Biophysical Properties of Lipids and Dynamic Membranes

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region of the membrane; the electrostatic charge of anioniclipids mediates interactions with cationic regions of mem-

bilayer asymmetry is produced primarily in the trans-Golgi network (TGN), but also at the plasma membrane

Review TRENDS in Cell Biology Vol.xxx No.x

TICB-382; No of Pages 9brane-associated proteins; and specific interactions withselected lipids, such as polyphosphoinositides, are impor-tant for spatial organization of their protein ligands.

The chemical compositions of the two leaflets of the lipidbilayer are complex and very different from each other. Forexample, nearly all anionic lipids in eukaryotic cells facethe cytoplasm, whereas most lipids with large glycosylatedheadgroups are exposed to the extracellular environment.Differences in bilayer asymmetry between eukaryotic andprokaryotic membranes are essential for the activity ofendogenous antimicrobial factors that rupture bacterialmembranes but are harmless to eukaryotic cells [1]. Thechemical composition of the bilayer affects its mechanical

by several proteins that require ATP hydrolysis [46]. ATPbinding cassette (ABC) transporters seem to move (flop)phospholipids from the inner to the outer leaflet. Amino-phospholipid translocases that control movement andretrieval (flipping) of lipids to the inner leaflet includeP-type ATPases. Several candidate flippases have beenidentified in yeast, with various specificities for differentlipids [7]. One such ATPase, Drs2p, a transmembraneprotein implicated in protein transport from the TGN,localizes to the TGN and specifically translocates phospha-tidylserine (PS) but not other lipids. A third class ofproteins, scramblases, dissipate the transbilayer asymme-try [8]. These proteins, which are even less definitivelycharacterized than are aminophospholipid translocases,are generally found to be activated by increased intracel-lular Ca2+ levels but do not require ATP for activity.

Corresponding authors: Janmey, P.A. ([email protected]);Kinnunen, P.K.J. ([email protected]).

Available online xxxxxx.

www.sciencedirect.com 0962-8924/$ see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.tcb.2006.08.009

Please cite this article as: P.A. Janmey, P.K.J. Kinnunen, Biophysical properties of lipids and dynamic membranes, TRENDS in Cell Biology (2006), doi:10.1016/j.tcb.2006.08.009Special issue: Membrane Dynamics

Biophysical propertdynamic membraneP.A. Janmey1 and P.K.J. Kinnunen2

1 Institute for Medicine and Engineering, Departments of PhysioPhiladelphia, PA 19104, USA2Helsinki Biophysics & Biomembrane Group, Department of MFIN-00014 Helsinki, Finland

The lipid bilayer is a 3D assembly with a rich variety ofphysical features that modulate cell signaling and pro-tein function. Lateral and transverse forces within themembrane are significant and change rapidly as themembrane is bent or stretched and as new constituentsare added, removed or chemically modified. Recent stu-dies have revealed how differences in structure betweenthe two leaflets of the bilayer and between differentareas of the bilayer can interact togetherwithmembranedeformation to alter the activities of transmembranechannels and peripheral membrane binding proteins.Here, we highlight some recent reports that the physicalproperties of the membrane can help control thefunction of transmembrane proteins and the motor-dependent elongation of internal organelles, such asthe endoplasmic reticulum.

IntroductionThe lipid bilayer of a cell membrane might seem to be apassive film that blocks flow of water and solutes and inwhich the truly regulatory elements proteins areinserted.But thevarietyof lipidsand their controlled spatialorganization, which define the biophysical properties of themembrane, have anactive role in cell function. For example,the length and degree of saturation of the lipid acyl chainsdetermine the thickness and ordering of the hydrophobices of lipids ands

y, Physics, Bioengineering, University of Pennsylvania,

cal Chemistry, Institute of Biomedicine, University of Helsinki,

properties and, conversely, application of forces to themembrane can alter its chemical composition. Some essen-tial aspects of bilayer structure are summarized in Box 1.

It has long been recognized that mechanical forces canhave physiologically relevant effects on cells [2,3], andrecent studies begin to suggest how the lipid bilayer actsin concert with transmembrane and peripherally boundproteins to detect and respond to forces. A brief summary ofthe mechanical properties of lipid bilayers is shown inBoxes 2,3.

Here, we highlight a few areas of cell biology in whichthe physical properties of membranes, and not only che-mically specific lipidprotein interactions, have recentlybeen identified as essential for proper cell function andintracellular signaling. These examples include the role ofmembrane tension in gating of mechanically sensitivechannels, stress-activation of enzyme activity, and howthe biophysical properties of the membrane can effectmembrane bending and stretching.

Transverse lipid asymmetryThe bilayer in a typical eukaryotic cell has a thickness of5 nm and a continuous surface area of hundreds of squaremicrons, containing hundreds of different lipid types and>108 individual molecules. The types of lipids in the innerand outer leaflets are very different (see Box 1). The trans-

2 Review TRENDS in Cell Biology Vol.xxx No.x

TICB-382; No of Pages 9Box 1. Lipid asymmetry in eukaryotic cell membranes

Hundreds of different lipid species are present in the plasmamembrane [5,6,37]. Many of the rare but important signalingphospholipids, such as polyphosphoinositides, seem to be exclu-sively generated or delivered to the cytoplasmic face (inner leaflet) ofthe plasma membrane and to specific classes of internal membranes.By contrast, cholesterol accounts for a large fraction of both the innerand outer leaflets but seems to be more abundant in the outer leaflet(Figure I). This asymmetry is not strictly conserved, and different celltypes, organelles and cells at different states of activity are likely tochange the lipid distribution.Within the lipid bilayer, disorder is introduced by differences in

chain length and saturation of the hydrophobic chains in themembrane interior and the lateral distribution of different lipidswithin each leaflet, which can alter the biophysical properties ofLoss of trans-bilayer asymmetry to expose PS on theouter surface is often a sign of injury and leads to activationof blood coagulation or recognition by phagocytes of cellsundergoing apoptosis. Intriguingly, cancer cells and vas-cular endothelial cells in tumors also expose PS, causingincreased coagulation and thrombosis in cancer patients[8,9]. Acidic phospholipids on the outer membrane can alsoprovide an environment that is sufficiently different fromsurfaces composed of lipids with no net charge and cho-lesterol to trigger the formation of amyloid-type fibers byseveral proapoptotic, cytotoxic and antimicrobial proteinsand peptides [1012].

Whereas creating and maintaining trans-bilayer asym-metry requires ATP and essentially depends on specificproteins, the mechanism of scrambling the lipids is lessclear and might in some contexts occur by physical ratherthan specific biochemical actions in the membrane. Forexample, whereas the first identified scramblase proteinsrequire intracellular calcium increases for activity, somescrambling activities might function in concert with

Figure I. The lipids found in the plasma membrane. Abbreviations: PI, pho

PIP3, phosphatidylinositol (3,4,5)-trisphosphate.

Please cite this article as: P.A. Janmey, P.K.J. Kinnunen, Biophysical properties of lipids and

www.sciencedirect.comthe membrane. Whether lipids within each leaflet are randomlydistributed or organized into domains is a crucial, unresolved andcontentious issue in membrane biochemistry, with many implicationsfor cell signaling. Proteins also constitute 50% of the cross-sectionalarea of the membrane, and peripheral proteins interact with bothextracellularly and cytoplasmically directed lipids.One consequence of trans-bilayer asymmetry is sequestration of

acidic phospholipids away from the external face of the membrane,a feature that distinguishes eukaryotic lipid outer membranes fromprokaryotic ones, which are highly anionic. Other consequencesare likely to arise from differences in membrane bending andstretching moduli (see Box 2) of different lipid compositions andfrom differences in permeability to water and other smallmolecules.cholesterol, and other scrambling mechanisms can betriggered by factors that promote lateral sequestrationof inner leaflet polyphosphoinositides. For examplepolyamines [13] and a phosphoinositide-specific peptidebased on the phosphatidylinositol (4,5)-bisphosphate(PtdIns(4,5)P2)-regulatory site of gelsolin [14] bothstrongly promote exposure of PS at the outer leaflet with-out increasing cytosolic Ca2+ levels. A purely physicalmechanism for lipid scrambling has recently been pro-posed [15], based on changes in lymphocyte PS exposureduring changes in cell volume leading to changes in mem-brane lipid packing. In this mechanism, shown in Figure 1,imposition of membrane curvature dilates one leaflet whilecompressing the other depending on whether the curva-ture is concave or convex. Compression of inner leafletsrich in PS apposed to dilated outer leaflets lowers theactivation energy for spontaneous translocation (flopping)of PS and phosphatidylethanolamine (PE) to the outerleaflet, and compensatory flipping transitions of PC occurat regions of opposite curvature. This example illustrates

sphatidylinositol; PIP, phosphatidylinositol phosphate; PIP2, PtdIns(4,5)P2;

dynamic membranes, TRENDS in Cell Biology (2006), doi:10.1016/j.tcb.2006.08.009

Review TRENDS in Cell Biology Vol.xxx No.x 3

TICB-382; No of Pages 9Box 2. Forces controlling membrane shape

Membrane tension

The cell membrane tends to maintain a specific lipid packing densityand therefore an optimal surface pressure on the order of 30 mNm1.Increasing the lipid spacing by osmotic swelling, for example, isstrongly resisted, and leads to rupturewhen themembrane is strainedslightly above its optimal packing. Compression within the plane ofthe membrane would also be resisted, but the membrane buckles outof plane before significant compression occurs.

Spontaneous curvature and bending stiffnesshow physical features of the membrane, in this case theincrease in free energy of the membrane as one leaflet isstretched while the other is compressed, lead to selectivemovement of lipid molecules from one leaflet to the other toequalize lateral tensions without necessarily activatingany specific protein-based lipid translocation complex.

Lateral lipid asymmetryThe lipid bilayer is also heterogeneous laterally, withvarious descriptions of this asymmetry having been putforward as evidence of rafts or other domains [16]. Forma-tion of specialized domains in the inner leaflet of the

The default shape for most membrane constituents is not flat.Instead, each lipid shape that deviates from a cylinder contributes aspontaneous curvature to the membrane.Molecules that have an overall inverted conical shape, such as

detergent molecules, lysophospholipids and polyphosphoinosi-tides, form structures with a positive curvature, such as micelles(Figure Ia). Cylindrical-shaped lipid molecules, such as phosphati-dylcholine and sphingomyelin, preferentially form flat bilayerstructures (Figure Ib). Lipid molecules that have an overall conicalshape, such as diacylglycerol and PE, with a small hydrophilic cross-section, form structures with a negative curvature, such as theinverted hexagonal phase of tubes with headgroups inside andhydrophobic tails outside (Figure Ic). The local shape of a membranedepends on which lipids are present and on how they are spatiallydistributed. Insertion or removal of lipids into the inner or outerleaflet leads to area mismatches that also alter curvature.Membranes resist bending because changing local curvature

alters both the headgroup spacing and the entropy of the hydro-phobic chains. Bending stiffness is characterized by two bendingmoduli quantifying stiffness in the two orthogonal radii of curvaturepossible for a planar membrane. For an initially flat membrane in thexy plane, one bending direction can be visualized in the z directionalong the x-axis and the other in the z direction along the y-axis. Thebending stiffness is strongly dependent on the nature of the lipidsand their spatial distribution.

Figure I. Structures formed by different lipids: (a) inverted conical lipids, such

as detergent molecules, lysophospholipids and polyphosphoinositides; (b)

cylindrical-shaped lipids, such as phosphatidylcholine and sphingomyelin; (c)

conical lipids, such as diacylglycerol and PE.


www.sciencedirect.complasma membrane are hypothesized to be important inrecruitment of signaling complexes to sites where trans-membrane receptors are activated, and in establishingpolarity necessary for directed cell locomotion. In contrastto the broad consensus and clear evidence for the generalfeatures of transmembrane asymmetry, the nature andeven existence of physiologically relevant lateral mem-brane domains is still controversial [17,18]. This lack ofconsensus is surprising, because lateral segregation ofcholesterol-induced microdomains in sphingomyelinbilayers and other synthetic mixed lipid systems [1925]was demonstrated soon after development of the fluidmosaic model of lipid membranes [26], and models ofdomains based on studies on cellular membranes werereported not long after [21,2730].

Inpart the difficulty in demonstrating that domains existin cellmembranes, asopposedtomodelmembranes inwhichthe evidence is clear, is that domains in cells are too small tovisualize by existing methods, and manipulations that ren-der them large enough to visualize are open to the criticismthat the manipulation itself caused them to form. Unliketrans-bilayer lipid movements, which are slow in theabsence of perturbations and therefore relatively easy tomaintain, lateral movements of lipids within a leaflet arevery rapid, and domains can form and disappear on amillisecond timescale, allowing measurements by spectro-scopic methods but perhaps not direct visualization.Furthermore, stabilization of domains could be due asmuchto protein-lipid binding as to lipidlipid interactions [31].However, the insights gained from model systems thatshow, for example, the often dominant effect of cholesterolor lipidswith long chain fatty acids onmixing or segregationof lipids inmixed systemsmight suggest strategies bywhichto detect or manipulate lipids domains in vivo. Currentproblems in studying lipid domains in cellmembranes, withemphasis on the technical challenges that limit visualiza-tion of these small domains, the special role of cholesterol[32,33] and other lipids in domain formation, and in theconceptual challenges to relate equilibrium phase diagramsof pure systems to small, transient domains in the cell, havebeen discussed in several recent reviews [31,3436].

Lateral membrane pressures and the regulation ofintegral membrane proteinsThe conformation of amphiphilic molecules is a compro-mise of free energies of their hydrophobic and hydrophilicparts. In a lipid bilayer this compromise results in neitherthe hydrophobic nor hydrophilic part of the phospholipidbeing in the lowest energy configuration that it would takeif it were not tied to its chemically incompatible partner.The hydrophilic headgroups at the surface of the mem-brane are crowded together more tightly than they wouldbe if free in solution. This frustration is evident when aheadgroup such as Ins(1,4,5)P3 is liberated from the mem-brane by a phospholipase and diffuses into the cell interiorto activate its cytoplasmic targets. The small diacylglycerolremaining in the membrane after PtdIns(4,5)P2 hydrolysiscan alter the membrane structure, because its small head-group renders it unstable in a flat bilayer; removal of

PtdIns(4,5)P2 from the plasma membrane has recentlybeen shown to be necessary for proper secretory vesicle



TICB-382; No of Pages 9Box 3. Forces within the lipid bilayer

Transverse forces

Membrane thickness is determined by the hydrophobic length of thelipid. The optimal membrane thickness depends on the chain length,the degree of saturation and the angle of tilt within the membrane.The transition from a thicker to a thinner membrane generatespacking disorders that increase elastic energy. Transmembraneproteins also have a specified length of hydrophobic contour thatcan differ from the optimal hydrophobic thickness of the bilayer. Thishydrophobic mismatch can lead to stretching or compression of lipidsand proteins within the membrane (Figure I) or to tilting of trans-bilayer helices to decrease the hydrophobic height. Insertion ofdifferent lipids in an isolated domain and insertion of proteins canboth affect the thickness of the membrane.fusion, perhaps because of the membrane perturbationthat follows release of the large PtdIns(4,5)P2 headgroup[37]. By contrast, the hydrophobic acyl chains of phospho-lipids are generally stretched out more than they would bewithout their hydrophilic anchors. The end to end distanceof, for example, a 16 carbon chain in a dipalmitoyl phos-phatidyl choline bilayer is much longer than the end to enddistance of hexadecane in bulk, and the loss of entropy thatcomes from straightening out the chain results in a sig-nificant lateral pressure within the lipid bilayer that varieswith the depth into the bilayer (Box 1). Structural andtheoretical work have provided quantitative estimates forhowmuch different regions of the phospholipid acyl chainsdeviate from a random configuration [38], and this devia-tion results in a lateral pressure gradient throughout thelipid bilayer [39] that can affect membrane curvature orthe structure of transmembrane proteins.

Even though the bilayer as a whole might be stable,each part of it is highly stressed. In general the

Line tensionWhen a heterogeneous population of membrane lipids separate intodomains, the border between domains results in lipid packing thatis different from that inside and outside the domain, resulting fromsuch effects as the differences in height between the domains. Thedeformation of molecules at the domain boundary that occurs mainlyto prevent exposure of hydrophobic regions to water costs energy,

Figure I. Schematic representation of the 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. Reproduced with permission from Refs. [68,69].


www.sciencedirect.comand this energy per length of the boundary is called the line tension.The magnitude of the line tension, which is generally not measurabledirectly, contributes to the parameters that determine domain sizeand stability.

Lateral pressures

A pressure due to loss of chain entropy within the hydrophobicdomain creates compressive forces within the bilayer, the magnitudeof which depends on the distance into the center of the bilayer, thenature of the hydrophobic chains (e.g. saturated, unsaturated, singlechains or sterols) and the membrane curvature.A compression force acts at the hydrophilic interface to crowd the

headgroup close enough to minimize exposure of the hydrophobicchains to water. These lateral forces are present even if no externalforce is applied to the membrane. Because such forces resultingfrom, for example, osmotic stress, membrane bending, or pulling ontransmembrane proteins deform the membrane, the lateral forcesare also affected and therefore the structure of proteins inserted in thebilayer can change. See Figure II.hydrophobichydrophilic interface exerts interfacialtension, pulling the molecules together; this is due to thehydrophobic effect minimizing contacts of the hydrocarbonparts with the aqueous phase, which is balanced by thesteric repulsion between theheadgroupsandentropic repul-sion between the acyl chains in the monolayer leaflets,exerting lateral pressure that tends to compress proteinsembedded within. As the forces acting on the system areconfined to very narrow zones of only few Angstroms withinthe bilayer, the prevailing pressures can be sufficient toinfluence transmembrane protein structure. These lateralstresses, which depend sensitively on lipid composition,curvature, pH, divalent cations, drugs and binding to pro-teins, are increasingly considered in models for how trans-membrane channels and other proteins, especially thosethat respond to force, can alter their configurations whenthey are stimulated [40]. Similar conformational changescan also be produced by transverse forces that result fromhydrophobic mismatch [41], as illustrated in Box 3.

Figure II. (a) The forces that act within the bilayer. Black lines represent the

hydrophobic chains and blue dots the hydrophilic headgroup. (b) The

corresponding lateral pressure, p(z), at different distances (z) across the bilayer

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

through the interior, which are greatest near the interfaces. When the areas

under the curves add to zero, the membrane is globally at rest. The red

arrows show how a mismatch in the thickness of a transmembrane protein and

the lipid bilayer acyl chains moves the regions of high pressure up or down

along the z-axis, and the blue arrows show how bending the membrane alters

the pressure gradient within the bilayer. Adapted from [39,69].


hydrophobic thickness. In the resting (closed) state, theprotein is in mechanical equilibrium with both lateral andtransverse forces within the lipid bilayer.

These membrane forces can change in several ways. Forexample, if the bilayer is dilated as in a patch clampexerting suction pressure or perhaps in a cell undergoinghypo-osmotic swelling, the lateral pressure within thebilayer decreases, with a resulting increase in the lateralcross-section of the protein, leading to channel opening.Two recent studies suggest alternative means to activatethe channel [42,43]. If the membrane bends, or its lipidcomposition changes in one or the other leaflet so as to alterlateral pressures or hydrophobic height, the pressure pro-files will also change and the protein conformationresponds. If the channel were to tilt within the membrane,perhaps because of the imposition of force on the proteinsbut not directly on the lipid, the spatial relation betweenprotein and membrane would also change and potentially


TICB-382; No of Pages 9Figure 1. Physical model for transmembrane lipid scrambling. In a flat membrane,

lipid asymmetry is built into the inner and outer leaflets by the work of flippases

and other enzymes. In the oversimplified view here one the outer leaflet is all

phosphatidylcholine (PC, blue) and the inner leaflet all phosphatidylserine (PS,

pink). When the membrane is bent in a concave shape, the area per lipid of the

outer leaflet increases while the area per lipid in the inner leaflet decreases. The

energy imbalance of lipids too crowded together (red) on one side and too far

apart on the other (light blue) to satisfy the optimal packing constraints of the lipids

lowers the activation energy for trans-bilayer flipping and lipids in the crowded

leaflet move to occupy space in the dilated leaflet. In a typical protrusion there are

regions of positive and negative curvature on both leaflets, so PS will flip to theAn example of how interfacial forces contribute to chan-nel function is provided by two recent theoretical modelsthat consider the changes in free energy as the spatialrelationship between a membrane channel and the mem-brane in which it is embedded changes. As shown inFigure 2, a typical transmembrane channel, for examplea mechanosensitive ion channel such as a TRP channel,has an asymmetric profile within the hydrophobic part ofthe lipid bilayer, characterized by the angle u and ahydrophobic height W that is less than the bilayer

Figure 2. Cross section of a mechanosensitive transmembrane complex such as a chan

parameters: the radius R, of the folded polypeptide, the thicknessW of its hydrophobic

perpendicular to the membrane surface. The hydrophobic mismatch, 2U, is the differ

thickness, 2a. Changes in the lateral bilayer forces in each leaflet can alter the polypeptid

[40] will subject it to a different force profile within the bilayer that can alter the foldin

outside and PC will flip to the inside, leading to lipid scrambling.


www.sciencedirect.comactivate the channel. Similar scenarios can be envisionedfor other classes of transmembrane proteins to providemechanisms that alter transmembrane protein functioneven in the absence of direct activation by a specific che-mical ligand. The theoretical models are consistent withexperimental data [42,43], and future work will probablyenable unambiguous distinction between physical andchemical activation mechanisms. In particular, quantita-tive measurements of the forces exerted on transmem-brane proteins due to shear flow, osmotic stress or cell-cell interactions need to be compared with the forces thatare estimated from modeling studies to be required toproduce the requisite changes in protein structure.

The possibility that lateral pressures in the membraneowing to lipid packing can alter protein function and affectcellular signals, including the potency of anesthetic agents[44], is reinforced by recent findings that some peptideshypothesized to alter ion channel activity by binding theeukaryotic cell channel protein are also potent antibacter-ial peptides [45]. It is possible that these small peptideshave specific and distinct protein ligands on prokaryoticand eukaryotic cells, and the finding that stereoisomers ofsteroids [46,47] can have distinct effects on ion channels

nel in its closed configuration. The geometry of the protein is described by three

domain, and the angle u that the hydrophobic domain boundary makes with a line

ence between the hydrophobic protein thickness, W, and the bilayer equilibriume structure embedded within, and tilting of the protein [42] or moving it up or down

g of the polypeptide. Adapted from Ref. [43].



TICB-382; No of Pages 9suggests some degree of chemical specificity. However,unique protein ligands for many amphipathic compoundsthat affect cell function have not yet been identified, and aneffect elicited by amphipathic molecules on membranebiophysics is also plausible as an explanation for theirbiological activities.

Elasticity of the membraneThe cell membrane resists deformation, and the magni-tude of this resistance to forces applied in various direc-tions is characterized by several elastic constants thatcharacterize resistance to different geometries of deforma-tion: shear, bending and stretching, discussed above. Thesephysical properties depend on the chemical composition ofthe bilayer and on the lateral and transverse asymmetries,discussed below.

Shear deformation and viscous flowShear deformations within the plane of the fluid bilayerpresent in eukaryotic cells meet no elastic resistancebecause the lipids and the transmembrane proteins canflow past each other. An underlying protein mesh, such asthe spectrinactin network, endows the membrane withresistance to shear, and the composite of 2D protein net-work and lipid bilayer together determine the remarkableviscoelastic properties of erythrocytes and other cells [48].The lack of resistance to shear places limits on how forcescan be applied, for example by motor proteins at themembrane surface. For example, myosin 1 [49,50], kinesin[51,52] and other motor proteins have specific binding sitesfor phosphoinositides or other acidic lipids residing in theinner leaflet, suggesting that these lipids anchor them tothe plasma membrane or the surface of a vesicle. Such ananchor could suffice to transport a vesicle within thecytoplasm as the motor walks along its track of actin ormicrotubules, but it is less clear whether this mechanismcan be used to displace the plasma membrane with respectto the cytoskeleton. Without a shear elastic modulus, aresistance to static deformation and not only to flow, thelipid part of the membrane might allow movement ofthe cytoskeleton if the motor moved rapidly enough forthe viscous resistance to be significant, but a slow move-ment would result in passive flow of the lipid to which themotor is anchored, with no relative motion of the cytoske-leton. This scenario is changed if the anchoring lipid isbound within a larger structure or sequestered within arigid domain. Studies of the types of movement generatedbymembrane-localizedmotors with and without linkage totransmembrane proteins therefore have the potential todetermine whether domains of increased mechanical sta-bility can form within the lipid bilayer or whether con-trolled movements of the membrane require a proteinlattice to stabilize the membrane.

Membrane bendingEven without proteins, lipid bilayer membranes resiststretching and bending with elastic constants that arephysiologically relevant. On the other hand, many mem-brane phospholipids, especially those in the inner leaflet,

introduce a spontaneous curvature because they prefer topack into curved but not flat bilayers. In addition to the


www.sciencedirect.combilayer sheets that form the plasma membrane andsurround internal organelles, membrane lipids can forma wide variety of structures, many of which are found inbiology [53]. A striking feature of the chemical compositionof cell membranes is that many if not most of their lipidconstituents are, by themselves, unable to form planarbilayer membranes. Phosphatidylcholine (PC) and PSare the common constituents of the outer and inner leaf-lets, respectively of eukaryotic cell plasma membrane anddo form flat or gently curved planar membranes in vitro,but PE, cholesterol and other abundant cellular lipids, andimportant rarer lipids such as phosphoinositides, diacyl-glycerol, ceramides and lyosphospholipids, cannot formbilayers except when mixed with other lipids (see Box2). The presence of these lipids in planar membranesdestabilizes them, and indeed this destabilization seemsto be essential for the biological function ofmembranes andfor their ability to undergo vesicle budding, fusion andother shape transformations. Therefore, local accumula-tion of these lipids in specialized domains will havemechanical as well as biochemical consequences. Localiza-tion of acidic lipids into fluid domains, for example, cantrigger phospholipase A2 activity [54], with resultingdestabilization of membrane structure as lysophospholi-pids accumulate.

An increment in the internal pressure of the bilayerhydrocarbon region by lipids such as diacylglycerol or PEwith unsaturated chains increases the tendency for themembrane to curl, while remaining lamellar. Such a stateis called frustrated as these lipids increase the tendency ofthe membrane to adopt a negative curvature while thelamellar state remains relatively flat to accommodateother contributions to its total free energy. One of thekey enzymes in cellular signaling cascades, protein kinaseC, a peripheral membrane protein, can be activated by thismembrane stress [55] because the lipid packing around itssubstrate is altered to allow the kinase to access the site ofphosphorylation. As another example, a novel type of aperipheral lipidprotein interaction called extended-lipidanchorage, has been described for cytochrome c, in whichhigh internal pressure in the membrane hydrocarbonregion promotes movement of the acyl chain to the mem-brane surface and further into a hydrophobic cavity insidethis protein, thus establishing a hydrophobic lipidproteininteraction in the absence of intercalation of the proteininto the bilayer [56]. These studies demonstrate that theactivity of membrane proteins can be regulated (i) by directlipid-protein interactions, with specific lipids acting asallosteric effectors, and (ii) by lipids influencing the phy-sical state of the membrane. Obviously, these two mechan-isms are not mutually exclusive for a given lipid.

Active remodeling of lipid bilayersEnzymes acting on membrane lipids can have pronouncedconsequences not only in causing changes in the lateraldistribution of lipids but also in producing changes in the3D organization of membranes. For example, removal ofthe phosphocholine headgroup of sphingomyelin by sphin-gomyelinase (SMase) yields ceramide, a lipid with very

different physicochemical properties that can alter spon-taneous curvature and lateral packing. Although


the ER, the binding of peripheral membrane proteins thatalter surface tensions, or changes in internal pressure canall impact the rate and direction of tube extension, evenwithout a change in the number of bound or activatedmotor proteins. Conversely, themechanical work of pullingout a tube might change the lipid composition at distinctregions within the tube compared with that in the parentvesicle if the membrane contains a mix of lipids that prefercurved surfaces that occur at the tip and the base of theextended tube [63].

Tube formation and fission into vesicles in vivo can use arange of proteins that alter or sense changes in membranemechanical properties. For example, proteins containingBAR domains, which are crescent shaped structures thatinteract electrostatically with curved membranes, areimplicated in the formation of membrane tubes [59]. Mem-brane tube formation is also facilitated by proteins homo-logous to Schizosaccharomyces pombe Cdc15 (PCHproteins), which bind to PS and PtdIns(4,5)P2 to deformthe membrane [64]. Additional changes to membranestructure occur when twisting motions within membranetubules coupled with the GTPase activity of dynaminincrease membrane tension, leading to fission [65]. SomeBAR domain proteins also bind dynamin, suggesting that

Figure 3. Shape changes in a membrane vesicle pulled by molecular motors. (a) A

confocal side-view image of a fluorescently labeled lipid vesicle shows a long

membrane tube pulled out of the vesicle by kinesin motor proteins that bind to the

membrane by biotinavidin links and translocate along microtubules that are

firmly attached (but not visualized) to the bottom surface. (b) A schematic

representation of the geometry and attachment sites at the tip of the tube. (c) A

diagram of the hypothetical arrangement of motors at the tip of the tube. Note the

accumulation of multiple motors at the tip of the protrusion that occurs as motors

slow down when they reach the end of the tube. Adapted from Ref. [62].


TICB-382; No of Pages 9sphingomyelin in the absence of cholesterol is miscible inphosphatidylcholine, ceramide has a profound tendency forsegregation into microdomains, driven by intermolecularhydrogen bonding [57]. This results in tight packing andreduced trans-to-gauche isomerization of the hydrocarbonchains, which changes acyl chain packing and increasesbending rigidity. As ceramide has a tendency to promotethe formation of the inverted hexagonal phase (Figure Ic inBox 2), the domains enriched in ceramide form projectionswith high curvature. Experiments using microinjection ofSMase on the surface of giant vesicles composed of phos-phatidylcholine and sphingomyelin have demonstrated theformation of smaller vesicles emerging from the largervesicle, consistent with this model. More specifically for-mation of either endocytotic vesicles into the internalcavity of giant liposomes or shedding of vesicles from theouter surface of the substrate liposome following the actionof SMase on the external or internal leaflet of the giantliposome was seen [58]. In this model system, the stressesinduced on the initially relatively flatmembrane due to thehydrolytic activity of SMase are sufficient to get eitherinward or outward budding of new vesicles depending onwhich side of the vesicle the enzyme is delivered. Theseresults show how reorganization of cellular membranescan be driven without ATP, simply by inducing enzymati-cally a phase transition of themembrane lipid composite inone leaflet that causes structural changes to the bilayer.

Membrane stretchingLipid bilayers strongly resist stretching because increasingthe average distance between head groups increases expo-sure of the hydrophobic domain to water. Some mem-branes, such as the plasma membrane of leukocytes,have much greater surface area than needed to enclosethe cell volume and so deformation of the membrane doesnot lead to bilayer stretching. Deformation of cellularmembranes, such as the formation of tubular invagina-tions from the plasma membrane, can be controlled bychanges in lipid composition and by the binding of specificproteins, such as those containing BAR domains, which arethought to bind the membrane to produce large tubularstructures [59]. Some organelle membranes might be nearthe limit at which further deformation is elasticallyresisted, and this resistance can influence changes inshape. For example, the membrane of the endoplasmicreticulum is pulled into tubes by motors that run alongmicrotubules [60]. This process has recently been repro-duced in vitro with a minimal set of proteins and purifiedlipids [61,62]. As shown in Figure 3, thin tubes can bepulled out of a large vesicular reservoir that can mimic thetubes pulled out of the ER. An important finding of thisstudy is that the force needed to pull out a pure lipid tube ison the order of 50 pN and therefore requires coordinatedpulling by multiple motors. The resistance of the mem-brane to deformation can be sufficiently large to stall themotors, stop tube elongation, and in some cases lead toelastic recoil of the tube. Therefore, tube extension in vivocould be initiated by decreasing membrane tension as wellas by activating motors, and extension might be stopped

when membrane tension reaches a high value or when themotor is inactivated. Changes in the lipid composition of


www.sciencedirect.comthe effects of these two classes of proteins on membranecurvature and stability are coordinated in vivo [59].


35 mN m1. The best examples of proteins controlled by


TICB-382; No of Pages 9lateral pressure are the phospholipases A2. Specific typesof phospholipase A2 from different species have maximalenzymatic activities in vitro at specific values of lipidlateral packing that can be tightly controlled in lipidmonolayers using Langmuir balances. Extrapolating fromthis approach, it is evident that equilibrium lateral pres-sure and thus phospholipase A2 activity can be controlledby stretching of membranes by osmotic pressure gradients.More specifically, a pressure of 3335 mN m1 is too highfor some enzymes, preventing proper insertion of the pro-tein in the lipid bilayer. However, subjecting the mem-brane to tension (for example as a result of osmotic swellingthat decreases lateral pressure) lowers pe, enabling phos-pholipase A2 to bind and orient itself in the surface in themanner required for the expression of its catalytic activity[66]. Importantly, this example demonstrates a fundamen-tal role for the physical state of the membrane in control-ling the activity of a membrane protein: a mechanical forceis directly converted into a biochemical signal, and themembrane is thus acting as an osmotic response element[67].

ConclusionThe biophysical features of the cell membrane are increas-ingly recognized to be important control elements in cellsignaling and membrane protein function. We have dis-cussed here a few examples where physical effects can beas important as specific biochemical reactions in the func-tion of the cell membrane. However, a separation betweenphysical and chemical events in the membrane is subtle.Nearly any chemical change in the membrane caused bylipid hydrolysis, trafficking or sequestration in the mem-brane has a physical consequence, manifested, for exam-ple, as a change in pressure or curvature. Likewise,mechanical work done on the membrane to bend or expandit will cause redistribution of the hundreds of distinct lipidspecies that form the bilayer. It seems likely that physicaland chemical features have evolved together to form thecomplexity of interactions responsible for cell function.

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Biophysical properties of lipids and dynamic membranesIntroductionTransverse lipid asymmetryLateral lipid asymmetryLateral membrane pressures and the regulation of integral membrane proteinsElasticity of the membraneShear deformation and viscous flowMembrane bendingActive remodeling of lipid bilayersMembrane stretchingConclusionReferences

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Biophysical Properties of Lipids and Dynamic Membranes