11.2 Membrane Dynamics in Chapter 11 Biological Membranes and Transport

11.2 Membrane Dynamics

One remarkable feature of all biological membranes is their plasticity — their ability to change shape without losing their integrity and becoming leaky. The bases for this property are the noncovalent interactions among lipids in the bilayer and the mobility allowed to individual lipids because they are not covalently anchored to one another. We turn now to the dynamics of membranes: the motions that occur and the transient structures that are allowed by these motions.

Acyl Groups in the Bilayer Interior Are Ordered to Varying Degrees

Although the lipid bilayer structure is stable, its individual phospholipid molecules have much freedom of motion (Fig. 11-17), depending on the temperature and the lipid composition. Below normal physiological temperatures, the lipids in a bilayer form a gel-like liquid-ordered $(L_{o})$ $left-parenthesis bold upper L Subscript bold o Baseline right-parenthesis$ state, in which all types of motion of individual lipid molecules are strongly constrained (Fig. 11-17a). Above physiological temperatures, individual hydrocarbon chains of fatty acids are in constant motion produced by rotation about the carbon–carbon bonds of the long acyl side chains and by lateral diffusion of individual lipid molecules in the plane of the bilayer. This is the liquid-disordered $(L_{d})$ $left-parenthesis bold upper L Subscript bold d Baseline right-parenthesis$ state (Fig. 11-17b). In the transition from the $L_{o}$ $upper L Subscript o$ state to the $L_{d}$ $upper L Subscript d$ state, the general shape and dimensions of the bilayer are maintained; what changes is the degree of motion (lateral and rotational) allowed to individual lipid molecules.

A two-part figure shows interconversion between bilayer lipids in a liquid-ordered state L subscript o, shown in part a, and a liquid-disordered state L subscript d, shown in part b. — FIGURE 11-17 Two extreme states of bilayer lipids. (a) In the liquid-ordered ( $L_{o}$ $upper L Subscript o$ ) state, polar head groups are uniformly arrayed at the surface, and the acyl chains are nearly motionless and packed with regular geometry. (b) In the liquid-disordered $(L_{d})$ $left-parenthesis upper L Subscript d Baseline right-parenthesis$ state, or fluid state, acyl chains undergo much thermal motion and have no regular organization. The state of membrane lipids in biological membranes is maintained somewhere between these extremes. [Information from H. Heller et al., *J. Phys. Chem.* 97:8343, 1993.]

FIGURE 11-17 Two extreme states of bilayer lipids. (a) In the liquid-ordered ( $L_{o}$ $upper L Subscript o$ ) state, polar head groups are uniformly arrayed at the surface, and the acyl chains are nearly motionless and packed with regular geometry. (b) In the liquid-disordered $(L_{d})$ $left-parenthesis upper L Subscript d Baseline right-parenthesis$ state, or fluid state, acyl chains undergo much thermal motion and have no regular organization. The state of membrane lipids in biological membranes is maintained somewhere between these extremes. [Information from H. Heller et al., *J. Phys. Chem.* 97:8343, 1993.]

At temperatures in the physiological range for a mammal (about 20 to 40 °C), long-chain saturated fatty acids (such as 16:0 and 18:0) tend to pack into an $L_{o}$ $upper L Subscript o$ gel phase, but the kinks in unsaturated fatty acids interfere with packing, favoring the $L_{d}$ $upper L Subscript d$ state (see Fig. 10-1). Shorter-chain fatty acyl groups are more mobile than longer-chain fatty acyl groups and thus also favor the $L_{d}$ $upper L Subscript d$ state. The sterol content of a membrane, which varies greatly with organism and organelle, is another important determinant of lipid state. Sterols (such as cholesterol) have paradoxical effects on bilayer fluidity: they interact with phospholipids containing unsaturated fatty acyl chains, compacting them and constraining their motion in bilayers. In contrast, their association with sphingolipids and phospholipids having long, saturated fatty acyl chains tends to make a bilayer fluid that would otherwise, without cholesterol, adopt the $L_{o}$ $upper L Subscript o$ state. In biological membranes composed of a variety of phospholipids and sphingolipids, cholesterol tends to associate with sphingolipids and to form regions in the $L_{o}$ $upper L Subscript o$ state surrounded by cholesterol-poor regions in the $L_{d}$ $upper L Subscript d$ state (see the discussion of membrane rafts below).

Transbilayer Movement of Lipids Requires Catalysis

At physiological temperatures, lateral diffusion in the plane of the bilayer is very rapid, but the movement of a lipid molecule from one leaflet of the bilayer to the other occurs very slowly, if at all, in most membranes (Fig. 11-18). Transbilayer — or “flip-flop” — movement requires that a polar or charged head group leave its aqueous environment and move into the hydrophobic interior of the bilayer, a process with a large, positive free-energy change. There are, however, situations in which such movement is essential. For example, in the ER, membrane glycerophospholipids are synthesized on the cytosolic face, whereas sphingolipids are synthesized or modified on the lumenal surface. To get from their site of synthesis to their eventual point of deposition, these lipids must undergo flip-flop diffusion.

A three-part figure shows three types of motion of single phospholipids in a bilayer. Part a shows uncatalyzed lateral diffusion, part b shows uncatalyzed transbilayer open parenthesis “flip-flop” close parenthesis diffusion, and part c shows catalyzed transbilayer translocations. — FIGURE 11-18 Motion of single phospholipids in a bilayer. (a) Lateral diffusion within a leaflet is very rapid, but (b) uncatalyzed movement from one leaflet to the other is very slow. (c) Three types of phospholipid translocators in the plasma membrane. PE is phosphatidylethanolamine; PS is phosphatidylserine.

Part a shows uncatalyzed lateral diffusion. A piece of plasma membrane has a red highlighted phospholipid in its top half, lined up with the surrounding phospholipids with its head above and two tails extending downward. The highlighted phospholipid has two phospholipids to its left and six phospholipids to its right. To its right, right- and left-pointing arrows show that it interconverts with an illustration to the right. Accompanying text reads, very fast open parenthesis 1 micrometer slash s close parenthesis. The right-hand illustration shows a similar piece of membrane but the highlighted phospholipid now has seven phospholipids to its left and one phospholipid to its right. Part b shows uncatalyzed transbilayer open parenthesis “flip-flop” close parenthesis diffusion. A piece of plasma membrane has a red highlighted phospholipid in its top half, lined up with the surrounding phospholipids with the head above and two tails extending downward. The highlighted phospholipid has four phospholipids to its left and four phospholipids to its right. An arrow curves clockwise from the highlighted phospholipid to point to the region beneath it. To its right, right- and left-pointing arrows show that it interconverts with an illustration to the right. Accompanying text reads, very slow open parenthesis t subscript one-half end subscript in days close parenthesis. The right-hand illustration shows a similar piece of membrane but the highlighted phospholipid now has its phosphate head below with the two tails extending upward and is lined up with the phospholipids in the bottom half of the membrane. The highlighted phospholipid still has four phospholipids to its left and four phospholipids to its right. Part c shows three catalyzed transbilayer translocations using three pieces of membrane, each with the region above labeled outside and the region below labeled inside. The left-hand catalyzed transbilayer translocation is labeled flippase open parenthesis P-type A T P ase close parenthesis moves P E and P S from outer to cytosolic leaflet. A piece of plasma membrane has a red highlighted phospholipid in its top half, lined up with the surrounding phospholipids with its head above and two tails extending downward. The highlighted phospholipid has two phospholipids to its left and three phospholipids to its right. N H 3 with a positive charge on N extends up from the spherical head of the red highlighted phospholipid. A vertical rectangle with rounded edges is positioned immediately to the right of the highlighted phospholipid and extends from above to below the membrane. A clockwise dashed arrow extends from the highlighted phospholipid, through the vertical rectangle, and back to point beneath the highlighted phospholipid. A curved arrow running through the bottom of the vertical rectangle shows that A T P is converted to A D P plus P subscript i. The middle catalyzed transbilayer translocation is labeled floppase open parenthesis P-type A B C transporter close parenthesis moves phospholipids from cytosolic to outer leaflet. A piece of plasma membrane has a red highlighted phospholipid in its bottom half, lined up with the surrounding phospholipids with its head below and two tails extending upward. The highlighted phospholipid has two phospholipids to its left and three phospholipids to its right. A vertical rectangle with rounded edges is positioned immediately to the right of the highlighted phospholipid and extends from above to below the membrane. A counterclockwise dashed arrow extends from the highlighted phospholipid, through the vertical rectangle, and back to point above the highlighted phospholipid. A curved arrow running through the bottom of the vertical rectangle shows that A T P is converted to A D P plus P subscript i. The right-hand catalyzed transbilayer translocation is labeled scramblase moves lipids in either direction, toward equilibrium. A piece of plasma membrane has a red highlighted phospholipid in its bottom half, lined up with the surrounding phospholipids with its head below and two tails extending upward. The highlighted phospholipid has two phospholipids to its left and three phospholipids to its right. A vertical rectangle with rounded edges is positioned immediately to the right of the highlighted phospholipid and extends from above to below the membrane. A counterclockwise dashed arrow extends from the highlighted phospholipid, through the vertical rectangle, and back to point above the highlighted phospholipid. On the right side of the vertical rectangle, there is a highlighted phospholipid in the top half of the membrane with two phospholipids to its right. A clockwise dashed arrow extends left from the upper highlighted phospholipid, curves through the vertical rectangle, and points beneath the highlighted phospholipid. The bottom of the vertical rectangle is labeled C a 2 plus.

Membrane proteins called flippases, floppases, and scramblases (Fig. 11-18c) facilitate the transbilayer movement (translocation) of individual lipid molecules. Like enzymes, these translocators act by providing a path that is energetically more favorable and therefore much faster than the uncatalyzed movement. The combination of asymmetric biosynthesis of membrane lipids, no uncatalyzed flip-flop diffusion, and the presence of selective, energy-dependent lipid translocators accounts for the transbilayer asymmetry in lipid composition discussed in Section 11.1.

Flippases catalyze translocation of the aminophospholipids phosphatidylethanolamine and phosphatidylserine from the extracellular to the cytoplasmic leaflet of the plasma membrane, contributing to the asymmetric distribution of phospholipids: phosphatidylethanolamine and phosphatidylserine primarily in the cytoplasmic leaflet, and the sphingolipids and phosphatidylcholine in the outer leaflet. Keeping phosphatidylserine out of the extracellular leaflet is important: its exposure on the outer surface triggers apoptosis (programmed cell death; see Chapter 12) and engulfment by macrophages that carry phosphatidylserine receptors. Flippases also act in the ER, where they move newly synthesized phospholipids from their site of synthesis in the cytosolic leaflet to the lumenal leaflet. Flippases consume about one ATP per molecule of phospholipid translocated, and they are structurally and functionally related to the P-type ATPases (active transporters) described in Section 11.3.

There are three other types of lipid-translocating activities: floppases, scramblases, and phosphatidylinositol transfer proteins. Floppases move plasma membrane phospholipids and sterols from the cytoplasmic leaflet to the extracellular leaflet and, like flippases, are ATP-dependent. Floppases are members of the ABC transporter family, described on page 395; all ABC transporters actively transport hydrophobic substrates outward across the plasma membrane. Each floppase specializes in movement of specific lipids: cholesterol, phosphatidylcholine, sphingomyelin, and phosphatidylserine. Scramblases are proteins that move any membrane phospholipid across the bilayer down its concentration gradient (from the leaflet where it has a higher concentration to the leaflet where it has a lower concentration); their activity is not dependent on ATP, although some require ${Ca}^{2 +}$ $Ca Superscript 2 plus$ . Scramblase activity leads to controlled randomization of the head-group composition on the two faces of the bilayer. The activity of some scramblases rises sharply with an increase in cytosolic ${Ca}^{2 +}$ $Ca Superscript 2 plus$ concentration, which may result from cell activation, cell injury, or apoptosis. Finally, a group of proteins that act primarily to move phosphatidylinositol lipids across lipid bilayers, the phosphatidylinositol transfer proteins, are believed to have important roles in lipid signaling and membrane trafficking.

Lipids and Proteins Diffuse Laterally in the Bilayer

Individual lipid molecules can move laterally in the plane of the membrane by changing places with neighboring lipid molecules; that is, they undergo Brownian movement within the bilayer (Fig. 11-18a). This rapid lateral diffusion in the plane of the bilayer tends to randomize the positions of individual molecules in a few seconds.

Lateral diffusion can be shown experimentally by attaching fluorescent probes to the head groups of lipids and using fluorescence microscopy to follow the probes over time (Fig. 11-19). In one technique, a small region $(5 μ m^{2})$ $left-parenthesis 5 mu m squared right-parenthesis$ of a cell surface with fluorescence-tagged lipids is bleached by intense laser radiation so that the irradiated patch no longer fluoresces when viewed with less-intense (nonbleaching) light in the fluorescence microscope. However, within milliseconds, the region recovers its fluorescence as unbleached lipid molecules diffuse into the bleached patch and bleached lipid molecules diffuse away from it. The rate of fluorescence recovery after photobleaching, or FRAP, is a measure of the rate of lateral diffusion of the lipids. Using the FRAP technique, researchers have shown that some membrane lipids diffuse laterally at rates of up to 1 μm/s. At this rate, a lipid molecule could move from one end of a eukaryotic cell to the other in a few seconds.

A figure shows how fluorescence recovery after photobleaching open parenthesis F R A P close parenthesis can be used to measure lateral diffusion rates of lipids in three steps. — FIGURE 11-19 Measurement of lateral diffusion rates of lipids by fluorescence recovery after photobleaching (FRAP). Lipids in the outer leaflet of the plasma membrane are labeled by reaction with a membrane-impermeant fluorescent probe (red) so that the surface is uniformly labeled when viewed with a fluorescence microscope. A small area is bleached with a laser, then recovers its fluorescence. With time, unbleached lipid molecules diffuse into the bleached region, and it again becomes fluorescent. The FRAP method can also be used to measure lateral diffusion of membrane proteins labeled with a fluorescent tag.

Step 1: A square labeled cell surface contains many, densely-packed red circles. Each phosphate head across the top layer of the lipid bilayer has a red dot attached above it. An arrow pointing down reads, intense laser beam bleaches small area. Step 2: The square is similar except that it has a white circle across most of its center. Wavy arrows point down at the phospholipid bilayer and that phosphate heads beneath them lack red circles. An arrow points down labeled, with time, unbleached phospholipids diffuse into bleached area. The square is similar except that there are now some red circles within the white circle. The white circle still contains fewer red circles than in step 1. The phospholipid bilayer now has red circles on some of the phosphate heads that lacked them in the previous step. Dotted arrows point from the left and right sides of the plasma membrane toward the center.

Another technique, single particle tracking, has allowed researchers to follow the movement of a single lipid molecule in the plasma membrane on a much shorter time scale. Results from these studies confirm that lipid molecules diffuse laterally with rapidity within small, discrete regions of the cell surface but that movement from one such region to a nearby region (“hop diffusion”) is rarer; membrane lipids behave as though corralled by fences that they can occasionally cross by hop diffusion (Fig. 11-20).

A figure shows how a fluorescently labeled lipid molecule moved with a start on the right from which it moves back and forth to fill in a purple circle. There is a blue circular region of lines to the left and slightly lower than the purple circle, then a narrower green band of lines that is circular at the top. There is a small orange circle above a small orange circle made of lines that lead to the point labeled finish. — FIGURE 11-20 Hop diffusion of individual lipid molecules. The motion of a single fluorescently labeled lipid molecule in a cell surface is recorded on video by fluorescence microscopy, with a time resolution of 25 µs (equivalent to 40,000 frames/s). The track shown here represents a molecule followed for 56 ms (2,250 frames); the trace begins in the purple area and continues through blue, green, and orange. The pattern of movement indicates rapid diffusion within a confined region (about 250 nm in diameter, shown by a single color), with occasional hops into an adjoining region. This finding suggests that the lipids are corralled by molecular fences that they occasionally jump. [Data from Takahiro Fujiwara, Ken Ritchie, Hideji Murakoshi, Ken Jacobson, and Akihiro Kusumi.]

Like membrane lipids, many membrane proteins are free to diffuse laterally in the plane of the bilayer and are in constant motion, as shown by the FRAP technique with fluorescence-tagged plasma membrane proteins. Some membrane proteins associate to form large aggregates (“patches”) on the surface of a cell or an organelle in which individual protein molecules do not move relative to one another; for example, acetylcholine receptors form dense, near-crystalline patches on neuronal plasma membranes at synapses. Other membrane proteins are anchored to internal structures that prevent their free diffusion. In the erythrocyte membrane, both glycophorin and the chloride-bicarbonate exchanger (p. 389) are tethered to spectrin, a filamentous cytoskeletal protein (Fig. 11-21). One possible explanation for the pattern of lateral diffusion of lipid molecules shown in Figure 11-20 is that membrane proteins immobilized by their association with spectrin form the “fences” that define the regions within which relatively unrestricted lipid motion can occur.

A figure shows the detailed structure and interaction of proteins in an erythrocyte plasma membrane, including chloride-bicarbonate exchange proteins, glycophorin, ankyrin, spectrin, and a junctional complex. — FIGURE 11-21 Restricted motion of the erythrocyte chloride-bicarbonate exchanger and glycophorin. The proteins span the membrane and are tethered to spectrin, a cytoskeletal protein, by another protein, ankyrin, limiting their lateral mobility. Ankyrin is anchored in the membrane by a covalently bound palmitoyl side chain (see Fig. 11-16). Spectrin, a long, filamentous protein, is cross-linked at junctional complexes containing actin. A network of cross-linked spectrin molecules attached to the cytoplasmic face of the plasma membrane stabilizes the membrane, making it resistant to deformation. This network of anchored membrane proteins may form the “corral” suggested by the experiment shown in Figure 11-20; the lipid tracks shown here are confined to different regions defined by the tethered membrane proteins. Occasionally a lipid molecule (green track) jumps from one corral to another (blue track), then another (red track).

The figure shows a piece of a cell that has been cut away so that the interior of the membrane is visible in a horizontal layer across the top and in a piece that curves around and back under on the right side and structures on the inside of the membrane are visible beneath. The membrane contains purple proteins labeled chloride-bicarbonate exchange proteins that make channels by forming crescents that curve away from the interior of the channel at the upper and lower right. When viewed on the outer or inner surface of the membrane, they form circular openings. The membrane also contains large proteins labeled glycophorin that have a large spherical portion above the membrane, outside of the cell that connects to a narrow piece that runs through the membrane to connect to a smaller spherical portion that extends into the cell. There are threads labeled spectrin made up of two intertwined strands that run along the interior of the membrane and encounter half-circle molecules of ankyrin that are sometimes paired, with one on each side of spectrin. Some pieces of spectrin join with rectangular structures with rounded ends labeled junctional complex open parenthesis actin close parenthesis. Spectrin separates the underside of the membrane into regions. A green back and forth line is labeled path of single lipid molecule and weaves back and forth to fill much of one of these open regions bounded by spectrin. The end of this green line crosses a spectrin strand and reaches another region enclosed by spectrin that contains red lines that fill it with a similar back and forth pattern. The red line crosses the end of a rectangle with rounded edges representing a junctional complex to reach a third region bounded by spectrin. This region is filled with a blue line that has a similar back and forth path.

Sphingolipids and Cholesterol Cluster Together in Membrane Rafts

We have seen that diffusion of membrane lipids from one bilayer leaflet to the other does not occur unless catalyzed and that the different lipid species of the plasma membrane are asymmetrically distributed in the two leaflets of the bilayer (Fig. 11-6). Even within a single leaflet, the lipid distribution is not uniform. Glycosphingolipids (cerebrosides and gangliosides), which typically contain long-chain saturated fatty acids, form transient clusters in the outer leaflet; these clusters largely exclude glycerophospholipids, which typically contain one unsaturated fatty acyl group and a shorter saturated acyl group. The long, saturated acyl groups of sphingolipids can form more compact, more stable associations with the long ring system of cholesterol than can the shorter, often unsaturated, chains of phospholipids. The cholesterol-sphingolipid microdomains of the plasma membrane make the bilayer slightly thicker and more ordered (less fluid) than neighboring regions, which are rich in phospholipids. These microdomains are more difficult to dissolve with nonionic detergents; they behave like liquid-ordered sphingolipid rafts adrift on an ocean of liquid-disordered phospholipids (Fig. 11-22). Proteins with relatively short hydrophobic helical sections (19 to 20 residues) cannot span the thicker bilayer in rafts, and thus they tend to be excluded. Proteins with longer hydrophobic helices (24 to 25 residues) segregate into the thicker bilayer regions of rafts, where the entire length of the helix is stabilized by the hydrophobic effect.

A figure shows the structure of a membrane microdomain open parenthesis raft close parenthesis and the surrounding membrane, including the positioning of a prenylated protein, acyl groups, cholesterol, caveolin, a G P I-linked protein, and a doubly acylated protein. — FIGURE 11-22 Membrane microdomains (rafts). Stable associations of sphingolipids and cholesterol in the outer leaflet produce a microdomain, slightly thicker than other membrane regions, that is enriched with specific types of membrane proteins. GPI-anchored proteins are prominent in the outer leaflet of these rafts, and proteins with one or several covalently attached long-chain acyl groups are common in the inner leaflet. Inwardly curved rafts called caveolae are especially enriched in proteins called caveolins (see Fig. 11-23).

The region above the piece of membrane is labeled outside and the region below is labeled inside. A bracket shows that the middle portion of a membrane is a raft, enriched in sphingolipids and cholesterol. The phospholipids outside of the raft and on the inner leaflet are entirely phosphatidylcholine, but the outer leaflet of raft contains 7 phosphatidylcholine molecules, 13 sphingolipids, and one phosphatidylinositol open parenthesis P I close parenthesis that has two red highlighted zigzag chains below and a chain of hexagons above connecting it to a protein labeled G P I-linked protein. There are many more long ovals of cholesterol in the region labeled raft than in other parts of the membrane. To the left of the raft region, there is an irregular sphere labeled prenylated protein. There are three zigzag chains extending vertically through the bottom half of the plasma membrane near the center, within the raft region, and these are labeled acyl groups open parenthesis palmitoyl, myristoyl close parenthesis. Beneath these chains, a horizontal strand of caveolin extends left to bend down slightly as it ends and right to curve upward to the center of the membrane, then back down to run horizontally before curving vertically down and back toward the left. Four phosphate heads behind the right horizontal piece of caveolin are purple to indicate phosphatidylserine. An irregularly shaped protein with two zigzag chains extending through the bottom half of the plasma membrane just before the right end of the raft is labeled doubly acylated protein.

Lipid rafts are remarkably enriched in two classes of integral proteins, with two specific types of covalently attached lipids. The integral proteins of one class have two long-chain saturated fatty acids (two palmitoyl groups, or a palmitoyl and a myristoyl group) covalently attached through Cys residues. Caveolin is one such protein; there are many others. Those of the second class, the GPI-anchored proteins, have a glycosyl phosphatidylinositol on their carboxyl-terminal residue (Fig. 11-16). Presumably, these lipid anchors, like the long, saturated acyl chains of sphingolipids, form more stable associations with the cholesterol and long acyl groups in rafts than they form with the surrounding phospholipids. (It is notable that other lipid-linked proteins, those with covalently attached isoprenyl groups such as farnesyl, are not preferentially associated with the outer leaflet of sphingolipid rafts; see Fig. 11-22.) The “raft” and “sea” domains of the plasma membrane are not rigidly separated; membrane proteins can move into and out of lipid rafts in a fraction of a second. But in the shorter time scale (microseconds) more relevant to many membrane-mediated biochemical processes, many of these proteins reside primarily in a raft.

The approximate fraction of the cell surface occupied by rafts can be as high as 50% in some cases; the rafts cover half of the ocean. Indirect measurements in cultured fibroblasts suggest a diameter of roughly 50 nm for an individual raft, which corresponds to a patch containing a few thousand sphingolipids and perhaps 10 to 50 membrane proteins. Because most cells express more than 50 different kinds of plasma membrane proteins, it is likely that a single raft contains only a subset of membrane proteins and that this segregation of membrane proteins is functionally significant. Their presence in a single raft would hugely increase the likelihood of their collision. Certain membrane receptors and signaling proteins, for example, seem to be segregated together in membrane rafts. Experiments show that signaling through these proteins can be disrupted by manipulations that deplete the plasma membrane of cholesterol and destroy lipid rafts.

Plasma membranes of many cells have specialized rafts called caveolae (“little caves”), which can represent about half of the total area of the plasma membrane (Fig. 11-23a). Closely associated with caveolae is caveolin, an integral protein with two globular domains connected by a hairpin-shaped hydrophobic domain that binds the protein to the cytoplasmic leaflet of the plasma membrane (Fig. 11-23b). Three palmitoyl groups attached to the carboxyl-terminal globular domain further anchor the protein to the membrane. Caveolins form dimers and associate with cholesterol-rich regions in the membrane. The presence of caveolin dimers forces the associated lipid bilayer to curve inward, forming caveolae. Caveolae involve both leaflets of the bilayer — the cytoplasmic leaflet, from which the caveolin globular domains project, and the extracellular leaflet, a cholesterol and sphingolipid raft with associated GPI-anchored proteins. Caveolae are implicated in a variety of cellular functions, including membrane trafficking within cells and the transduction of external signals into cellular responses.

A three-part figure shows caveolae in a plasma membrane in part a, a close-up of a single caveola in part b, and the effects of mechanical stress on a caveola and on a cell in part c. — FIGURE 11-23 Caveolin forces inward curvature of a membrane. (a) Caveolae are small invaginations in the plasma membrane. (b) Cartoon showing the location and role of a caveolin dimer in causing inward membrane curvature. Each caveolin monomer has a central hydrophobic domain and three long-chain acyl groups (red), which hold the molecule to the inside of the plasma membrane. When several caveolin dimers are concentrated in a small region (a raft), they force a curvature in the lipid bilayer, forming a caveola. Cholesterol molecules in the bilayer are shown in orange. (c) Flattening of caveolae allows the plasma membrane to expand in response to various stresses. [Information from R. G. Parton, *Nat. Rev. Mol. Cell Biol.* 8:185–194, 2007.]

Part a shows a piece of plasma membrane with rounded invaginations that have relatively narrow openings to the outside of the cell. An arrow points down to part b, where a close-up of one of these invaginations is shown. The invagination is labeled caveola and its membrane is lined with tan elongated ovals representing cholesterol along the inside, facing the opening within the invagination, and with both cholesterol and purple threads of calveolin along the outside, which faces the inside of the cell. A close-up of a piece of this membrane shows that the surface facing the opening has 16 sphingolipids with four interspersed phosphatidylcholine molecules and the surface facing the inside of the cell has all phosphatidylcholine except for four spheres of phosphatidylserine behind each caveolin strand. On the left, a horizontal piece of caveolin has three red zigzag chains that extend up through the bottom half of the plasma membrane. It extends to the left, where it bends down slightly before ending, and to the right, where it bends in to reach the region between the upper and lower halves of the membrane, then bends back to run horizontally along four spheres of phosphatidylserine that are each beneath a cholesterol molecule before bending down vertically and then curving slightly to the left. As it runs vertically, it is paired with a caveolin molecule to the right and the two together are labeled as caveolin dimer open parenthesis six fatty acyl, eight cholesterol moieties close parenthesis. The right-hand caveolin molecule runs vertically up to the plasma membrane, runs horizontally along four spheres of phosphatidylserine that are each beneath a cholesterol molecule before bending up to reach the region between the upper and lower halves of the plasma membrane and then bending down to run horizontally below three red zigzag chains that extend up through the bottom half of the plasma membrane and then bending down slightly below the membrane to end. Part c shows a simplified single caveola as a circular invagination with a relatively narrow opening to the outside of the cell and with membrane to the upper left and right. Mechanical stress pulls it to left and right and this results in a horizontal piece of membrane that has the straightened membrane from the caveola in its center. A cell with eight caveolae along its plasma membrane becomes an enlarged cell when all of the caveolae are straightened in this way. The enlarged cell has a smooth outer membrane consisting of alternating pieces of original outer membrane and pieces of caveola membrane from the starting cell.

Caveolae may also provide a means of expanding the cell surface. The lipid bilayer itself is not elastic, but if existing caveolae lose their associated caveolin as the result of a regulatory signal, the caveolae flatten into the plasma membrane (Fig. 11-23c). The effect is to add surface area, allowing the cell to expand without bursting in response to osmotic or other stress.

Membrane Curvature and Fusion Are Central to Many Biological Processes

Caveolins are not unique in their ability to induce curvature in membranes. Changes of curvature are central to one of the most remarkable features of biological membranes: their ability to undergo fusion with other membranes without losing their continuity. Within the eukaryotic endomembrane system, the membranous compartments constantly reorganize. Vesicles bud from the ER to carry newly synthesized lipids and proteins to other organelles and to the plasma membrane. Exocytosis, endocytosis, cell division, fusion of egg and sperm cells, and entry of a membrane-enveloped virus into a host cell all involve a membrane reorganization that requires the fusion of two membrane segments without loss of continuity (Fig. 11-24). Most of these processes begin with a local increase in membrane curvature.

A figure shows eight ways in which membrane fusion is important in cellular processes. These are budding of vesicles from Golgi complex, exocytosis, endocytosis, fusion of endosome and lysosome, viral infection, fusion of sperm and egg, fusion of small vacuoles open parenthesis plants close parenthesis, and separation of two plasma membranes at cell division. — FIGURE 11-24 Membrane fusion. The fusion of two membranes is central to a variety of cellular processes involving organelles and the plasma membrane.

The name of each process is accompanied by an illustration. Budding of vesicles from Golgi complex: five horizontal pieces of Golgi complex have an arrow pointing from the left side through a sphere. Exocytosis: the arrow passing through the sphere from the Golgi apparatus continues through a membrane. Endocytosis: an invagination in a membrane contains many small red dots and an arrow points through this into a cell. Fusion of endosome and lysosome: the arrow from endocytosis joins with an arrow from a yellow sphere, forming a structure with a small sphere with red dots on the left and a larger yellow sphere on the right. Fusion of sperm and egg: A sperm with an oval head and long tail is shown pushing into the plasma membrane and an arrow points into the cell. Fusion of small vacuoles open parenthesis plants close parenthesis: An open sphere is shown with three fused smaller spheres and two spheres outside of it with arrows pointing through two of the fused spheres into the center and toward one of the spheres outside. Separation of two plasma membranes at cell division: A piece of plasma membrane is open at the center with arrows pointing up and down from the open space.

Cardiolipin, present primarily in mitochondrial membranes of eukaryotes, but also a major component of the membrane lipids of bacteria, can create or recognize membrane curvature. It is cone-shaped — its head group is small relative to its four fatty acyl chains — so it can act as a wedge in the monolayer, contracting that monolayer relative to the other.

A figure shows ball-and-stick models of cardiolipin and phosphatidylcholine.

Cardiolipin has four yellow chains, two on the left and two on the right, extending from a central region that has red, orange, and gray groups. The overall structure is curved with the narrow head region that widens to the sides along the tails. It has a three-carbon chain with the central C bonded to O H. The left-hand C is bonded to O bonded to P double bonded to O, bonded to O minus, and bonded to O further bonded to C H 2 bonded to C H bonded to C H 2 on the left bonded to O bonded to C double bonded to O that is further bonded to a zigzag chain of 14 additional carbons and to O on the right bonded to C double bonded to O and further bonded to a zigzag chain of 8 additional carbons. The right-hand C is bonded to O bonded to P double bonded to O, bonded to O minus, and bonded to O bonded to C H 2 bonded to C H bonded to C H 2 above bonded to C double bonded to O and further bonded to a zigzag chain of 13 additional carbons and to O below bonded to C double bonded to O and further bonded to a zigzag chain of 14 additional carbons. Phosphatidylcholine has a narrow, relatively linear head region with silver, blue, orange, and red atoms that joins to two yellow tails that extend down to the left and right. It is relatively narrow. It has C H bonded to C H 2 above bonded to O bonded to P double bonded to O, bonded to O minus, and bonded to O further bonded to C H 2 bonded to C H 2 bonded to N plus bonded to 3 C H 3, bonded to O to the left bonded to C double bonded to O and further bonded to a zigzag chain of 17 additional carbons, and bonded to C H 2 to the right bonded to O bonded to C double bonded to O and further bonded to a zigzag chain of 15 additional carbons.

Membrane curvature is the result. In E. coli, cardiolipin is highly localized at the two poles of the rod-shaped cell, where its curvature is sharpest. It seems very likely that other membrane lipids will be found to influence local curvature of the bilayer.

A protein that is intrinsically curved may force a bilayer to curve by binding to it (Fig. 11-25); the binding energy provides the driving force for the increase in bilayer curvature. Alternatively, multiple subunits of a scaffold protein may assemble into curved supramolecular complexes and stabilize curves that spontaneously form in the bilayer. For example, a superfamily of proteins containing BAR domains (named for the first three members of the family that were identified: BIN1, amphiphysin, and RVS167) can assemble into a crescent-shaped scaffold that binds to the membrane surface, forcing or favoring membrane curvature. BAR domains consist of coiled coils that form long, thin, curved dimers with a positively charged concave surface that tends to form ionic interactions with the negatively charged head groups of membrane lipids ${PIP}_{2}$ $PIP Subscript 2$ and ${PIP}_{3}$ $PIP Subscript 3$ . The enzymatic formation of these inositol lipids can tag a plasma membrane area for creation of inward curvature by a BAR protein (Fig. 11-25). Some of these BAR proteins also have an amphipathic helix (having one polar face and one hydrophobic face; see Fig. 11-32) that inserts like a wedge into one leaflet of the bilayer, expanding its area relative to the other leaflet and thereby forcing curvature. Such a protein might also serve as a detector of preexisting membrane curvature due to differences in lipid composition of two leaflets of a bilayer.

A three-part figure shows three ways in which proteins can induce curvature in a membrane. Part a shows interactions between a protein with intrinsic curvature that interacts with negatively charged phospholipid head groups, part b shows a protein with one or several amphipathic helices inserted into a leaflet of the bilayer, and part c shows a curved membrane interacting with proteins with B A R domains that have formed a superstructure. — FIGURE 11-25 Three models for protein-induced curvature of membranes. [Information from (a, b) B. Qualmann et al., *EMBO J.* 30:3501, 2011, Fig. 1; (c) B. J. Peter et al., *Science* 303:495, 2004, Fig. 1A.]

Part a shows a curved membrane with 11 red spheres representing phosphate heads that each have two faded red tails extending into the upper part of the membrane. There are three blue phosphate heads between each red one. A crescent-shaped structure above fits against the curve of the circle and has a plus sign above each red sphere. Text below reads, a protein with intrinsic curvature and with a high density of positive charge on its concave surface interacts with negatively charged phospholipid head groups, favoring curvature of the bilayer. Part b shows a similar membrane with 9 red spheres and with a similar crescent-shaped structure above but with the phospholipid in the top half of the membrane more tightly packed than those in the bottom half. The crescent-shaped structure overlaps the top half of the membrane to the left and right of the region with red spheres and has a horizontal helix in each of these regions that is aligned with the phosphate heads of the membrane. There are plus symbols in the crescent-shaped structure above each red phosphate head. Text below reads, a protein with one or several amphipathic helices inserted into one leaflet of the bilayer crowds the lipids on that leaflet, forcing the membrane to bend. Part c shows a curved piece of membrane with many helices above it. The helices form a rectangular structure above the center of the membrane and extend lower to the left and right, where the contact the top of the membrane. Text below reads, proteins with B A R domains can polymerize into a superstructure that favors and maintains the curvature.

Septins make up a family of GTP-binding proteins (14 genes in humans) that polymerize at curved regions of the plasma membrane and participate in cellular processes such as cell division, exocytosis, phagocytosis, and apoptosis. All septins have an amphipathic helix that can submerge its hydrophobic side in one leaflet of the bilayer, forcing one leaflet of the bilayer to expand laterally, either causing or sensing local curvature in the membranes. Studies of cells with mutations in this helix show its biological importance in vesicle trafficking and neurotransmitter release.

Specific fusion of two membranes requires mediation by fusion proteins. Fusion proteins ensure (1) that the two membranes recognize each other; (2) that their surfaces become closely apposed, which requires removal of the water molecules normally associated with the polar head groups of lipids; (3) that their bilayer structures become locally disrupted, resulting in fusion of the outer leaflets of the two membranes (hemifusion); and (4) that their bilayers fuse to form a single continuous bilayer. The fusion occurring in receptor-mediated endocytosis, or regulated secretion, also requires (5) that the process is triggered at the appropriate time or in response to a specific signal. Fusion proteins mediate these events, bringing about specific recognition and a transient local distortion of the bilayer structure that favors membrane fusion. (Note that these fusion proteins are unrelated to the products encoded by two fused genes, also called fusion proteins, discussed in Chapter 9.)

A well-studied example of membrane fusion occurs at synapses, when intracellular (neuronal) vesicles loaded with neurotransmitter fuse with the plasma membrane. Yeast cells provide another experimentally accessible system in which vesicles fuse with the plasma membrane, releasing their secretion products. Both processes involve a family of proteins called SNAREs (snap receptors; Fig. 11-26). SNAREs in the cytoplasmic face of the intracellular vesicle are called v-SNAREs (v for vesicle); those in the target membrane with which the vesicle fuses (the plasma membrane during exocytosis) are t-SNAREs (t for target). The protein NSF (N-ethylmaleimide sensitive factor) regulates the interactions among SNAREs. During fusion, a v-SNARE and a t-SNARE bind to each other and undergo a structural change that produces a bundle of long, thin rods made up of helices from both SNAREs and two helices from the protein SNAP25 (Fig. 11-26). The two SNAREs initially interact at their ends, then zip up into the bundle of helices. This structural change pulls the two membranes into contact and initiates the fusion of their lipid bilayers. An alternative designation of SNARE types is based on structural features of the proteins: R-SNAREs have an Arg residue critical to their function, and Q-SNAREs have a critical Gln residue. Typically, R-SNAREs act as v-SNAREs, and Q-SNAREs act as t-SNAREs.

A figure shows the steps involved in membrane fusion to release neurotransmitters at a synapse. — FIGURE 11-26 Membrane fusion during neurotransmitter release at a synapse. The secretory vesicle membrane contains the v-SNARE synaptobrevin (red). The target (plasma) membrane contains the t-SNAREs syntaxin (blue) and SNAP25 (violet). When a local increase in [ ${Ca}^{2 +}$ $Ca Superscript 2 plus$ ] signals release of neurotransmitter, the v-SNARE, SNAP25, and t-SNARE interact, forming a coiled bundle of four α helices, pulling the two membranes together and disrupting the bilayer locally. This leads first to hemifusion, joining the outer leaflets of the two membranes, then to complete membrane fusion and neurotransmitter release. When fusion is complete, the SNARE complex is disassembled. [Information from Y. A. Chen and R. H. Scheller, *Nat. Rev. Mol. Cell Biol.* 2:98, 2001.]

FIGURE 11-26 Membrane fusion during neurotransmitter release at a synapse. The secretory vesicle membrane contains the v-SNARE synaptobrevin (red). The target (plasma) membrane contains the t-SNAREs syntaxin (blue) and SNAP25 (violet). When a local increase in [ ${Ca}^{2 +}$ $Ca Superscript 2 plus$ ] signals release of neurotransmitter, the v-SNARE, SNAP25, and t-SNARE interact, forming a coiled bundle of four α helices, pulling the two membranes together and disrupting the bilayer locally. This leads first to hemifusion, joining the outer leaflets of the two membranes, then to complete membrane fusion and neurotransmitter release. When fusion is complete, the SNARE complex is disassembled. [Information from Y. A. Chen and R. H. Scheller, *Nat. Rev. Mol. Cell Biol.* 2:98, 2001.]

A secretory vesicle is shown in the cytosol above a plasma membrane. The vesicle contains many small, circular neurotransmitter molecules. A small red oval runs across the vesicle membrane at the lower left and is joined with a red thread that runs down and then bends to the left. A similar small red oval runs across the vesicle at the lower right and is joined with a thread that runs down and then bends to the right, where it is labeled v-S N A R E. The plasma membrane has a small red oval crossing its membrane on the left that joins to a blue thread above that curves smoothly up and to the left. A similar small red oval crosses the membrane to the right and joins to a blue thread above that curves smoothly up and to the right, where it is labeled t-S N A R E. To the right of the left-hand blue thread, there is a purple horizontal thread just below the top of the plasma membrane, in the yellow region. It has purple strands extending up from its left and right that smoothly curve up and to the left. A similar purple structure is embedded in the membrane just to the left of the right-hand blue thread with its strands extending up and to the right. This purple structure is labeled S N A P 25. Accompanying text reads, neurotransmitter-filled vesicle approaches the plasma membrane. An arrow points down to show the vesicle closer to the plasma membrane. The red, purple, and blue threads on the left run horizontally to the left and then intertwine. The red, purple, and blue threads on the right run horizontally to the right and then intertwine. Accompanying text reads, v-S N A R E and t-S N A R E bind to each other, zipping up from the amino termini and drawing the two membranes together. An arrow points down to show the vesicle in contact with the membrane and with the threads on each side fully intertwined. A small vertical space is visible between the bottom layer of phosphate heads of the vesicle membrane and the top layer of phosphate heads of plasma membrane where the bottom center of the vesicle and top center of the plasma membrane meet. Accompanying text reads, zipping causes curvature and lateral tension on bilayers, favoring hemifusion between outer leaflets. An arrow points down to show the vesicle membrane fused with the plasma membrane. The yellow lipid layer is continuous between the two and no blue phosphate layers separate them. Accompanying text reads, hemifusion: outer leaflets of both membranes come into contact. An arrow points down to show that there is now an opening between the space in the vesicle and the region beneath the membrane. A single membrane comes in from the lower left, bends upward where there is a red oval with intertwined strands to its left, forms a rounded region above, then bends more sharply downward at the bottom right of the rounded region where there is a red oval with intertwined strands to its right, before leaving to the lower right. Spheres representing neurotransmitter molecules are shown moving through the opening to leave the rounded region. Accompanying text reads, complete fusion creates a fusion pore. An arrow points down to show that the sides of the circle have moved to the left and right. There is now a membrane that runs horizontally, then bends upward where there is a red oval with intertwined threads that extend to the upper left, then runs horizontally, then bends downward, reaches another red oval with intertwined threads that extend to the upper right, then runs horizontally to the right. Neurotransmitters are shown throughout the open region. Accompanying text reads, pore widens; vesicle contents are released outside cell.

The complex of SNAREs and SNAP25 is the target of several powerful neurotoxins. Clostridium botulinum toxin is a bacterial protease that cleaves specific bonds in SNARE proteins, preventing neurotransmission and causing paralysis and death. Because of its very high specificity for these proteins, purified botulinum toxin has served as a powerful tool for dissecting the mechanism of neurotransmitter release in vivo and in vitro. Used in small amounts, botulinum toxin (Botox) is used in medicine to treat disorders of eye and neck muscles, as well as cosmetically for the removal of skin wrinkles. Tetanus toxin, produced by the bacterium Clostridium tetani, also is a protease with high specificity for SNARE proteins. It causes painful muscle spasms and rigidity of voluntary muscles — hence the characteristic symptom “lockjaw.”

Integral Proteins of the Plasma Membrane Are Involved in Surface Adhesion, Signaling, and Other Cellular Processes

Several families of integral proteins in the plasma membrane provide specific points of attachment between cells or between a cell and proteins of the extracellular matrix. Integrins are surface adhesion proteins that mediate a cell’s interaction with the extracellular matrix and with other cells, including some pathogens. Integrins also carry signals in both directions across the plasma membrane, integrating information about the extracellular and intracellular environments. All integrins are heterodimeric proteins composed of two unlike subunits, α and β, each anchored to the plasma membrane by a single transmembrane helix. The large extracellular domains of the α and β subunits combine to form a specific binding site for extracellular proteins such as collagen and fibronectin, which contain a common determinant of integrin binding, the sequence Arg–Gly–Asp (RGD).

Other plasma membrane proteins involved in surface adhesion are the cadherins, which undergo homophilic (“with the same kind”) interactions with identical cadherins in an adjacent cell. Selectins have extracellular domains that, in the presence of ${Ca}^{2 +}$ $Ca Superscript 2 plus$ , bind specific polysaccharides on the surface of an adjacent cell. Selectins are present primarily in the various types of blood cells and in the endothelial cells that line blood vessels (see Fig. 7-29). They are an essential part of the blood-clotting process.

SUMMARY 11.2 Membrane Dynamics

Lipids in a biological membrane can exist in liquid-ordered or liquid-disordered states. Membrane fluidity is affected by temperature, fatty acid composition, and sterol content.
Flip-flop diffusion of lipids between the inner and outer leaflets of a membrane occurs only when the diffusion is specifically catalyzed by flippases, floppases, scramblases, or PI transporters.
Proteins and lipids can diffuse laterally within the plane of the membrane, but this mobility is limited by interactions of membrane proteins with internal cytoskeletal structures and interactions of lipids with lipid rafts.
One class of lipid rafts is enriched for sphingolipids and cholesterol with a subset of membrane proteins that are GPI-linked or attached to several long-chain fatty acyl moieties. Caveolins are integral proteins that associate with the inner leaflet of the plasma membrane, forcing it to curve inward to form caveolae, which are involved in membrane transport, signaling, and the expansion of plasma membranes.
Proteins containing BAR domains cause local membrane curvature and mediate the fusion of two membranes, which accompanies processes such as endocytosis, exocytosis, and viral invasion. Septins are proteins that sense or cause membrane curvature. Membrane fusion depends on SNARE proteins, which draw two membranes close together and favor fusion.
Integrins, cadherins, and selectins are transmembrane proteins of the plasma membrane that act both to attach cells to each other and to carry messages between the extracellular matrix and the cytoplasm.