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1.  Dynamin-driven membrane fission reveals catalytic principle of membrane remodeling 
Science (New York, N.Y.)  2013;339(6126):1433-1436.
Biological membrane fission is conducted by protein-driven stress. To create such membrane stress the GTPase dynamin-1, protein orchestrating membrane fission in endocytosis, assembles into helical scaffolds that constrict the necks of endocytic vesicles. We found that under constant GTP turnover two-rung dynamin scaffold is sufficient to produce fission of lipid nanotubes. Analyzing membrane fission by short dynamin scaffolds, we reveal a catalytic cycle which translates constriction stresses into fission. Upon constriction, coordinated membrane wedging by the scaffold facilitates reversible merger of the inner leaflet of the nanotube, the hemifission. Modeling of this reversible step identifies a low-energy path based on geometric coupling of the scaffold and the membrane. The final translation of the metastable hemifission into complete fission is stochastically linked to disassembly of the scaffold. This catalytic conversion of localized stresses into membrane remodeling suggests a novel paradigm for fission and fusion of cellular membranes.
doi:10.1126/science.1233920
PMCID: PMC3980720  PMID: 23520112
2.  Advances in Analysis of Low Signal-to-Noise Images Link Dynamin and AP2 to the Functions of an Endocytic Checkpoint 
Developmental cell  2013;26(3):279-291.
SUMMARY
Numerous endocytic accessory proteins (EAPs) mediate assembly and maturation of clathrin-coated pits (CCPs) into cargo-containing vesicles. Analysis of EAP function through bulk measurement of cargo uptake has been hampered due to potential redundancy among EAPs and, as we show here, the plasticity and resilience of clathrin-mediated endocytosis (CME). Instead, EAP function is best studied by uncovering the correlation between variations in EAP association to individual CCPs and the resulting variations in maturation. However, most EAPs bind to CCPs in low numbers, making the measurement of EAP association via fused fluorescent reporters highly susceptible to detection errors. Here, we present a framework for unbiased measurement of EAP recruitment to CCPs and their direct effects on CCP dynamics. We identify dynamin and the EAP-binding α-adaptin appendage domain of the AP2 adaptor as switches in a regulated, multistep maturation process and provide direct evidence for a molecular checkpoint in CME.
doi:10.1016/j.devcel.2013.06.019
PMCID: PMC3939604  PMID: 23891661
3.  Integrated Electron Microscopy: Super-Duper Resolution 
PLoS Biology  2013;11(8):e1001639.
New developments in electron microscopy used in combination reveal the elegant architecture of cellular structures at very high resolution.
Since its inception, electron microscopy (EM) has revealed that cellular membranes are organized into structurally distinct subdomains, created by localized protein and lipid assemblies to perform specific complex cellular functions. Caveolae are membrane subdomains that function as signaling platforms, endocytic carriers, sensors of membrane tension, and mechanical stress, as well as in lipid homeostasis. They were first discovered almost 60 years ago by pioneering electron microscopists. While new and exciting developments in SUPER-resolution fluorescent light microscopy facilitate studies of the spatial organization of fluorescently labeled protein components, these techniques cannot reveal the underlying cellular structures. Thus, equally exciting are developments in EM: genetically encoded probes for protein localization at sub-10 nm resolution, more powerful instruments that allow imaging of larger cell volumes, and computational methods for reconstructing three-dimensional images. Used in combination, as done by Ludwig et al. in the current issue of PLOS Biology, these tools reveal high-resolution insights into the composition and organization of the caveolae coat and the formation of these specialized structures. Together, these advances are contributing to a resurgence in EM.
doi:10.1371/journal.pbio.1001639
PMCID: PMC3754885  PMID: 24013596
4.  Context Dependent Pro-Angiogenic Function of Bone Morphogenetic Protein Signaling is Mediated by Disabled Homolog 2 
Developmental cell  2012;23(2):441-448.
Bone Morphogenetic Proteins (BMPs) have diverse functions during development in vertebrates. We have recently shown that BMP2 signaling promotes venous specific angiogenesis in zebrafish embryos. However, factors that confer a context dependent pro-angiogenic function of BMP2 signaling within endothelial cells need to be identified. Here, we report that Disabled homolog 2 (Dab2), a cargo specific adaptor protein for Clathrin, is essential to mediate the pro-angiogenic function of BMP2 signaling. We find that inhibition of Dab2 attenuates internalization of BMP receptors and abrogates the pro-angiogenic effects of BMP signaling in endothelial cells. Moreover, inhibition of Dab2 decreases phosphorylation of SMAD-1, 5, and 8, indicating that Dab2 plays an essential role in determining the outcome of BMP signaling within endothelial cells, and may provide a molecular basis for a context dependent pro-angiogenic function of BMP2 signaling.
doi:10.1016/j.devcel.2012.07.007
PMCID: PMC3659797  PMID: 22898784
5.  Dynamin-Catalyzed Membrane Fission Requires Coordinated GTP Hydrolysis 
PLoS ONE  2013;8(1):e55691.
Dynamin is the most-studied membrane fission machinery and has served as a paradigm for studies of other fission GTPases; however, several critical questions regarding its function remain unresolved. In particular, because most dynamin GTPase domain mutants studied to date equally impair both basal and assembly-stimulated GTPase activities, it has been difficult to distinguish their respective roles in clathrin-mediated endocytosis (CME) or in dynamin catalyzed membrane fission. Here we compared a new dynamin mutant, Q40E, which is selectively impaired in assembly-stimulated GTPase activity with S45N, a GTP-binding mutant equally defective in both basal and assembly-stimulated GTPase activities. Both mutants potently inhibit CME and effectively recruit other endocytic accessory proteins to stalled coated pits. However, the Q40E mutant blocks at a later step than S45N, providing additional evidence that GTP binding and/or basal GTPase activities of dynamin are required throughout clathrin coated pit maturation. Importantly, using in vitro assays for assembly-stimulated GTPase activity and membrane fission, we find that the latter is much more potently inhibited by both dominant-negative mutants than the former. These studies establish that efficient fission from supported bilayers with excess membrane reservoir (SUPER) templates requires coordinated GTP hydrolysis across two rungs of an assembled dynamin collar.
doi:10.1371/journal.pone.0055691
PMCID: PMC3561337  PMID: 23383266
6.  Hotspots organize clathrin-mediated endocytosis by efficient recruitment and retention of nucleating resources 
Traffic (Copenhagen, Denmark)  2011;12(12):1868-1878.
The formation of clathrin-coated pits (CCPs) at the plasma membrane has been reported to sometimes occur repeatedly at predefined sites. However, defining such CCP `hotspots' structurally and mechanistically has been difficult due to the dynamic and heterogeneous nature of CCPs. Here we explore the molecular requirements for hotspots using a global assay of CCP dynamics. Our data confirmed that a subset of CCPs is nucleated at spatially distinct sites. The degree of clustering of nucleation events at these sites is dependent on the integrity of cortical actin, and the availability of certain resources, including the adaptor protein AP-2 and the phospholipid PI(4,5)P2. We observe that modulation in the expression of FCHo1 and 2, which have been reported to initiate CCPs, affect only the number of nucleations. Modulation in the expression levels of other accessory proteins, such as SNX9, affects the spatial clustering of CCPs but not the number of nucleations. Based on these findings we distinguish two classes of accessory proteins in clathrin-mediated endocytosis (CME): nucleation factors and nucleation organizers. Finally, we observe that clustering of transferrin receptors spatially randomizes pit nucleation and thus reduces the role of hotspots. Based on these data, we propose that hotspots are specialized cortical actin patches that organize CCP nucleations from within the cell by more efficient recruitment and/or retention of resources required for CCP nucleation partially due to the action of nucleation organizers.
doi:10.1111/j.1600-0854.2011.01273.x
PMCID: PMC3298691  PMID: 21883765
clathrin; endocytosis; hotspot; SNX9; FCHo1; FCHo2
7.  Common Membrane Trafficking Defects of Disease Associated Dynamin 2 Mutations 
Traffic (Copenhagen, Denmark)  2011;12(11):1620-1633.
Dynamin (Dyn) is a multidomain and multifunctional GTPase best known for its essential role in clathrin-mediated endocytosis (CME). Dyn2 mutations have been linked to two human diseases, Centronuclear Myopathy (CNM) and Charcot-Marie-Tooth (CMT) disease. Paradoxically, although Dyn2 is ubiquitously expressed and essential for embryonic development, the disease-associated Dyn2 mutants are autosomal dominant, but result in slowly progressing and tissue-specific diseases. Thus, although the cellular defects that cause disease remain unclear, they are expected to be mild. To gain new insight into potential pathogenic mechanisms we utilized mouse Dyn2 conditional knock-out cells combined with retroviral-mediated reconstitution to mimic both heterozygous and homozygous states and characterized cellular phenotypes using quantitative assays for several membrane trafficking events. Surprisingly, none of the four mutants studied exhibited a defect in CME, but all were impaired in their ability to support p75/neurotrophin receptor export from the Golgi, the raft-dependent endocytosis of cholera toxin, and clathrin-independent endocytosis of EGFR. While it will be important to study these mutants in disease-relevant muscle and neuronal cells, given the importance of neurotrophic factors and lipid rafts in muscle physiology, we speculate that these common cellular defects might contribute to the tissue-specific diseases caused by a ubiquitously expressed protein.
doi:10.1111/j.1600-0854.2011.01250.x
PMCID: PMC3188678  PMID: 21762456
Charcot-Marie-Tooth disease; Centronuclear Myopathy; Clathrin-mediated endocytosis; EGF receptor; p75/neurotrophin receptor; lipid rafts
8.  A pseudo-atomic model of the dynamin polymer identifies a hydrolysis-dependent powerstroke 
Cell  2011;147(1):209-222.
Summary
The GTPase dynamin catalyzes membrane fission. Though this process requires dynamin assembly, G domain dimerization and stimulated GTP hydrolysis, the underlying structural interactions and conformational changes remain a mystery. Here we present the GMPPCP-bound structures of the truncated human dynamin 1 helical polymer at 12.2Å and a fusion protein linking human dynamin 1’s catalytic G domain to its GTPase effector domain (GG) at 2.2Å. Newly resolved density features in the polymer reconstruction and the unique conformation of GGGMPPCP allowed us to position crystallized dynamin fragments in the assembled structure and define their connectivity. The resulting model shows that G domain dimers only form between tetramers in sequential rungs of the dynamin helix. Using chemical crosslinking, we demonstrate that dynamin tetramers are dimers of domain-swapped dimers. Structural comparison of GGGMPPCP to the GG transition-state complex identifies a hydrolysis-dependent powerstroke that may play a role in membrane remodeling events necessary for fission.
doi:10.1016/j.cell.2011.09.003
PMCID: PMC3185303  PMID: 21962517
10.  Measuring the hierarchy of molecular events during clathrin-mediated endocytosis 
Traffic (Copenhagen, Denmark)  2011;12(7):815-825.
A well-orchestrated hierarchy of molecular events is required for successful initiation and maturation of clathrin-coated pits (CCPs). Nevertheless, CCPs display a broad range of lifetimes. This dynamic heterogeneity could either reflect differences in the temporal hierarchy of molecular events, or similar CCP maturation processes with variable kinetics. To address this question, we have used multi-channel image acquisition and automated analysis of CCP dynamics in combination with a new method to quantify the time courses of recruitment of endocytic factors to CCPs of different lifetimes. Using this approach we have extracted the kinetics of recruitment and disassembly of fluorescently labelled clathrin and/or AP-2 throughout the entire lifetime of temporally-defined CCP cohorts. Based on these analyses, we can (i) directly correlate recruitment profiles of these two proteins, (ii) define 5 distinct CCP maturation phases, i.e. initiation, growth, maturation, separation and departure, (iii) distinguish events with absolute versus fractional timing and (iv) provide information on the spatial distribution of fluorophores during CCP maturation. Emerging from these analyses is a more clearly defined role for AP-2 in determining the temporal hierarchy for clathrin recruitment and CCP maturation. This method provides a new means to identify other such hierarchies during CCP maturation.
doi:10.1111/j.1600-0854.2011.01197.x
PMCID: PMC3115502  PMID: 21447041
Clathrin-mediated endocytosis; AP-2; total internal reflection microscopy; intensity time courses; recruitment; automated analysis; segmentation
11.  Phosphatidylinositol-(4,5)-bisphosphate regulates clathrin-coated pit initiation, stabilization, and size 
Molecular Biology of the Cell  2011;22(14):2588-2600.
Phosphatidylinositol-(4,5)-bisphosphate (PIP2) is the main lipid binding partner of proteins involved in clathrin-mediated endocytosis (CME). Total internal reflection fluorescence microscopy coupled to computational image analysis revealed that the balance of PIP2 synthesis in the bulk plasma membrane and its local turnover within clathrin-coated pits control multiple distinct yet only partly overlapping stages of CME.
Clathrin-mediated endocytosis (CME) is the major mechanism for internalization in mammalian cells. CME initiates by recruitment of adaptors and clathrin to form clathrin-coated pits (CCPs). Nearly half of nascent CCPs abort, whereas others are stabilized by unknown mechanisms and undergo further maturation before pinching off to form clathrin-coated vesicles (CCVs). Phosphatidylinositol-(4,5)-bisphosphate (PIP2), the main lipid binding partner of endocytic proteins, is required for CCP assembly, but little is currently known about its contribution(s) to later events in CCV formation. Using small interfering RNA (siRNA) knockdown and overexpression, we have analyzed the effects of manipulating PIP2 synthesis and turnover on CME by quantitative total internal reflection fluorescence microscopy and computational analysis. Phosphatidylinositol-4-phosphate-5-kinase cannot be detected within CCPs but functions in initiation and controls the rate and extent of CCP growth. In contrast, the 5′-inositol phosphatase synaptojanin 1 localizes to CCPs and controls early stabilization and maturation efficiency. Together these results suggest that the balance of PIP2 synthesis in the bulk plasma membrane and its local turnover within CCPs control multiple stages of CCV formation.
doi:10.1091/mbc.E11-04-0362
PMCID: PMC3135483  PMID: 21613550
12.  A new role for the dynamin GTPase in the regulation of fusion pore expansion 
Molecular Biology of the Cell  2011;22(11):1907-1918.
The role of dynamin GTPase activity in controlling fusion pore expansion and postfusion granule membrane topology was investigated. The experiments show that, in addition to playing a role in endocytosis, GTPase activity of dynamin regulates the rapidity of fusion pore expansion from tens of milliseconds to seconds after fusion.
Dynamin is a master regulator of membrane fission in endocytosis. However, a function for dynamin immediately upon fusion has also been suspected from a variety of experiments that measured release of granule contents. The role of dynamin guanosine triphosphate hydrolase (GTPase) activity in controlling fusion pore expansion and postfusion granule membrane topology was investigated using polarization optics and total internal reflection fluorescence microscopy (pTIRFM) and amperometry. A dynamin-1 (Dyn1) mutant with increased GTPase activity resulted in transient deformations consistent with rapid fusion pore widening after exocytosis; a Dyn1 mutant with decreased activity slowed fusion pore widening by stabilizing postfusion granule membrane deformations. The experiments indicate that, in addition to its role in endocytosis, GTPase activity of dynamin regulates the rapidity of fusion pore expansion from tens of milliseconds to seconds after fusion. These findings expand the membrane-sculpting repertoire of dynamin to include the regulation of immediate postfusion events in exocytosis that control the rate of release of soluble granule contents.
doi:10.1091/mbc.E11-02-0101
PMCID: PMC3103406  PMID: 21460182
13.  Local clustering of transferrin receptors promotes clathrin-coated pit initiation 
The Journal of Cell Biology  2010;191(7):1381-1393.
The relationship between cargo accumulation and clathrin-coated pit initiation and maturation is examined by direct visualization of receptor-engaged clathrin-coated pits.
Clathrin-mediated endocytosis (CME) is the major pathway for concentrative uptake of receptors and receptor–ligand complexes (cargo). Although constitutively internalized cargos are known to accumulate into maturing clathrin-coated pits (CCPs), whether and how cargo recruitment affects the initiation and maturation of CCPs is not fully understood. Previous studies have addressed these issues by analyzing the global effects of receptor overexpression on CME or CCP dynamics. Here, we exploit a refined approach using expression of a biotinylated transferrin receptor (bTfnR) and controlling its local clustering using mono- or multivalent streptavidin. We show that local clustering of bTfnR increased CCP initiation. By tracking cargo loading in individual CCPs, we found that bTfnR clustering preceded clathrin assembly and confirmed that bTfnR-containing CCPs mature more efficiently than bTfnR-free CCPs. Although neither the clustering nor the related changes in cargo loading altered the rate of CCP maturation, bTfnR-containing CCPs exhibited significantly longer lifetimes than other CCPs within the same cell. Together these results demonstrate that cargo composition is a key source of the differential dynamics of CCPs.
doi:10.1083/jcb.201008117
PMCID: PMC3010081  PMID: 21187331
14.  Clathrin-mediated Endocytosis: A Universe of New Questions 
Molecular Biology of the Cell  2010;21(22):3818-3819.
doi:10.1091/mbc.E10-05-0386
PMCID: PMC2982115  PMID: 21079028
16.  G domain dimerization controls dynamin's assembly-stimulated GTPase activity 
Nature  2010;465(7297):435-440.
Dynamin is an atypical GTPase that catalyzes membrane fission during clathrin-mediated endocytosis. The mechanisms of dynamin’s basal and assembly-stimulated GTP hydrolysis are unknown, though both are indirectly influenced by the GTPase effector domain (GED). Here we present the 2.0Å resolution crystal structure of a minimal GTPase-GED fusion protein (GG) constructed from human dynamin 1, which has dimerized in the presence of the transition state mimic GDP.AlF4−. The structure reveals dynamin’s catalytic machinery and explains how assembly-stimulated GTP hydrolysis is achieved through G domain dimerization. A sodium ion present in the active site suggests that dynamin uses a cation to compensate for the developing negative charge in the transition state in the absence of an arginine finger. Structural comparison to the rat dynamin G domain reveals key conformational changes that promote G domain dimerization and stimulated hydrolysis. The structure of the GG dimer provides new insight into the mechanisms underlying dynamin-catalyzed membrane fission.
doi:10.1038/nature09032
PMCID: PMC2879890  PMID: 20428113
17.  Phosphatidic Acid Plays a Regulatory Role in Clathrin-mediated Endocytosis 
Molecular Biology of the Cell  2010;21(16):2944-2952.
We have manipulated the activities of PLD and DGK, enzymes that regulate PA biosynthesis, and directly measured their effects on cellular PA levels and on clathrin-mediated endocytosis (CME). We report a previously unappreciated complexity in PA regulation and show that PA selectively regulates CME of EGF but not transferrin.
Clathrin-mediated endocytosis (CME) is the main route of internalization of receptor-ligand complexes. Relatively little is known about the role of specific lipids in CME, in particular that of phosphatidic acid (PA). We examined the effect of altering cellular PA levels on CME by manipulating the activities and/or levels of either phospholipase D (PLD1 and PLD2) or diacylglycerol kinase (DGK), two enzyme classes involved in PA production. DGK inhibition resulted in a dramatic reduction of cellular PA, measured directly using an enzyme-coupled reaction, which resulted in a decreased rate of EGFR internalization measured biochemically. This corresponded to a decreased rate of clathrin-coated pit (CCP) initiation and increased lifetimes of productive CCPs, as determined by quantitative live-cell total internal reflection fluorescence microscopy. Unexpectedly, PLD inhibition caused an increase in cellular PA, suggesting that PLD activity negatively regulates PA synthesis by other more productive pathways. Consistent with opposite effects on cellular PA levels, PLD inhibition had opposite effects on EGFR internalization and CCP dynamics, compared with DGK inhibition. Importantly, the constitutive internalization of transferrin receptors was unaffected by either treatment. These findings demonstrate that PA plays a regulatory rather than obligatory role in CME and differentially regulates ligand-stimulated CME of EGFR.
doi:10.1091/mbc.E10-05-0421
PMCID: PMC2921119  PMID: 20573978
18.  Dissecting dynamin’s role in clathrin-mediated endocytosis 
Biochemical Society transactions  2009;37(Pt 5):1022-1026.
The GTPase dynamin is essential for clathrin-mediated endocytosis (CME), but its exact function and mechanism of action has been controversial. Here we review findings that have led to current models for dynamin function, either as a mechanochemical enzyme driving membrane fission or as a regulatory GTPase monitoring rate limiting steps in CME. However, these models are not mutually exclusive and subsequent studies have provided evidence for both dynamin functions. Here we present recent evidence derived from divergent in vivo and in vitro approaches that dynamin plays a dual role in CME, functioning at early stages as a fidelity monitor to regulate clathrin-coated pit maturation and at later stages to directly catalyze membrane fission and clathrin-coated vesicle formation.
doi:10.1042/BST0371022
PMCID: PMC2879887  PMID: 19754444
clathrin-mediated endocytosis; dynamin; fission; vesicle formation
19.  The Palade Symposium: Celebrating Cell Biology at Its Best 
Molecular Biology of the Cell  2010;21(14):2367-2370.
A symposium was held at the University of California, San Diego, to honor the contributions of Nobel Laureate, George Palade, to cell biology. The speakers included Günter Blobel, on the structure and function of nuclear pore complexes; Peter Walter, on the unfolded protein response in health and disease; Randy Schekman, on human disease-linked mutations in the COPII machinery; Scott Emr, on the regulation of plasma membrane composition by selective endocytosis; Roger Kornberg, on the structure and function of the transcription machinery; Peter Novick, on the regulation of rab GTPases along the secretory pathway; Jim Spudich, on the mechanism of the enigmatic myosin VI motor; and Joe Goldstein, on the function of the Niemann-Pick C (NPC)-linked gene products, NPC1 and NPC2, in cholesterol transport. Their work showcased the multidisciplinary nature, diversity, and vitality of cell biology. In the words of George Palade, their talks also illustrated “how cell biology could be used to understand disease and how disease could be used to discover normal cell biology.” An integrated understanding of the cellular machinery will be essential in tackling the plethora of questions and challenges posed by completion of the human genome and for understanding the molecular mechanisms underlying human disease.
doi:10.1091/mbc.E10-03-0179
PMCID: PMC2903666  PMID: 20505070
20.  Cargo- and adaptor-specific mechanisms regulate clathrin-mediated endocytosis 
The Journal of Cell Biology  2010;188(6):919-933.
Clathrin-coated pit size and dynamic behavior varies with low density lipoprotein receptor (LDLR) expression levels in a manner dependent on the LDLR-specific adaptors, Dab2 and ARH.
Clathrin-mediated endocytosis of surface receptors and their bound ligands (i.e., cargo) is highly regulated, including by the cargo itself. One of the possible sources of the observed heterogeneous dynamics of clathrin-coated pits (CCPs) might be the different cargo content. Consistent with this, we show that CCP size and dynamic behavior varies with low density lipoprotein receptor (LDLR) expression levels in a manner dependent on the LDLR-specific adaptors, Dab2 and ARH. In Dab2-mCherry–expressing cells, varying LDLR expression leads to a progressive increase in CCP size and to the appearance of nonterminal endocytic events. In LDLR and ARH-mCherry–expressing cells in addition to an increase in CCP size, turnover of abortive CCPs increases, and the rate of CCP maturation decreases. Altogether, our results underscore the highly dynamic and cargo-responsive nature of CCP assembly and suggest that the observed heterogeneity is, in part, related to compositional differences (e.g., cargo and adaptors) between CCPs.
doi:10.1083/jcb.200908078
PMCID: PMC2845073  PMID: 20231386
21.  Signal recognition particle (SRP) and SRP receptor: A new paradigm for multi-state regulatory GTPases 
Biochemistry  2009;48(29):6696-6704.
The GTP-binding proteins or GTPases comprise a superfamily of proteins that provide molecular switches in numerous cellular processes. The ‘GTPase switch’ paradigm, in which a GTPase acts as a bimodal switch that is turned ‘on’ and ‘off’ by external regulatory factors, has been used to interpret the regulatory mechanism of many GTPases for over two decades. Nevertheless, recent work has unveiled an emerging class of ‘multi-state’ regulatory GTPases that do not adhere to this classical paradigm. Instead of relying on external nucleotide exchange factors or GTPase activating proteins to switch between the ‘on’ and ‘off’ states, these GTPases have the intrinsic ability to exchange nucleotides and to sense and respond to upstream and downstream factors. In contrast to the bimodal nature of the ‘GTPase switch’, these GTPases undergo multiple conformational rearrangements, allowing multiple regulatory points to be built into a complex biological process to ensure the efficiency and fidelity of the pathway. We suggest that these multi-state regulatory GTPases are uniquely suited to provide spatial and temporal control over complex cellular pathways that require multiple molecular events to occur in a highly coordinated fashion.
doi:10.1021/bi9006989
PMCID: PMC2883566  PMID: 19469550
22.  Conserved Functions of Membrane Active GTPases in Coated Vesicle Formation 
Science (New York, N.Y.)  2009;325(5945):1217-1220.
Coated vesicles concentrate and package cargo molecules to mediate their efficient transport between intracellular compartments. Cytosolic coat proteins such as clathrin and adaptor complexes, and COPI and COPII coatomer complexes self-assemble to deform the membrane and interact directly with cargo molecules to capture them in nascent buds. The GTPases Arf, Sar1 and dynamin are core components of the coated vesicle machinery. These GTPases, which associate with and dissociate from donor membranes in a GTP-dependent manner, can also actively remodel membranes. Recent evidence suggests that although structurally diverse, Arf-family GTPases and dynamin may play mechanistically similar roles as fidelity monitors that govern cargo packaging and coated vesicle maturation, and as components of the fission machinery to mediate vesicle release.
doi:10.1126/science.1171004
PMCID: PMC2864031  PMID: 19729648
23.  Membrane Insertion of the Pleckstrin Homology Domain Variable Loop 1 Is Critical for Dynamin-catalyzed Vesicle Scission 
Molecular Biology of the Cell  2009;20(22):4630-4639.
The GTPase dynamin catalyzes the scission of deeply invaginated clathrin-coated pits at the plasma membrane, but the mechanisms governing dynamin-mediated membrane fission remain poorly understood. Through mutagenesis, we have altered the hydrophobic nature of the membrane-inserting variable loop 1 (VL1) of the pleckstrin homology (PH) domain of dynamin-1 and demonstrate that its stable insertion into the lipid bilayer is critical for high membrane curvature generation and subsequent membrane fission. Dynamin PH domain mutants defective in curvature generation regain function when assayed on precurved membrane templates in vitro, but they remain defective in the scission of clathrin-coated pits in vivo. These results demonstrate that, in concert with dynamin self-assembly, PH domain membrane insertion is essential for fission and vesicle release in vitro and for clathrin-mediated endocytosis in vivo.
doi:10.1091/mbc.E09-08-0683
PMCID: PMC2777094  PMID: 19776347
24.  A partnership between dynamin and lipids defines dynamics and intermediates of membrane fission 
Cell  2008;135(7):1276-1286.
SUMMARY
The GTPase dynamin is critically involved in membrane fission during endocytosis. How does dynamin use the energy of GTP hydrolysis for membrane remodeling? By monitoring the ionic permeability through lipid nanotubes (NT) interacting with dynamin and GTP we detected cyclic localized squeezing and relaxation of NT, ultimately yielding leak-free fission stochastically. Our calculations revealed that critical local narrowing of NT induces cooperative lipid tilting, leading to self-merger of the inner monolayer of NT (hemi-fission), consistent with the absence of leakage. We found that dynamin pre-assembled on NT without GTP produces stable curvature which, depending upon lipid composition, approaches the hemi-fission threshold. GTP addition causes gradual disassembly of dynamin, triggering either spontaneous fission or curvature relaxation. We propose that dynamin transmits GTP’s energy to periodic curvature stress caused by an assembly of a limited curvature scaffold, which next relaxes and rewinds to its initial state upon cooperative GTP hydrolysis.
doi:10.1016/j.cell.2008.11.028
PMCID: PMC2768395  PMID: 19084269
25.  Real-time Visualization of Dynamin-catalyzed Membrane Fission and Vesicle Release 
Cell  2008;135(7):1263-1275.
The GTPase dynamin assembles at the necks of budded vesicles in vivo and functions in membrane fission. We have developed fluid supported bilayers with excess membrane reservoir, (SUPER) templates, to assay vesicle formation and membrane fission. Consistent with previous studies, in the absence of GTP, dynamin assembles in spirals forming long membrane tubules. GTP addition triggers disassembly, but not membrane fission arguing against models that fission is mediated by concerted and global GTP-driven conformational changes. In contrast, under physiological conditions in the constant presence of GTP, dynamin mediates membrane fission. Under these conditions, fluorescently-labeled dynamin cooperatively organizes into self-limited assemblies that continuously cycle at the membrane and drive vesicle release. When visualized at the necks of emergent vesicles, self-limited dynamin assemblies display intensity fluctuations and persist for variable time periods before fission. Thus, self-limited assemblies of dynamin generated in the constant presence of GTP catalyze membrane fission.
doi:10.1016/j.cell.2008.11.020
PMCID: PMC2673235  PMID: 19084268
Dynamin; supported bilayers; membrane reservoir; membrane fission; GTPase activity

Results 1-25 (51)