The boundary that makes a cell possible was measured before anyone had seen it, using red blood cells, acetone and a shallow trough of water.
The previous lesson ended with the claim that a cell holds a composition different from its surroundings and must therefore have a boundary. This lesson asks what the boundary is made of, how thick it is, and how well it works. The last question turns out to matter most, because the answer is "far too well".
Why oil and water settle the question before any experiment
Charles Ernest Overton, working in Zurich in the 1890s, tested thousands of compounds for how readily they entered plant cells and found one predictor that beat everything else: how well the compound dissolved in olive oil. Substances that partition into oil crossed easily, substances that stay in water did not, and this held regardless of molecular size within the range he tested. Overton concluded in 1899 that the cell boundary is made of something oily, probably a lipid, and he was right thirty years before there was any direct evidence.
The reason a lipid boundary forms at all is not that lipids attract each other, which is the intuitive but wrong explanation. It is that water is strongly self-attracting, through hydrogen bonds, and a hydrocarbon chain sitting in water forces the surrounding molecules into an ordered cage that costs entropy. Push the hydrocarbons together and that ordered water is released. The driving force is the freed water, not the oil, which is why the effect is called the hydrophobic effect and why it gets stronger, not weaker, as temperature rises over the ordinary range.
The size of the effect can be read off a measurement. Phospholipids in water aggregate above a critical concentration of roughly M, and the free energy of moving one lipid from solution into an aggregate is , which at 310 K is kJ/mol. Compare a single-chain detergent such as sodium dodecyl sulfate, whose critical concentration is about 8 mM, giving kJ/mol. Two tails instead of one is worth roughly five times the binding energy, and that factor is the whole difference between a soap, which forms small micelles and dissolves membranes, and a phospholipid, which forms sheets and makes cells. At kJ/mol a lipid essentially never leaves the membrane it is in. The structure is not held together by anything; it is held together by water refusing to accommodate it.
Geometry decides sheet rather than sphere. A single-tailed detergent is a wedge, wide head and narrow tail, and wedges pack into a small sphere. A two-tailed phospholipid is closer to a cylinder, and cylinders pack into a flat sheet. A sheet has edges where hydrocarbon meets water, which is expensive, so a large enough sheet closes on itself into a sealed vesicle. A cell membrane is therefore self-assembling, self-sealing and self-repairing, none of which had to be designed.
Two molecules thick, measured in 1925
Evert Gorter and François Grendel, in Leiden in 1925, did the experiment that fixed the thickness. They took red blood cells from several mammals, counted them, extracted their lipids with acetone, and spread the extract as a single layer on the surface of water in a Langmuir trough, where a movable barrier compresses the film until it just becomes continuous. Reading the area of the film and dividing by the total surface area of the cells they had started with, they got a ratio close to two, and concluded that the membrane is a lipid layer two molecules thick.
The conclusion is correct and the experiment was flawed twice over. Acetone does not extract all membrane lipid, so their film was too small by roughly a third. And they estimated a red cell's surface area at about 99 µm², whereas the biconcave disc actually has about 145 µm², so their denominator was too small as well. Correct only the area and the ratio falls to , which would have suggested something other than a bilayer. Correct the extraction too, dividing the film area by 0.7, and it returns to . Two compensating errors gave the right answer, which is a good reason to be suspicious of clean results and a good reason not to dismiss them either.
The modern picture agrees. The hydrocarbon core of a bilayer is about 3 nm thick and the whole structure with head groups is 4 to 5 nm, so a membrane is roughly one thousandth the width of the cell it encloses. Each lipid occupies about 0.65 nm² of surface, so a red cell with 140 µm² of membrane and two leaflets carries lipid molecules, and the measured figure is close to that.
Example. Suppose Gorter and Grendel had used a modern lipid extraction, recovering everything, but had kept their 99 µm² figure for the cell area. What ratio would they have found, and what would they have concluded?
Their film area would have been µm² per cell, and dividing by the assumed 99 µm² gives 2.86. A ratio approaching three invites the conclusion that the membrane is three lipid layers thick, which is not a structure that makes chemical sense, since a middle layer would have to bury its head groups in hydrocarbon or its tails in water. The instructive part is what a careful reader should do with a puzzling ratio: check the denominator first. Cell surface area is the hardest quantity in the experiment to measure and the easiest to get wrong, because it depends on knowing the cell's shape rather than just its volume, and a biconcave disc has far more surface than the sphere of the same volume. The lipid film area, by contrast, is read directly off a calibrated trough.
Now you. Bacterial membranes contain no cholesterol, while an animal plasma membrane may be up to forty per cent cholesterol by molecule. Given that cholesterol is a rigid flat ring system with a single small hydroxyl group, what would you predict it does when inserted between phospholipids?
Answer
It should sit with its hydroxyl at the head group level and its rigid rings alongside the upper part of the fatty acid chains, and it should have two effects that sound contradictory and are not. Below the melting temperature of the lipids it gets in the way of orderly packing, so it keeps the membrane from freezing into a rigid gel; above it, the rigid ring restrains chain motion, so it stiffens a membrane that would otherwise be too fluid. Cholesterol therefore buffers fluidity against temperature rather than raising or lowering it, which is what a cell held at a fixed 37 degrees Celsius needs less than a cell that experiences weather. It also plugs the transient gaps between chains, and measured permeability to water and small solutes falls substantially when cholesterol is added. Bacteria solve the same problem differently, with hopanoids or by adjusting how many of their fatty acid chains are unsaturated, which is a real example of two lineages converging on a requirement rather than on a molecule.
A mosaic, and a fluid one
For thirty years after Gorter and Grendel the standing model was Danielli and Davson's 1935 sandwich: a lipid bilayer coated on both faces with sheets of protein. Electron microscopy in the late 1950s appeared to confirm it, since a thin section stained with osmium showed two dark lines with a light one between, and J. David Robertson generalised this "unit membrane" to every membrane in the cell.
It was wrong, and two lines of evidence killed it. Membrane proteins, once anyone managed to purify them, turned out to be largely hydrophobic on the outside, which is the wrong surface chemistry for something that lies on a membrane rather than in it. And freeze-fracture electron microscopy, which splits a frozen membrane between its two leaflets, showed the interior studded with particles 6 to 10 nm across, exactly what proteins crossing the bilayer would look like from inside.
The fluidity was demonstrated by Larry Frye and Michael Edidin in 1970 with an experiment of unusual clarity. They fused a mouse cell to a human cell to make a single hybrid, then labelled mouse surface proteins with an antibody carrying one fluorescent dye and human surface proteins with another. Immediately after fusion the hybrid was half red and half green. Within forty minutes at 37 degrees Celsius the two colours were completely intermixed over the whole surface. Nothing had been synthesised or destroyed in that time; the proteins had simply diffused.
Seymour Singer and Garth Nicolson assembled this into the fluid mosaic model in 1972: a two-dimensional lipid solvent in which proteins float, some spanning the bilayer, some anchored to one face. The lateral diffusion coefficient of a lipid is about 1 µm²/s, so with in two dimensions a lipid crosses a 2 µm bacterium in s. Flipping from one leaflet to the other is a different matter entirely, because it requires dragging a polar head group through the hydrocarbon core, and spontaneous flip-flop takes hours to days. That asymmetry is the reason the two leaflets can have different compositions and keep them.
They do. Phosphatidylserine, which carries a negative charge, is held almost entirely on the cytoplasmic face by dedicated flippase enzymes. When a cell dies by apoptosis the flippases stop and a scramblase runs, phosphatidylserine appears on the outer face, and macrophages recognise it and eat the cell. A lipid on the wrong side of a 5 nm sheet is a death certificate, which is a useful reminder that the membrane is not only a container.
The mosaic is dense. Protein makes up about half the mass of a typical plasma membrane, rises to 76 per cent in the inner mitochondrial membrane, which is a machine rather than a wall, and falls to 18 per cent in myelin, which is insulation and wants to be nothing but lipid.
Making more of it
A dividing cell has to double its membrane, and it cannot do so the way it doubles a protein. Lipids are synthesised by enzymes embedded in a membrane that already exists, using glycerol phosphate and activated fatty acids, and the product is inserted into the leaflet the enzyme faces. No cell builds a bilayer from nothing. Every membrane in every cell is an enlarged piece of a membrane that was there before, which is Virchow's principle from the first lesson applied to a structure rather than to a cell.
Two consequences follow. Because synthesis happens on one face, a flippase must move roughly half the new lipid across to keep the two leaflets growing together, which is the same machinery that maintains the asymmetry described above. And because a eukaryotic cell makes almost all its lipid in the endoplasmic reticulum, that lipid has to be distributed to every other compartment, which happens both by vesicles and at close contact sites where two membranes come within about 20 nm and lipids are handed across by transfer proteins.
Example. An E. coli cell is a rod 2 µm long and 0.8 µm across, and doubles every 20 minutes. Estimate how many lipid molecules per second its inner membrane requires.
The surface area is the cylinder plus the two caps, µm², which is nm². At 0.65 nm² per lipid in each of two leaflets that is lipids. Doubling in 1200 s therefore requires about lipid molecules a second, and a gram-negative bacterium with a second, outer membrane needs roughly twice that. The number is worth having because it puts membrane synthesis on the same footing as the protein synthesis rate derived in the previous lesson, around 2500 proteins a second: lipid assembly is a major continuous manufacturing activity rather than a trickle, and a cell devotes a serious fraction of its acetyl-CoA to it.
Now you. Some antibiotics, and the antifungal drugs of the azole class, target lipid synthesis rather than protein or DNA synthesis. Given the numbers above, why is that a reasonable target, and what determines whether such a drug can be selective?
Answer
It is reasonable because membrane synthesis is continuous, fast and essential: a cell that cannot make lipid cannot grow or divide, and unlike a metabolic enzyme there is no alternative route and nothing to scavenge from the medium in most cases. Selectivity depends entirely on whether the target enzyme differs between pathogen and host. The azoles work because fungi build ergosterol into their membranes while animals build cholesterol, and the enzymes making the two diverge enough for a small molecule to inhibit one; the same divergence explains why azoles nonetheless have real interactions with human steroid metabolism, since the enzyme families are related. Where no such divergence exists the approach fails, which is why there are few useful drugs against the shared early steps of fatty acid synthesis. The general rule for any antimicrobial target is not "is it essential" but "is it essential and different", and being essential alone guarantees only toxicity.
The problem this creates
Everything so far is good news. Now the bad news, which sets the agenda for the next lesson.
Measure how fast a substance crosses a pure lipid bilayer with no proteins in it, expressed as a permeability coefficient in centimetres per second, and the range is extraordinary. Water crosses at roughly , urea at , glucose at about , chloride at , potassium at , and sodium at around . From water to sodium is a factor of , eleven orders of magnitude, across the same 5 nm of material.
The reason is electrostatic. Moving an ion out of water, whose relative permittivity is about 80, into hydrocarbon, whose permittivity is about 2, strips away the shell of oriented water molecules that stabilises its charge. The Born model estimates the cost as
and putting in a sodium radius of 0.095 nm gives 356 kJ/mol. That is an overestimate, because the model treats the membrane as infinitely thick and ignores the ion polarising its surroundings, and more careful treatments give 150 to 200 kJ/mol. It does not matter which figure is used. The fraction of ions with enough thermal energy to cross goes as , and even the generous 150 kJ/mol gives .
So the membrane is not a filter with a preference. It is a wall, and the things it excludes most completely are exactly the ions the previous lesson said a cell must concentrate, along with sugars, amino acids, nucleotides and every phosphorylated intermediate of metabolism. A cell with a bare lipid bilayer around it would be sealed in and would starve.
Example. Two facts sit oddly together: a lipid bilayer is highly permeable to water, and water is a small polar molecule that hydrogen bonds strongly. Why does water cross when sodium, which is about the same size, does not?
Because the barrier is charge, not size or polarity. Water is neutral overall, so there is no Born energy to pay; the cost of moving it into hydrocarbon is only the loss of a few hydrogen bonds, worth some tens of kilojoules per mole in total, and a small fraction of molecules pay that at any moment. A sodium ion carries a full elementary charge, and the energy needed to strip its hydration shell scales as the square of that charge. The general rule for a bilayer is that permeability tracks the oil-water partition coefficient, which is Overton's 1899 result restated, and charge is what destroys a partition coefficient. This also predicts something checkable: an ion that can spread its charge over a large organic molecule should cross easily. Tetraphenylborate and similar hydrophobic ions do exactly that, and are used in the laboratory as artificial charge carriers precisely because they violate the pattern for the reason the theory says they should.
Now you. Some cells, including those lining the kidney tubule, move water far faster than a plain bilayer allows. Peter Agre found the reason in 1992 and shared a Nobel Prize for it. What kind of thing must it be, and what awkward requirement must it meet?
Answer
It must be a protein channel, since a bilayer's own permeability cannot be tuned by the cell but the number of proteins in it can. Agre's aquaporins are exactly that, and a single one passes about water molecules per second. The awkward requirement is selectivity in the hard direction: a pore wide enough for water is wide enough for a proton, and protons move through a hydrogen-bonded chain of water molecules by relay rather than by travelling, so a water-filled tube through a membrane would short out any proton gradient the cell was maintaining. The structure solves it by placing two asparagine residues at the pore's midpoint that force a passing water molecule to reorient, breaking the hydrogen-bonded chain and stopping the relay. Why that matters so much only becomes clear in the lesson on respiration, where the entire energy supply of the cell turns out to be stored as a proton gradient across a membrane.
What the boundary has bought and what it has cost
A bilayer is a remarkable piece of engineering that nobody engineered. It costs nothing to maintain, since water assembles it. It seals itself when punctured, because an edge is expensive. It is two dimensional, so anything embedded in it finds a partner far faster than it would in three dimensions. It is fluid enough to let a cell change shape, divide, and fuse vesicles with itself, and it holds its two faces distinct for days.
Against that, it is impermeable to almost everything of interest, which means every useful traffic across it has to be built as a separate protein, specified by a gene and paid for in energy. That is not a design flaw. It is the source of all the control a cell has. A wall with no doors keeps nothing out that matters, but a wall with doors that the cell opens and closes is the difference between a droplet and an organism.
The next lesson is the doors: the channels that let selected ions run downhill at a hundred million a second, the carriers that ferry sugars, and the pumps that push ions uphill and thereby build the gradients everything else will spend. It ends with a voltage across the membrane that can be calculated from measured concentrations, and with a bill, in ATP, that the lessons after it have to find a way to pay.