19 research outputs found
Using ApoE Nanolipoprotein Particles To Analyze SNARE-Induced Fusion Pores
Here we introduce ApoE-based nanolipoprotein
particle (NLP)a
soluble, discoidal bilayer mimetic of ∼23 nm in diameter, as
fusion partners to study the dynamics of fusion pores induced by SNARE
proteins. Using <i>in vitro</i> lipid mixing and content
release assays, we report that NLPs reconstituted with synaptic v-SNARE
VAMP2 (vNLP) fuse with liposomes containing the cognate t-SNARE (Syntaxin1/SNAP25)
partner, with the resulting fusion pore opening directly to the external
buffer. Efflux of encapsulated fluorescent dextrans of different sizes
show that unlike the smaller nanodiscs, these larger NLPs accommodate
the expansion of the fusion pore to at least ∼9 nm, and dithionite
quenching of fluorescent lipid introduced in vNLP confirms that the
NLP fusion pores are short-lived and eventually reseal. The NLPs also
have capacity to accommodate larger number of proteins and using vNLPs
with defined number of VAMP2 protein, including physiologically relevant
copy numbers, we find that 3–4 copies of VAMP2 (minimum 2 per
face) are required to keep a nascent fusion pore open, and the SNARE
proteins act cooperatively to dilate the nascent fusion pore
A Half-Zippered SNARE Complex Represents a Functional Intermediate in Membrane Fusion
SNARE
(soluble <i>N</i>-ethylmaleimide-sensitive factor
attachment protein receptor) proteins mediate fusion by pulling biological
membranes together via a zippering mechanism. Recent biophysical studies
have shown that t- and v-SNAREs can assemble in multiple stages from
the N-termini toward the C-termini. Here we show that functionally,
membrane fusion requires a sequential, two-step folding pathway and
assign specific and distinct functions for each step. First, the N-terminal
domain (NTD) of the v-SNARE docks to the t-SNARE, which leads to a
conformational rearrangement into an activated half-zippered SNARE
complex. This partially assembled SNARE complex locks the C-terminal
(CTD) portion of the t-SNARE into the same structure as in the postfusion
4-helix bundle, thereby creating the binding site for the CTD of the
v-SNARE and enabling fusion. Then zippering of the remaining CTD,
the membrane-proximal linker (LD), and transmembrane (TMD) domains
is required and sufficient to trigger fusion. This intrinsic property
of the SNAREs fits well with the action of physiologically vital regulators
such as complexin. We also report that NTD assembly is the rate-limiting
step. Our findings provide a refined framework for delineating the
molecular mechanism of SNARE-mediated membrane fusion and action of
regulatory proteins
High-Throughput Monitoring of Single Vesicle Fusion Using Freestanding Membranes and Automated Analysis
In
vivo membrane fusion primarily occurs between highly curved
vesicles and planar membranes. A better understanding of fusion entails
an accurate in vitro reproduction of the process. To date, supported
bilayers have been commonly used to mimic the planar membranes. Soluble <i>N</i>-ethylmaleimide-sensitive factor attachment protein receptor
(SNARE) proteins that induce membrane fusion usually have limited
fluidity when embedded in supported bilayers. This alters the kinetics
and prevents correct reconstitution of the overall fusion process.
Also, observing content release across the membrane is hindered by
the lack of a second aqueous compartment. Recently, a step toward
resolving these issues was achieved by using membranes spread on holey
substrates. The mobility of proteins was preserved but vesicles were
prone to bind to the substrate when reaching the edge of the hole,
preventing the observation of many fusion events over the suspended
membrane. Building on this recent advance, we designed a method for
the formation of pore-spanning lipid bilayers containing t-SNARE proteins
on Si/SiO<sub>2</sub> holey chips, allowing the observation of many
individual vesicle fusion events by both lipid mixing and content
release. With this setup, proteins embedded in the suspended membrane
bounced back when they reached the edge of the hole which ensured
vesicles did not bind to the substrate. We observed SNARE-dependent
membrane fusion with the freestanding bilayer of about 500 vesicles.
The time between vesicle docking and fusion is ∼1 s. We also
present a new multimodal open-source software, Fusion Analyzer Software,
which is required for fast data analysis
High-Throughput Monitoring of Single Vesicle Fusion Using Freestanding Membranes and Automated Analysis
In
vivo membrane fusion primarily occurs between highly curved
vesicles and planar membranes. A better understanding of fusion entails
an accurate in vitro reproduction of the process. To date, supported
bilayers have been commonly used to mimic the planar membranes. Soluble <i>N</i>-ethylmaleimide-sensitive factor attachment protein receptor
(SNARE) proteins that induce membrane fusion usually have limited
fluidity when embedded in supported bilayers. This alters the kinetics
and prevents correct reconstitution of the overall fusion process.
Also, observing content release across the membrane is hindered by
the lack of a second aqueous compartment. Recently, a step toward
resolving these issues was achieved by using membranes spread on holey
substrates. The mobility of proteins was preserved but vesicles were
prone to bind to the substrate when reaching the edge of the hole,
preventing the observation of many fusion events over the suspended
membrane. Building on this recent advance, we designed a method for
the formation of pore-spanning lipid bilayers containing t-SNARE proteins
on Si/SiO<sub>2</sub> holey chips, allowing the observation of many
individual vesicle fusion events by both lipid mixing and content
release. With this setup, proteins embedded in the suspended membrane
bounced back when they reached the edge of the hole which ensured
vesicles did not bind to the substrate. We observed SNARE-dependent
membrane fusion with the freestanding bilayer of about 500 vesicles.
The time between vesicle docking and fusion is ∼1 s. We also
present a new multimodal open-source software, Fusion Analyzer Software,
which is required for fast data analysis
Interaction parameters (diffusion coefficients and mobile fractions) of Syn1A and cdVAMP2 in a sponge phase, in the absence or presence of Munc18-1.
<p>FITC-labeled molecules are indicated with a star. Two-sample <i>t</i>-tests on fast diffusion components: cdVAMP2* vs. Syn1A+cdVAMP2* (p<0.05); cdVAMP2* vs. Syn1A+cdVAMP2*+Munc18 (non-significant); Syn1A+cdVAMP2* vs. Syn1A+cdVAMP2*+Munc18 (p<0.05). Two-sample <i>t</i>-test on slow diffusion components: Syn1A* vs. Syn1A+cdVAMP2* (non-significant).</p
High-Throughput Monitoring of Single Vesicle Fusion Using Freestanding Membranes and Automated Analysis
In
vivo membrane fusion primarily occurs between highly curved
vesicles and planar membranes. A better understanding of fusion entails
an accurate in vitro reproduction of the process. To date, supported
bilayers have been commonly used to mimic the planar membranes. Soluble <i>N</i>-ethylmaleimide-sensitive factor attachment protein receptor
(SNARE) proteins that induce membrane fusion usually have limited
fluidity when embedded in supported bilayers. This alters the kinetics
and prevents correct reconstitution of the overall fusion process.
Also, observing content release across the membrane is hindered by
the lack of a second aqueous compartment. Recently, a step toward
resolving these issues was achieved by using membranes spread on holey
substrates. The mobility of proteins was preserved but vesicles were
prone to bind to the substrate when reaching the edge of the hole,
preventing the observation of many fusion events over the suspended
membrane. Building on this recent advance, we designed a method for
the formation of pore-spanning lipid bilayers containing t-SNARE proteins
on Si/SiO<sub>2</sub> holey chips, allowing the observation of many
individual vesicle fusion events by both lipid mixing and content
release. With this setup, proteins embedded in the suspended membrane
bounced back when they reached the edge of the hole which ensured
vesicles did not bind to the substrate. We observed SNARE-dependent
membrane fusion with the freestanding bilayer of about 500 vesicles.
The time between vesicle docking and fusion is ∼1 s. We also
present a new multimodal open-source software, Fusion Analyzer Software,
which is required for fast data analysis
High-Throughput Monitoring of Single Vesicle Fusion Using Freestanding Membranes and Automated Analysis
In
vivo membrane fusion primarily occurs between highly curved
vesicles and planar membranes. A better understanding of fusion entails
an accurate in vitro reproduction of the process. To date, supported
bilayers have been commonly used to mimic the planar membranes. Soluble <i>N</i>-ethylmaleimide-sensitive factor attachment protein receptor
(SNARE) proteins that induce membrane fusion usually have limited
fluidity when embedded in supported bilayers. This alters the kinetics
and prevents correct reconstitution of the overall fusion process.
Also, observing content release across the membrane is hindered by
the lack of a second aqueous compartment. Recently, a step toward
resolving these issues was achieved by using membranes spread on holey
substrates. The mobility of proteins was preserved but vesicles were
prone to bind to the substrate when reaching the edge of the hole,
preventing the observation of many fusion events over the suspended
membrane. Building on this recent advance, we designed a method for
the formation of pore-spanning lipid bilayers containing t-SNARE proteins
on Si/SiO<sub>2</sub> holey chips, allowing the observation of many
individual vesicle fusion events by both lipid mixing and content
release. With this setup, proteins embedded in the suspended membrane
bounced back when they reached the edge of the hole which ensured
vesicles did not bind to the substrate. We observed SNARE-dependent
membrane fusion with the freestanding bilayer of about 500 vesicles.
The time between vesicle docking and fusion is ∼1 s. We also
present a new multimodal open-source software, Fusion Analyzer Software,
which is required for fast data analysis
High-Throughput Monitoring of Single Vesicle Fusion Using Freestanding Membranes and Automated Analysis
In
vivo membrane fusion primarily occurs between highly curved
vesicles and planar membranes. A better understanding of fusion entails
an accurate in vitro reproduction of the process. To date, supported
bilayers have been commonly used to mimic the planar membranes. Soluble <i>N</i>-ethylmaleimide-sensitive factor attachment protein receptor
(SNARE) proteins that induce membrane fusion usually have limited
fluidity when embedded in supported bilayers. This alters the kinetics
and prevents correct reconstitution of the overall fusion process.
Also, observing content release across the membrane is hindered by
the lack of a second aqueous compartment. Recently, a step toward
resolving these issues was achieved by using membranes spread on holey
substrates. The mobility of proteins was preserved but vesicles were
prone to bind to the substrate when reaching the edge of the hole,
preventing the observation of many fusion events over the suspended
membrane. Building on this recent advance, we designed a method for
the formation of pore-spanning lipid bilayers containing t-SNARE proteins
on Si/SiO<sub>2</sub> holey chips, allowing the observation of many
individual vesicle fusion events by both lipid mixing and content
release. With this setup, proteins embedded in the suspended membrane
bounced back when they reached the edge of the hole which ensured
vesicles did not bind to the substrate. We observed SNARE-dependent
membrane fusion with the freestanding bilayer of about 500 vesicles.
The time between vesicle docking and fusion is ∼1 s. We also
present a new multimodal open-source software, Fusion Analyzer Software,
which is required for fast data analysis
Interaction between SNARE proteins in a sponge phase.
<p>The full length Syn1A protein and the cytoplasmic domain of FITC-labeled VAMP2 protein (cdVAMP2*) were reconstituted into two separate sponge phases (at the respective lipid-to-protein molar ratios of 20,000 and 80,000). The Syn1A sponge was pre-incubated for 1 hour at room temperature with or without Munc18-1 protein (1:1 molar ratio between Syn1A and Munc18-1). The Syn1A ± Munc18-1 sponge and the cdVAMP2* sponge were then mixed and allowed to interact for 1 hour at room temperature. In the absence of Munc18-1 (blue fitting curve), cdVAMP2* displays two diffusion coefficients: a slow diffusion coefficient (2.0 ± 0.1 μm<sup>2</sup>/s) corresponding to cdVAMP2* bound to Syn1A in the sponge membrane and a fast diffusion coefficient (9.7 ± 1.4 μm<sup>2</sup>/s) corresponding to free (unbound) cdVAMP2* in the sponge channels. In the presence of Munc18-1 (red fitting curve), cdVAMP2* displays a single, fast, diffusion coefficient (6.4 ± 1.1 μm<sup>2</sup>/s), as observed when it is added to a protein-free sponge phase, showing that Munc18-1 inhibits the interaction between Syn1A and cdVAMP2* (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158457#pone.0158457.t003" target="_blank">Table 3</a>).</p
Formation of Giant Unilamellar Proteo-Liposomes by Osmotic Shock
Giant unilamellar vesicles (GUVs),
composed of a phospholipid bilayer,
are often used as a model system for cell membranes. However, the
study of proteo-membrane interactions in this system is limited as
the incorporation of integral and lipid-anchored proteins into GUVs
remains challenging. Here, we present a simple generic method to incorporate
proteins into GUVs. The basic principle is to break proteo-liposomes
with an osmotic shock. They subsequently reseal into larger vesicles
which, if necessary, can endure the same to obtain even larger proteo-GUVs.
This process does not require specific lipids or reagents, works under
physiological conditions with high concentrations of protein, the
proteins remains functional after incorporation. The resulting proteo-GUVs
can be micromanipulated. Moreover, our protocol is valid for a wide
range of protein substrates. We have successfully reconstituted three
structurally different proteins, two trans-membrane proteins (TolC
and the neuronal t-SNARE), and one lipid-anchored peripheral protein
(GABARAP-Like 1 (GL1)). In each case, we verified that the protein
remains active after incorporation and in its correctly folded state.
We also measured their mobility by performing diffusion measurements
via fluorescence recovery after photobleaching (FRAP) experiments
on micromanipulated single GUVs. The diffusion coefficients are in
agreement with previous data