# Structural Biochemistry/Membrane Protein stresses in fusion and fission
## Membrane Fusion

Membrane fusion is defined as a progress of two separated membranes that would be unified into one continuous membrane through the progress of intermediate transformations. At the beginning of the progress, only contacting monolayers of membranes would merge into one, while the distal membranes would be separated. Hence, the fusion stalk, which is the lipid bridge, connects the two monolayers membranes and result in the first stage of fusion, hemifusion. Eventually, this fusion stalk would allow the unification between the distal monolayer membranes. And such unification would result in the formation of the fusion pore that would merge the separated distal monolayers. As a result, that would be the last step of the unification between monolayers membranes.

## Membrane Fission

Membrane fission occurs when there's a separation of membrane into two separated membranes. This progress takes place through the membrane neck. Hence, fission occurs when there's a self-merger between the inner monolayer of the membrane neck. This step results in the formation of the fission stalk. And the fission progress would end when there's the self-unification between the outer monolayers.

### BAR Proteins

Cellular membrane has the ability to execute very important functions like locomotion and receptor turnover. It is necessary for cells to sense changes in the environment and transport signals across the membranes. To perform this, cells need to reconstruct the shape of its membrane with relatively high spatial and temporal accuracy due to the dependency from cellular processes—such as cell division, endocytosis, and cell migration. Therefore, membranes have the power in remodeling the overall shape of the cell. For this to occur, membrane-bending proteins are required and membrane curvature are closely examined. The sign, or sometimes known as the direction, of the curvature is considered to be an arbitrary measure. From a cytosol, plasma membrane invaginations predominantly contain segments with positive curvature. On the contrary, protrusions consist of membranes with a negative curvature. Membrane curvature is critical in the investigation of membrane fusion and fission, which is the analysis of activity of membrane proteins, and the unity of proteins to the location of membrane-cytosol interface. Membrane curvature is not known to be a passive characteristic of the cellular membrane; instead, it has risen to be a regulated state due to its active influences of diverse processes of membrane fusion and fission.
Examples of such membrane proteins include the Bin/amphiphysin/Rvs (BAR) domain, one of the main membrane-bending protein superfamily. The BAR domains lack sequence motifs of characteristic signature in their primary structure making it not entirely recognizable. However, at the structural level, BAR domains are considerably conserved, containing a three helix coiled core which creates a curved homodimers or heterodimers. This gives an overall ‘banana shape’.
The formation of homodimers or heterodimers are still relatively a mystery in the BAR domain. However, researches have shown that a mishap of the dimerization interface contribute malfunction in BAR proteins in terms of remodeling. The nonfunctionality happens due to proper interaction of the BAR domains with the negatively-charged lipid headgroups like phosphinositides, which require the specific positioning of a positively-charged residues in the context of a banana- shaped dimer.
Most BAR proteins contain an extra domain, such as a src-hology 3 (SH3) domain, that permit BAR proteins to communicate with proline-rich domain containing proteins. Therefore, the BAR domain proteins are a type of scaffolding proteins that systemize a selection of proteins intp a “curvature-dependent” form. The three general categories of BAR proteins are classical BAR domain, IMD/Inverse BAR domain, and the FCH domain (F-BAR). Classical BAR domains are typically found in arfaptin and contain the highest level of intrinsic curvature. It extends to smaller divisions depending on additional emembrane binding domains like an amphipathic N-terminal helix or the phox domain. All classical BAR domains are known to provide and encourage positive membrane curvature.
An example of the F-BAR domain is the Cdc42-interacting protein 4. This time of protein domain signifies the largest and most diverse domains. It is further broken down into six subcategories. Moreover, the intrinsic curvature of this particular protein domain ranges from high to almost planar. Due to this, proteins of this kind are allowed to support a large range of membrane curvatures. The IMD/Inverse BAR domain contain a negative curvature and symbolize a mechanism for cells to generate extrusions. A similar protein domain to IMD/Inverse BAR domain is PinkBAR domain, which contains no intrinsic curvature. The absence of intrinsic curvature for PinkBAR domains help to form scaffolds on flat membrane surfaces. Bar proteins function a large role in membrane scaffolding, organelle creation, organismal patterning, and disease. Most of the functions associated with BAR domains narrow down to the intrinsically curved dimers of all BAR domains. The shape of the BAR domain dimers correlate to its curvature, which inherently depicts its ability to bend membranes.
The molecular mechanism of membrane bending for BAR domains arises in the intrinsic curvature of the dimers. The Bar Domain dimers encourage membrane bending by imposing its shape on the membrane substrate, a process known as the ‘scaffolding’ mechanism. Another membrane bending mechanism includes the presence of amphipathic wedges, which can sense membrane curvature, into the bilayer. Such wedges can also encourage formation through the concerted displacement of lipids in the leaflet proximal at its present location. Additionally, substrate selection could potentially cause experimental bias; the composition bilayer and pre-existing curvature for the BAR domain determines the likelihood of recruiting and remodeling for the BAR domain. Some BAR proteins that have amphipathic wedges are known to play a significant role in membrane fission. Furthermore, membrane bending and fission are inversely related.
The property of BAR domains to be involved in bilayers and recruit specific interactive partners requires access to and control of its target. Studies have indicated that BAR proteins show an inclination for membranes with a specified curvatures in vitro, and such membrane preferences correspond to the intrinsic curvature of the BAR domain. Also, amphipathic sequences detect and bind to locations where curvature stress creates packing defects in the region of the lipid headgroup. The amount of defects correlates with the degree of curvature. As a result, the ability of BAR domain proteins to sense the defects is a method to control differential binding to membranes of various curvatures.
Despite the developing progress in gaining an understanding of membrane transport and signaling, studies of how the cells reshape their membranes have provided information on the structural and functional features of the membrane bilayer. However, the molecular mechanisms of which membrane-bending domains process and stabilize membrane curvature and how the events are coordinated remains elusive. For one, the structural complexity of the membrane bilayer is constantly influenced to be susceptible to reconstruct and recruit specific membrane remodelers. Second, the deficiency of standardized methods to investigate membrane remodeling and the tools to computationally examine the complicated lipid mixtures that is generally located in cellular membranes.

## Similarity of the membrane fusion and membrane fission

One common feature of the membrane fusion and membrane fission is the creation of the membrane stalk. In fusion, there is the formation of the fusion stalk which is the merging between the monolayer membranes. In fission, there is also the formation of the fission stalk which is the unification of the inner monolayers of the membrane neck. Scientists have done research on the pre-stalk fusion. They believe this progress requires one lipid molecule to be inserted between the two opposing hydrocarbon membranes. As a result, there would be the formation of the lipid bridge between the membranes.

## Differences between membrane fusion and membrane fission

One difference is the opposite sequences of shapes of the fission and fusion progress. Membrane fusion results in the unification of the monolayers membranes, while fission results in the separation of the membranes. Hence, fission allows the separated membranes to have a greater curvatures and stronger bent of the membranes. However, fusion results in a smaller curvatures which allows the bending of the membranes to relax. In conclusion, membrane bending would favor membrane fission, while membrane fusion would results in membrane unbending. The second difference is the self-connectivity between the membrane fusion and membrane fission. Since fission occurs when there's a separation of the continuous membrane, that would result in the limited area for each separated membranes. However, the unification of membranes, fusion, would increase the area over the entire membrane. Therefore, fission is not favored by self-connectivity, while fusion is supported by the forces that result in the merging of the membranes.

## Membrane remodeling

Proteins are the forces that generate the merging between the monolayer membranes or the separation of the monolayer membrane. Hence, membrane remodeling is the reconstructing of the lipid bilayer which is determined by the membrane proteins. There are two physical requirements for membrane remodeling. First, the free energy before the beginning of the remodeling must be higher than the energy after the remodeling. This means that there must be a release of energy that would allow for the reconstructing of the membranes. In this case, we use the term "relaxation of the free energy" to summarize the first requirement. Second, the intermediate energy must be low enough to be overcome by the thermal fluctuations. The progress of membrane remodeling is determined by the proteins. They are proteins that provide the free energy for this spontaneous reaction. Hence, the proteins can change the physical structures of the lipid bilayers and the lipid composition of the monolayers membrane. These physical changes would result in the membrane remodeling.

## Free energy-elastic energy

The free energy used in membrane remodeling is called elastic energy. Elastic energy is created by the three different forms of membranes. They are membrane bending, stretching, and tilting of the lipid chains. First, membrane bending is determined by the curvatures of the membranes surface. The bending of the membrane, curvature, is the shape of the membranes. Mathematically, curvature is defined by two radii R$_1$ and R$_2$ of the arcs plane. We use the equations of inverse radii c$_1$= 1/R$_1$ and c$_2$ = 1/R$_2$ to calculate the sum and product of the curvatures. As a result, those values would determine the shapes of the lipid bilayer. The sum and the product of curvatures are called the total and Gaussian curvatures. And they both require free energy (F) to generate membrane remodeling. The sum of the curvatures has F$_B$, the free energy of the total curvature. This value is based on the membrane shape. And it can change because there are multiple stages from the beginning of the membrane remodeling to the fusion or fission progress. Hence, fission would result in the increase of this free energy, while fission would decrease the free energy F$_B$. Second, membrane stretching depends on the fusion pore. This fusion pore would allow the increase in the area of the surface membrane when fusions occurs. Lastly, membrane titling depends on the merging of the lipid hydrocarbon chains when there is the formation of the fusion stalk or fission stalk.

## Generating fusion by curvation

In general, protein-curvature generates fusion by identifying a lipid bilayer area at the top of the membrane. Then the protein would bend the membrane into cylindrical shapes. Proteins choose the area on top of the membrane because there would be "relaxation of elastic energy" when the fusion reaction occurs. Hence, there are two requirements for this fusion reaction to occur. First, the lipid bilayer area must be at the external area where fusion takes place. If it is not at the external area, that would stabilize curvature and results in the non-spontaneous reaction when there is no release of free energy at the end of the reaction. Second, the curvature must be large enough in order generate the fusion reaction. There are two mechanisms that are examples of this fusion reaction. The first mechanism is called the hydrophobic insertion or wedging mechanism. This reaction requires hydrophobic proteins to be inserted into the lipid bilayer chains. Hence, there would be the process of expanding the polar head of the monolayers membrane. As a result, the proteins would gather and interact with the SNARE complexes.Then Ca$^{2+}$ inside the hydrophobic proteins would be inserted into the lipid matrix and that would cause the bending of the lipid region. As a result, there would be changes in the shapes of the membranes such that it can be either cylindrical or conical belt that has protein-free end cap at its ends. Lastly, the fusion reaction will take place at these ends of the membranes when they release free energy during bending. The second mechanism is called the force transmission. Here, SNARE complex has syntaxin and synaptobrevin that are factors that allow the fusion reaction to take place. The SNARE complex will start at the N terminus of the lipid bilayer chain and merge the monolayer in the zipper like fashion. As a result, it would form the stable four-helix bundle which is called the core SNARE complex between the monolayer membranes.

## Generating fission by curvature

Proteins generate fission through the release of the free energy in the membrane neck. There are two mechanisms that illustrate this fission reaction. The first mechanism is called the scaffolding mechanism. It requires protein complexes to drive the membrane scaffolding. One of the examples of membrane scaffolding is its role in the release of enveloped viruses. As a result, scaffolding would lead to the bending of the membranes into cylindrical shapes. And that would results in the "relaxation of elastic energy". The second mechanism is called the hydrophobic insertion. Like fusion, hydrophobic insertion in fission also requires hydrophobic proteins to be inserted between the lipid matrix. For instance, the protein, BAR domain, would cause the bending of the lipid region. As a result, there would be changes in the shapes of the membranes which then drive the spontaneous reaction in fission

## Generating membrane remodeling by membrane tension

Tension inside the lipid bilayer membrane would cause the reaction of the membrane remodeling to occur. This is based on the process of protein scaffolding. And scaffolding would create a lipid bridge between the monolayers and result in the first stage of fusion which is called the hemifusion. Then there would be the formation of the fusion pore which link the distal monolayers and that would complete the remodeling process.
Endosomes in the cell have complexes that help sort cellular packages for transport. These are known as ESCRT. They have been found to have a role in pathology, by affecting cytokinesis, virus envelope budding, and vesicular biogenesis. Removing receptors from the cell membranes and delivering them to lysosomes is needed for controlling signaling in cells. The molecular machinery that sorts all the cellular packages in the endosomes has been greatly researched and progress has been made about its structure and basic function. Target receptors are ubiquitylated and through cell-mediate endocytosis, they are sent to an endosome. Epidermal Growth Factor (EGFR) is a receptor that is made to degrade, but others are recycled back to the membrane or to the endosomal-golgi bodies via tubules. The cellular packages that are assigned to be degraded are placed into vesicles and they bud into the lumen of endosomes, which go to the multivesicular bodies and then fuse further with lysosomes. The ESCRT complex is composed of 5 subunits, which are regulated by molecules sequentially to the endosomal membranes. However, the ESCRT assembly at the endosomes remains unclear. ESCRT protein complex is sent to the middle of the cell during cytokinesis by directly interacting with the phosphoprotein CEP55, which is a requirement for membrane fission.
Source: Divergent pathways lead to ESCRT-IIIcatalyzed membrane fission Suman Peel1, Pauline Macheboeuf2, Nicolas Martinelli2 and Winfried Weissenhorn2 1 Department of Biochemistry, School of Medical Sciences, University Walk, University of Bristol, Bristol BS8 1TD, UK 2Unit of Virus Host Cell Interactions (UVHCI) UMI 3265 Universite´ Joseph Fourier-EMBL-CNRS, 6 rue Jules Horowitz 38042 Grenoble, France

## Reference

Kozlov,Michael. McMahon, Harvey. Chernomordik, Leonid. Protein-driven membrane stresses in fusion and fission. PubMed. 11/15/12.
Mim, Carsten; Unger, Vinzenz M. 'Membrane curvature and its generation by BAR proteins'. Trends in biochemical sciences doi:10.1016/j.tibs.2012.09.001 (volume 37 issue 12 pp.526 - 533).