Fusion of lipid membranes: an alternative pathway

Electropores promote metastable interbilayer splays, lowering the energy barrier for peripore stalk formation even at membrane separations >1 nm, conditions under which strong hydration repulsion would otherwise hinder stalk formation.

“Our proposed membrane fusion pathway centers on electropores, which promote metastable interbilayer splays, lowering the energy barrier for peripore stalk formation even at membrane separations >1 nm -conditions under which strong hydration repulsion would otherwise hinder stalk formation 4,7,37 .”

• cites [4] sourcePartly supported

  The passage labeled Abstract (page unavailable) discusses stalk formation, lipid-tail splaying, and the balance between stalk elastic energy and hydration repulsion at close membrane spacing. It does not say that electropores create metastable interbilayer splays or lower the barrier for peripore stalk formation at separations greater than 1 nm. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [7] sourcePartly supported

  In “Step III: lipid tail splaying and fusion stalk formation” (page unavailable), the paper says lipid-tail splaying precedes stalk formation and that perturbing the membrane interface can lower the energy barrier. “Step II: tight docking” also discusses repulsive forces. The cited passages do not establish that electropores promote metastable interbilayer splays or peripore stalks at separations greater than 1 nm. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [37] sourceSupported

• AssumptionRests on an assumption: The biased molecular dynamics simulations and potential-of-mean-force calculations accurately model the free energy landscape of lipid bilayer fusion.
• AssumptionRests on an assumption: The POPC bilayer simulation system is representative of biological lipid membranes for the purpose of inferring a general fusion mechanism.

Lipid headgroups forming membrane surfaces are highly hydrophilic and hydrated, imposing a high energy barrier for two membranes to fuse due to hydration repulsion.

“The lipidic headgroups forming the membranes' surface are highly hydrophilic and hydrated, imposing a high energy barrier for two membranes to fuse due to hydration repulsion [4][5][6][7] .”

• cites [4] sourcePartly supported

  The passage on close membrane apposition (labelled Abstract in our copy; no page given) says that intermembrane hydration repulsion builds as membrane spacing decreases and that a forming stalk can release this hydration energy. It does not explicitly say that the lipid headgroups are highly hydrophilic and hydrated, and it describes hydration repulsion’s role in hemifusion as likely rather than stating the full causal explanation as fact. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [5] sourcePartly supported

  In “Conditions of lipid bilayer fusion” (page unavailable), the paper says dehydration can bring bilayers close enough to promote fusion. In “Analysis of fusogenic conditions” (page unavailable), it describes a large energy cost from intermembrane repulsion at close contact. Our copy does not specifically identify highly hydrophilic, hydrated lipid headgroups or attribute that repulsion to hydration. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [6] sourceSupported
• cites [7] sourceSupported

Intermembrane splayed lipids are thought to template the reorientation of surrounding lipids, promoting formation of the stalk, a nanometric lipid bridge connecting the two proximal monolayers.

“Such intermembrane splayed lipids are thought to template the reorientation of surrounding lipids, promoting formation of the next intermediate, the stalk: a nanometric lipid bridge connecting the two proximal monolayers [14][15][16][17] .”

• cites [14] sourceSupported
• cites [15] 1983, Possible mechanism of membrane-fusion, BiofizikaNot checked yet

  Not checked in the free run.

• cites [16] sourcePartly supported

  The Introduction describes the fusion stalk as a local lipid connection between the proximal monolayers, supporting the description of the stalk. The Structure of Fusion Stalk section describes chain tilt and curvature, but neither passage says that intermembrane splayed lipids template the reorientation of surrounding lipids or thereby promote stalk formation. Page numbers were not available in our copy. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites [17] sourceNot checked — no full text of the source

An alternative pathway for membrane fusion arises from electroporation induced by transmembrane potentials in the absence of proteins.

“Here we uncover an alternative pathwaymembrane fusion arising from electroporation induced by transmembrane potentials 8,9 in absence of proteins.”

• cites [8] sourceSupported
• cites [9] sourcePartly supported

  The paper reports transmembrane-field-induced electroporation in membrane-only simulations (MD simulation method; page unavailable). Its Conclusions describe the resulting stalk-like bridges as precursors to hemifusion, not as demonstrated membrane fusion. The claimed pathway to fusion therefore goes beyond what this source establishes. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

The enhancement of ΔΦ0 arises from overlapping electric double layers of contact sites, increasing the probability of electroporation preceding the peripore-stalk-zipper pathway.

“This enhancement arises from overlapping electric double layers of contact sites, increasing the probability of electroporation 8,12 preceding the peripore-stalk-zipper pathway.”

• cites [8] sourcePartly supported

  The Results and Discussion section ('Membrane Potential Fluctuations and Transient Pore Formation'; page unavailable) reports that larger transmembrane potentials stabilize pores, supporting the proposed increase in pore-formation probability. It does not attribute an enhancement of ΔΦ0 to overlapping electric double layers at contact sites or place electroporation before a peripore-stalk-zipper pathway. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [12] sourcePartly supported

  The Introduction (page unavailable) says transient membrane-potential gradients can lower the energy barrier for pore formation, supporting the general link between electrostatics and pore formation. It does not attribute a ΔΦ0 enhancement to overlapping electric double layers at contact sites or place electroporation before a peripore-stalk-zipper pathway. (LLM, conf=0.76)

  Experimental — accuracy not yet measured

This topologically and energetically distinct pathway for membrane fusion is of particular interest because transmembrane potentials, of similar magnitude to those (ca. 0.1 V/nm) leading to electropores in lipid bilayers, may occur naturally across lipid membranes of vesicles and cells.

“This topologically and energetically distinct pathway for membrane fusion is of particular interest because transmembrane potentials, of similar magnitude to those (ca.0.1V/nm) leading to electropores in lipid bilayers, may occur naturally across lipid membranes of vesicles and cells, as recently demonstrated 8,12 , as well as under external application 9,34 .”

• cites [8] sourcePartly supported

  Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  Experimental — accuracy not yet measured
• cites [12] sourcePartly supported

  Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  Experimental — accuracy not yet measured
• cites [9] sourcePartly supported

  Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  Experimental — accuracy not yet measured
• cites [34] sourceNot checked — no full text of the source

Electroporation of opposing membranes promotes membrane fusion via formation of an intermembrane splayed lipid and peripore stalk, without any protein involvement.

“Using biased molecular dynamics simulations to carry out extensive potential-of-mean-force calculations 10 , we show how electroporation of opposing membranes promotes membrane fusion via formation of an intermembrane splayed lipid and peripore stalk, without any protein involvement.”

• cites [10] sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

• AssumptionRests on an assumption: The biased molecular dynamics simulations and potential-of-mean-force calculations accurately model the free energy landscape of lipid bilayer fusion.
• AssumptionRests on an assumption: The POPC bilayer simulation system is representative of biological lipid membranes for the purpose of inferring a general fusion mechanism.

Electropores lower the energy barrier for peripore-stalk formation.

“Electropores lower the energy barrier for peripore-stalk formation.”

• AssumptionRests on an assumption: The biased molecular dynamics simulations and potential-of-mean-force calculations accurately model the free energy landscape of lipid bilayer fusion.
• AssumptionRests on an assumption: The POPC bilayer simulation system is representative of biological lipid membranes for the purpose of inferring a general fusion mechanism.

Zipping of the electropore rims to form the final full-fusion pore is directly observed.

“We further confirm this mechanism by observing directly the process of zipping of the electropore rims to form the final full-fusion pore.”

• AssumptionRests on an assumption: The biased molecular dynamics simulations and potential-of-mean-force calculations accurately model the free energy landscape of lipid bilayer fusion.
• AssumptionRests on an assumption: The POPC bilayer simulation system is representative of biological lipid membranes for the purpose of inferring a general fusion mechanism.

The peripore stalk involves both the proximal and distal monolayers of the two membranes and bridges the whole membranes rather than solely the proximal leaflets.

“The peripore stalk involves both the proximal and distal monolayers of the two membranes: it is not a hemifusion stalk, as we shall see, since it bridges the whole membranes rather than solely the proximal leaflets (hemi-membranes).”

The predictions are validated by direct observation of bilayer fusion or non-fusion in the respective presence or absence of transmembrane potentials across GUV membranes.

“We validate our predictions by direct observation of bilayer fusion or non-fusion in the respective presence or absence of transmembrane potentials across GUV membranes.”

ΔG stalk at D_PO4 ≈ 0.92 nm under electroporated condition is comparable to that at D_PO4 ≈ 0.61 nm for the unporated condition.

“As summarized in Fig. 2I, ΔG stalk at D PO4 ≈ 0.92 nm, under electroporated condition (Fig. 2D-F) is comparable to that at D PO4 ≈ 0.61 nm for the unporated condition (Fig. 2A-C).”

• AssumptionRests on an assumption: The biased molecular dynamics simulations and potential-of-mean-force calculations accurately model the free energy landscape of lipid bilayer fusion.
• AssumptionRests on an assumption: The POPC bilayer simulation system is representative of biological lipid membranes for the purpose of inferring a general fusion mechanism.

Before contact, the two GUVs exhibit transient membrane potential fluctuations exceeding 300 mV, sufficient for electroporation.

“Before contact, the two GUVs exhibit transient membrane potential fluctuations (Fig. 4B I) exceeding 300 mV, sufficient for electroporation.”

Spontaneous ΔΦ0 fluctuations are visible prior to, during, and after fusion.

“Spontaneous ΔΦ 0 fluctuations are visible prior to, during, and after fusion.”

The fused GUV has a somewhat reduced ΔΦ0 compared to the initial 2 GUVs, due to the exchange of divalent ions made possible by the transient nanopore formation.

“The fused GUV has a somewhat reduced ΔΦ 0 compared to the initial 2 GUVs, due to the exchange of divalent ions made possible by the transient nanopore formation (Fig. 4B IV, Fig. S10).”

Statistical analysis of over 5000 PMF profiles of splay formation shows that the energy landscape is highly sensitive to a lipid's vertical position.

“Statistical analysis of over 5000 PMF profiles of splay formation shows that the energy landscape is highly sensitive to a lipid's vertical position.”

• AssumptionRests on an assumption: The biased molecular dynamics simulations and potential-of-mean-force calculations accurately model the free energy landscape of lipid bilayer fusion.
• AssumptionRests on an assumption: The POPC bilayer simulation system is representative of biological lipid membranes for the purpose of inferring a general fusion mechanism.

In each system, a small fraction of splayed lipids lack a finite ΔG, indicating instability of the splayed state.

“In each system, a small fraction of splayed lipids lack a finite ΔG, indicating instability of the splayed state (Fig. S6A).”

• AssumptionRests on an assumption: The biased molecular dynamics simulations and potential-of-mean-force calculations accurately model the free energy landscape of lipid bilayer fusion.
• AssumptionRests on an assumption: The POPC bilayer simulation system is representative of biological lipid membranes for the purpose of inferring a general fusion mechanism.

GUV fusion is enabled by spontaneous transmembrane potentials.

“Fusion of giant unilamellar vesicles (GUVs) is enabled by spontaneous transmembrane potentials.”

As the two GUVs come in contact, a localized rise in ΔΦ0 is observed.

“As the two GUVs come in contact, a localized rise in ΔΦ 0 is observed (Fig. 4B II.Measured ΔΦ 0 histograms shown in Fig. S10).”

The peripore stalk structure is representative of the morphology observed across a range of midplane separations.

“The peripore stalk structure shown in Fig. 2E is representative of the morphology observed across a range of midplane separations (Figs.S1-S2).”

• AssumptionRests on an assumption: The biased molecular dynamics simulations and potential-of-mean-force calculations accurately model the free energy landscape of lipid bilayer fusion.
• AssumptionRests on an assumption: The POPC bilayer simulation system is representative of biological lipid membranes for the purpose of inferring a general fusion mechanism.

The dynamic progression from two shape-deformed contacted GUVs to a single GUV is completed within one second.

“The dynamic progression from two shape-deformed contacted GUVs (Fig. 4A III) to a single GUV is completed within one second (Movie S2).”

White-light imaging shows a fusion sequence of two GUVs composed of DPhPS:DPhPC lipids, from approach to complete fusion.

“White-light imaging (Fig. 4A) shows a fusion sequence of two GUVs composed of DPhPS:DPhPC lipids, from approach to complete fusion.”

GUV fusion occurs immediately afterwards, within the time resolution of the video.

“GUV fusion occurs immediately afterwards, within the time resolution of the video.”

The distribution of the splay formation energy barrier ΔG is comparable in both electroporated and unporated systems (~20 kBT), despite the large disparity in D_PO4.

“Notably, the distribution of the splay formation energy barrier ΔG (Fig. S7) is comparable in both systems (~20k B T), despite the large disparity in D PO4 , supporting our earlier findings in Fig. 2.”

• AssumptionRests on an assumption: The biased molecular dynamics simulations and potential-of-mean-force calculations accurately model the free energy landscape of lipid bilayer fusion.
• AssumptionRests on an assumption: The POPC bilayer simulation system is representative of biological lipid membranes for the purpose of inferring a general fusion mechanism.

Once a peripore stalk forms, it zips together two opposing overlapping electropores, going from the initial stalk/electropores configuration to the final bilayer fusion pore.

“Once a peripore stalk forms, we see clearly, in fig.2J, the time progression, illustrated by the MD simulation snapshots, of how it zips together two opposing overlapping electropores, going from the initial stalk/electropores configuration (fig.2J(i)) to the final bilayer fusion pore (fig.2J(ii)), as indicated in the schematic cartoon and the time-series of the zipping process.”

• AssumptionRests on an assumption: The biased molecular dynamics simulations and potential-of-mean-force calculations accurately model the free energy landscape of lipid bilayer fusion.
• AssumptionRests on an assumption: The POPC bilayer simulation system is representative of biological lipid membranes for the purpose of inferring a general fusion mechanism.

Both electroporated and porefree processes exhibit similar stalk-formation barriers (ΔG stalk ≈ 50 kJ/mol ≈ 20 kBT).

“Both electroporated and porefree processes exhibit similar stalk-formation barriers (ΔG stalk ≈ 50 kJ/mol ≈ 20 k B T; Fig. 2 G-H), though, notably, at significantly-different bilayer separations D PO4 across the midplane.”

• AssumptionRests on an assumption: The biased molecular dynamics simulations and potential-of-mean-force calculations accurately model the free energy landscape of lipid bilayer fusion.
• AssumptionRests on an assumption: The POPC bilayer simulation system is representative of biological lipid membranes for the purpose of inferring a general fusion mechanism.

PMF profiles of splay formation typically exhibit two distinct energy barriers: ΔG for initiating splay formation and ΔG for retraction of a splayed lipid back into the lamellar state.

“The resulting PMF profiles typically exhibit two distinct energy barriers, as indicated in Fig. 3C: ΔG, corresponding to the barrier for initiating splay formation, and ΔG, associated with the retraction of a splayed lipid back into the lamellar state.”

• AssumptionRests on an assumption: The biased molecular dynamics simulations and potential-of-mean-force calculations accurately model the free energy landscape of lipid bilayer fusion.
• AssumptionRests on an assumption: The POPC bilayer simulation system is representative of biological lipid membranes for the purpose of inferring a general fusion mechanism.

Peripore stalks expand annularly, enclosing the electropores and forming full fusion pores through a zipping process driven by the reduction of pore edge tension.

“These peripore stalks then expand annularly, enclosing the electropores and forming full fusion pores through a”

In control experiments with GUVs made of net charge-neutral zwitterionic lipids kept in identical solutions, trans-membrane potential distributions are absent.

“As a control, the same experiments were performed with GUVs made of net charge-neutral zwitterionic lipids kept in identical solutions, for which trans-membrane potential distributions are absent (Figs.4C-E, where Fig. 4E shows absence of SH contrast, confirming absence of transmembrane potential).”

These electropores give rise to the initial hydrophobic lipidic connection between the membranes, a peripore stalk.

“These pores, referred to below as the electropores, give rise to the initial hydrophobic lipidic connection between the membranes, a peripore stalk, Fig. 1B(3).”

The main mechanism of fusion remains the lipid-mediated topological rearrangements, with proteins largely acting to promote the membrane configurations required for these steps.

“Yet the main mechanism of fusion remains the lipid-mediated topological rearrangements, with proteins largely acting to promote the membrane configurations required for these steps 19,20 (Fig. 1 A(1)).”

• cites [19] sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

• cites [20] sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

Complete fusion proceeds by formation and expansion of a fusion pore within the hemifusion diaphragm, yielding a fully fused membrane.

“Complete fusion then proceeds by formation and expansion of a fusion pore within this diaphragm, yielding a fully fused membrane 5,7 , as illustrated in Fig. 1A.”

• cites [5] sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

• cites [7] sourceNot checked yet

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A consistent conclusion from molecular dynamics simulations based on lipid-only systems is that fusogenic proteins are indispensable because they facilitate the otherwise unfavorable intermediates of splayed lipids, stalks, and fusion pores.

“A consistent conclusion is that fusogenic proteins are indispensable because they facilitate the otherwise unfavorable intermediates of splayed lipids, stalks, and fusion pores.”

• cites [13] sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

• cites [14] sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

• cites [18] sourceNot checked — no full text of the source

An alternative physicochemical mechanism for fusion between interacting lipid membranes operates independently of fusion proteins, even at the earliest stages, without requiring sub-nanometric membrane contact.

“Here we describe and validate an alternative physicochemical mechanism for fusion between interacting lipid membranes, which operates independently of fusion proteins, even at the earliest stages, without requiring sub-nanometric membrane contact.”

Fusogenic proteins, most notably SNAREs, are key drivers of membrane fusion because by pulling membranes together to under 1 nm they reduce lipid hydration and lower the hydration-repulsion energy barrier.

“Fusogenic proteins-most notably SNAREs-are widely recognized as key drivers of membrane fusion because by pulling the membranes together to under 1 nm they reduce lipid hydration and thus lower the hydration-repulsion energy barrier 7,13 .”

• cites [7] sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

• cites [13] sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

Fusion is initiated by the formation of pores in the opposing membranes stimulated by the electric field.

“Rather, fusion is initiated by the formation of pores 21 in the opposing membranes stimulated by the electric field.”

• cites [21] sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

Pulling membranes together greatly increases the likelihood that lipids adopt the splayed state, in which one acyl chain inserts into the opposing membrane.

“This greatly increase the likelihood that lipids adopt the splayed state, in which one acyl chain inserts into the opposing membrane-an energetically costly early fusion intermediate 14 .”

• cites [14] sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

Enhanced splayed-lipid formation is attributed to increased topological complexity when pores are present, which broadens the energy landscape.

“We attribute this enhanced splayed-lipid formation to the increased topological complexity when pores are present, which broadens the energy landscape.”

Previous cornerstone studies established that the splayed state formation is an essential precursor to stalk formation.

“Previous cornerstone studies established that the splayed state formation, as in Fig. 1, and also Fig. 3A, is an essential precursor to stalk formation 13,14,18 .”

• cites [13] sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

• cites [14] sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

• cites [18] sourceNot checked — no full text of the source

A possible simple interpretation is that peripore stalk formation is enabled by the high curvature of the lipid monolayer covering the rim of an electropore.

“A possible simple interpretation of the computational results in macroscopic physical terms is that the formation of the peripore stalk is enabled by the high curvature of the lipid monolayer covering the rim of an electropore.”

A novel, energetically viable membrane-membrane fusion pathway is driven by electroporation, where electropores, formed under a transmembrane potential, facilitate peripore stalk formation at membrane separations comparable with or greater than 1 nm-prior to the onset of hydration repulsion-and thus achievable without the need for fusogenic proteins.

“We describe a novel, energetically viable membrane-membrane fusion pathway driven by electroporation, where electropores, formed under a transmembrane potential, facilitate peripore stalk formation at membrane separations comparable with or greater than 1nm-prior to the onset of hydration repulsion, and thus achievable without the need for fusogenic proteins.”

These results directly validate the proposed mechanism of membrane fusion due to electroporation revealed by the MD simulations.

“These results directly validate the proposed mechanism of membrane fusion due to electroporation revealed above by the MD simulations.”

The high curvature of the electropore rim may strongly reduce the energy of hydration repulsion between the rims of opposing electropores compared to that between two flat membranes.

“Consequently, this curvature may strongly reduce the energy of the hydration repulsion between the rims of the opposing electropores compared to that between two flat membranes, analogous to the electrostatic repulsion between two strongly curved charged surfaces (e.g., cylinders) versus flat planes of equal charge density.”