Geometry of the Contact Zone between Fused Membrane-Coated Beads Mimicking Cell-Cell Fusion

Savić, Filip; Kliesch, Torben-Tobias; Verbeek, Sarah F.; Bao, Chunxiao; Thiart, Jan; Kros, Alexander; Geil, Burkhard; Janshoff, Andreas

doi:10.1016/j.bpj.2016.04.026

Cited by 4 publications

(8 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Note that this procedure renders efficiencies rather low since fusion relaxes tension and naturally limits the amount of fused vesicles. The method to use the fluorescence intensity of the lipid dye from the incubated LUVs as a reporter for fusion (lipid mixing) was validated with conventional vesicle fusion assays and turned out to be in good agreement with previous work 31 , 35 . In Fig.…”

Section: Resultssupporting

confidence: 78%

“…Additional FRAP experiments performed on LUVs attached to the membrane patch targeting to bleach A594 confirmed that lipids are shared between the SLB and the LUVs. We can therefore assume that some vesicles are arrested in a stable hemifusion intermediate and do not fuse with the membrane patch, which we attribute to lack of free volume within the bilayer patch 35 . The tensed SLB (ROI 1) in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…A natural drawback of using PDMS membranes is the presence of a solid support that may interfere with essential steps in successful fusion and also alters the energy landscape since adhesion energy needs to be spend in order to peel of the membrane 35 . Therefore, we also employed an assay based on sessile GUVs with adjustable tension.…”

Section: Resultsmentioning

confidence: 99%

See 2 more Smart Citations

Membrane tension increases fusion efficiency of model membranes in the presence of SNAREs

Kliesch

Dietz

Turco

et al. 2017

Sci Rep

Self Cite

View full text Add to dashboard Cite

The large gap in time scales between membrane fusion occurring in biological systems during neurotransmitter release and fusion observed between model membranes has provoked speculations over a large number of possible factors that might explain this discrepancy. One possible reason is an elevated lateral membrane tension present in the presynaptic membrane. We investigated the tension-dependency of fusion using model membranes equipped with a minimal fusion machinery consisting of syntaxin 1, synaptobrevin and SNAP 25. Two different strategies were realized; one based on supported bilayers and the other one employing sessile giant liposomes. In the first approach, isolated patches of planar bilayers derived from giant unilamellar vesicles containing syntaxin 1 and preassembled SNAP 25 (ΔN-complex) were deposited on a dilatable PDMS sheet. In a second approach, lateral membrane tension was controlled through the adhesion of intact giant unilamellar vesicles on a functionalized surface. In both approaches fusion efficiency increases considerably with lateral tension and we identified a threshold tension of 3.4 mN m−1, at which the number of fusion events is increased substantially.

show abstract

Section: Resultssupporting

confidence: 78%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Membrane tension increases fusion efficiency of model membranes in the presence of SNAREs

Kliesch

Dietz

Turco

et al. 2017

Sci Rep

Self Cite

View full text Add to dashboard Cite

show abstract

“…If both leaflets fuse, as indicated by a cumulative step height in a single trace larger than 4-5 nm also very large attractive forces of up to 8 nN occur due to the strong van der Waals attraction between silica surfaces ( Fig. 2B) (25). Hemifusion with a cumulative step height between 2 and 5 nm was associated with adhesion forces around 1 nN, substantially larger than forces associated with docking.…”

Section: Resultsmentioning

confidence: 99%

SNARE-mediated membrane fusion trajectories derived from force-clamp experiments

Oelkers

Witt

Halder

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

Fusion of lipid bilayers is usually prevented by large energy barriers arising from removal of the hydration shell, formation of highly curved structures, and, eventually, fusion pore widening. Here, we measured the force-dependent lifetime of fusion intermediates using membrane-coated silica spheres attached to cantilevers of an atomicforce microscope. Analysis of time traces obtained from force-clamp experiments allowed us to unequivocally assign steps in deflection of the cantilever to membrane states during the SNARE-mediated fusion with solid-supported lipid bilayers. Force-dependent lifetime distributions of the various intermediate fusion states allowed us to propose the likelihood of different fusion pathways and to assess the main free energy barrier, which was found to be related to passing of the hydration barrier and splaying of lipids to eventually enter either the fully fused state or a long-lived hemifusion intermediate. The results were compared with SNARE mutants that arrest adjacent bilayers in the docked state and membranes in the absence of SNAREs but presence of PEG or calcium. Only with the WT SNARE construct was appreciable merging of both bilayers observed.embrane fusion plays a pivotal role in many fundamental biological processes, comprising viral infection, cell-cell fusion during fertilization, tissue formation, and intracellular transport during exo-and endocytosis (1). Among the best-studied biological examples is the Ca 2+ -regulated fusion of synaptic vesicles with the presynaptic plasma membrane in neurons and chromaffin cells (2, 3). Fusion in these cells is catalyzed by concerted action of SNARE proteins through formation of a tetrameric coiled-coil complex that releases a sufficient amount of free energy to lower the barriers for membrane merging. One major energy barrier is associated with the strong repulsive hydration forces when two smooth bilayers approach each other. Other energy-costly contributions originate from lipid splaying as the initiation of stalk formation, expansion of the stalk structure driven by the strong curvature associated with membrane destabilization, hemifusion diaphragm expansion, and, finally, pore formation (4-8). The free energy associated with complex formation and folding of SNAREs was reported to be ∼35 k B T, close to the energy required to create the highly curved transition structures (40-50 k B T) (9, 10). Albeit a part of this free energy might be dissipated as heat, this coincidence of matching energy is also supported by experimental studies showing that only very few SNARE complexes are sufficient to induce fusion in artificial systems (5,6,(11)(12)(13). Although precise data for thermodynamics and kinetics of SNARE zippering and unzippering are now available (11,14,15), few studies address how the energy landscape of membrane fusion is shaped by participation of SNAREs. were among the first to measure fusion events in the absence of proteins as a function of external force using a surface force apparatus, and Moy and coworkers (19) m...

show abstract

“…It has the advantage of deformability, opening possibilities for membrane stretching [88,89]. Examples of applications of non-planar solid supports are wavy glass substrates [90] and glass beads [91,92].…”

Section: Solid-supported Membranesmentioning

confidence: 99%

Effects of arginine derivatives and oligopeptides on the physical properties of model membranes

Verbeek¹

View full text Add to dashboard Cite

show abstract

Geometry of the Contact Zone between Fused Membrane-Coated Beads Mimicking Cell-Cell Fusion

Cited by 4 publications

References 25 publications

Membrane tension increases fusion efficiency of model membranes in the presence of SNAREs

Membrane tension increases fusion efficiency of model membranes in the presence of SNAREs

SNARE-mediated membrane fusion trajectories derived from force-clamp experiments

Effects of arginine derivatives and oligopeptides on the physical properties of model membranes

Contact Info

Product

Resources

About