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Biophysical properties of lipids and dynamic membranes

P.A. Janmey1 and P.K.J. Kinnunen2

1Institute for Medicine and Engineering, Depts. of Physiology, Physics, Bioengineering,

University of Pennsylvania, Philadelphia, PA 19104, janmey@mail.med.upenn.edu

2Helsinki Biophysics & Biomembrane Group, Department of Medical Chemistry,

Institute of Biomedicine, University of Helsinki, FIN-00014 Finland,

Paavo.Kinnunen@Helsinki.Fi

Abstract

The lipid bilayer is a three-dimensional assembly with a rich variety of physical features

that modulate cell signaling and protein function. Even though it is globally fluid, the

large number of different amphipathic molecules that make up the cell membrane have

different physical-chemical properties that have to compromise and coordinate within the

self-assembled bilayer they share. As a result, lateral and transverse forces within the

membrane are significant and rapidly change as the membrane is bent or stretched and as

new constituents are added, removed, or chemically modified. Many of these physical

effects have an impact on functions critical to cell structure and function.

Introduction

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The lipid bilayer of a cell membrane might appear to be a passive film that blocks flow of

water and solutes and in which the truly regulatory elements - proteins - are inserted. But

the variety of lipids, their controlled spatial organization, and many other data suggest

that they play an active role in cell function, and much of this role depends on the

biophysical properties of the membrane. The appearance of a self-assembled vesicle,

perhaps formed by isoprene derivatives 1 was a turning point in evolution because it

allowed macromolecules and other solutes to be enclosed, separated, and concentrated in

a small volume distinct from that of its surroundings. The more complex functions such

as selective entry or exit of solutes, and transmitting or transducing signals from the

outside to the inside of cells is generally ascribed primarily or entirely to proteins that use

the lipid bilayer only as an amphipathic film in which to insert. However, despite their

small size, there is evidence that lipids play many roles in cell biology, some of which

depend on their physical state.

Transverse lipid asymmetry

The bilayer in a typical eukaryotic cell has a thickness of 5 nm and can have a

continuous surface area of hundreds of square microns, containing >108 individual lipids.

The chemical compositions of the inner (facing the cytoplasm) and outer leaflets of the

plasma membrane and those of internal organelles, are very different. Significant

metabolic energy goes into maintaining this bilayer asymmetry, in part through the

function of ATP consuming aminophospholipid translocases 2 that retrieve

phosphatidylserine (PS) and other anionic phospholipids from the outer leaflet so that this

face of the cell contains almost exclusively zwitterionic phospholipids whereas the inner

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leaflet is highly enriched in acidic lipids. Loss of this asymmetry to expose PS on the

outer surface is often a sign of injury leading to activation of blood coagulation or of cell

apoptosis and can be triggered by numerous factors including agents that promote lateral

sequestration of inner leaflet polyphosphoinositides 3. Intriguingly, cancer cells and

vascular endothelial cells in tumors also expose PS, causing augmented coagulation and

thrombosis in cancer patients 4, 5. Acidic phospholipids create not only a high negative

surface charge density but also provide a highly acidic environment, with a surface pH of

approx. 5.2 estimated for a membrane containing 20 mol% of PS, for instance 6. Such

membranes provide an environment that is dramatically different from bilayers composed

of zwitterionic lipids and cholesterol, for instance, and have been shown to trigger the

formation of amyloid-type fibers by a number proapoptotic, cytotoxic, and antimicrobial

proteins and peptides 7-9.

Lateral lipid asymmetry

The lipid bilayer is also heterogeneous laterally, with various descriptions of this

asymmetry as evidence of rafts or other domains. The basis for lateral mixing/demixing

is in the lipid-lipid interactions, which are manifested in the so-called phase diagrams.

Along these lines the lateral segregation of cholesterol-induced microdomains in

sphingomyelin bilayers and other mixed lipid systems 10-16 was demonstrated already in

the early investigations soon after development of the fluid mosaic model of lipid

membranes 17, substantiating similar conclusions reached on the basis of studies on

cellular membranes 12, 18-21. An early model of a lipid membrane domain, now often

called a raft, shown in Figure 1, was based in part on differential changes in spectra

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obtained from membrane probes with preference for ordered or disordered phases caused

when saturated or unsaturated fatty acids, or other membrane active molecules were

added to the cell membrane 21. The importance of cholesterol and sphingolipids in

stabilizing outer leaflet domains and in the formation of detergent-insoluble lipid

fractions has also been demonstrated for lipid domain formation 22, 23. Studies comparing

cholesterol to its metabolic and probably evolutionary precursor lanosterol 24 point to the

unique enhanced ability of cholesterol to stabilize the so-called liquid ordered membrane

phase which is the basis for raft formation 25.

Current problems in studying lipid domains in cell membranes, with emphasis on the

technical challenges that limit visualization of these small domains and in the conceptual

challenges to relate equilibrium phase diagrams of pure systems to small, transient

domains in the cell, have been discussed in several recent reviews 26-28. A recent

advance is the use of atomic force microscopy to visualize domains of chemically

unmodified lipids in supported membranes because of the height difference between

domains 29, 30, but significant challenges remain to apply this technique to intact cells. A

crucial unknown in cell membrane domains is whether their formation is driven entirely

by the physical chemical principles that drive self-assembly of amphiphiles in water, or

whether transient chemical interactions - hydrogen bonding or specific ionic attractions -

also are important. The potential for specificity in domain formation of selected lipids

is suggested by models of cholesterol -phosphatidyl choline pair formation in the outer

leaflet 31 and hydrogen bonding networks possible for a small number of rare lipids

involved in signaling, such as phosphoinositides 32, 33 and phosphatidic acid 34, 35, in the

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inner leaflet. Differentiating specific lipid interactions from steric, hydrophobic, dipolar,

and electrostatic interactions common to them all may help resolve some of the

uncertainty whether, how, and when lipid domains form.

Lateral membrane pressures

The conformation of amphiphilic molecules is always a compromise of free energies of

their hydrophobic and hydrophilic parts. In a lipid bilayer this compromise results in

neither the hydrophobic nor hydrophilic part of the phospholipid being in the lowest

energy configuration it would take if it were not tied to its chemically incompatible

partner. The hydrophilic head groups at the surface of the membrane are crowded

together more tightly than they would be if free in solution. This frustration is evident

when a headgroup such as IP3 is liberated from the membrane by a phospholipase and

diffuses into the cell interior to activate its cytoplasmic targets. In contrast, the

hydrophobic acyl chains are generally stretched out more than they would be without

their hydrophilic anchors. The end to end distance of, for example, a 16 carbon chain in

a dipalmitoyl phosphatidyl choline bilayer is much longer than the end to end distance of

hexadecane in bulk, and the loss of entropy due to straightening out the chain results in a

significant lateral pressure within the lipid bilayer that varies with the depth into the

bilayer (see Box 1). Decades of structural and theoretical work have provided

quantitative estimates for how much different regions of the phospholipid acyl chains

deviate from a random configuration 36, and this deviation results in a lateral pressure

gradient throughout the lipid bilayer 37.

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Even though the bilayer as a whole may be in a stable state, or near equilibrium, each

part of it is highly stressed. In general the hydrophobic/hydrophilic interface exerts

interfacial tension, due to the hydrophobic effect minimizing contacts of the hydrocarbon

parts with the aqueous phase, which is balanced by the steric repulsion between the

headgroups and entropic intermolecular repulsion between the acyl chains in the

monolayer leaflets, exerting lateral pressure that would tend to compress proteins

embedded within. As the forces acting on the system are confined to very narrow zones

of only few Ångstroms within the bilayer the prevailing pressures can be ultimately very

high, reaching hundreds of atm. These lateral stresses, which depend sensitively on lipid

composition, curvature, pH, divalent cations, drugs, and binding to proteins is

increasingly considered in models for how transmembrane proteins, especially those that

respond to force, can alter their configurations when they are stimulated 38. A schematic

diagram showing how changes in lateral pressures activate or inactivate transmembrane

proteins through structural changes is shown in Figure 2. Similar conformational

changes can be produced by transverse forces due to hydrophobic mismatch 39, as

illustrated in Figure b.

Membrane stress and antimicrobial agents

The physical aspects of the cell membrane are increasingly recognized to be crucial for

understanding the function of antimicrobial peptides and other agents. Antimicrobial

peptides represent the first line of defense, innate immunity, against invading

microorganisms by all eukaryotic cells, from plants to human. They vary greatly in

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primary and secondary structure but have in common a net positive charge and

amphiphilicity, with a hydrophobic face that allows their partitioning into the interface of

cell membranes. Although the precise mechanisms of these agents are not known, they

are unlikely to require specific receptors in the bacteria they kill, since most of them have

very broad specificities, often killing Gram-positive and Gram-negative bacteria with

similar efficiency. Further, peptides composed of D-amino acids are equally effective as

the natural L-peptides 40. This apparent lack of specificity is likely to be important for

their function since attacks on the generic physical properties of the membrane cannot be

evaded by mutations that alter specific membrane components, and bacteria are much

less capable of developing resistance to antimicrobial peptides than to antibiotic agents

that attack specific bacterial proteins. Indeed antimicrobial agents need not be peptides,

and some organisms such as shark express cationic steroids such as squalamine 41 that

have antimicrobial efficiency and broad antibacterial spectra similar to those of cationic

peptides 42. One model to explain the strong but selective membrane destabilizing effects

of antimicrobial agents is shown in Figure 3. Cationic amphiphilic antimicrobial

molecules dock to the anionic surface of bacteria, changing the lateral pressure as the

structures at the hydrophilic-hydrophobic interface are altered. Eventually, the forces

stabilizing the interface are weakened until the bilayer loses integrity and allows

uncontrolled flow of solutes across the membrane.

The possibility that lateral pressures in the membrane due to lipid packing can alter

protein function and affect cellular signals including anesthetic effects 43 is reinforced by

recent findings that some peptides hypothesized to alter ion channel activity by binding

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the eukaryotic cell channel protein are also potent antibacterial peptides 44. While it is

possible that these small peptides have specific and distinct protein ligands on both

prokaryotic and eukaryotic cells, a common effect elicited by changes in membrane

biophysics is also plausible. On the other hand different biological effects of

stereoisomers of steroids 45, 46 suggest that separating bilayer effects from specific

binding to chiral elements in proteins will be complicated.

Elasticity of the membrane

The cell membrane resists deformation, and the magnitude of this resistance to forces

applied in various directions is characterized by several elastic constants.

Shear deformation and viscous flow

Shear deformations within the plane of the fluid bilayer, which is usually presumed to be

the primary or only phase present in eukaryotic cells, meet no elastic resistance since the

lipids and the transmembrane proteins can flow past each other. An underlying protein

mesh such as the spectrin-actin network endows the membrane with resistance to shear,

and the composite of 2-D protein network and lipid bilayer together determine the

remarkable viscoelastic properties of erythrocytes and other cells 47. The lack of

resistance to shear places limits on how forces can be applied by motor proteins at the

membrane surface. For example, myosin 1 48, 49, kinesin 50, 51, and other motor proteins

have specific binding sites for phosphoinositides or other acidic lipids suggesting that

these lipids may anchor them to the plasma membrane or the surface of a vesicle. Such

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an anchor may suffice to transport a vesicle within the cytoplasm as the motor walks

along its track, but it is less clear if this mechanism can be used to displace the plasma

membrane with respect to the cytoskeleton. Without a shear elastic modulus, the lipid

part of the membrane might provide resistance and therefore allow movement of the

cytoskeleton if the motor moved rapidly enough for the viscous resistance to be

significant, but a slow movement would result in passive flow of the lipid to which the

motor is anchored, with no relative motion of the cytoskeleton. This scenario is changed

if the anchoring lipid is bound within a larger structure or sequestered within a rigid

domain.

Membrane bending

Even without proteins, lipid bilayer membranes resist stretching and bending with elastic

constants that are physiologically relevant. Bending is characterized by two curvatures c1

and c2 , or equivalently radii of curvature R1= 1/c1 and R2 = 1/c2 in orthogonal directions.

These two curvatures define two quantities, a mean curvature H = (c1 + c2) / 2 and a

Gaussian curvature K = c1c2. The mean and Gaussian curvatures at each point of the

membrane define its topology, which in addition to lamellar membranes can include a

wide variety of forms, many of which are found in biology 52. A striking feature of the

chemical composition of cell membranes is that many if not most of its lipid constituents

are, by themselves, unable to form planar bilayer membranes. Phosphatidylcholine and

phosphatidylserine are the common constituents of the outer and inner leaflets,

respectively of eukaryotic cell plasma membrane and do form flat or gently curved planar

membranes in vitro, but phosphatidylethanolamine, cholesterol, and other abundant

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cellular lipids, and important rarer lipids such as phosphoinositides, diacylglycerol,

ceramides, and lysophospholipids cannot form bilayers except when mixed with other

lipids (see Figure a). The presence of these lipids in planar membranes destabilizes them,

and indeed this destabilization appears to be essential to the biological function of

membranes and for their ability to undergo vesicle budding, fusion, and other shape

transformations. Therefore, local accumulation of these lipids in specialized domains

will have mechanical as well as biochemical consequences.

An increment in the internal pressure of the bilayer hydrocarbon region by lipids such as

diacylglycerol or phosphatidylethanolamine with unsaturated chains increases the

tendency for the membrane to curl, while the membrane remains lamellar. Such a state is

called ‘frustrated’ as these lipids increase the membrane’s tendency to adopt a negative

curvature while the lamellar state remains. One of the key enzymes in cellular signaling

cascades, protein kinase C, a peripheral membrane protein, can be activated by this

membrane stress 53. As another example, a novel type of a peripheral lipid-protein

interaction, ‘extended-lipid anchorage’ has been described for cytochrome c, in which

high internal pressure in the membrane hydrocarbon region promotes movement of the

acyl chain to the membrane surface and further into a hydrophobic cavity inside this

protein, thus establishing a hydrophobic lipid-protein interaction in the absence of

intercalation of the protein into the bilayer 54. The above studies demonstrate that the

activity of membrane proteins can be regulated (i) by direct lipid-protein interactions,

with specific lipids acting as allosteric effectors, and (ii) by lipids influencing the

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physical state of the membrane. Obviously, these two mechanisms are not mutually

exclusive for a given lipid.

Membrane negative curvature also promotes membrane fusion. After two opposing

membranes are brought together by specific proteins overcoming barriers due to

undulations and steric protrusions, rapid fusion for PS containing bilayers can be

triggered by Ca2+, which causes dehydration of the headgroup of this phospholipid and

causes its subsequent effective shape to promote membrane negative spontaneous

curvature. These latter two factors are known to be critical determinants in the events

favoring the merging of adhering bilayers. Physical aspects of bilayer fusion have been

extensively reviewed elsewhere (e.g.55).

Enzymes acting on membrane lipids can have pronounced consequences not only in

causing changes in the lateral distribution of lipids but also in producing changes in the 3-

D organization of membranes. Along these lines removal of the phosphocholine

headgroup of sphingomyelin by sphingomyelinase (SMase) yields ceramide, a lipid with

very different physicochemical properties. While sphingomyelin in the absence of

cholesterol is miscible in phosphatidylcholine, ceramide has a profound tendency for

segregation into microdomains, driven by intermolecular hydrogen bonding 56. This

results in tight packing and reduced trans->gauche isomerization of the hydrocarbon

chains, with augmented bending rigidity. As ceramide has a tendency to promote the

formation of the inverted hexagonal phase, the domains enriched in ceramide form cusps.

Experiments using microinjection of SMase on the surface of giant vesicles composed of

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phosphatidylcholine and sphingomyelin have demonstrated the formation of smaller

vesicles consistent with this model. More specifically formation of either endocytotic

vesicles into the internal cavity of giant liposomes or shedding of vesicles from the outer

surface of the substrate liposome following the action of SMase on the external or

internal leaflet of the giant liposome was seen 56. These results show how reorganization

of cellular membranes can be driven without ATP, simply by inducing enzymatically a

phase transition of the membrane lipids.

Membrane stretching

Lipid bilayers strongly resist stretching because increasing the average distance between

head groups increases exposure of the hydrophobic domain to water. Some membranes,

such as the plasma membrane of leukocytes, have much greater surface area than needed

to enclose the cell volume and so deformation of the membrane does not lead to bilayer

stretching. However, internal organelle membranes are often near the limit where further

deformation is elastically resisted, and this resistance can influence shape changes. For

example, the membrane of the endoplasmic reticulum is pulled into tubes by motors that

run along microtubules 57. This process has recently been reproduced in vitro with a

minimal set of proteins and purified lipids 58, 59. As shown in Figure 4, thin tubes can be

pulled out of a large vesicular reservoir that may mimic the tubes pulled out of the ER.

An important finding of this study is that the force needed to pull out a pure lipid tube is

on the order of 50 pN, and therefore requires coordinate pulling by multiple motors. The

membrane resistance to deformation can be sufficiently large to stall the motors, stop

tube elongation, and in some cases lead to elastic recoil of the tube. Therefore,

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regulation of tube extension in vivo could be regulated by changes in membrane tension

as efficiently as by regulation of the motor proteins that pull on the tubes. Changes in

the lipid composition of the ER, the binding of peripheral membrane proteins that alter

surface tensions, or changes in internal pressure can all impact the rate and direction of

tube extension even without a change in the number of bound or activated motor proteins.

Conversely, the mechanical work of pulling out a tube changes the lipid composition

within the tube compared to that in the parent vesicle 60.

The lipid lateral packing also bears on the control of the activity of membrane proteins,

with several enzymes and mechanosensitive ion channels exhibiting an optimum of

membrane lateral packing density required for activity. The equilibrium lateral pressure

πe of biomembranes has been estimated at approximately 33-35 mN/m, which is already

inhibitory for some phospholipases, for instance. Subjecting the membrane to tension (for

example due to osmotic swelling decreasing lateral pressure) lowers πe, enabling

phospholipase A2 to exert catalytic activity 61. This example demonstrates a fundamental

role for the membrane physical state in controlling the activity of a peripheral membrane

protein, a mechanical force being directly converted into a biochemical signal,

membranes thus acting as an osmotic response element.

Conclusion

The biophysical features of the cell membrane are increasingly recognized to be

important control elements in cell signaling and membrane protein function. A few

examples where physical effects may be as important as specific biochemical reactions in

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the function of the cell membrane are discussed here. However, a separation between

physical and chemical events in the membrane is subtle. Nearly any chemical change in

the membrane caused by lipid hydrolysis, trafficking or sequestration in the membrane

has a physical consequence manifested, for example, as a change in pressure, or

curvature. Likewise, mechanical work done on the membrane to bend or expand it will

cause redistribution of the hundreds of distinct lipid species that form the bilayer. It

seems likely that physical and chemical features have evolved together to form the

complexity of interactions responsible for cell function.

Figure Legends

Figure 1 Organization of structurally distinct domains within the plasma membrane.

Lipids with saturated chains are hypothesized to preferentially partition into ordered

domains (darker regions) whereas lipids with unsaturated chains partition into disordered

domains. From ref 21.

Figure 2. Activation of a transmembrane protein by changes in bilayer lateral stresses.

A slice through a bilayer membrane containing an intrinsic protein is viewed in two

different conformational states, r and t. At right, the cross-sectional area profile A(z) of

each of the two states is plotted as a function of depth z within the membrane of thickness

h. Each conformation, for example, an open or closed channel, is in mechanical

equilibrium with the forces exerted by the membrane lipids. As these forces change, due

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to such effects as curvature, redistribution or enzymatic modification, the probability of

being in state r or s will also change. From ref 37.

Figure 3. Model of membrane destabilization by a cationic antimicrobial agent. Some

antimicrobial agents that do not appear to form specific pores, are hypothesized to

function by a “carpet” mechanism. A single cationic amphiphile with positive charges

denoted as small protruding circles docks electrostatically on the negatively charged

surface of the bacterium. Each such binding event alters the structure and local

electrostatic charge of the hydrophilic part of the membrane interface and may affect the

interfacial tension, but need not affect membrane permeability. Additional binding

events eventually lead to sufficient membrane destabilization that the bilayer integrity is

lost and the permeability barrier breaks down. From ref 62.

Figure 4. Shape changes in a membrane vesicle pulled by molecular motors. (A)

Confocal side-view image of a membrane tube pulled out of a lipid vesicle by kinesin

motor proteins bound to the membrane by biotin-avidin links and translocating along

microtubules at the bottom surface (B) Schematic representation of the geometry and

attachment sites at the tip of the tube. (C, D) molecular linkages and arrangement of

motors at the tip of the tube. From ref 59.

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References

1. Pereto, J., et al. (2004) Ancestral lipid biosynthesis and early membrane

evolution. Trends Biochem Sci 29, 469-477

2. Bevers, E.M., et al. (1999) Lipid translocation across the plasma membrane of

mammalian cells. Biochim Biophys Acta 1439, 317-330

3. Bucki, R., et al. (2000) Phosphatidylinositol 4,5-bisphosphate domain inducers

promote phospholipid transverse redistribution in biological membranes. Biochemistry

39, 5838-5844

4. Zwaal, R.F., et al. (2005) Surface exposure of phosphatidylserine in pathological

cells. Cell Mol Life Sci 62, 971-988

5. Utsugi, T., et al. (1991) Elevated expression of phosphatidylserine in the outer

membrane leaflet of human tumor cells and recognition by activated human blood

monocytes. Cancer Res 51, 3062-3066

6. McLaughlin, S. (1989) The electrostatic properties of membranes. Annu Rev

Biophys Biophys Chem 18, 113-136

7. Segrest, J.P., et al. (1976) Amyloid A: amphipathic helixes and lipid binding.

Biochemistry 15, 3187-3191

8. Zhao, H., et al. (2004) Formation of amyloid fibers triggered by

phosphatidylserine-containing membranes. Biochemistry 43, 10302-10307

9. Zhao, H., et al. (2005) Binding of endostatin to phosphatidylserine-containing

membranes and formation of amyloid-like fibers. Biochemistry 44, 2857-2863

17

10. Suurkuusk, J., et al. (1976) A calorimetric and fluorescent probe study of the gel-

liquid crystalline phase transition in small, single-lamellar

dipalmitoylphosphatidylcholine vesicles. Biochemistry 15, 1393-1401

11. Galla, H.J., and Sackmann, E. (1975) Chemically induced phase separation in

mixed vesicles containing phosphatidic acid. An optical study. J Am Chem Soc 97, 4114-

4120

12. Stier, A., and Sackmann, E. (1973) Spin labels as enzyme substrates.

Heterogeneous lipid distribution in liver microsomal membranes. Biochim Biophys Acta

311, 400-408

13. Shimshick, E.J., and McConnell, H.M. (1973) Lateral phase separations in binary

mixtures of cholesterol and phospholipids. Biochem Biophys Res Commun 53, 446-451

14. Shimshick, E.J., et al. (1973) Lateral phase separations in membranes. J Supramol

Struct 1, 285-294

15. Trauble, H., and Sackmann, E. (1972) Studies of the crystalline-liquid crystalline

phase transition of lipid model membranes. 3. Structure of a steroid-lecithin system

below and above the lipid-phase transition. J Am Chem Soc 94, 4499-4510

16. Sackmann, E., and Trauble, H. (1972) Studies of the crystalline-liquid crystalline

phase transition of lipid model membranes. I. Use of spin labels and optical probes as

indicators of the phase transition. J Am Chem Soc 94, 4482-4491

17. Singer, S.J., and Nicolson, G.L. (1972) The fluid mosaic model of the structure of

cell membranes. Science 175, 720-731

18. Klausner, R.D., et al. (1979) Ionophore A23187 disrupts membrane structure by

modifying protein-lipid interactions. Nature 281, 82-83

18

19. Karnovsky, M.J., et al. (1982) The concept of lipid domains in membranes. J Cell

Biol 94, 1-6

20. Karnovsky, M.J., et al. (1982) Lipid domains in membranes. Ann N Y Acad Sci

401, 61-75

21. Klausner, R.D., et al. (1980) Lipid domains in membranes. Evidence derived

from structural perturbations induced by free fatty acids and lifetime heterogeneity

analysis. J Biol Chem 255, 1286-1295

22. Zuckermann, M.J., et al. (2004) Modeling lipid-sterol bilayers: applications to

structural evolution, lateral diffusion, and rafts. Methods Enzymol 383, 198-229

23. Harder, T., and Simons, K. (1997) Caveolae, DIGs, and the dynamics of

sphingolipid-cholesterol microdomains. Curr Opin Cell Biol 9, 534-542

24. Bloch, K.E. (1979) Speculations on the evolution of sterol structure and function.

CRC Crit Rev Biochem 7, 1-5

25. Miao, L., et al. (2002) From lanosterol to cholesterol: structural evolution and

differential effects on lipid bilayers. Biophys J 82, 1429-1444

26. London, E. (2005) How principles of domain formation in model membranes may

explain ambiguities concerning lipid raft formation in cells. Biochim Biophys Acta 1746,

203-220

27. Kenworthy, A.K. (2005) Where do we go from here? Meeting Report on the

Biophysical Society Discussion on 'Probing Membrane Microdomains', October 28-31,

2004, Asilomar, CA, USA. Traffic 6, 518-523

28. Mukherjee, S., and Maxfield, F.R. (2004) Membrane domains. Annu Rev Cell Dev

Biol 20, 839-866

19

29. McQuaw, C.M., et al. (2005) Lateral heterogeneity of

dipalmitoylphosphatidylethanolamine-cholesterol Langmuir-Blodgett films investigated

with imaging time-of-flight secondary ion mass spectrometry and atomic force

microscopy. Langmuir 21, 807-813

30. Shaw, J.E., et al. (2006) Correlated fluorescence-atomic force microscopy of

membrane domains: structure of fluorescence probes determines lipid localization.

Biophys J 90, 2170-2178

31. McConnell, H. (2005) Complexes in ternary cholesterol-phospholipid mixtures.

Biophys J 88, L23-25

32. Redfern, D.A., and Gericke, A. (2005) pH-dependent domain formation in

phosphatidylinositol polyphosphate/phosphatidylcholine mixed vesicles. J Lipid Res 46,

504-515

33. Redfern, D.A., and Gericke, A. (2004) Domain formation in phosphatidylinositol

monophosphate/phosphatidylcholine mixed vesicles. Biophys J 86, 2980-2992

34. Kooijman, E.E., et al. (2005) What makes the bioactive lipids phosphatidic acid

and lysophosphatidic acid so special? Biochemistry 44, 17007-17015

35. Demel, R.A., et al. (1992) Monolayer characteristics and thermal behaviour of

phosphatidic acids. Chem Phys Lipids 60, 209-223

36. Sonne, J., et al. (2005) Methodological problems in pressure profile calculations

for lipid bilayers. J Chem Phys 122, 124903

37. Cantor, R. (1997) Lateral Pressures in Cell Membranes: A Mechanism for

Modulation of Protein Function. J. Phys. Chem. B 101, 1723-1725

20

38. Kung, C. (2005) A possible unifying principle for mechanosensation. Nature 436,

647-654

39. Sackmann, E. (1984) Physical basis for trigger processes and membrane

structures. In Biological Membranes (Chapman, D., ed), 105–143, Academic Press

40. Suchyna, T.M., et al. (2004) Bilayer-dependent inhibition of mechanosensitive

channels by neuroactive peptide enantiomers. Nature 430, 235-240

41. Wehrli, S.L., et al. (1993) Structure of the novel steroidal antibiotic squalamine

determined by two-dimensional NMR spectroscopy. Steroids 58, 370-378

42. Ding, B., et al. (2002) Correlation of the antibacterial activities of cationic peptide

antibiotics and cationic steroid antibiotics. J Med Chem 45, 663-669

43. Mohr, J.T., et al. (2005) Anesthetic potency of two novel synthetic polyhydric

alkanols longer than the n-alkanol cutoff: evidence for a bilayer-mediated mechanism of

anesthesia? J Med Chem 48, 4172-4176

44. Jung, H.J., et al. (2006) Lipid membrane interaction and antimicrobial activity of

GsMTx-4, an inhibitor of mechanosensitive channel. Biochem Biophys Res Commun 340,

633-638

45. Romanenko, V.G., et al. (2002) Modulation of endothelial inward-rectifier K+

current by optical isomers of cholesterol. Biophys J 83, 3211-3222

46. Li, W., et al. (2006) Enantiomers of neuroactive steroids support a specific

interaction with the GABA-C receptor as the mechanism of steroid action. Mol

Pharmacol

47. Discher, D.E. (2000) New insights into erythrocyte membrane organization and

microelasticity. Curr Opin Hematol 7, 117-122

21

48. Tang, N., et al. (2002) Dynamics of myo1c (myosin-ibeta) lipid binding and

dissociation. J Biol Chem 277, 42763-42768

49. Takeda, T., and Chang, F. (2005) Role of fission yeast myosin I in organization of

sterol-rich membrane domains. Curr Biol 15, 1331-1336

50. Klopfenstein, D.R., and Vale, R.D. (2004) The lipid binding pleckstrin homology

domain in UNC-104 kinesin is necessary for synaptic vesicle transport in Caenorhabditis

elegans. Mol Biol Cell 15, 3729-3739

51. Klopfenstein, D.R., et al. (2002) Role of phosphatidylinositol(4,5)bisphosphate

organization in membrane transport by the Unc104 kinesin motor. Cell 109, 347-358

52. Snedden, J., and Templer, R. (1995) Polymorphism of lipid-water systems. In

Structure and dynamics of membranes (Lipowsky, R., and Sackman, E., eds), 97-160,

Elsevier

53. Drobnies, A.E., et al. (2002) CTP:phosphocholine cytidylyltransferase and

protein kinase C recognize different physical features of membranes: differential

responses to an oxidized phosphatidylcholine. Biochim Biophys Acta 1564, 82-90

54. Tuominen, E.K., et al. (2002) Phospholipid-cytochrome c interaction: evidence

for the extended lipid anchorage. J Biol Chem 277, 8822-8826

55. Cevc, G., and Richardsen, H. (1999) Lipid vesicles and membrane fusion. Adv

Drug Deliv Rev 38, 207-232

56. Holopainen, J.M., et al. (1998) Sphingomyelinase induces lipid microdomain

formation in a fluid phosphatidylcholine/sphingomyelin membrane. Biochemistry 37,

17562-17570

22

57. Waterman-Storer, C.M., and Salmon, E.D. (1998) Endoplasmic reticulum

membrane tubules are distributed by microtubules in living cells using three distinct

mechanisms. Curr Biol 8, 798-806

58. Roux, A., et al. (2002) A minimal system allowing tubulation with molecular

motors pulling on giant liposomes. Proc Natl Acad Sci U S A 99, 5394-5399

59. Leduc, C., et al. (2004) Cooperative extraction of membrane nanotubes by

molecular motors. Proc Natl Acad Sci U S A 101, 17096-17101

60. Roux, A., et al. (2005) Role of curvature and phase transition in lipid sorting and

fission of membrane tubules. Embo J 24, 1537-1545

61. Lehtonen, J.Y., and Kinnunen, P.K. (1995) Phospholipase A2 as a

mechanosensor. Biophys J 68, 1888-1894

62. Savage, P.B., et al. (2002) Antibacterial properties of cationic steroid antibiotics.

FEMS Microbiol Lett 217, 1-7

63. van den Brink-van der Laan, E., et al. (2004) Nonbilayer lipids affect peripheral

and integral membrane proteins via changes in the lateral pressure profile. Biochim

Biophys Acta 1666, 275-288

64. Dumas, F., et al. (1999) Is the protein/lipid hydrophobic matching principle

relevant to membrane organization and functions? FEBS Lett 458, 271-277

65. Jensen, M.O., and Mouritsen, O.G. (2004) Lipids do influence protein function-

the hydrophobic matching hypothesis revisited. Biochim Biophys Acta 1666, 205-226

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Box 1. Forces acting within the cell membrane

Membrane tension

The cell membrane tends to maintain a specific lipid packing density, and therefore an

optimal surface pressure on the order of 40 mN/m. Increasing the lipid spacing by

osmotic swelling, for example, is strongly resisted, and leads to rupture when the

membrane is strained slightly above its optimal packing. Compression within the plane

of the membrane would also be resisted, but the membrane will buckle out of plane

before significant compression occurred.

Shear stiffness

Fluid bilayers have insignificant elastic resistance to shear deformation since the lipids

are not bound to each other. Viscous resistance of bulk lipid is apprroximately an order

of magnitude greater than that of the cytosol.

Curvature and Bending stiffness.

Membranes resist bending because changing local curvature alters both the headgroup

spacing and the entropy of the hydrophobic chains. Bending stiffness is characterized by

two bending moduli quantifying stiffness in the two orthogonal radii of curvature

possible for a planar membrane. The bending stiffness is strongly dependent on the

nature of the lipids and their spatial distribution. The default shape for most membrane

constituents is not flat. Instead, each lipid shape that deviates from a cylinder (see Fig

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a) contributes a spontaneous curvature to the membrane. Insertion or removal of lipids

into either the inner or outer leaflet leads to area mismatches that also alter curvature.

Figure a. Effect of lipid shape on membrane spontaneous curvature. (A) Molecules that

have an overall inverted conical shape, such as detergent molecules, lysophospholipids

and polyphosphoinositides form structures with a positive curvature, such as micelles.

(B) Cylindrical-shaped lipid molecules such as phosphatidyl choline and sphingomyelin

preferentially form flat bilayer structures. (C) Lipid molecules having an overall conical

shape, with a small hydrophilic cross-section form structures with a negative curvature.

The local shape of a membrane depends on which lipids are present and on how they are

spatially distributed. From 63

Electrostatic forces

Eukaryotic cells have no cationic lipids, and no strong electrostatic attractions between

lipids within the membrane. However, significant dipolar interactions occur between

zwitterionic lipids in which the positive and negative charges are well separated. In the

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inner leaflet of the plasma membrane, the presence of anionic lipids generates a

membrane potential, and in the absence of multivalent counterions, repulsive

electrostatic interactions within the plane of the bilayer occur. However, divalent metal

ions and multivalent ions including cationic proteins and polyamines can lead to

attractive interactions and lipid clustering.

Transverse forces

Membrane lipids have a limited hydrophobic length, and therefore a specified membrane

thickness. The optimal membrane thickness depends on the covalent chain length, the

degree of saturation, and the angle of tilt within the membrane. The transition from

higher to lower membrane profiles generates packing disorders that increase elastic

energy. Transmembrane proteins also have a specified length of hydrophobic contour

that may differ from the optimal hydrophobic thickness of the bilayer. This co-called

hydrophobic mismatch can lead to stretching or compression of both lipid and protein

within the membrane (see Figure b) or to tilting of transbilayer helixes to decrease the

hydrophobic height.

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Figure b. Schematic representation hydrophobic mismatch between a membrane protein

of hydrophobic length dP in a lipid bilayer in which the unperturbed hydrophobic

thickness dL is smaller (top) or larger (bottom) than dP. The influence of the protein,

extends over a certain distance from the protein surface and progressively vanishes so

that the bilayer recovers its unperturbed thickness dL. From 64, 65.

Line tension

When membrane lipids demix into domains, the border between domains results in lipid

packing that is different from that inside and outside the domain, resulting from such

effects as differences in height between the domains. The energy at the interface

contributes to the parameters that determine domain size and stability.

Lateral pressures

An extensional pressure due to loss of chain entropy within the hydrophobic domain

creates compressive forces within the bilayer, the magnitude of which depends on the

distance into the center of the bilayer, the nature of the hydrophobic chains (e.g.

saturated, unsaturated, single chains or sterols) and membrane curvature.

A compression force acts at the hydrophilic interface in order to crowd the headgroup

close enough to minimize exposure of the hydrophobic chains to water. See Fig c.

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Figure c. On the left are shown the forces that act on the lipid within the bilayer. Black

lines representing the hydrophobic chains and blue dots the hydrophilic headgroup. At

the right is shown the corresponding lateral pressure, π(z) at different distances (z) across

the bilayer thickness. Strong tensions at the interfaces are balanced by positive pressures

through the interior, largest near the interfaces. The areas under the curves add to zero,

and the membrane is globally at rest. The effects of hydrophobic mismatch due to

insertion of a transmembrane protein are largely to move the hydrophilic-hydrophobic

interface up or down, whereas the effect of curvature and chain unsaturation is to alter the

pressures within the membrane interior. Adapted from 37, 65.

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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