8 research outputs found
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
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
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
A Programmable DNA Origami Platform to Organize SNAREs for Membrane Fusion
Soluble <i>N</i>-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)
complexes are the core molecular machinery of membrane fusion, a fundamental
process that drives inter- and intracellular communication and trafficking.
One of the questions that remains controversial has been whether and
how SNAREs cooperate. Here we show the use of self-assembled DNA-nanostructure
rings to template uniform-sized small unilamellar vesicles containing
predetermined maximal number of externally facing SNAREs to study
the membrane-fusion process. We also incorporated lipid-conjugated
complementary ssDNA as tethers into vesicle and target membranes,
which enabled bypass of the rate-limiting docking step of fusion reactions
and allowed direct observation of individual membrane-fusion events
at SNARE densities as low as one pair per vesicle. With this platform,
we confirmed at the single event level that, after docking of the
templated-SUVs to supported lipid bilayers (SBL), one to two pairs
of SNAREs are sufficient to drive fast lipid mixing. Modularity and
programmability of this platform makes it readily amenable to studying
more complicated systems where auxiliary proteins are involved