From the time cells were first discovered in the mid-1600s, scientists knew that there must be some sort of outer wrapping around the cell to hold the contents of the cell together. Although it was too thin for them to see with simple light microscropes, scientists called this outer wrapping a membrane (in Latin, membrana), which means a thin layer of skin or tissue. From the 17th century until around the 1960s, the outer membrane of cells was thought to be a simple passive barrier. We now understand that the plasma membrane is a very dynamic part of the cell and that is much more than just a barrier. Yes, it does restrict many molecules from entering (or leaving) the cell, but it is also designed so that some molecules can very quickly move through the membrane, and thus enter or leave the cell with ease.
Our scientific understanding of membranes began with the American statesman Benjamin Franklin. In 1774, Franklin observed the effects of oil on a surface of water and found that the oil does not mix with the water but rather spreads over the water’s surface to create a thin film:
I fetched out a cruet of oil and dropped a little of it on the water. I saw it spread itself with surprising swiftness upon the surface… Though not more than a teaspoonful, produced an instant calm over a space several yards square which spread amazingly and extended itself gradually till it reached the [other] side, making all that quarter of the pond, perhaps half an acre, as smooth as a looking glass.
More than a century later, in 1890, Lord Rayleigh repeated Franklin’s experiments while studying at Cambridge University in England. He and other scientists developed tools and mathematical methods for calculating the surface area covered by the oil film. Although these early studies didn’t directly focus on membranes or even cells, they were very important because they described the repulsion that occurs when water-insoluble fluids, such as oil, come in contact with water. It was this insight – that oil and water repel each other – that led scientists to wonder if the cell membrane might somehow be made of a substance that repels water. This way, it could keep fluids outside the cell from passing through, while also preventing the fluids inside the cell from leaking out. The fact that, when viewed under a microscope, animal cells look similar to spheres of oil helped to popularize the view that cells were somehow surrounded by an oily film.
Discovering membrane structure
It took several more decades before scientists came to understand the structural features of the membrane that allow it to repel water. This understanding came in three major steps. First, chemists observed that all known types of cells contain molecules called lipids that are hydrophobic, or water-insoluble. If cells are mostly water, how do they also contain water-insoluble things? Scientists then imagined that maybe a water-insoluble outer surrounding might be the answer. If the outer membrane was made of water-insoluble lipids, the membrane would restrict water and water-soluble molecules from passing through, while hydrophobic molecules (water-insoluble) could pass through the membrane. They had further evidence to back up this idea – oxygen gas is hydrophobic but can pass through cell membranes easily.
The second major advance came in 1931 with the invention of the electron microscope, which resolved a six-year debate in the scientific community. In 1924, two competing scientists came up with opposite conclusions about the structure of the membrane. A Danish-American scientist named Hugo Fricke performed calculations involving the surface area of those cells, and their capacity for electric charge. Based on these calculations, he found that the layer of lipids surrounding the cell is 3.3 mm thick (Fricke, 1924). Although his measurements were dramatically accurate, lack of understanding of the structure of lipids led him and others to the conclusion that the layer of lipids around the cell could only be one layer thick. Meanwhile, two Dutch scientists, Evert Gorter and François Grendel approached the question a different way. They extracted all of the lipids from a sample of red blood cells and allowed them to spread out on a watery surface, much like Ben Franklin had done with the oil. They found that when the lipids spread out as one layer, the area that they covered was almost exactly twice the surface of the red blood cells themselves (Gorter & Grendel, 1925). Thus, Gorter and Grendel concluded that the lipid surface surrounding the cells must be two layers. It turns out that the limited technology of the time led to two major errors in their work. First, they did not completely extract all of the lipids from the red blood cells. Second, they underestimated the surface of the red blood cell because they were unaware of its double-concave shape. However, the two mistakes acted to cancel each other out almost exactly and their conclusions were correct.
When the electron microscope was invented in 1931 by the German scientists Max Knoll and Ernst Ruska, two thin lines could easily be seen surrounding all cells (Knoll & Ruska, 1970). This was dramatic and convincing evidence that the membrane consists of a double layer of lipids. Even more dramatically, the electron microscope revealed that the cell membrane also had visible structures embedded in it (Figure 1).
The third advance in the understanding of membranes came when it was realized that the membrane is a “fluid” structure in which component molecules are in constant and rapid motion. Although several key measurements and experiments contributed to this breakthrough in our understanding, perhaps the most dramatic was a cell fusion experiment conducted by Larry Frye and Michael Edidin at Johns Hopkins University in 1970 (Frye & Edidin, 1970). For this clever experiment, the scientists grew human cells in one dish and mouse cells in another. They used a technique, brand new at the time, to attach a fluorescent labels to some of the proteins on the outside of cells. They labeled some of the proteins in the human cells with a fluorescent blue dye, while labeling the proteins on the mouse cells with a red dye. Then, they used a virus to trick the cells into fusing together. These hybrid cells that were half human, half mouse did not survive for very long, but they did live just long enough to show us something about membranes. At first, just after the cells had fused, all of the blue label was segregated on one half of the hybrid cell, while the red label was on the other half. However, very, quickly, the labels began to intermix with each other and within 40 minutes, the blue and red labels were evenly distributed throughout the surface of the hybrid cell (Figure 2).
The quick mixing of the fluorescent labels means that the proteins that are on the surface of the cell are not fixed in place – they can and do diffuse rapidly around the exterior of the cell, while still being embedded in the plasma membrane. This realization led to the development of the fluid-mosaic model of membrane structure, which was first fully articulated by S. J. Singer and Garth L. Nicolson in 1972 (Singer & Nicolson, 1972). Singer and Nicolson explained the plasma membrane as a bilayer, two layers of lipid molecules, with protein molecules embedded in the layers. They compared this to a mosaic of colored tiles that are inlaid to form a design or picture. However, in this case, the tiles are the molecules of lipid and protein, and they are not fixed in place – they move about through diffusion. Another way to imagine the surface of the membrane is to picture the surface of the ocean on a rough and windy day. The lipid molecules are like the ocean water and the proteins are bobbing around like “icebergs…floating in a sea of lipid” (Singer & Nicolson, 1972). See Figure 3 to see an illustration of the concept.
Amphipathic nature of cell membranes
Since 1972, we have learned a great deal about the molecular components of biological membranes and our current understanding of the very complex and dynamic nature of membranes is a far cry from the static film that was once imagined. By far, the most important structural feature of the membrane is the amphipathic nature of the lipids that make up the bulk of the membrane. It turns out that the lipids that comprise membranes are not purely hydrophobic. These special lipids have a charged phosphate group at one end which makes this region of the molecule water-soluble, or hydrophilic.
Thus, these phospholipid molecules have water-soluble head groups and water-insoluble tail groups, creating an amphipathic overall structure (Figure 4). Soaps and detergents are also amphipathic, which not only explains how they dissolve easily in water, but also how they dissolve oils and greases in water, the key to their effectiveness as cleaning agents.
The amphipathic nature of the phospholipid molecules is important because it explains how these molecules establish a two-layered membrane. Two rows of lipid molecules self-assemble in opposite orientations (Figure 5). The hydrophobic tail regions tuck together to create a water-free inner environment, and the hydrophilic head regions face outward where they are free to interact with water, the principle solvent both inside and outside of cells.
Types of molecules in cell membranes
But membranes are more than simple bilayers. The experiment by Frye and Edidin involved proteins that float in the plasma membrane. It turns out that the membrane has many different kinds of molecules floating in it, not just proteins. For example, most animal cell membranes contain cholesterol, a completely different kind of lipid. Cholesterol functions to regulate the fluidity of the membrane and also prevent freezing and cracking of the cell membrane at low temperatures. (That animal cells have cholesterol in their membranes but plant cells do not explains why all cholesterol in our diets come from animal products, not plant ones.) In addition, some lipid groups have the phosphate head group replaced by a carbohydrate group. These are called glycolipids. Similarly, some of the proteins that are in membranes also have carbohydrate groups attached to them and are called glycoproteins. Both glycolipids and glycoproteins are important “cell markers” used by cells to identify themselves to other cells.
Some proteins are fully integrated into the membrane and are called integral membrane proteins or transmembrane proteins, since they “span” both layers of the membrane. Transmembrane proteins are useful to the cell because they can interact with molecules on the outside of the cell and relay information about the extracellular environment to the interior of the cell. Other proteins are more loosely attached on the inside or outside of the membrane and are called peripheral membrane proteins. Peripheral membrane proteins are often used by the cell during signal transduction – the process by which a cell responds to a signal from another cell. In addition, while most proteins are free to float around the membrane as we saw with the hybrid cell experiment, some proteins are attached to part of the cytoskeleton and are thus anchored in one place. This anchoring can serve as a crucial structural component of the cell and its attachment to other cells or to the tissue matrix. Figure 6 below gives a more complete picture of the many kinds of molecules that are found in biological membranes.
As explained in our module The Discovery and Structure of Cells, the outer plasma membrane is not the only membrane in the cell. Many interior organelles have membranes as well, including the nucleus, mitochondrion, chloroplast, endoplasmic reticulum, Golgi body, lysosome and peroxisome. These membranes are all very similar. They all are composed of a sea of phospholipids with proteins and other components floating within. The main differences are that the specific phospholipids that make up the membranes are somewhat different and the floating components within the membranes are different. Each organelle, including the plasma membrane, has a unique signature of proteins floating in the phospholipid bilayer.
Now to the question of what the plasma membrane actually does. First and most obvious is that the plasma membrane is indeed a selective barrier. It allows the chemical activities inside the cell to proceed mostly undisturbed by events outside the cell. The famous cell biologist Gerald Weissmann emphasized the importance of this role:
In the beginning, there must have been a membrane! Whatever flash of lightning there was that organized purines, pyrimidines, and amino acids into macromolecules capable of reproducing themselves it would not have yielded cells [except] for the organizational trick afforded by the design of a membrane wrapping.
The lipid nature of the membrane allows it to serve as a good barrier. Lipids are water-insoluble and repel water, thus they are an ideal medium to separate the watery inside and outside of a cell. Anything that is water-soluble, even tiny single atoms such as H+ ions, will not easily pass through a lipid bilayer. However, water-insoluble molecules may pass freely; these include small molecules such as oxygen and carbon dioxide, and large water-insoluble hormones such as estrogen, testosterone, cortisol, thyroid hormone, and vitamin D. For these reasons, membranes are said to be semipermeable barriers. They do not let water or water-soluble molecules pass, but they do allow diffusion of water-insoluble (lipid soluble) molecules.
However, membranes are more than passive barriers. This is made clear by the many molecules that cannot pass through simple bilayers very quickly, but can pass into and out of cells. Water is the best example. As the understanding of membranes developed in the scientific community, a conundrum emerged. The phospholipid bilayer structure should not be very permeable to water, but when cells are studied in the laboratory, most are very permeable to water. How could this be? Scientists went so far as to build synthetic membranes using exactly the kinds and quantities of phospholipids found in specific types of cells. These synthetic membranes had very low water permeability, while the cells they modeled had very high water permeability. The hypothesis at the time was that there must be some sort of pore or channel in membranes through which water can pass, but all evidence for this was indirect. Channels for ions had been discovered, but the way that cells move water in and out remained a mystery.
This changed in 1992 when Peter Agre and colleagues reported their accidental discovery of channels called aquaporins (Preston et al., 1992). These channels are embedded in the plasma membrane and allow water to pass into and out of the cell (Figure 7). Agre and colleagues were not in the business of studying water transport. They were studying the Rhesus (Rh) factors that are present on red blood cells and result in blood incompatibility complications. In trying to isolate and purify these Rh factors, they noticed a “contaminant” in their test tubes – a membrane protein that they were not trying to study but which kept getting in the way. When they noticed that this protein is one of the most abundant proteins on the surface of the red blood cell, they decided to take a closer look and eventually realized that this “contaminant” was a protein that scientists had been looking for decades. Over the next few years, a whole family of related aquaporin proteins was discovered, and these proteins have a nearly identical structure in humans, fruit flies, fungi, and plants, indicating an ancient origin and strong conservation throughout more than a billion years of evolution.
Interestingly, a research group from Romania led by Gheorghe Benga had likely made this discovery at least six years before Agre, but they had not fully isolated nor identified the protein. Nevertheless, controversy has been raised over the issue of proper credit because Benga’s work almost certainly describes the same protein and had been published publically years before, both in a US journal and an international one. Nevertheless, Agre and colleagues did not to cite this work in their publications or Nobel Prize lectures, and most of the scientific community overlooked them as well. It should be noted that, working in an Eastern Bloc country as the collapse of the Soviet Union approached, Benga and his colleagues did not have the prestige or resources that Agre and his colleagues enjoyed at Johns Hopkins University. It is conceivable that, had Benga been working in a more internationally prestigious institution and/or with more financial resources, he may have shared the Nobel Prize in 2003.
The discovery of aquaporins highlights how proteins embedded in the plasma membrane can act as gatekeepers and govern the entry of molecules into and out of the cell. The membrane has many such gatekeepers and, like aquaporin, that are very specific. For example, aquaporin allows water molecules in and out freely, but other molecules much less so. Closely related molecules can pass through, but with much less efficiency (Figure 8). For example, urea, ammonia, and alcohol can each pass through aquaporins and indeed these channels are the main route through which these molecules are absorbed by most cells. However, they pass through more than a million times more slowly than water does. The structure of aquaporins reveals how they achieve this selectivity. Within the tunnel-like chamber through which water molecules pass, there are structural features that fit only a molecule with the size, shape, and partial-charge distribution that water has. Thus, while molecules similar in size and charge to water sometimes can pass through, they pass through at a much lower rate than water itself.
The examples of aquaporins and CFTR show how the plasma membrane can be selective about what enters and leaves the cell. As cell biologist Daniel Mazia put it:
The cell membrane is not a wall or a skin or a sieve. It is an active and responsive part of the cell; it decides what is inside and what is outside, and what the outside does to the inside.
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