Cell membranes are made up of two lipid monolayers. In plasma and organelle membranes there is one monolayer facing the extracellular space or the lumen of the organelle, respectively, whereas the other monolayer usually faces the cytosol. Lipids, carbohydrates and peripheral proteins are present in different types and proportions when both monolayers are compared. Moreover, transmembrane proteins are placed in the membrane with a precise orientation. This unequal distribution of molecules between both monolayers is referred as membrane asymmetry, and was known even before the fluid mosaic model of membrane was proposed in 1972.
It is essential for the cell to generate and maintain membrane asymmetry. For example, in the plasma membrane, the outer monolayer contains most of the lipids having choline, such as phosphatidylcholine and sphingomyelin, whereas the inner monolayer contains more phosphatidylethanolamine, phosphatidylinositol and phosphatidylserine. This is useful for the cell because it sets an unequal electrical charges distribution at both sides of the membrane, which helps to generate the membrane potential. Furthermore, it allows the association of specific proteins with a particular surface of the membrane due to the distinct environment created by the electro-chemical properties of lipid heads, which are different between both sides. The location of some lipids in a particular monolayer is important in this respect. For example, phosphatidylinositol is mostly located in the inner monolayer of the plasma membrane and can be split into two molecules by some phospholipases; then one of the two molecules travels through the cytosol working as a second messenger. Furthermore, carbohydrates are located in the outer monolayer of the plasma membrane, contributing to the asymmetry. The membrane asymmetry also provides physical properties to membranes. For example, in the plasma membrane, it is thought that it is easier the formation of vesicles toward the cytosol, i.e. membrane invaginations are easier toward the cytosol.
Disorganization of the membrane asymmetry usually brings damages to the cell. For example, during apoptosis (programmed cell death), the outer monolayer of the plasma membrane exhibits lipids that are typical of the inner monolayer. These lipids are recognized by macrophages that finally remove the cell. There are proteins in the plasma membrane of healthy cells surveying for phosphatidylserine in the outer monolayer. When they find one, it is quickly returned to the inner monolayer. The lost of asymmetry is also important for blood coagulation. Some virus with a membrane envelope show phosphatidylserine in the outer surface so that they can be incorporated more easily by phagocytosis or macropinocytosis.
Although membrane asymmetry is largely related to the lipid distribution, there is a differential distribution of carbohydrates and proteins between the two monolayers of cell membranes. Carbohydrates are mostly present in the outer monolayer of the plasma membrane, forming the so-called glycocalyx, and in the luminal monolayer of endosomes and lysosomes. Carbohydrates work as recognition sites in the surface of the plasma membrane, and as a protection layer in the luminal surface of organelle membranes. Proteins also contribute to membrane asymmetry because they are precisely arranged in membranes. For example, transmembrane proteins in the plasma membrane are precisely oriented, with one cytosolic domain and one extracellular domain. This is very important for receptors that need to expose the recognition site to the extracellular space to recognize their ligands.
Where and how is asymmetry generated?
Lipids (Figure 1). The asymmetry of lipids is mainly generated in the Golgi apparatus, but also in other compartments. Curiously, in the endoplasmic reticulum, where most of the lipids are synthesized, there is a very similar distribution of lipids between both monolayers. It is hard for lipids with large heads to move from one monolayer to the other (known as "flip-flop" movement) because of the barrier created by the hydrophobic environment of fatty acid chains. However, lipids with smaller polar heads, like cholesterol, diacylglycerol, ceramide or protonated fatty acids, swaps between monolayers more frequently. Lipids with larger polar heads may save the hydrophobic barrier thanks to specific transporters, or translocases, found in membranes. There are three types of translocases: flippases, floppases and scramblases (Figure 2). These proteins move lipids between both monolayers, thus modifying the membrane asymmetry. Flippases move lipids to the inner monolayer (cytosolic), floppases to the outer monolayer (extracellular or luminal), and scramblases exchange lipids between both monolayers. Scramblases are the only one that does not need ATP. The asymmetry is maintained in the cell membranes because "flip-flop" movement is rare. For instance, sphingolipids are not exchanged between monolayers and remain in the monolayer where they were synthesized: luminal monolayer of the Golgi apparatus, which will be later the outer monolayer of the plasma membrane. More than 80 % of the plasma membrane sphingolipids are located in the outer monolayer.
Translocases are selectively distributed through the membranes of cell compartments providing a particular local asymmetry. Moreover, the distribution and activity of members of flippases and floppases families are also regulated. As mentioned previously, apoptosis changes the asymmetry of plasma membrane initiating phagocytosis. This break of membrane asymmetry is produced by the activation of a scramblase located in the plasma membrane. However, the scramblases located in the endoplasmic reticulum are always active and is responsible of the almost symmetry found in the reticulum membranes. In the erythrocyte membrane, there are scramblases that are activated after massive calcium entry and led to blood coagulation.
Carbohydrates are found in the outer monolayer of the plasma membrane and in the luminal surface of endosomal/lysosomal system. Carbohydrates are synthesized in the membranes of the endoplasmic reticulum and Golgi apparatus. In the endoplasmic reticulum, the initial steps of carbohydrates synthesis starts in the cytosolic surface, but is finished in the luminal surface. In the Golgi apparatus, all the synthesis processes are done in the luminal surface.
Proteins arrangement in the membrane is established during synthesis process in the endoplasmic reticulum. However, proteins associated to the cytosolic monolayer are synthesized in the cytosol by free ribosomes.
2. Fission and fusion
A very useful feature of membranes is their ability to fission and get fused again. It provides an extraordinary plasticity for remodeling membrane bound compartments, which means grow, division, fusion of the compartment, and be able of releasing and recruiting vesicles. For instance, it is indispensable for vesicle trafficking, and for cell division, particularly in cytokinesis, where plasma membrane increases in size, breaks, and get sealed again to form the two independent cells. Membrane fission and fusion are largely ruled by proteins, like SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor), but the physicochemical properties of lipids are also involved.
A number of natural processes and experimental conditions may lead to membrane breakages. For example, some tissues are under natural mechanical forces that compromise the plasma membrane integrity, as it usually happens in muscular tissues. Cardiomyocytes undergo frequent minor breakages of their plasma membranes that are repaired quickly. In the laboratory, cell cloning protocols need to introduce large glass micropipettes into the oocytes across the plasma membrane, electroporation of membranes is needed to introduce DNA or vectors, handling of cells in cultures produces mechanical loads that break cell membranes. Holes in the plasma membrane are lethal for the cell if they are not sealed in a few seconds. Cells have repairing mechanisms to avoid the loss of the internal content, and keep and maintain an internal environment different from the external one. In some tissues where cells do not proliferate, like the nervous tissue, replacing the damaged cells is not an option. Therefore, they must have effective membrane repairing mechanisms. There are two mechanisms for repairing membranes depending on the size of the damage.
When the breakages are small (smaller than 0.2 µm) (Figure 3), the physicochemical properties of lipids are able to repair the damage. At the edge of the membrane gap, lipids change to an unstable arrangement that pushes the edges against each other, eventually closing the hole. How quick is this sealing depends on the membrane tension, which in turn depends on the anchoring points between plasma membrane and both cytoskeleton and extracellular matrix. When a gap is made in the plasma membrane, calcium enters into the cytosol propelled by concentration gradient. This calcium increase in the cytosol partially disorganizes the cytoskeleton around the damaged area, decreasing the number of contacts between cytoskeleton and plasma membrane. There is less membrane tension, and, therefore, it is favored a faster sealing of the gap. ESCRT proteins, which are involved in the formation of intraluminal vesicles in the multivesicular bodies, are also involved in the repairing of smaller membrane breakages (less than 100 nm). In in vitro membrane models, it has been observed very small holes (a few nm) which are frequently produced consequence of the normal lipid movement by fluidity, and are quickly sealed by the lipids properties. This is not regarded as membrane damage and repair but a normal behavior of the membrane.
When damages are larger (more than 0.2 - 0.5 µm), the edges of the gap are too far from each other and no auto-sealing is possible. In this situation, a cellular response is initiated (Figure 4). The repairing process must last no more than a few seconds, because after that period cell dies. The large gap allows the entering of a huge amount of calcium that changes the cellular behavior. If the calcium concentration is not restored by sealing the breakage, apoptosis is activated and there is cytoplasm lost. It has been shown that sea urchin oocytes are not capable of repairing their membranes in calcium free environments, and therefore die. It is curious that neither sodium, potassium or chlorine ions do not participate in this repairing. The initial increase of calcium starts the repairing mechanisms, which include the fusion of membrane bound compartments located in the cell cortex with the plasma membrane, working like a patch. The compartments involved are endosomes, lysosomes, vesicles and other organelles. Lysosomes are thought to be important in this process. Endocytosis and endoplasmic reticulum disorganization would help forming more compartment used for the sealing. In addition, some proteins get oxidized and make bridges between vesicles near the gap, keeping close for patching. Calcium also activate enzymes that help in the sealing process by removing the cytoskeleton of the affected area and making easy the movement of organelles in the area. Actin filaments get organized like a ring around the borders of the gap. This ring gets smaller in diameter dragging the edges of the gap and new membrane to the center of the breakage. From an evolutionary point of view, it has been suggested that this repairing mechanism was later modified during evolution and used for fusion and fission of inner membranes and for vesicular trafficking.
The mechanism of fusion of inner compartments to form one large compartment before the fusion with the plasma membrane to repair the gap, suggested by McNeil, has not experimental support. This supposed inner large compartment has not been observed at electron microscopy and membrane action potential measurements fit best with a progressive fusion of many small inner compartments with the plasma membrane. Moreover, the sealing is faster in those cells with many membrane-bound organelles.
Cells and tissues show mechanisms to get adapted to repetitive mechanical stretching: extracellular matrix changes its molecular content, increase the number of adhesion molecules in plasma membranes, synthetize more intermediate filaments of the cytoskeleton, and increase the number and size of membrane-bound organelles. Cellular responses to mechanical stress can be studied in cell cultures. It has been observed that stretching of about 10 to 15 % increase the membrane surface by fusion of internal organelles. This usually happen in the urinary bladder epithelium, which frequently undergoes large stretching. If cells in cultures are stretched twice, a faster repairing process can be observed the second time when compared with the first one. Furthermore, the amount of vesicles released by Golgi apparatus is larger than usual, so that the cell may respond more efficiently when membranous compartments need to be fused between each other. The cell attachment to the extracellular matrix is also strengthened. The proeins of the cytoskeleton and extracellular matrix increase in number to withstand repetitive mechanical tensions.
Membrane repairing and cellular regeneration are not the same. Membrane repairing mechanisms are started after a membrane damage. Cell regeneration is a mechanism are started when a large part of the cell is lost, so that the damaged cell must recover, synthesize and assemble molecules a great part of the cytoplasm to get the normal state.
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