromo-9H-carbazol-9-yl)-3-(dimethylamino)propan-2-ol) (1) is a carbazole-based compound reported as a specific and relatively potent inhibitor of the N-WASP actin remodelling complex (S-isomer EC50 = 4.35 µM; R-isomer EC50 = 3.44 µM). An NMR solution structure showed that wiskostatin interacts with a cleft in the regulatory GTPase binding domain of N-WASP. However, numerous studies have reported wiskostatin's actions on membrane transport and cytokinesis that are independent of the N-WASP-Arp2/3 complex pathway, but offer limited alternative explanation. The large GTPase, dynamin has established functional roles in these pathways. This study reveals that wiskostatin and its analogues, as well as other carbazole-based compounds, are inhibitors of helical dynamin GTPase activity and endocytosis. We characterise the effects of wiskostatin on in vitro dynamin GTPase activity, in-cell endocytosis, and determine the importance of wiskostatin functional groups on these activities through design and synthesis of libraries of wiskostatin analogues. We also examine whether other carbazole-based scaffolds frequently used in research or the clinic also modulate dynamin and endocytosis. Understanding off-targets for compounds used as research tools is important to be able to confidently interpret their action on biological systems, particularly when the target and off-targets affect overlapping mechanisms (e.g. cytokinesis and endocytosis). Herein we demonstrate that wiskostatin is a dynamin inhibitor (IC50 20.7 ± 1.2 µM) and a potent inhibitor of clathrin mediated endocytosis (IC50 = 6.9 ± 0.3 µM). Synthesis of wiskostatin analogues gave rise to 1-(9H-carbazol-9-yl)-3-((4-methylbenzyl)amino)propan-2-ol (35) and 1-(9H-carbazol-9-yl)-3-((4-chlorobenzyl)amino)propan-2-ol (43) as potent dynamin inhibitors (IC50 = 1.0 ± 0.2 µM), and (S)-1-(3,6-dibromo-9H-carbazol-9-yl)-3-(dimethylamino)propan-2-ol (8a) and (R)-1-(3,6-dibromo-9H-carbazol-9-yl)-3-(dimethylamino)propan-2-ol (8b) that are amongst the most potent inhibitors of clathrin mediated endocytosis yet reported (IC50 = 2.3 ± 3.3 and 2.1 ± 1.7 µM, respectively).

Dynamin is primed at endocytic sites for ultrafast endocytosis.

Dynamin mediates fission of vesicles from the plasma membrane during endocytosis. Typically, dynamin is recruited from the cytosol to endocytic sites, requiring seconds to tens of seconds. However, ultrafast endocytosis in neurons internalizes vesicles as quickly as 50 ms during synaptic vesicle recycling. Here, we demonstrate that Dynamin 1 is pre-recruited to endocytic sites for ultrafast endocytosis. Specifically, Dynamin 1xA, a splice variant of Dynamin 1, interacts with Syndapin 1 to form molecular condensates on the plasma membrane. Single-particle tracking of Dynamin 1xA molecules confirms the liquid-like property of condensates in vivo. When Dynamin 1xA is mutated to disrupt its interaction with Syndapin 1, the condensates do not form, and consequently, ultrafast endocytosis slows down by 100-fold. Mechanistically, Syndapin 1 acts as an adaptor by binding the plasma membrane and stores Dynamin 1xA at endocytic sites. This cache bypasses the recruitment step and accelerates endocytosis at synapses.

Microtubule assembly by tau impairs endocytosis and neurotransmission via dynamin sequestration in Alzheimer's disease synapse model.

Elevation of soluble wild-type (WT) tau occurs in synaptic compartments in Alzheimer's disease. We addressed whether tau elevation affects synaptic transmission at the calyx of Held in slices from mice brainstem. Whole-cell loading of WT human tau (h-tau) in presynaptic terminals at 10-20 µM caused microtubule (MT) assembly and activity-dependent rundown of excitatory neurotransmission. Capacitance measurements revealed that the primary target of WT h-tau is vesicle endocytosis. Blocking MT assembly using nocodazole prevented tau-induced impairments of endocytosis and neurotransmission. Immunofluorescence imaging analyses revealed that MT assembly by WT h-tau loading was associated with an increased MT-bound fraction of the endocytic protein dynamin. A synthetic dodecapeptide corresponding to dynamin 1-pleckstrin-homology domain inhibited MT-dynamin interaction and rescued tau-induced impairments of endocytosis and neurotransmission. We conclude that elevation of presynaptic WT tau induces de novo assembly of MTs, thereby sequestering free dynamins. As a result, endocytosis and subsequent vesicle replenishment are impaired, causing activity-dependent rundown of neurotransmission.

Dephospho-dynamin 1 coupled to activity-dependent bulk endocytosis participates in epileptic seizure in primary hippocampal neurons.

BACKGROUND AND PURPOSE: Epilepsy is a severe neurological and mental disorder, and not all patients adequately respond to the current treatments. Dynamin 1 plays a key role in synaptic endocytosis and the modulation of neurological function. MATERIAL AND METHODS: Cultured hippocampal neurons were used in the study. First, the viability of neurons was determined by the CCK-8 assay after culturing in magnesium-free medium, DMSO, dynasore (dynamin agonist), and PIP2 (dynamin antagonist). Then, the effect of dynasore on seizure activity was evaluated. Next, we tested the levels of phospho-dynamin 1/total dynamin 1 and dynamin 1 mRNA in the control group and four epilepsy groups. Moreover, the uptake of tetramethylrhodamine-dextran in the different groups was measured. RESULTS: Dephospho-dynamin 1 expression was significantly increased in hyperexcitable neurons, while there was no change in total dynamin 1 level. The level of dephospho-dynamin 1 in hyperexcitable neurons was reduced when cultured with dynasore but increased with PIP2 treatment. Activity-dependent bulk endocytosis (ADBE) was upregulated in hyperexcitable neurons. Along with a decrease in dephospho-dynamin 1 level, ADBE was also downregulated with dynasore treatment, while PIP2 did not affect ABDE. The close link between the dephosphorylation status of dynamin 1 and ADBE suggests that ADBE activation depends on dynamin 1 dephosphorylation. CONCLUSION: Dephospho-dynamin 1 triggers ADBE to meet the requirements of high-frequency discharges during epileptic seizures.

miR-199b-5p-AKAP1-DRP1 Pathway Plays a Key Role in ox-LDL-induced Mitochondrial Fission and Endothelial Apoptosis.

BACKGROUND: Atherosclerosis (AS) remains prevalent despite hyperlipidemia-lowering therapies. Although multiple functions of miR-199b-5p have been implicated in cancers, its role in endothelial apoptosis and AS remains unclear. This study aimed to examine the role of miR-199b-5p in mitochondrial dynamics and endothelial apoptosis. METHODS: Human umbilical vein endothelial cells (HUVECs) treated with oxidized low-density lipoprotein (ox-LDL) were subjected to other treatments, followed by a series analysis. We found that ox-LDL-treated HUVECs were associated with miR-199b-5p downregulation, increased reactive oxygen species level, reduced adenosine triphosphate (ATP) production, mitochondrial fission, and apoptosis, whereas enhanced miR-199b-5p expression or applied mitochondrial division inhibitor 1 (Mdivi-1) markedly reversed these changes. RESULTS: Mechanistically, A-kinase anchoring protein 1 (AKAP1) was confirmed as a downstream target of miR-199b-5p by dual-luciferase activity reporter assay. AKAP1 overexpression reversed the anti-apoptotic effects of miR-199b-5p through the enhanced interaction of AKAP1 and dynamin protein 1 (DRP1) in ox-LDL-treated HUVECs. Moreover, miR-199b-5p downregulation, AKAP1 upregulation, and excessive mitochondrial fission were verified in human coronary AS endothelial tissues. CONCLUSION: The miR-199b-5p-dependent regulation of AKAP1/DRP1 is required to inhibit hyperlipidemia- induced mitochondrial fission and endothelial injury and may be a promising therapeutic target for AS.

Dynamin 1 controls vesicle size and endocytosis at hippocampal synapses.

Following calcium-triggered vesicle exocytosis, endocytosis regenerates vesicles to maintain exocytosis and thus synaptic transmission, which underlies neuronal circuit activities. Although most molecules involved in endocytosis have been identified, it remains rather poorly understood how endocytic machinery regulates vesicle size. Vesicle size, together with the transmitter concentration inside the vesicle, determines the amount of transmitter the vesicle can release, the quantal size, that may control the strength of synaptic transmission. Here, we report that, surprisingly, knockout of the GTPase dynamin 1, the most abundant brain dynamin isoform known to catalyze fission of the membrane pit's neck (the last step of endocytosis), not only significantly slowed endocytosis but also increased the synaptic vesicle diameter by as much as ∼40-64% at cultured hippocampal synapses. Furthermore, dynamin 1 knockout increased the size of membrane pits, the precursor for endocytic vesicle formation. These results suggest an important function of dynamin other than its well-known fission function - control of vesicle size at the pit formation stage.

Lipopolysaccharide Accelerates Neuropilin-1 Protein Degradation by Activating the Large GTPase Dynamin-1 in Macrophages.

Neuropilin-1 (Nrp1) is highly expressed in macrophages and plays a critical role in acute and chronic inflammation-associated diseases, such as sepsis, type II diabetes, and metabolic syndrome. Therefore, it is of importance to understand the regulation of Nrp1. It is known that lipopolysaccharide (LPS) downregulates Nrp1 mRNA levels through the NF-κB signaling in macrophages. However, whether and how LPS regulates Nrp1 protein degradation remain unknown. Here, we show that LPS promotes Nrp1 protein decay through a lysosome-dependent manner. Liver kinase B1 (LKB1)-Rab7 does not mediate this process. However, the large GTPase dynamin-1 (Dyn1) but not Dyn2 is involved in LPS-accelerated Nrp1 degradation. Mechanistically, LPS activates Dyn1 by attenuating p-Dyn1 (Ser774) levels, implying increased Nrp1 endocytosis and consequent degradation. As a result, blocking Nrp1 degradation by Dyn1 siRNA attenuates LPS-induced inflammatory response. Collectively, our study shows that LPS promotes Nrp1 protein degradation via a Dyn1-dependent pathway, revealing a previously uncovered role of Dyn1 in LPS-promoted Nrp1 protein decay.

T0901317, an Agonist of Liver X Receptors, Attenuates Neuronal Apoptosis in Early Brain Injury after Subarachnoid Hemorrhage in Rats via Liver X Receptors/Interferon Regulatory Factor/P53 Upregulated Modulator of Apoptosis/Dynamin-1-Like Protein Pathway.

METHODS: Subarachnoid hemorrhage (SAH) models of Sprague-Dawley rats were established with perforation method. T0901317 was injected intraperitoneally 1-hour post-SAH. GSK2033, an inhibitor of LXRs, and interferon regulatory factor (IRF-1) CRISPR activation were injected intracerebroventricularly to evaluate potential signaling pathway. The severity of SAH, neurobehavior test in both short- and long-term and apoptosis was measured with Western blot and immunofluorescence staining. RESULTS: Expression of LXR-α and IRF-1 increased and peaked at 24 h post-SAH, while LXR-ß remained unaffected in SAH+vehicle group compared with Sham group. Post-SAH T0901317 treatment attenuated neuronal impairments in both short- and long-term and decreased neuronal apoptosis, the expression of IRF-1, P53 upregulated modulator of apoptosis (PUMA), dynamin-1-like protein (Drp1), Bcl-2-associated X protein (Bax) and cleaved caspase-3, and increasing B-cell lymphoma 2 (Bcl-2) at 24 h from modeling. GSK2033 inhibited LXRs and reversed T0901317's neuroprotective effects. IRF-1 CRISPR activation upregulated the expression of IRF-1 and abolished the treatment effects of T0901317. CONCLUSION: T0901317 attenuated neuronal apoptosis via LXRs/IRF-1/PUMA/Drp1 pathway in SAH rats.

Dynamin regulates L cell secretion in human gut.

BACKGROUND: The mechanochemical enzyme dynamin mediates endocytosis and regulates neuroendocrine cell exocytosis. Enteroendocrine L cells co-secrete the anorectic gut hormones glucagon-like peptide 1 (GLP-1) and peptide YY (PYY) postprandially and is a potential therapeutic target for metabolic diseases. In the present study, we aimed to determine if dynamin is implicated in human L cell secretion. METHODS: Western blot was performed on the murine L cell line GLUTag. Static incubation of human colonic mucosae with activators and inhibitors of dynamin was carried out. GLP-1 and PYY contents of the secretion supernatants were assayed using ELISA. RESULTS AND CONCLUSION: s: Both dynamin I and II are expressed in GLUTag cells. The dynamin activator Ryngo 1-23 evoked significant GLP-1 and PYY release from human colonic mucosae while the dynamin inhibitor Dynole 3-42 significantly inhibited release triggered by known L cell secretagogues. Thus, the cell signaling regulator dynamin is able to bi-directionally regulate L cell hormone secretion in the human gut and may represent a novel target for gastrointestinal-targeted metabolic drug development.

Flexible pivoting of dynamin pleckstrin homology domain catalyzes fission: insights into molecular degrees of freedom.

The neuronal dynamin1 functions in the release of synaptic vesicles by orchestrating the process of GTPase-dependent membrane fission. Dynamin1 associates with the plasma membrane-localized phosphatidylinositol-4,5-bisphosphate (PIP2) through the centrally located pleckstrin homology domain (PHD). The PHD is dispensable as fission (in model membranes) can be managed, even when the PHD-PIP2 interaction is replaced by a generic polyhistidine- or polylysine-lipid interaction. However, the absence of the PHD renders a dramatic dampening of the rate of fission. These observations suggest that the PHD-PIP2-containing membrane interaction could have evolved to expedite fission to fulfill the requirement of rapid kinetics of synaptic vesicle recycling. Here, we use a suite of multiscale modeling approaches to explore PHD-membrane interactions. Our results reveal that 1) the binding of PHD to PIP2-containing membranes modulates the lipids toward fission-favoring conformations and softens the membrane, and 2) PHD associates with membrane in multiple orientations using variable loops as pivots. We identify a new loop (VL4), which acts as an auxiliary pivot and modulates the orientation flexibility of PHD on the membrane-a mechanism that we believe may be important for high-fidelity dynamin collar assembly. Together, these insights provide a molecular-level understanding of the catalytic role of PHD in dynamin-mediated membrane fission.

Cdk5 and GSK3ß inhibit fast endophilin-mediated endocytosis.

Endocytosis mediates the cellular uptake of micronutrients and cell surface proteins. Fast Endophilin-mediated endocytosis, FEME, is not constitutively active but triggered upon receptor activation. High levels of growth factors induce spontaneous FEME, which can be suppressed upon serum starvation. This suggested a role for protein kinases in this growth factor receptor-mediated regulation. Using chemical and genetic inhibition, we find that Cdk5 and GSK3ß are negative regulators of FEME. They antagonize the binding of Endophilin to Dynamin-1 and to CRMP4, a Plexin A1 adaptor. This control is required for proper axon elongation, branching and growth cone formation in hippocampal neurons. The kinases also block the recruitment of Dynein onto FEME carriers by Bin1. As GSK3ß binds to Endophilin, it imposes a local regulation of FEME. Thus, Cdk5 and GSK3ß are key regulators of FEME, licensing cells for rapid uptake by the pathway only when their activity is low.

Spatial decoding of endosomal cAMP signals by a metastable cytoplasmic PKA network.

G-protein-coupled receptor-regulated cAMP production from endosomes can specify signaling to the nucleus by moving the source of cAMP without changing its overall amount. How this is possible remains unknown because cAMP gradients dissipate over the nanoscale, whereas endosomes typically localize micrometers from the nucleus. We show that the key location-dependent step for endosome-encoded transcriptional control is nuclear entry of cAMP-dependent protein kinase (PKA) catalytic subunits. These are sourced from punctate accumulations of PKA holoenzyme that are densely distributed in the cytoplasm and titrated by global cAMP into a discrete metastable state, in which catalytic subunits are bound but dynamically exchange. Mobile endosomes containing activated receptors collide with the metastable PKA puncta and pause in close contact. We propose that these properties enable cytoplasmic PKA to act collectively like a semiconductor, converting nanoscale cAMP gradients generated from endosomes into microscale elevations of free catalytic subunits to direct downstream signaling.

Altered Fast Synaptic Transmission in a Mouse Model of DNM1-Associated Developmental Epileptic Encephalopathy.

Developmental epileptic encephalopathies (DEEs) are severe seizure disorders that occur in infants and young children, characterized by developmental delay, cognitive decline, and early mortality. Recent efforts have identified a wide variety of genetic variants that cause DEEs. Among these, variants in the DNM1 gene have emerged as definitive causes of DEEs, including infantile spasms and Lennox-Gastaut syndrome. A mouse model of Dnm1-associated DEE, known as "Fitful" (Dnm1Ftfl ), recapitulates key features of the disease, including spontaneous seizures, early lethality, and neuronal degeneration. Previous work showed that DNM1 is a key regulator of synaptic vesicle (SV) endocytosis and synaptic transmission and suggested that inhibitory neurotransmission may be more reliant on DNM1 function than excitatory transmission. The Dnm1Ftfl variant is thought to encode a dominant negative DNM1 protein; however, the effects of the Dnm1Ftfl variant on synaptic transmission are largely unknown. To understand these synaptic effects, we recorded from pairs of cultured mouse cortical neurons and characterized all four major connection types [excitation of excitation (E-E), inhibition of inhibition (I-I), E-I, I-E]. Miniature and spontaneous EPSCs and IPSCs were larger, but less frequent, at all Dnm1Ftfl synaptic types, and Dnm1Ftfl neurons had reduced expression of excitatory and inhibitory SV markers. Baseline evoked transmission, however, was reduced only at inhibitory synapses onto excitatory neurons, because of a smaller pool of releasable SVs. In addition to these synaptic alterations, Dnm1Ftfl neurons degenerated later in development, although their activity levels were reduced, suggesting that Dnm1Ftfl may impair synaptic transmission and neuronal health through distinct mechanisms.

Dynamin 1 is important for microtubule organization and stabilization in glomerular podocytes.

Dynamin 1 is a neuronal endocytic protein that participates in vesicle formation by scission of invaginated membranes. Dynamin 1 is also expressed in the kidney; however, its physiological significance to this organ remains unknown. Here, we show that dynamin 1 is crucial for microtubule organization and stabilization in glomerular podocytes. By immunofluorescence and immunoelectron microscopy, dynamin 1 was concentrated at microtubules at primary processes in rat podocytes. By immunofluorescence of differentiated mouse podocytes (MPCs), dynamin 1 was often colocalized with microtubule bundles, which radially arranged toward periphery of expanded podocyte. In dynamin 1-depleted MPCs by RNAi, α-tubulin showed a dispersed linear filament-like localization, and microtubule bundles were rarely observed. Furthermore, dynamin 1 depletion resulted in the formation of discontinuous, short acetylated α-tubulin fragments, and the decrease of microtubule-rich protrusions. Dynamins 1 and 2 double-knockout podocytes showed dispersed acetylated α-tubulin and rare protrusions. In vitro, dynamin 1 polymerized around microtubules and cross-linked them into bundles, and increased their resistance to the disassembly-inducing reagents Ca2+ and podophyllotoxin. In addition, overexpression and depletion of dynamin 1 in MPCs increased and decreased the nocodazole resistance of microtubules, respectively. These results suggest that dynamin 1 supports the microtubule bundle formation and participates in the stabilization of microtubules.

Dynasore Blocks Ferroptosis through Combined Modulation of Iron Uptake and Inhibition of Mitochondrial Respiration.

Ferroptosis is a form of regulated necrosis characterized by a chain-reaction of detrimental membrane lipid peroxidation following collapse of glutathione peroxidase 4 (Gpx4) activity. This lipid peroxidation is catalyzed by labile ferric iron. Therefore, iron import mediated via transferrin receptors and both, enzymatic and non-enzymatic iron-dependent radical formation are crucial prerequisites for the execution of ferroptosis. Intriguingly, the dynamin inhibitor dynasore, which has been shown to block transferrin receptor endocytosis, can protect from ischemia/reperfusion injury as well as neuronal cell death following spinal cord injury. Yet, it is unknown how dynasore exerts these cell death-protective effects. Using small interfering RNA suppression, lipid reactive oxygen species (ROS), iron tracers and bona fide inducers of ferroptosis, we find that dynasore treatment in lung adenocarcinoma and neuronal cell lines strongly protects these from ferroptosis. Surprisingly, while the dynasore targets dynamin 1 and 2 promote extracellular iron uptake, their silencing was not sufficient to block ferroptosis suggesting that this route of extracellular iron uptake is dispensable for acute induction of ferroptosis and dynasore must have an additional off-target activity mediating full ferroptosis protection. Instead, in intact cells, dynasore inhibited mitochondrial respiration and thereby mitochondrial ROS production which can feed into detrimental lipid peroxidation and ferroptotic cell death in the presence of labile iron. In addition, in cell free systems, dynasore showed radical scavenger properties and acted as a broadly active antioxidant which is superior to N-acetylcysteine (NAC) in blocking ferroptosis. Thus, dynasore can function as a highly active inhibitor of ROS-driven types of cell death via combined modulation of the iron pool and inhibition of general ROS by simultaneously blocking two routes required for ROS and lipid-ROS driven cell death, respectively. These data have important implications for the interpretation of studies observing tissue-protective effects of this dynamin inhibitor as well as raise awareness that off-target ROS scavenging activities of small molecules used to interrogate the ferroptosis pathway should be taken into consideration.

Early and nonredundant functions of dynamin isoforms in clathrin-mediated endocytosis.

Dynamin GTPases (Dyn1 and Dyn2) are indispensable proteins of the core clathrin-mediated endocytosis (CME) machinery. Best known for their role in fission at the late stages of CME, many studies have suggested that dynamin also plays a regulatory role during the early stages of CME; however, detailed studies regarding isoform-specific early regulatory functions of the dynamins are lacking. With a recent understanding of the regulation of Dyn1 in nonneuronal cells and improved algorithms for highly sensitive and quantitative analysis of clathrin-coated pit (CCP) dynamics, we have evaluated the differential functions of dynamin isoforms in CME using domain swap chimeras. We report that Dyn1 and Dyn2 play nonredundant, early regulatory roles during CME in nonneuronal cells. The proline/arginine-rich domain of Dyn2 is important for its targeting to nascent and growing CCPs, whereas the membrane-binding and curvature-generating pleckstrin homology domain of Dyn1 plays an important role in stabilizing nascent CCPs. We confirm the enhanced ability of dephosphorylated Dyn1 to support CME, even at substoichiometric levels compared with Dyn2. Domain swap chimeras also revealed previously unknown functional differences in the GTPase and stalk domains. Our study significantly extends the current understanding of the regulatory roles played by dynamin isoforms during early stages of CME.

Electrophysiological Methods for Detection of Membrane Leakage and Hemifission by Dynamin 1.

Membrane fusion and fission are indispensable parts of intracellular membrane recycling and transport. Electrophysiological techniques have been instrumental in discovering and studying fusion and fission pores, the key intermediates shared by both processes. In cells, electrical admittance measurements are used to assess in real time the dynamics of the pore conductance, reflecting the nanoscale transformations of the pore, simultaneously with membrane leakage. Here, we described how this technique is adapted to in vitro mechanistic analyses of membrane fission by dynamin 1 (Dyn1), the protein orchestrating membrane fission in endocytosis. We reconstitute the fission reaction using purified Dyn1 and biomimetic lipid membrane nanotubes of defined geometry. We provide a comprehensive protocol describing simultaneous measurements of the ionic conductance through the nanotube lumen and across the nanotube wall, enabling spatiotemporal correlation between the nanotube constriction by Dyn1, leading to fission and membrane leakage. We present examples of "leaky" and "tight" fission reactions, specify the resolution limits of our method, and discuss how our results support the hemi-fission conjecture.

BTBD9 and dopaminergic dysfunction in the pathogenesis of restless legs syndrome.

Restless legs syndrome (RLS) is characterized by an urge to move legs, usually accompanied by uncomfortable sensations. RLS symptoms generally happen at night and can be relieved by movements. Genetic studies have linked polymorphisms in BTBD9 to a higher risk of RLS. Knockout of BTBD9 homolog in mice (Btbd9) and fly results in RLS-like phenotypes. A dysfunctional dopaminergic system is associated with RLS. However, the function of BTBD9 in the dopaminergic system and RLS is not clear. Here, we made use of the simple Caenorhabditis elegans nervous system. Loss of hpo-9, the worm homolog of BTBD9, resulted in hyperactive egg-laying behavior. Analysis of genetic interactions between hpo-9 and genes for dopamine receptors (dop-1, dop-3) indicated that hpo-9 and dop-1 worked similarly. Reporter assays of dop-1 and dop-3 revealed that hpo-9 knockout led to a significant increase of DOP-3 expression. This appears to be evolutionarily conserved in mice with an increased D2 receptor (D2R) mRNA in the striatum of the Btbd9 knockout mice. Furthermore, the striatal D2R protein was significantly decreased and Dynamin I was increased. Overall, activities of DA neurons in the substantia nigra were not altered, but the peripheral D1R pathway was potentiated in the Btbd9 knockout mice. Finally, we generated and characterized the dopamine neuron-specific Btbd9 knockout mice and detected an active-phase sleepiness, suggesting that dopamine neuron-specific loss of Btbd9 is sufficient to disturb the sleep. Our results suggest that increased activities in the D1R pathway, decreased activities in the D2R pathway, or both may contribute to RLS.