Lipid bilayer fusion
In
Fusion is involved in many cellular processes, particularly in
Lipid mechanism
There are four fundamental steps in the fusion process, although each of these steps actually represents a complex sequence of events.[1] First, the involved membranes must aggregate, approaching each other to within several nanometers. Second, the two bilayers must come into very close contact (within a few angstroms). To achieve this close contact, the two surfaces must become at least partially dehydrated, as the bound surface water normally present causes bilayers to strongly repel at this distance. Third, a destabilization must develop at one point between the two bilayers, inducing a highly localized rearrangement of the two bilayers. Finally, as this point defect grows, the components of the two bilayers mix and diffuse away from the site of contact. Depending on whether hemifusion or full fusion occurs, the internal contents of the membranes may mix at this point as well.
The exact mechanisms behind this complex sequence of events are still a matter of debate. To simplify the system and allow more definitive study, many experiments have been performed in vitro with synthetic lipid vesicles. These studies have shown that divalent
In the fusion process, the lipid head group is not only involved in charge density, but can affect dehydration and defect nucleation. These effects are independent of the effects of ions. The presence of the uncharged headgroup phosphatidylethanolamine (PE) increases fusion when incorporated into a phosphatidylcholine bilayer. This phenomenon has been explained by some as a dehydration effect similar to the influence of calcium.[8] The PE headgroup binds water less tightly than PC and therefore may allow close apposition more easily. An alternate explanation is that the physical rather than chemical nature of PE may help induce fusion. According to the stalk hypothesis of fusion, a highly curved bridge must form between the two bilayers for fusion to occur.[9] Since PE has a small headgroup and readily forms inverted micelle phases it should, according to the stalk model, promote the formation of these stalks.[10] Further evidence cited in favor of this theory is the fact that certain lipid mixtures have been shown to only support fusion when raised above the transition temperature of these inverted phases.[11][12] This topic also remains controversial, and even if there is a curved structure present in the fusion process, there is debate in the literature over whether it is a cubic, hexagonal or more exotic extended phase.[13]
Fusion proteins
The situation is further complicated when considering fusion in vivo since biological fusion is almost always regulated by the action of
Fusion in laboratory practice
In studies of molecular and cellular biology it is often desirable to artificially induce fusion. Although this can be accomplished with the addition of calcium as discussed earlier, this procedure is often not feasible because calcium regulates many other biochemical processes and its addition would be a strong confound. Also, as mentioned, calcium induces massive aggregation as well as fusion. The addition of
Alternatively, SNARE-inspired model systems can be used to induce membrane fusion of lipid vesicles. In those systems membrane anchored complementary DNA,[20][21][22] PNA,[23] peptides,[24] or other molecules[25] "zip" together and pull the membranes into proximity. Such systems could have practical applications in the future, for example in drug delivery.[26] The probably best investigated system[27] consists of coiled-coil forming peptides of complementary charge (one is typically carrying an excess of positively charged lysins and is thus termed peptide K, and one negatively charged glutamic acids called peptide E).[28] Interestingly, it was discovered that not only the coiled-coil formation between the two peptides is necessary for membrane fusion to occur, but also that the peptide K interacts with the membrane surface and cause local defects.[29]
Assays to measure membrane fusion
There are two levels of fusion: mixing of membrane lipids and mixing of contents. Assays of membrane fusion report either the mixing of membrane lipids or the mixing of the aqueous contents of the fused entities.
Assays for measuring lipid mixing
Assays evaluating lipid mixing make use of concentration dependent effects such as nonradiative energy transfer, fluorescence quenching and pyrene excimer formation.
- NBD-Rhodamine Energy Transfer:[30] In this method, membrane labeled with both NBD (donor) and Rhodamine (acceptor) combine with unlabeled membrane. When NBD and Rhodamine are within a certain distance, the Förster resonance energy transfer (FRET) happens. After fusion, resonance energy transfer (FRET) decreases when the average distance between probes increases, while NBD fluorescence increases.
- Pyrene Excimer Formation: Pyrene monomer and excimer emission wavelengths are different. The emission wavelength of monomer is around 400 nm and that of excimer is around 470 nm. In this method, membrane labeled with Pyrene combines with unlabeled membrane. Pyrene self associates in membrane and then excited pyrene excites other pyrene. Before fusion, the majority of the emission is excimer emission. After fusion, the distance between probes increases and the ratio of excimer emission decreases.[citation needed]
- Octadecyl Rhodamine B Self-Quenching:[31] This assay is based on self-quenching of octadecyl rhodamine B. Octadecyl rhodamine B self-quenching occurs when the probe is incorporated into membrane lipids at concentrations of 1–10 mole percent[32] because Rhodamine dimers quench fluorescence. In this method, membrane labeled Rhodamine combines with unlabeled membrane. Fusion with unlabeled membranes resulting in dilution of the probe, which is accompanied by increasing fluorescence.[33][34] The major problem of this assay is spontaneous transfer.
Assays for measuring content mixing
Mixing of aqueous contents from vesicles as a result of lysis, fusion or physiological permeability can be detected fluorometrically using low molecular weight soluble tracers.
- Fluorescence quenching assays with ANTS/DPX:[35][36] ANTS is a polyanionic fluorophore, while DPX is a cationic quencher. The assay is based on the collisional quenching of them. Separate vesicle populations are loaded with ANTS or DPX, respectively. When content mixing happens, ANTS and DPX collide and fluorescence of ANTS monitored at 530 nm, with excitation at 360 nm is quenched. This method is performed at acidic pH and high concentration.
- Fluorescence enhancement assays with Tb3+/DPA:[37][38] This method is based on the fact that chelate of Tb3+/DPA is 10,000 times more fluorescent than Tb3+ alone. In the Tb3+/DPA assay, separate vesicle populations are loaded with TbCl3 or DPA. The formation of Tb3+/DPA chelate can be used to indicate vesicle fusion. This method is good for protein free membranes.[citation needed]
- Single molecule DNA assay.[39] A DNA hairpin composed of 5 base pair stem and poly-thymidine loop that is labeled with a donor (Cy3) and an acceptor (Cy5) at the ends of the stem was encapsulated in the v-SNARE vesicle. We separately encapsulated multiple unlabeled poly-adenosine DNA strands in the t-SNARE vesicle. If the two vesicles, both ~100 nm in diameter, dock and a large enough fusion pore forms between them, the two DNA molecules should hybridize, opening up the stem region of the hairpin and switching the Förster resonance energy transfer (FRET) efficiency (E) between Cy3 and Cy5 from a high to a low value.
See also
- Interbilayer Forces in Membrane Fusion
- Fusion mechanism
- Cell fusion
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