Molecules are not static, but rather are dynamic. This also applies to molecular aggregates. Liposomes and bilayers in general must be somewhat dynamics, otherwise they might be impenetrable barriers across which nothing could pass. Cell membranes must separate the outside of a cell from the inside, but they must also allow passage of molecules and even ions across the membrane. What is the evidence that membranes are dynamic?

First, lipids can diffuse laterally in the membrane. This can be shown as follows. Make a liposome from phosphatidylethanolamine PE, which has been labeled with TNBS (Trinitrobenzensulfonate). The NH₂ on the head group of PE can attach the TNBS which undergoes nucleophilic aromatic substitution with the expulsion of the SO₃^-2. The TNB group absorbs UV light. Next, using a fluorescent microscope, the fluorescence intensity of a region of the surface can be recorded. Then shine a laser on a small area of the surface, which can photobleach the fluorescence in the area. Over time, fluorescence can be detected from the region again. The rate at which it returns is a measure of the lateral diffusion of the labeled lipids into the region. Lipids can undergo lateral diffusion at a rate of about 2 mm/s. This implies that the lipids can transit the surface of a bacteria in 1 sec.

Transverse, or flip-flop diffusion (movement of a phospholipid from one leaftlet to the other, not within a given leaflet) should be more difficult. Experimentally this is investigated as shown in the diagrams below.

Liposomes experiments:  To test flip-flop diffusion in an artificial membrane, liposome are made with a mixture of PC and a PC derivative with a nitroxide spin label (has a single unpaired electron). Both inner and outer leaflets of the membrane have the labeled PC. Like a proton in NMR spectroscopy, a single electron has a spin which can give rise to an electron-spin resonance (ESR) signal (as a proton gives rise to a nuclear magnetic resonance signal) when irradiated with the appropriate frequency electromagnetic radiation (microwave frequency for ESR, radio frequency for NMR) in the presence of a magnetic field. The liposomes are kept at 0^oC and the ESR signal is determined. Ascorbic acid, a water soluble vitamin and antioxidant/ reducing agent, is added to the liposomes. This reduces the spin labeled PC in the outer leaflet, but not the inner leaftlet of the bilayer since ascorbic acid can not enter the liposome or otherwise interact with it.  This reduces the ESR signal to a lower, constant value. The sample is divided into two. One sample is left at 0^oC, the other is raised to 30^oC. The ESR signal is recorded as a function of time. The 0^oC prep shows no change in ESR with time, while the 30^oC prep ESR signal decreases with time. This decrease must result from flip-flop diffusion of labeled PC from the inner leaflet to the outer, and subsequent reduction by ascorbic acid.  These experiments in experimental bilayer systems like liposomes shows that flip-flop diffusion is orders of magnitude slower than lateral diffusion.

Figure:  Flip/Flop diffusion in liposomes A: Making vesicles with ESR active PC analog only in outer leaflet

Figure:  Flip/Flop diffusion in liposomes B:   Raising the temperature to initiate flip/flopdiffusion

Cell Experiments:  An analogous experiment can be done with bacteria. Radiolabeled ³² PO₄ is added to cells for one minute, which leads to the labeling of newly synthesized phospholipid (PL) which locates to the inner leaflet. The cells are then split into two samples. One sample is reacted immediately  with TNBS, which will label only PE in the outer leaflet. The other sample is incubated 3 minutes (to allow PL synthesis) and then reacted  with TNBS. After a short labeling period, the cells are destroyed by adding organic solvents which prevents new lipids biosynthesis.  The lipids are extracted into the solvent and then subjected to TLC.

The lipids can be labeled in three  ways. Some will be labeled with ³² P alone, some with TNBS alone, and some with both ³²P and TNBS. TLC (or other techniques such as HPLC or GC) can easily separate PC and TNBS-labeled PC since they have different structures and hence will migrate to different places on a TLC plate.   No routine chromatography technique could, however, separate PC and ³² P-PC, since their molecular structure is the same, the only difference being in the nuclei of the P (different number of neutrons).  

Those lipids with double labels (TNB and ³² P) must have flipped from the inner leaftlet to the outer leaftlet where they could be labeled with TNBS. The cells incubated for 3 minutes before the addition of TNBS have a much higher level of doubly labeled PL's. Quantitating these data as a function of differing time of incubation at elevated temperatures show that the rate of flip-flop diffusion is much higher in cells than liposomes, which suggests that the process is catalyzed by an enzyme (flipase) in cells.

Figure:  Flip/Flop diffusion in bacterial cells A: Labeling inner leaflet phospholipids with ³² P

Figure:  Flip/Flop diffusion in bacterial cells B:   Labeling Cells in A with TNBS to detect Flip/Flop

Lipids in membranes are often distributed asymmetrically.  The inner and outer leaflet of a biological membrane usually have different PL compositions.  For example, in red blood cell membranes, the outer leaflet is composed mostly of sphingomylein (SM) and PC, while the inner leaflet it composed mostly of PE and phosphatidyl serine (PS).  This phospholipid contains the amino acid serine linked through its side chain (-CH₂OH) to phosphate in position 3 of diacylglyerol.   With a negative charge on the phosphate and carboxylate and a positive charge on the amine of PS, this phospholipid is acidic with a net negative charge.  In fact, all the PS is located in the inner leaftet!  This observation will become important latter on, when we discuss programmed cell death.  A dying cell will expose PS to the outside.  This is in fact one of the markers of a dying cell.

The membrane lipid composition in an average mammalian cell

In addition, lipids of a given type often cluster within a leaftlet to form lipid "rafts" which is analogous to a phase separation.  Divalent cations like calcium, which can bind to negatively charged PLs like PS, can cause "rafts" of PS to form, which is an example of lateral asymmetry within a leaftlet of a bilayer.  Rafts also appear to be enriched in cholesterol and lipids with saturated fatty acids, which would lead to regions of enhanced packing and reduced fluidity.  Rafts probably bind or exclude binding of other biological molecules like proteins.   Recent studies have shown that the Ebola virus interacts with lipid rafts in the process of entering and exiting the infected cell.   Rafts are also involved in how cells sense and respond to their environment.  Signaling molecules on the outside of the cell can bind receptor proteins in the membrane.  As we will see later, conformational changes in the receptor protein signals the inside of the cells that the receptor is bound with a ligand.  Once bound, the receptor can move in the membrane and often cluster in outer leaflet rafts that contain cholesterol and sphingholipids. Inner leaflet rafts have also been observed.  

Lipid Conformational Transitions

If a liposome preparation is placed in a sensitive calorimeter and the temperature slowly increased, it is observed that the liposome preparation absorbs a significant amount of heat at a temperature characteristic of the PL which compose the liposome. This is analogous to what would happen if solid water was heated. At the melting point of water, an increment of heat is required, the heat of fusion, to break H-bonds and cause melting. Likewise the heat of vaporization is observed when H-bonds are broken on the liquid-gas transition. These transition are associated with non-covalent processes, namely, breaking H-bonds. 

Figure:  Heat absorption and water: phase transitions

Figure:  Differential Scanning Calorimeter (DSC):  Phase transition for DPPC (Dipalmitoyl phosphatidylcholine)

The transition observed with liposomes is caused by conformational chains in the packing of the acyl chains of the PLs, as the acyl chains change  from trans to gauche conformations.  Before the transition, the acyl chains are more tightly packed in the gel phase, and after they are less tightly packed in the liquid crystalline phase, since many chains are in the gauche conformation. 

Differences in Gel and Liquid Crystalline Phases in Phospholipids Bilayers

The midpoint of the phase transition is called the melting point, Tm. Vesicles in the liquid crystalline phase are more fluid, dynamic, and hence more permeable. Note that the liposomes have not been destroyed but simply have undergone a phase change, much like ice turning to liquid water.   Liposomes made of different PL have different Tm. 

Vesicles make from PLs with bigger head groups have a lower Tm, since they are less "stable".  For example, the Tm for vesicles of  di-16:0 versions of PA, PE, and PC have Tm's of 67, 63, and 41 degrees C, respectively. Cholesterol is also a ubiquitous component of animal cell membranes. Its size will allow it to fit into either leaftlet with its polar OH pointed to the outside. One function of cholesterol in membranes is to keep the membrane fluid at any reasonable temperature. When a membrane is at a temperature less than the Tm, it is ordinarily in a gel, not liquid crystalline phase. The cholesterol helps prevent ordered packing of the acyl chains of the PL's, which increases their freedom of motion. Hence the fluidity and permeability of the membrane is increased. At temperatures greater than the Tm, the rigid ring of cholesterol reduces the freedom of the acyl chains to rotation, and hence decreases the number of chains in the gauche conformation. This decreases the fluidity and permeability.

Large Scale Conformational Transitions

Large scale conformational transitions can occur in lipid bilayers.  These include bud formation, formation of vesicles which split off from the membrane, and fusion of membrane vesicles, all of which are important biologically.  To some extent, the tendency to engage in these larger shape transitions depends on the local curvature of the vesicle, which depends on the local phospholipid composition and fluidity of the membrane.  We have previously seen that segregation of lipids into rafts (or domains) within a leaflet depends on the phospholipid content.  Baumgart et al. have made giant unilamellar vesicles (GUV) containing a mixture of three lipids: sphingomyelin (SM), dioleylphosphatidylcholine (DOPC), and cholesterol (C) to study transitions in the bilayers.  SM and C segregate laterally into a "liquid phase" domain characterized by significant order (Lo) while DOPC forms a "liquid phase" with more disorder (Ld).  They could detect the different phases through fluorescence microscopy of GUV's labeled with the fluorophores.  The first, N-lissamine rhodamine dipalmitoylphosphatidylethanolamine (red fluorescence), partitions almost exclusively into the Ld phase.  The second, perylene (blue fluorescence), partitioned with great selectivity into the Lo phase.  The results of their studies are shown in the figures below.  This images provide stunning visualization of lipid domains (rafts) including those in the shapes of circles, stripes, and rings.  Their works shows that the boundary between the domains is important in determining lipid structures.  Structures are favored which reduce the perimeter at the boundaries of the phases.  It appears that the Ld phase (and associated lipids) is found preferentially in area of high membrane curvature, while the Lo phase is found is regions of lower curvature.  the two figures below are reprinted with permission of Nature and the authors:   Baumgart,  Hess,  and Webb, W. Imaging coexisting fluid domains in biomembrane models coupling curvature and line tension.  Nature. 425, pg 821 (2003)

Figure:  Equatorial cross sections of GUVs with multiple phases

 Figure: GUVs structures with multiple phases

Membrane Permeability

Finally, one can measure the permeability of ions and molecules across the bilayer.  Permeabilities are correlated to the partition coefficient of the molecule or ion in organic solvents. If the molecule readily dissolves in a nonpolar solvent, it is more likely to pass through the hydrophobic barrier of the membrane. Obviously the size of the molecule would play a part as well. Hence charged ions like Na⁺ and K⁺ have low permeabilities.  Cl^- has a higher permeability, since the charge density on the larger Cl anion is smaller.   Water has a surprisingly high permeability although it is polar. It is small and can enter down deep into the head group region through sequential H-bonding which must assist its transfer across the membrane.

Figure: Permeability of liposome bilayers