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Membrane fusion intermediate in which contacting membrane leaflets have merged while the aqueous compartments remain separate. A lipid mixing assay therefore need not establish full membrane fusion.

Membrane shape affects the energetic cost and geometry of a bud, neck or protrusion. Proteins, lipid composition and mechanical forces can change curvature; curvature alone does not perform the topological reconnection required for membrane fusion or membrane fission.

Membrane fusion joins initially separate lipid bilayers, changing connections between membrane compartments. It requires controlled rearrangement of both leaflets while avoiding uncontrolled leakage.

The recovered vesicle fractions contain bands at the expected positions for the supplied SNARE proteins. The v-SNARE preparation contains its principal band, and the t-SNARE preparation contains both t-SNARE and the SNAP25 doublet. Thus protein-containing vesicle fractions have been recovered, consistent with successful reconstitution.

The SDS-PAGE experiment does not establish the orientation of the proteins, their proportion facing outward, the stoichiometry in an individual vesicle, or membrane fusion. Nor does a stained band prove that all of its protein is correctly folded and active. Those are separate functional and membrane topology questions.

After warming, more labelled v-SNARE remains full length: lane 2 is approximately 80–85% of the reference signal, compared with about 40–45% in lane 1. Formation of a protected SNARE complex provides a consistent explanation: the susceptible part of v-SNARE is less accessible after complex assembly.

Protection is not itself a unique readout of membrane fusion. Some protein may face the vesicle lumen, and SNARE complexes can protect protein without proving that the two aqueous compartments have merged. The inference depends on comparing these alternatives with the detergent and partner-free controls.

NBD and rhodamine are initially close together in the labelled vesicle lipid bilayer, allowing Förster resonance energy transfer and suppressing donor fluorescence. Mixing labelled lipid bilayer with unlabelled lipid bilayer dilutes the dyes, decreases energy transfer and increases the NBD signal.

The rise with the complementary SNARE proteins, but little rise with protein-free vesicles, therefore supports SNARE-dependent lipid mixing. It is consistent with membrane fusion, but does not by itself distinguish complete fusion from hemifusion or other causes of dye dilution. A separate content mixing assay with a leakage control would test merger of the aqueous compartments.

Cold preincubation can accumulate docked, fusion-ready intermediates. On warming, that population proceeds promptly to lipid mixing, whereas newly combined vesicles must first encounter, dock and assemble productive SNARE complexes. This explains the faster initial rise and larger signal at the displayed endpoint without requiring a different total amount of fluorescent lipid.

The original reconstitution experiments likewise distinguish docking from subsequent membrane fusion.^[ref]

Triton disperses the lipid bilayer into detergent-containing particles, separating the fluorescent lipids enough to remove most fluorescence quenching. Both traces approach the maximum-dilution reference even though their earlier lipid-mixing histories differed. The final fluorescence jump is detergent-induced dequenching, not an additional burst of physiological membrane fusion. It provides a useful normalization endpoint for the available dye signal.

The robust starting point is a topological accounting problem. An archaeal host related to Asgard archaea and an Alphaproteobacteria-related partner must become a cell with an internal mitochondrion, a cell nucleus separated from the cytoplasm, and an interconnected endomembrane system. Their modern relatives are not unchanged ancestors. Several historical sequences can satisfy these endpoints, so the required transformations should be separated from one particular engulfment story.

The bacterial partner must become enclosed within the host's overall cellular boundary and maintained through host reproduction. Its descendants retain inner and outer mitochondrial membranes, requiring preservation or remodelling of ancestral bacterial envelope topology rather than fusion of every membrane into one. If entry used engulfment, host membrane extension, closure and a fission event would enclose the partner, and an additional host-derived surrounding membrane would have to be removed or incorporated into the evolving system. If protrusions grew around an external partner and fused, an alternative sequence could internalize it without a fully developed modern phagocytosis pathway. The modern mitochondrial envelope alone does not decide between these routes.

The host also needs a plasma membrane separating its final cytoplasm from the environment. Producing internal compartments requires membrane curvature, membrane growth and controlled membrane fusion or membrane fission. A purely continuous infolding of the surface has an extracellular-facing lumen; invagination changes geometry but does not allow a molecule to cross a lipid bilayer. Subsequent sealing and traffic can produce an internal lumen while preserving this orientation. This distinction is developed under topology of membrane compartments.

For the cell nucleus, two membranes surround the chromosome-containing compartment, with the outer nuclear envelope continuous with the endoplasmic reticulum. Openings through the envelope become selective nuclear pore complexes, while the chromosome compartment must communicate with the cytoplasm for transport without losing its identity. In an outside-in scenario, internal membranes expand around the chromosomes and fuse. In an inside-out scenario, protrusions from an ancestral chromosome-containing body expand and merge into a surrounding cytoplasm; the original boundary contributes to the nuclear boundary. The latter is an explicit alternative hypothesis, not an observed reconstruction of the event.^[ref]

Culturing an Asgard archaeon revealed extensive protrusions and close metabolic associations, motivating an entangle–engulf–endogenize proposal. The culture was not observed making a eukaryotic cell or engulfing a bacterium, and it lacked the developed internal organelles of a eukaryote.^[ref] The evidence establishes plausibility of some components, not the order of every ancestral membrane event.

The lipid divide adds a biochemical problem: typical archaeal membranes and typical bacterial/eukaryotic membranes differ in lipid linkage and glycerol stereochemistry. Replacement, coexistence or redistribution of lipid-synthesis pathways must be compatible with continuing membrane function; an abrupt wholesale replacement is not forced. Finally, membrane remodelling must be coupled to regulated protein targeting, metabolite exchange and division, so that the mitochondrion becomes an inherited endosymbiont and the new compartments persist. These requirements constrain any scenario even where the historical route remains uncertain.

A trans-SNARE complex links SNARE proteins anchored in different lipid bilayers. Its formation can bring membranes together before complete membrane fusion.