Single vesicle assaying of SNARE-synaptotagmin-driven fusion reveals fast and slow modes of both docking and fusion and intrasample heterogeneity.

Abstract:

:Lipid mixing between vesicles functionalized with SNAREs and the cytosolic C2AB domain of synaptotagmin-1 recapitulates the basic Ca(2+) dependence of neuronal exocytosis. However, in the conventional ensemble lipid mixing assays it is not possible to discriminate whether Ca(2+) accelerates the docking or the fusion of vesicles. Here we report a fluorescence microscopy-based assay to monitor SNARE-mediated docking and fusion of individual vesicle pairs. In situ measurement of the concentration of diffusing particles allowed us to quantify docking rates by a maximum-likelihood approach. This analysis showed that C2AB and Ca(2+) accelerate vesicle-vesicle docking with more than two orders of magnitude. Comparison of the measured docking rates with ensemble lipid mixing kinetics, however, suggests that in most cases bilayer fusion remains the rate-limiting step. Our single vesicle results show that only ∼60% of the vesicles dock and only ∼6% of docked vesicles fuse. Lipid mixing on single vesicles was fast (t(mix) < 1 s) while an ensemble assay revealed two slow mixing processes with t(mix) ∼ 1 min and t(mix) ∼ 20 min. The presence of several distinct docking and fusion pathways cannot be rationalized at this stage but may be related to intrasample heterogeneities, presumably in the form of lipid and/or protein composition.

journal_name

Biophys J

journal_title

Biophysical journal

authors

Christensen SM,Mortensen MW,Stamou DG

doi

10.1016/j.bpj.2010.12.3730

subject

Has Abstract

pub_date

2011-02-16 00:00:00

pages

957-67

issue

4

eissn

0006-3495

issn

1542-0086

pii

S0006-3495(11)00007-5

journal_volume

100

pub_type

杂志文章
  • Consistent picture of the reversible thermal unfolding of hen egg-white lysozyme from experiment and molecular dynamics.

    abstract::Synchrotron radiation circular dichroism, Fourier transform infrared, and nuclear magnetic resonance spectroscopies, and small-angle x-ray scattering were used to monitor the reversible thermal unfolding of hen egg white lysozyme. The results were compared with crystal structures and high- and low-temperature structur...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/j.bpj.2010.07.060

    authors: Meersman F,Atilgan C,Miles AJ,Bader R,Shang W,Matagne A,Wallace BA,Koch MH

    更新日期:2010-10-06 00:00:00

  • Determination of ensemble-average pairwise root mean-square deviation from experimental B-factors.

    abstract::Root mean-square deviation (RMSD) after roto-translational least-squares fitting is a measure of global structural similarity of macromolecules used commonly. On the other hand, experimental x-ray B-factors are used frequently to study local structural heterogeneity and dynamics in macromolecules by providing direct i...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/j.bpj.2009.11.011

    authors: Kuzmanic A,Zagrovic B

    更新日期:2010-03-03 00:00:00

  • Atomic detail peptide-membrane interactions: molecular dynamics simulation of gramicidin S in a DMPC bilayer.

    abstract::Molecular dynamics simulations have been performed of the sequence-symmetric cyclic decapeptide antibiotic gramicidin S (GS), in interaction with a hydrated dimyristoylphosphatidylcholine (DMPC) bilayer, and the results compared with a "control" simulation of the system in the absence of GS. Following experimental evi...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/S0006-3495(00)76424-1

    authors: Mihailescu D,Smith JC

    更新日期:2000-10-01 00:00:00

  • Determining force dependence of two-dimensional receptor-ligand binding affinity by centrifugation.

    abstract::Analyses of receptor-ligand interactions are important to the understanding of cellular adhesion. Traditional methods of measuring the three-dimensional (3D) dissociation constant (Kd) require at least one of the molecular species in solution and hence cannot be directly applied to the case of cell adhesion. We descri...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/S0006-3495(98)77807-5

    authors: Piper JW,Swerlick RA,Zhu C

    更新日期:1998-01-01 00:00:00

  • Bending free energy from simulation: correspondence of planar and inverse hexagonal lipid phases.

    abstract::Simulations of two distinct systems, one a planar bilayer, the other the inverse hexagonal phase, indicate consistent mechanical properties and curvature preferences, with single DOPE leaflets having a spontaneous curvature, R0 = -26 Å (experimentally ~ -29.2 Å) and DOPC leaflets preferring to be approximately flat (R...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/j.bpj.2013.03.048

    authors: Sodt AJ,Pastor RW

    更新日期:2013-05-21 00:00:00

  • Determination of the orientation distribution of adsorbed fluorophores using TIRF. II. Measurements on porphyrin and cytochrome c.

    abstract::The theory for determination of the orientation of adsorbed fluorescent molecules using total internal reflection fluorescence, as explained in part I of this series, is illustrated by measurements on adsorbed tetramethylpyridinium porphyrin (H2TMPyP) and porphyrin cytochrome c molecules. The results are encouraging, ...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/S0006-3495(95)80440-6

    authors: Bos MA,Kleijn JM

    更新日期:1995-06-01 00:00:00

  • Calculating the bulk modulus for a lipid bilayer with nonequilibrium molecular dynamics simulation.

    abstract::Nonequilibrium molecular dynamics (NEMD) computer simulations are used to calculated the bulk modulus for a dimyristoylphosphatidylcholine bilayer. A methodology is developed whereby NEMD can be effectively used to calculate material properties for complex systems that undergo long time-scale conformational changes. I...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/S0006-3495(02)75479-9

    authors: Ayton G,Smondyrev AM,Bardenhagen SG,McMurtry P,Voth GA

    更新日期:2002-03-01 00:00:00

  • Stimulation of Nipah Fusion: Small Intradomain Changes Trigger Extensive Interdomain Rearrangements.

    abstract::Nipah is an emerging paramyxovirus that is of serious concern to human health. It invades host cells using two of its membrane proteins-G and F. G binds to host ephrins and this stimulates G to activate F. Upon activation, F mediates virus-host membrane fusion. Here we focus on mechanisms that underlie the stimulation...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/j.bpj.2016.09.002

    authors: Dutta P,Siddiqui A,Botlani M,Varma S

    更新日期:2016-10-18 00:00:00

  • The kinetics of the hydrogen/deuterium exchange of epidermal growth factor receptor ligands.

    abstract::Five highly homologous epidermal growth factor receptor ligands were studied by mass spectral analysis, hydrogen/deuterium (H/D) exchange via attenuated total reflectance Fourier transform-infrared spectroscopy, and two-dimensional correlation analysis. These studies were performed to determine the order of events dur...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1529/biophysj.107.125856

    authors: Iloro I,Narváez D,Guillén N,Camacho CM,Guillén L,Cora E,Pastrana-Ríos B

    更新日期:2008-05-15 00:00:00

  • Fabrication of a new substrate for atomic force microscopic observation of DNA molecules from an ultrasmooth sapphire plate.

    abstract::A new stable substrate applicable to the observation of DNA molecules by atomic force microscopy (AFM) was fabricated from a ultrasmooth sapphire (alpha-Al2O3 single crystal) plate. The atomically ultrasmooth sapphire as obtained by high-temperature annealing has hydrophobic surfaces and could not be used for the AFM ...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/S0006-3495(98)77878-6

    authors: Yoshida K,Yoshimoto M,Sasaki K,Ohnishi T,Ushiki T,Hitomi J,Yamamoto S,Sigeno M

    更新日期:1998-04-01 00:00:00

  • Detecting ultraviolet damage in single DNA molecules by atomic force microscopy.

    abstract::We report detection and quantification of ultraviolet (UV) damage in DNA at a single molecule level by atomic force microscopy (AFM). By combining the supercoiled plasmid relaxation assay with AFM imaging, we find that high doses of medium wave ultraviolet (UVB) and short wave ultraviolet (UVC) light not only produce ...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1529/biophysj.107.108209

    authors: Jiang Y,Ke C,Mieczkowski PA,Marszalek PE

    更新日期:2007-09-01 00:00:00

  • Parameter inference for biochemical systems that undergo a Hopf bifurcation.

    abstract::The increasingly widespread use of parametric mathematical models to describe biological systems means that the ability to infer model parameters is of great importance. In this study, we consider parameter inferability in nonlinear ordinary differential equation models that undergo a bifurcation, focusing on a simple...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1529/biophysj.107.126086

    authors: Kirk PD,Toni T,Stumpf MP

    更新日期:2008-07-01 00:00:00

  • Theory of threshold fluctuations in nerves. I. Relationships between electrical noise and fluctuations in axon firing.

    abstract::Relations describing threshold fluctuation phenomena in nerves are derived by calculating the approximate response of the Hodgkin-Huxley (HH) axon to electrical noise. We use FitzHugh's reduced phase space approximation and describe the dynamics of a noisy nerve by a two-dimensional brownian motion. The theory predict...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/S0006-3495(71)86277-X

    authors: Lecar H,Nossal R

    更新日期:1971-12-01 00:00:00

  • Microtubule Dynamics, Kinesin-1 Sliding, and Dynein Action Drive Growth of Cell Processes.

    abstract::Recent experimental studies of the role of microtubule sliding in neurite outgrowth suggested a qualitative model, according to which kinesin-1 motors push the minus-end-out microtubules against the cell membrane and generate the early cell processes. At the later stage, dynein takes over the sliding, expels the minus...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/j.bpj.2018.08.046

    authors: Oelz DB,Del Castillo U,Gelfand VI,Mogilner A

    更新日期:2018-10-16 00:00:00

  • Transmembrane helix stability: the effect of helix-helix interactions studied by Fourier transform infrared spectroscopy.

    abstract::We have measured, using infrared spectroscopy, the hydrogen/deuterium exchange rates of the amide protons in the photosynthetic antenna of Rhodospirillum rubrum. These measurements were made not only on the intact protein in detergent solution but also on two dissociated forms (B820 and B777). We have, on the basis of...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/S0006-3495(98)74022-6

    authors: Sturgis J,Robert B,Goormaghtigh E

    更新日期:1998-02-01 00:00:00

  • Modeling feedback loops of the Mammalian circadian oscillator.

    abstract::The suprachiasmatic nucleus governs daily variations of physiology and behavior in mammals. Within single neurons, interlocked transcriptional/translational feedback loops generate circadian rhythms on the molecular level. We present a mathematical model that reflects the essential features of the mammalian circadian ...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1529/biophysj.104.040824

    authors: Becker-Weimann S,Wolf J,Herzel H,Kramer A

    更新日期:2004-11-01 00:00:00

  • Stability and robustness of an organelle number control system: modeling and measuring homeostatic regulation of centriole abundance.

    abstract::Control of organelle abundance is a fundamental unsolved problem in cell biology. Mechanisms for number control have been proposed in which organelle assembly is actively increased or decreased to compensate for deviations from a set-point, but such phenomena have not been experimentally verified. In this report we ex...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1529/biophysj.107.107052

    authors: Marshall WF

    更新日期:2007-09-01 00:00:00

  • Association thermodynamics and conformational stability of beta-sheet amyloid beta(17-42) oligomers: effects of E22Q (Dutch) mutation and charge neutralization.

    abstract::Amyloid fibrils are associated with many neurodegenerative diseases. It was found that amyloidogenic oligomers, not mature fibrils, are neurotoxic agents related to these diseases. Molecular mechanisms of infectivity, pathways of aggregation, and molecular structure of these oligomers remain elusive. Here, we use all-...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/j.bpj.2009.09.062

    authors: Blinov N,Dorosh L,Wishart D,Kovalenko A

    更新日期:2010-01-20 00:00:00

  • The force exerted by a single kinesin molecule against a viscous load.

    abstract::Kinesin is a motor protein that uses the energy derived from the hydrolysis of ATP to power the transport of organelles along microtubules. To probe the mechanism of this chemical-to-mechanical energy transduction reaction, the movement of microtubules across glass surfaces coated with kinesin was perturbed by raising...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/S0006-3495(94)80537-5

    authors: Hunt AJ,Gittes F,Howard J

    更新日期:1994-08-01 00:00:00

  • Calcium-Dependent Structural Dynamics of a Spin-Labeled RyR Peptide Bound to Calmodulin.

    abstract::We have used chemical synthesis, electron paramagnetic resonance (EPR), and circular dichroism to detect and analyze the structural dynamics of a ryanodine receptor (RyR) peptide bound to calmodulin (CaM). The skeletal muscle calcium release channel RyR1 is activated by Ca2+-free CaM and inhibited by Ca2+-bound CaM. T...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/j.bpj.2016.10.025

    authors: Her C,McCaffrey JE,Thomas DD,Karim CB

    更新日期:2016-12-06 00:00:00

  • Chlorophyll ring deformation modulates Qy electronic energy in chlorophyll-protein complexes and generates spectral forms.

    abstract::The possibility that the chlorophyll (chl) ring distortions observed in the crystal structures of chl-protein complexes are involved in the transition energy modulation, giving rise to the spectral forms, is investigated. The out-of-plane chl-macrocycle distortions are described using an orthonormal set of deformation...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1529/biophysj.107.104554

    authors: Zucchelli G,Brogioli D,Casazza AP,Garlaschi FM,Jennings RC

    更新日期:2007-09-15 00:00:00

  • Glutamate receptor-channel gating. Maximum likelihood analysis of gigaohm seal recordings from locust muscle.

    abstract::Gigaohm recordings have been made from glutamate receptor channels in excised, outside-out patches of collagenase-treated locust muscle membrane. The channels in the excised patches exhibit the kinetic state switching first seen in megaohm recordings from intact muscle fibers. Analysis of channel dwell time distributi...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/S0006-3495(90)82367-5

    authors: Bates SE,Sansom MS,Ball FG,Ramsey RL,Usherwood PN

    更新日期:1990-07-01 00:00:00

  • Electro-optical property of extremely stretched skinned muscle fibers.

    abstract::Skinned fibers of frog semitendinosus muscle could easily be stretched up to 8 mum or more in sarcomere length. Such extremely stretched fibers gave quite sharp optical diffraction patterns. The intensities of all observable diffraction lines were found to increase on application of electric field (10 similar to 100 V...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/s0006-3495(75)85799-7

    authors: Umazume Y,Fujime S

    更新日期:1975-02-01 00:00:00

  • Partial least-squares functional mode analysis: application to the membrane proteins AQP1, Aqy1, and CLC-ec1.

    abstract::We introduce an approach based on the recently introduced functional mode analysis to identify collective modes of internal dynamics that maximally correlate to an external order parameter of functional interest. Input structural data can be either experimentally determined structure ensembles or simulated ensembles, ...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/j.bpj.2012.07.022

    authors: Krivobokova T,Briones R,Hub JS,Munk A,de Groot BL

    更新日期:2012-08-22 00:00:00

  • Equal sensitivity of the two strands of phiX174 replicative DNA to breakage by ionizing radiation.

    abstract::The supertwisted, double-stranded, replicative intermediate of øX174 DNA (RFI) has been used to determine whether one of the two strands of the double helix is uniquely sensitive to induction of single-strand breaks by ionizing radiation. This could result from a particularly sensitive base sequence or a transfer of e...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/s0006-3495(73)85970-3

    authors: Achey PM,Courtney DG

    更新日期:1973-01-01 00:00:00

  • Theory for ligand rebinding at cell membrane surfaces.

    abstract::Conditions for which a ligand reversibly bound to a cell surface dissociates and then rebinds to the surface have been theoretically examined. The coupled differential equations that describe reaction at the interface between sites on a plane and three-dimensional solution have been described previously (Thompson, N. ...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/S0006-3495(98)77836-1

    authors: Lagerholm BC,Thompson NL

    更新日期:1998-03-01 00:00:00

  • The effect of diffusion on the binding of membrane-bound receptors to coated pits.

    abstract::We have formulated a kinetic model for the primary steps that occur at the cell membrane during receptor-mediated endocytosis. This model includes the diffusion of receptor molecules, the binding of receptors to coated pits, the loss of coated pits by invagination, and random reinsertion of receptors and coated pits. ...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/S0006-3495(85)83879-0

    authors: Keizer J,Ramirez J,Peacock-López E

    更新日期:1985-01-01 00:00:00

  • Substrate dynamics in enzyme action: rotations of monosaccharide subunits in the binding groove are essential for pectin methylesterase processivity.

    abstract::The dynamical behavior of biomacromolecules is a fundamental property regulating a large number of biological processes. Protein dynamics have been widely shown to play a role in enzyme catalysis; however, the interplay between substrate dynamics and enzymatic activity is less understood. We report insights into the r...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/j.bpj.2013.02.049

    authors: Mercadante D,Melton LD,Jameson GB,Williams MA,De Simone A

    更新日期:2013-04-16 00:00:00

  • Translational repression contributes greater noise to gene expression than transcriptional repression.

    abstract::Stochastic effects in gene expression may result in different physiological states of individual cells, with consequences for pathogen survival and artificial gene network design. We studied the contributions of a regulatory factor to gene expression noise in four basic mechanisms of negative gene expression control: ...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/j.bpj.2008.09.052

    authors: Komorowski M,Miekisz J,Kierzek AM

    更新日期:2009-01-01 00:00:00

  • Conductance transients onto dendritic spines in a segmental cable model of hippocampal neurons.

    abstract::Dendritic shaft (Zd) and spine (Zsp) input impedances were computed numerically for sites on hippocampal neurons, using a segmental format of cable calculations. The Zsp values for a typical spine appended onto a dendritic shaft averaged less than 2% higher than the Zd values for the adjacent dendritic shaft. Spine sy...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/S0006-3495(84)84001-1

    authors: Turner DA

    更新日期:1984-07-01 00:00:00