1,963 research outputs found
Synaptic proteins promote calcium-triggered fast transition from point contact to full fusion.
The molecular underpinnings of synaptic vesicle fusion for fast neurotransmitter release are still unclear. Here, we used a single vesicle-vesicle system with reconstituted SNARE and synaptotagmin-1 proteoliposomes to decipher the temporal sequence of membrane states upon Ca(2+)-injection at 250-500 μM on a 100-ms timescale. Furthermore, detailed membrane morphologies were imaged with cryo-electron microscopy before and after Ca(2+)-injection. We discovered a heterogeneous network of immediate and delayed fusion pathways. Remarkably, all instances of Ca(2+)-triggered immediate fusion started from a membrane-membrane point-contact and proceeded to complete fusion without discernible hemifusion intermediates. In contrast, pathways that involved a stable hemifusion diaphragm only resulted in fusion after many seconds, if at all. When complexin was included, the Ca(2+)-triggered fusion network shifted towards the immediate pathway, effectively synchronizing fusion, especially at lower Ca(2+)-concentration. Synaptic proteins may have evolved to select this immediate pathway out of a heterogeneous network of possible membrane fusion pathways.DOI:http://dx.doi.org/10.7554/eLife.00109.001
Fast Membranes Hemifusion via Dewetting between Lipid Bilayers
The behavior of lipid bilayer is important to understand the functionality of
cells like the trafficking of ions between cells. Standard procedures to
explore the properties of lipid bilayer and hemifused states typically use
either supported membranes or vesicles. Both techniques have several
shortcoming in terms of bio relevance or accessibility for measurements. In
this article the formation of individual free standing hemifused states between
model cell membranes is studied using an optimized microfluidic scheme which
allows for simultaneous optical and electrophysiological measurements. In a
first step, two model membranes are formed at a desired location within a
microfluidic device using a variation of the droplet interface bilayer (DiB)
technique. In a second step, the two model membranes are brought into contact
forming a single hemifused state. For all tested lipids, the hemifused state
between free standing membranes form within hundreds of milliseconds, i.e.
several orders of magnitude faster than reported in literature. The formation
of a hemifused state is observed as a two stage process, whereas the second
stage can be explained as a dewetting process in no-slip boundary condition.
The formed hemifusion states are long living and a single fusion event can be
observed when triggered by an applied electric field as demonstrated for
monoolein
Field theoretic study of bilayer membrane fusion: I. Hemifusion mechanism
Self-consistent field theory is used to determine structural and energetic
properties of metastable intermediates and unstable transition states involved
in the standard stalk mechanism of bilayer membrane fusion. A microscopic model
of flexible amphiphilic chains dissolved in hydrophilic solvent is employed to
describe these self-assembled structures. We find that the barrier to formation
of the initial stalk is much smaller than previously estimated by
phenomenological theories. Therefore its creation it is not the rate limiting
process. The barrier which is relevant is associated with the rather limited
radial expansion of the stalk into a hemifusion diaphragm. It is strongly
affected by the architecture of the amphiphile, decreasing as the effective
spontaneous curvature of the amphiphile is made more negative. It is also
reduced when the tension is increased. At high tension the fusion pore, created
when a hole forms in the hemifusion diaphragm, expands without bound. At very
low membrane tension, small fusion pores can be trapped in a flickering
metastable state. Successful fusion is severely limited by the architecture of
the lipids. If the effective spontaneous curvature is not sufficiently
negative, fusion does not occur because metastable stalks, whose existence is a
seemingly necessary prerequisite, do not form at all. However if the
spontaneous curvature is too negative, stalks are so stable that fusion does
not occur because the system is unstable either to a phase of stable radial
stalks, or to an inverted-hexagonal phase induced by stable linear stalks. Our
results on the architecture and tension needed for successful fusion are
summarized in a phase diagram.Comment: in press, Biophys.J. accepted versio
Field Theoretic Study of Bilayer Membrane Fusion III: Membranes with Leaves of Different Composition
We extend previous work on homogeneous bilayers to calculate the barriers to
fusion of planar bilayers which contain two different amphiphiles, a
lamellae-former and a hexagonal former, with different compositions of the
twoin each leaf. Self-consistent field theory is employed, and both standard
and alternative pathways are explored. We first calculate these barriers as the
amount of hexagonal former is increased equally in both leaves to levels
appropriate to the plasma membrane of human red blood cells. We follow these
barriers as the composition of hexagonal-formers is then increased in the cis
layer and decreased in the trans layer, again to an extent comparable to the
biological system. We find that, while the fusion pathway exhibits two barriers
in both the standard and alternative pathways, in both cases the magnitudes of
these barriers are comparable to one another, and small, on the order of 13 kT.
As a consequence, one expects that once the bilayers are brought sufficiently
close to one another to initiate the process, fusion should occur rapidly.Comment: 9 figure
Field Theoretic Study of Bilayer Membrane Fusion: II. Mechanism of a Stalk-Hole Complex
We use self-consistent field theory to determine structural and energetic
properties of intermediates and transition states involved in bilayer membrane
fusion. In particular, we extend our original calculations from those of the
standard hemifusion mechanism, which was studied in detail in the first paper
of this series, to consider a possible alternative to it. This mechanism
involves non-axial stalk expansion, in contrast to the axially symmetric
evolution postulated in the classical mechanism. Elongation of the initial
stalk facilitates the nucleation of holes and leads to destabilization of the
fusing membranes via the formation of a stalk-hole complex. We study properties
of this complex in detail, and show how transient leakage during fusion,
previously predicted and recently observed in experiment, should vary with
system architecture and tension. We also show that the barrier to fusion in the
alternative mechanism is lower than that of the standard mechanism by a few
over most of the relevant region of system parameters, so that this
alternative mechanism is a viable alternative to the standard pathway
A New Mechanism of Model Membrane Fusion Determined from Monte Carlo Simulation
We have carried out extensive Monte Carlo simulations of the fusion of tense
apposed bilayers formed by amphiphilic molecules within the framework of a
coarse grained lattice model. The fusion pathway differs from the usual stalk
mechanism. Stalks do form between the apposed bilayers, but rather than expand
radially to form an axial-symmetric hemifusion diaphragm of the trans leaves of
both bilayers, they promote in their vicinity the nucleation of small holes in
the bilayers. Two subsequent paths are observed: (i) The stalk encircles a hole
in one bilayer creating a diaphragm comprised of both leaves of the other
intact bilayer, and which ruptures to complete the fusion pore. (ii) Before the
stalk can encircle a hole in one bilayer, a second hole forms in the other
bilayer, and the stalk aligns and encircles them both to complete the fusion
pore. Both pathways give rise to mixing between the cis and trans leaves of the
bilayer and allow for transient leakage.Comment: revised version, accepted to Biophys. J. (11 figures
Fusion pore conductance to determine the effects of mutating the structure of influenza virus hemagglutinin
Enveloped viruses, such as influenza, infect cells by fusing their viral envelope with the cell membrane. The fusion pore is a macromolecular structure that links two membranes that are fusing. This paper will focus on the fusion pore initiated by the hemagglutinin (HA) protein of influenza virus upon infection of a host cell. Mutations in the HA protein can alter the time-course and structure of the developing fusion pore. While there is a clear relationship between HA's structure and the dynamic opening of the pore, the initial 3D structure of the fusion pore as it first begins to form remains unknown. We have attempted to address this unanswered question by measuring fusion pore conductance - a one dimensional electrophysiological measurement - at millisecond time resolution for both wild type and mutant HA proteins, using an automated patch clamp apparatus. Correlating the entire life history of the fusion pore with the snapshots we get from 3D imaging (cryo-electron tomography) would allow us to capture the initial pore opening, as well as better understand the effect that mutating the structure of HA has on influenza viral infection. At this time, we have not yet been able to observe the fusion event; however, we do believe that future experimentation using fusion pore conductance to investigate the effects of HA's structure on influenza viral infection are both promising and necessary
New mechanism of membrane fusion
We have carried out Monte Carlo simulation of the fusion of bilayers of
single chain amphiphiles which show phase behavior similar to that of
biological lipids. The fusion mechanism we observe is very different from the
``stalk'' hypothesis. Stalks do form on the first stage of fusion, but they do
not grow radially to form a hemifused state. Instead, stalk formation
destabilizes the membranes and results in hole formation in the vicinity of the
stalks. When holes in each bilayer nucleate spontaneously next to the same
stalk, an incomplete fusion pore is formed. The fusion process is completed by
propagation of the initial connection, the stalk, along the edges of the
aligned holes.Comment: 4 pages, 3 figure
Initiation and dynamics of hemifusion in lipid bilayers
One approach to the understanding of fusion in cells and model membranes
involves stalk formation and expansion of the hemifusion diaphragm. We predict
theoretically the initiation of hemifusion by stalk expansion and the dynamics
of mesoscopic hemifusion diaphragm expansion in the light of recent experiments
and theory that suggested that hemifusion is driven by intra-membrane tension
far from the fusion zone. Our predictions include a square root scaling of the
hemifusion zone size on time as well as an estimate of the minimal tension for
initiation of hemifusion. While a minimal amount of pressure is evidently
needed for stalk formation, it is not necessarily required for stalk expansion.
The energy required for tension induced fusion is much smaller than that
required for pressure driven fusion
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