Structural Biochemistry/Membrane Fusion

Introduction
The fusion of two cells results in the coming together of the membranes of the two cells at one location which allows for the exchange of material. It is one of the most common ways for materials to enter or exit a cell and the process eventually leads to one continuous membrane surrounding the contents of the two cells. The membrane of the cells are made up of a double layer of phospholipids which interact and creates a connection between two cells. The fusion of the two cell membranes can be seen through the technique of x-ray diffraction. The technique of x-Ray diffraction confirms the theory that the cells form an hourglass shaped structure called a "stalk" during fusion. This stalk eventually grows to connect the two membranes and the two membranes eventually merge into one large one. The fusion of two membranes is catalyzed by proteins. These proteins act by recognizing other cells that have the potential to be fused and can also initiate the process by pulling the membranes close together to eliminate lipid and water interactions.

For membrane fusion to occur, the process must be thermodynamically favorable, that is, the free energy of the complex before remodeling must be lower than that of the complex afterwards. It is said that the process must be energetically "downhill" in order for the process to be feasible. The intermediate structures must also be low enough to overcome thermal fluctuations in a reasonable amount of time. The driving force of the reaction is the relaxation of elastic energy.

The amount of energy required for membrane fusion to occur is roughly 40kBT which is about the same amount of energy to hydrolyze a few ATP molecules.

Exocytotic Membrane Fusion
This is the process in which a cell's internal organelles fuse with the cytoplasmic side of the cellular membrane. It is an important process for the release of hormones and neurotransmitters as well as other biological compounds. This fusion process typically occurs in three steps. Step 1 consists of the internal cellular component that is to be released migrating towards the exterior of the cellular membrane. Step 2 is the merging of the membranes, and step 3 consists of the merging of the interior components such as molecules within a vesicle. The overall process is effected by different factors. An important factor that determines the ability of exocytotic membrane fusion is pH. Another factor is the hydrolysis of ATP as the fusion itself requires energy. Other factors include: transmembrane electrical potential, Osmotic forces, Proteinases, and Calmodulin.

Membrane Fusion Mechanics
Membrane fusion begins with the local merger of two separate cellular monolayer membranes, while the remote monolayer membranes remain separate. This initial connection between the two cellular membranes is call a fusion stalk and represents the first stage in the process of membrane fusion between two separate cellular membranes, called hemifusion. Stalk evolution eventually lead to the fusion of the remote monolayer of the two cellular membranes. The outcome of this merger is a fusion pore that connects the volumes of the previously separated cellular membranes. The fusion pore has the ability to expand or contract depending on the biological conditions. Stalk formation in the membrane fusion requires the temporary disruption of the two cellular membranes, which is unfavorable and opposed by the hydrophobic forces working to maintain a continuous lipid assembly on the cellular membrane. One theory that solves this contradiction is the involvement of one lipid molecule in the cellular membrane building a bridge between the two separate cellular membranes. In the pre-stalk fusion intermediate, one lipid molecule splays its two hydrocarbon chains into the opposing membrane and begins building a developing lipid bridge between the two separate cellular membranes. This chain-splay mechanism to counteract the hydrophobic conditions of the cellular membrane has been demonstrated countless times with simulations under conditions of partially dehydration of the membrane contact. This shows that the process of membrane fusion is ran by the strong lipid bridge force pushing between the two separate cellular membranes together. As a result of fusion, the overall curvature of the merged membrane can partially relax the bending and is reduced opposed to the former two cellular membranes. Physical forces driving unbent membranes favor membrane fusion due to the self-connectivity of two cellular membranes opposed to one small and strongly bent cellular membrane. The lipids of the merged cellular membrane can redistribute over the increased membrane area instead of being limited in one compacted cellular membrane.

Membrane Elastic Energy
In membrane remodeling there are three different energies that play a major role: membrane bending, membrane stretching, and tilting of the lipid hydrocarbon chain. Membrane bending energy is dependent on the curvature of the membrane surface. The free energy at any point on the membrane surface can be different due to the variations of lipids and protein compositions. Membrane fusion decreases the free energy of membrane bending. The second type of elastic energy is membrane stretching. This type of energy originates from the lateral tension from the membrane stretch that can drive the expansion of the fusion pore. The last type can be considered as the tilting of the hydrocarbon chain of the lipid molecules between the two cellular membranes during fusion.

SNARE complex


This is a complex made of proteins that is involved in membrane fusion. The proteins involved are called SNARE proteins which act to regulate vesicle fusion. SNARE stands for soluble N- ethylmaleimide-sensitive-factor attachment protein receptor.The SNARE complex consists of proteins that forms a four-helix bundle. They are often located on the plasma membrane and help to draw certain membranes together to initiate the fusion process. These proteins are found in all eukaryotic cells and help determine the compartment in which the vesicle will fuse. There are two kinds of SNARE proteins. The first one is the v-SNARE where the "v" stands for vesicle. The second one is the t-SNARE where the "t" stands for target. The v-SNARE which is part of the transport vesicle is an integral protein and will bind to a t-SNARE on the target membrane which will result in fusion of the target membrane to the transport vesicle. The SNARE complex plays a vital role in neurotransmission. For neurotransmission to occur, there needs to be fusion of the presynaptic plasma membrane with vesicles containing the neurotransmitter molecules. SNARE proteins must meet certain conditions in order to be active in cell fusion. The first condition is that the SNARE proteins must be located on two different membranes. Second, these membranes must be able to reach each other. Third, heavy and light chains must get together on the membrane and form t-SNAREs. Fourth, the SNAREs must be able to link the two membranes. Fifth, the anchors and linkers must be functional. Lastly, the membranes surfaces msut be compatible for fusion.

SNARE proteins can be assigned to three protein families. The families are the syntaxins, the VAMPs, and the SNAP-25 family. One of the distinguishing features that all SNARE proteins have is their coiled-coil domains. The SNARE complex itself is formed from a coil of syntaxin, a coil of VAMP, and two coils of SNAP-25. The overall mechanism of the membrane fusion is that a vesicle will land on on a membrane with the help of Rab proteins and bring the SNARE proteins closer to each other. The SNARE core complex then brings the two membranes together and creates tension within the two membranes. When the membranes get closer and closer, hemifusion can occur then a fusion pore opening and expansion allowing for membrane fusion. The SNARE proteins basically provide the driving force as well as stabilizes the transition states of the process.



One of the many important uses of membrane fusion and the SNARE complex is within the bodies nervous system. It is important for chemical synaptic transmission where neurotransmitters bound by pre-synaptic vesicles are eventually released be a calcium dependent mechanism into the synaptic cleft after membrane fusion occurs. Before fusion is able to occur, the vesicle is transported to the specific target membrane. A "fusion trigger" such as Ca2+ directs fusion for completion after the vesicle has gone through priming. In order for this fusion to occur, the SNARE complex is important in overcoming repulsive ionic forces as well as getting rid of hydration between the membranes lipid bilayers. In recent years the SNARE proteins have been reclassified as R-SNAREs and Q-SNARES instead of the v and t SNAREs. R-SNAREs are argining containing while Q-SNAREs are glutamine containing.

Hydrophobic insertion (wedging) mechanism
One mechanism of membrane fusion is the Hydrophobic insertion (wedging) mechanism. Proteins are intermediates to the process of membrane fusion and certain proteins can induce membrane fusion which in turn causes elastic stress. Examples of proteins that can drive this process are lipid-modifying enzymes, flippases and rigid protein scaffolding. The core essence of this mechanism lies in the insertion of hydrophobic or amphiphatic regions into the shallow membrane causes the expansion of the polar head region which induces protein curvature. Proteins that are capable of this include epsins, small G-proteins, and N-bar domains and proteins capable of inserting small hydrophobic loops. Further research deals with finding which structurally related proteins can drive membrane remodeling.

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