Introduction and Goals
Cell membranes are an essential feature of any cell; they physically separate the cell from its environment, including other cells. In this tutorial we will discuss some of the functions that membranes carry out, and describe the macromolecules that comprise membranes and how they affect the general structure and properties of membranes. In addition, we will also discuss some experimental approaches used to study the properties of membranes.
By the end of this tutorial you should know:
- The general functions of membranes
- The properties of the lipid bilayer
- The composition and distribution of lipids in the bilayer
- How lipid fluidity is affected by lipid composition
- The types of proteins associated with the lipid bilayer
- How lipid and protein composition are determined
- How lipid and protein diffusion in the lipid bilayer are studied
Membrane Structure and Function
The roles of a biological membrane
Biological membranes vary in their precise composition, however, there are general activities and properties common to all membranes. Membranes play four general roles in the cell (Figure 1). First, and foremost, the membrane surrounding the cell, which is referred to as the plasma membrane, defines the boundary of the cell and acts as a permeability barrier that restricts the movement of substances in or out of the cell. In a eukaryotic cell, in addition to the plasma membrane, there also are membranes that define organelles such as the mitochondria and nucleus. Their membranes also act as permeability barriers, so the contents of the organelles cannot mix freely with the contents of the cytoplasm.
Second, membranes organize and compartmentalize specific activities within or around the cell. This is accomplished by their association with proteins, which have distinct activities with different organelle membranes or distinct regions of the plasma membrane. For example, there are specific enzymes embedded in the endoplasmic reticulum (ER) membrane that modify proteins by the addition of a polysaccharide.
Third, membranes regulate the transport of molecules in and out of the cell, and between organelles and the cytoplasm. This activity is regulated by specific proteins embedded in the membrane, which allow selective movement of ions, glucose, and other small molecules. The regulation of transport across a membrane will be discussed in subsequent tutorials on passive and active transport.
Fourth, the plasma membrane receives signals from the environment, including other cells. In most cases, these are extracellular signals in the form of small molecules or proteins that are detected by specific receptors embedded in the membrane, and result in a change in the cell. This process of receiving a chemical signal and transmitting it to the cell is referred to as signal transduction; this topic will be discussed in greater detail in subsequent tutorials.
Membranes are a Mosaic of Lipids and Proteins
The major components of a biological membrane are lipids and proteins (Figure 2). All membranes are organized as a lipid bilayer, with proteins embedded in, or associated with, the bilayer. Carbohydrates are also present, although much less abundant, and are found as sugars linked to either lipids or proteins in the membrane.
Lipid Composition of a Membrane
The three major types of lipids that form the lipid bilayer are phospholipids, glycolipids and sterols (Figure 3). The phospholipids (introduced in the tutorial entitled Properties of Macromolecules II) are the predominant type of lipid, and it is their amphipathic nature (having both polar heads and long-chain hydrocarbon tails) that causes them to form a bilayer spontaneously in water. The configuration of the lipids in the bilayer is tail-to-tail; the polar heads are exposed to water on the extracellular and intracellular sides of the membrane, and the long-chain fatty acids are shielded from the water. A few common phospholipids in mammalian cells are phosphatidylethanolamine, phosphatidylserine and phosphatidylcholine, which all contain unsaturated fatty acid chains. In the plasma membrane of animal cells, cholesterol is also a common component of the lipid bilayer.
Different membranes have different proportions of specific lipids. For instance, the plasma membrane of a liver cell has a much higher proportion of cholesterol than the membranes of the mitochondria; whereas, the membranes of the mitochondria have a higher proportion of phosphatidylethanolamine than the plasma membrane. In addition, there is asymmetry across the lipid bilayer (Figure 4). Glycolipids are found almost exclusively on the outer monolayer of the plasma membrane, and phosphatidylserines are generally restricted to the inner monolayer. Membrane asymmetry is established during membrane synthesis, and is maintained. Most lipids are arranged randomly throughout a single monolayer, and move freely throughout the monolayer. In the plasma membrane of cells, however, there can be small microdomains enriched for particular lipids, and often, membrane proteins as well. These so-called lipid rafts are rich in sphingolipids, which contain long-chain saturated fatty acids and cholesterol. The lipid raft has a bilayer that is thicker than the bulk of the membrane, and can lead to the accumulation of certain types of membrane proteins to the raft. Lipid rafts function to cluster the lipids and proteins used in signal transduction.
The lipid composition of any given membrane can be determined by thin-layer chromatography (TLC). This technique separates lipids in a mixture based on their differential affinity for a stationary phase and a moving phase. Lipids are extracted from isolated membranes with a mixture of organic solvents. This lipid mixture is then spotted onto a glass plate coated with the solid phase (usually silica). The plate is then dipped into a solvent that is a mixture of water and organic solutions. As the solvent travels up the plate, different lipids will migrate away from the spot of origin at different rates; polar lipids will travel more slowly and be found closer to the origin, and nonpolar lipids (e.g. cholesterol) will travel more rapidly and be found further from the origin (see animation).
The Lipid Bilayer is Fluid
The lipid bilayer is a dynamic structure. In a typical phospholipid bilayer, an individual lipid will change position with its neighbors in the same monolayer as often as 107 times per second. In addition, lipids can move a great distance within a monolayer, termed lateral diffusion. There is little exchange of lipids between monolayers, termed transverse diffusionor flip-flop. There are specific enzymes that catalyze the flip-flop of lipids from one monolayer to another, but this is usually tightly regulated.
The mobility of lipids within a biological membrane can be measured by fluorescence recovery after photobleaching (FRAP) (Figure 5). In this procedure lipids in the membrane are uniformly labeled with a fluorescent molecule and a laser beam is used to irradiate a small area of the membrane, bleaching out the fluorescence in that area. This results in a small, dark, nonfluorescent spot in the membrane. The membrane is then allowed to recover; very rapidly the dark spot disappears and the membrane is uniformly fluorescent again, indicating that there was lateral movement of labeled lipids into the area that had been irradiated.
Membrane fluidity is determined by several factors: temperature, the length of the fatty acid chains, the level of saturation of the fatty acid chains, and the presence of cholesterol. Artificial bilayers, composed of a single phospholipid, can be used to measure the transition temperature of that phospholipid. This is the temperature below which the lipid bilayer will gel (freeze) and above which it will become more fluid (melt). Shorter hydrocarbon chains and more double bonds increase membrane fluidity by decreasing the transition temperature. Cholesterol tends to make membranes less fluid at higher temperatures, but paradoxically, high concentrations of cholesterol make membranes more fluid at lower temperatures.
Protein Composition of a Membrane
The lipid bilayer provides the basic structure of a membrane, however, the proteins associated with the membrane carry out the specific functions of that membrane. For instance, the plasma membrane of a cell contains proteins that are receptors for extracellular signals and that transmit signals to the interior of the cell. The inner membranes of mitochondria contain distinct protein complexes that function in electron transport and ATP synthesis during oxidative phosphorylation. The diversity of proteins associated with different membranes is tremendous and depends on the type of cell and the type of membrane. There are, however, only a few ways that a protein can associate with the membrane. Figure 6 illustrates a typical plasma membrane. Integral membrane proteins (usually hydrophobic alpha helices) are partially embedded in the lipid bilayer. Transmembrane proteinsare a class of integral membrane proteins that traverse the lipid bilayer, either in a single pass or multiple passes. Proteins can associate with the lipid bilayer without being inserted into it directly. Lipid-anchored proteins have a covalently linked lipid that is inserted in one side of the lipid bilayer. On the inner side (the cytosolic side) of the bilayer, the protein is usually covalently linked to a fatty acid or prenyl. On the outer side (the extracellular side) of the bilayer, proteins are covalently linked to a glycosylphosphatidylinositol (GPI). Peripheral proteins are juxtaposed to the lipid bilayer but do not interact with it directly; rather, they bind to another membrane protein.
The organization of protein complexes in the lipid bilayer can be visualized by freeze-fracture electron microscopy. In this procedure, cells are rapidly frozen and fractured with a diamond knife. The fracture tends to run along the gap between the two monolayers of the lipid bilayer, splitting the lipid bilayer into two pieces. This exposes the interior of the outer monolayer (the E face) and the interior of the inner monolayer (the P face) (Figure 7). These are then shadowed with platinum and subjected to electron microscopy. If the proteins exposed in the two halves of the bilayer are of sufficient mass, they will be detected as particles studding the membrane and leave a gap in the opposing half of the lipid bilayer. This approach does not detect all proteins associated with the lipid bilayer, nor does it determine the composition of proteins in the lipid bilayer. In order to determine the protein composition of a membrane, the proteins must be dissociated from the membrane. The most common way of disrupting the lipid bilayer and solubilizing the proteins is through the use of detergents such as sodium dodecyl sulfate (SDS). Once membrane proteins are isolated from the lipid bilayer, they can be analyzed in a variety of ways.
Proteins Can be Separated by SDS PAGE
Perhaps the most widely used technique for analyzing protein composition is SDS polyacrylamide gel electrophoresis (SDS PAGE). SDS PAGE separates proteins based on their molecular weights (see animation). The proteins are subjected to an electrical field and they migrate toward the positive pole of the field at a rate that is proportional to the molecular weight of the protein. A mixture of proteins is mixed with the detergent SDS and heated, causing the proteins to become denatured and uniformly coated with SDS. As a result, all proteins acquire a net negative charge. The protein/SDS mixture is loaded onto a polyacrylamide gel and an electrical field is generated by a power supply attached to the gel. The negatively charged proteins will begin to move toward the positive pole of the electric field and migrate down the gel. The polyacrylamide gel is mesh-like, which restricts the migration of large polypeptides but allows the rapid migration of smaller polypeptides. Eventually the largest polypeptides will have moved only a short distance from the top of the gel and the smallest polypeptides will have moved toward the bottom of the gel. A stain is then used to visualize the proteins in the gel, which will appear as discrete bands of different intensities, reflecting different amounts of each protein. The molecular weight of any protein can be determined by its mobility relative to known molecular weight markers. Using this technique, one can determine the number of proteins associated with any particular membrane, identify individual proteins (based on their molecular weight), and determine the relative amounts of proteins.
Protein Mobility Varies in the Lipid Bilayer
Proteins, like lipids, are able to move laterally in the lipid bilayer, albeit not as rapidly. This was elegantly shown in an experiment using the fusion of human cells and mouse cells (Figure 8). These cells were induced to fuse by the presence of Sendai virus. Initially the hybrid cells showed asymmetric distribution of human and mouse plasma membrane proteins; within minutes, however, the proteins from the two different cells began to mix. After forty minutes, there was a complete intermingling of human proteins and mouse proteins, indicating mobility of proteins in the plasma membranes.
All proteins do not diffuse through the plasma membrane at the same rate. Some proteins are clustered in lipid rafts, or are associated with other proteins that may restrict diffusion. In addition, in some epithelial cells there are features of the plasma membrane, so-called tight junctions, that serve to create four domains within the plasma membrane and restrict protein movement.
The orientation of a protein relative to a lipid bilayer is fixed. Membrane proteins are inserted into the lipid bilayer during protein synthesis and there is no transverse diffusion. Some proteins may be located exclusively on the extracellular or intracellular sides of the bilayer, whereas others traverse the lipid bilayer and have regions on both sides of the bilayer. Plasma membrane proteins are frequently glycosylated on specific amino acids. These regions of the proteins are almost always on the extracellular side of the lipid bilayer, where they play an important role in cell recognition and cell adhesion.
Biological membranes have several general functions, including delineation of boundaries and permeability barriers, organization and localization of activities, regulation of transport, and signal detection. Membranes are composed of a lipid bilayer with proteins associated with, or embedded in, the bilayer. The most predominant lipids in the bilayer are the phospholipids and the glycolipids, which usually are distributed asymmetrically between the two sides of the bilayer, and sterols, such as cholesterol, which are distributed uniformly. Lipid composition can be determined by thin-layer chromatography (TLC), which separates lipids based on solvent affinity. The length and level of saturation of the hydrocarbon bonds of membrane lipids, and the amount of cholesterol, determine the fluidity of the membrane. Lipids diffuse laterally through the lipid bilayer very rapidly, but they rarely move transversely. The flip-flop of lipids is mediated and regulated by specific enzymes. The movement of lipids in the bilayer can be measured by fluorescence recovery after photobleaching (FRAP).
The diversity of proteins associated with a membrane is great. It is the proteins that carry out many of the functions of the membrane (e.g. transport, signal detection and many distinct enzymatic activities). There are several categories of membrane proteins, classified by their association with the lipid bilayer, including integral proteins, lipid-anchored proteins and peripheral proteins. The organization of proteins embedded in the lipid bilayer can be visualized with freeze-fracture electron microscopy. The protein complexity of a membrane can be determined by SDS polyacrylamide gel electrophoresis (SDS PAGE), which is an electrophoretic method of separating proteins based on their molecular weight. Some proteins in the bilayer can diffuse laterally but not reverse their orientation in the lipid bilayer. Movement of proteins in the plasma membrane was demonstrated by human/mouse cell fusion experiments.