Update of the 1972 Singer-Nicolson Fluid-Mosaic Model of Membrane Structure

DISCOVERIES 2013, Oct-Dec; 1(1): e3 DOI: 10.15190/d.2013.3 Updated Fluid-Mosaic Model of cellular membranes

REVIEW

Garth L. Nicolson1,*

1The Institute for Molecular Medicine, Department of Molecular Pathology, Huntington Beach, CA, USA *Corresponding author: Prof. Emeritus Garth L. Nicolson, Ph.D, The Institute for Molecular Medicine, P.O. Box 9355, S. Laguna Beach, CA 92652 USA. Email: [email protected] Citation: Nicolson GL. Update of the 1972 Singer-Nicolson Fluid-Mosaic Model of Membrane Structure. Discoveries 2013, Oct-Dec; 1(1): e3. DOI: 10.15190/d.2013.3

ABSTRACT Keywords: membrane domains; extracellular The Fluid-Mosaic Membrane Model of cell matrix; lipid rafts; membrane-associated cytoske- membrane structure was based on thermodynamic leton; membrane asymmetry; membrane dynamics; principals and the available data on component lateral mobility within the membrane plane [Singer Abbreviations: Bin/amphiphysin/Rvs family of SJ, Nicolson GL. The Fluid Mosaic Model of the lipid-binding molecules (BAR); Fluid-Mosaic Mem- structure of cell membranes. Science 1972; 175: brane Model (F-MMM) 720-731]. After more than forty years the model remains relevant for describing the basic nano-scale Introduction structures of a variety of biological membranes. The Fluid Mosaic Membrane Model of biological More recent information, however, has shown the membrane structure was envisioned as a basic importance of specialized membrane domains, such framework model for interpreting existing data on as lipid rafts and protein complexes, in describing membrane proteins and lipids, and their dynamics1. the macrostructure and dynamics of biological At this time, the accepted model for cellular membranes. In addition, membrane-associated membrane structure was a static tri-layer model of cytoskeletal structures and extracellular matrix also protein-lipid-protein2, later refined as the Unit play roles in limiting the mobility and range of Membrane Model3. The tri-layer membrane models motion of membrane components and add new were based on the lipid bilayer proposal of Gorter layers of complexity and Grendel4, with and hierarchy to the added unfolded An updated Fluid-Mosaic Membrane Model, original model. An protein beta-sheets updated Fluid-Mosaic incorporating membrane domains and on either side of the Membrane Model is restraints on the mobility/range of motion of lipid bilayer and described, where more membrane components. bound to it by emphasis has been electrostatic and placed on the mosaic nature of cellular membranes other forces. A few trans-membrane proteins were where protein and lipid components are more added later (Robertson, 1981)5 to reconcile crowded and limited in their movements in the observations on ion transport and freeze-fractured membrane plane by lipid-lipid, protein-protein and images of cell membranes6. Alternatively, lipo- lipid-protein interactions as well as cell-matrix, cell- protein Subunit Membrane Models were proposed cell and cytoskeletal interactions. These interactions without a lipid bilayer matrix7,8. are important in restraining membrane components The Fluid Mosaic Model1 was successful and maintaining the unique mosaic organization of because it was based on the natural bilayer formation cell membranes into functional, dynamic domains. of membrane glycerophospholipids and the www.discoveriesjournals.org/discoveries 1 Updated Fluid-Mosaic Model of cellular membranes amphipathic structures of integral globular model and did not question its fundamental membrane proteins that allowed their intercalation structural principles19,22-27. into the hydrophobic lipid bilayer matrix. Only this It is now widely accepted that there are model considered the different categories of proteins limitations in the Fluid Mosaic Model as originally (integral and peripheral) and the ability of proposed1 in explaining the domain structures components in membranes to rapidly move laterally present in membranes, especially those membranes and change their topographic distributions within a found in specialized tissues and cells. Thus, the fluid membrane matrix (Figure 1)1. The bilayer model has been refined and renamed as the ‘Fluid- structure of membrane phospholipids and their Mosaic Membrane Model’ (F-MMM) to highlight lateral motions in the membrane plane were the important role of mosaic, aggregate and domain consistent with data on membrane structure and have structures in membranes and restraints on the free been the subjects of a number of reviews over the lateral movements of certain components. Early years (the early literature can be found in9-15). For modifications of the basic F-MMM included the example, Edidin12 reviewed the history of membrane interactions of extracellular matrix and membrane- lipid structural proposals over the last century and associated cytoskeletal components with cell concluded that cellular membranes must contain a membranes and their influences on the mobility and phospholipid bilayer matrix. Also, the proposal that distribution of trans-membrane glycoproteins9. Also, integral or intrinsic proteins exist as globular the possibility that less mobile lipid-protein or lipid- structures imbedded into the lipid bilayer (in contrast lipid domains might exist in membranes as frozen or to peripheral or extrinsic membrane proteins that are semi-frozen islands in a sea of fluid phospholipids also present but are not bound to membranes by was also proposed9. As will be discussed below, the hydrophobic interactions) remains supported by hypothesis that trans-membrane interactions with overwhelming evidence9-19. membrane-associated structures influences Although the Fluid Mosaic Model has been membrane dynamics was important in explaining the remarkably consistent with data collected on integration of structure, component mobility, and biological membranes since the early 1960s9-21, it membrane function9,10,28. The relatively recent was inevitable that the original model could not discoveries of lipid rafts and other specialized explain all of the data in every subsequent study. membrane domains, membrane-associated ‘fences’ However, the model quickly evolved from the and membrane ‘fenceposts,’ and their possible original 1972 model, and the revisions that occurred functions in controlling and restraining membrane within a few years took into account many questions protein distribution and mobility continued this that were raised about the 1972 model9. Most of trend10,12,23-37. these criticisms came decades after the original

Figure 1. The Singer-Nicolson Fluid- Mosaic Membrane Model of cell membrane structure as proposed in 1972. In this view of a cell membrane the solid bodies with stippled cut surfaces represent globular integral membrane proteins randomly distributed in the plane of the membrane. Some integral membrane proteins form specific integral protein complexes, as shown in the figure. Integral proteins are represented in a fluid lipid bilayer. The model does not contain other membrane- associated structures or membrane domains (from Singer and Nicolson1).

www.discoveriesjournals.org/discoveries 2 Updated Fluid-Mosaic Model of cellular membranes

Models of cell membranes produced a few years proposed1,14, the basic nano-scale structural after the 1972 model were much less homogeneous organization of membranes has remained relatively looking than the original model9. They contained consistent with current evidence9,10,15,18,21-24,40-45, with additional information not included in the original some modifications26,27,32,34,35,40,45. This will be model, such as protein and lipid aggregations and discussed below. segregation into domains, cytoskeletal and matrix Cell membranes are dynamic structures that are interactions, among other features (some of these are quite susceptible to deformation. For example, shown in Figure 2 of Nicolson9). Importantly, membranes deform when confronted with forces less revisions of the F-MMM retained all of the basic than those driven by the hydrophobic effect46. elements of the original model. In newer models of Membrane deformation depends on the energies of biological membranes, the arrangement of compo- lipid tilt and splay, which in turn are dependent on nents into more compact structures and domains lipid composition46. Lipids that support positive maximizes the mosaic nature of these structures (the spontaneous curvature can reverse the effects of historical development of the F-MMM and lipids that support negative spontaneous curvature, supporting data from the previous century, are and this may be important in membrane fusion and discussed in10). other dynamic membrane-membrane interactions47. Different lipids and integral membrane proteins Physical Principles and Cell Membranes must adjust to each other’s hydrophobic structures. Singer14 drew attention to the work of Kauzmann38 Thus, Israelachvili40 proposed that the F-MMM and the important concept of hydrophobic required refinement to account for these differences. interactions in maintaining cell membrane structure. Further, Mouritsen and Bloom48 suggested that Hydrophobic structures self-associate to exclude sorting of lipids and proteins was based on the water interactions, whereas hydrophilic structures interactions of their hydrophobic regions and to a interact with the aqueous, ionic environment. lesser degree their hydrophilic interactions in order Membrane phospholipids self-assemble to form to prevent mismatches between lipids and proteins. bilayers due to the energy provided by the This concept was incorporated into the Mattress hydrophobic effect and van der Waals forces39. Model of membranes that described how variations Membrane integral globular proteins interact with in the hydrophobic parts of lipids and proteins drive membrane lipids, mainly their acyl tails, due to associations or hydrophobic matching between hydrophobic forces and much less to hydrophilic different types of membrane components to prevent interactions between the lipid head groups and membrane distortions48. This concept will be protein hydrophilic groups15,39,40. As originally considered again in another section.

Figure 2. Different examples of cell membrane integral membrane protein lateral mobility as envisioned by Jacobson and colleagues in 1995. Integral membrane protein lateral movements are described as: transient confinement by obstacle clusters (A); transient confinement by the cytoskeleton (B); directed motion by attachment to the cytoskeleton (C); and free, random diffusion in the membrane plane (D) (from Jacobson et al.23).

www.discoveriesjournals.org/discoveries 3 Updated Fluid-Mosaic Model of cellular membranes

Membrane distortions, such as deformation, other functional components of membranes. curvature, compression and expansion, are driven by Disruption of the normal membrane asymmetry is different forces and components within associated with cell activation (activation of cell membranes46-51. For example, certain soluble adhesion, aggregation, apoptosis, recognition by proteins can bind to membranes and cause phagocytic cells, among other events), and it can deformation. Examples are proteins that contain also be associated with pathologic processes58. BAR (Bin/ amphiphysin/ Rvs) domains that form It follows that a number of lipid transporters crescent-shaped α-helical bundles that bind to have been discovered that are important in membranes via electrostatic and hydrophobic maintaining lipid asymmetry57-59. Examples include interactions. BAR proteins can generate membrane the inner membrane-directed, ATP-dependent curvature by scaffolding to the inner surface of the transporters (‘flippases’), outer membrane-directed, membrane, causing it to bend to the curvature of the ATP-independent transporters (‘floppases’), and the protein52. When inserted into a membrane some bidirectional, ATP-indepen-dent transporters proteins alter their shape by undergoing folding (‘scramblases’)58,59. The existence of these transitions to form α-helices that wedge between phospholipid transporters in maintaining the proper membrane components, thus deforming the phospholipid asymmetries in cellular membranes membrane and causing membrane curvature53. suggests that maintenance of membrane asymmetry is functionally important. Cell Membrane Asymmetry Membrane integral protein asymmetry is Biological membranes are asymmetric structures1,9- easier to explain, but certainly no less complex15- 11,18,54-56. The asymmetric nature of cell membranes 17,64. It is probably initiated at the time of protein was actually known well before the original F- synthesis during the initial insertion of the MMM was proposed (for example, Stoeckenius and polypeptide chains into the membrane mediated by Engelman56). One reason for this is that the free molecular gatekeepers called translocons16,17. Since energy required to flip membrane amphipathic lipids the energy required to flip integral globular and proteins across the hydrophobic membrane membrane proteins across a hydrophobic barrier is interior is substantial; thus, cell membrane flip-flop enormous, integral membrane protein asymmetry that could result in symmetric structures should be does not have to be actively maintained after rare1,13,14. biosynthesis. Membrane lipid asymmetry is essential in guiding membrane curvature and other aspects of Membrane Proteins and Membrane-Associated membrane structure50. The compositional differences Proteins between the inner and outer leaflets of cell As discussed in the original publication on the F- membranes suggest that the outer leaflet is curvature MMM1, it was important to distinguish between the neutral, while the inner leaflet may have a integral (or intrinsic) proteins that are tightly bound preference for negative curvature46. The finding of to membranes by mainly hydrophobic forces and asymmetric distributions of various phospholipids intercalated into the membrane hydrophobic matrix between the inner and outer leaflets of cell and peripheral (or extrinsic) proteins that are loosely membranes has proved to be relatively bound to membranes by electrostatic or other non- monotonous54-60. For example, the enrichment of hydrophobic interactions. Numerous examples of amine- and serine-containing phospholipids found both types of membrane proteins abound, and this on the inner surface and choline-containing has been reviewed in more detail elsewhere14-17. I phospholipids and spingomyelins on the outer have discussed the importance of integral membrane surface of cell membranes creates increased affinity proteins in defining basic membrane nano-scale of cholesterol to the outer bilayer leaflet, and this structure of cell membranes; however, peripheral might have some advantage in terms of membrane membrane proteins also have an important role, but associations into domains and maintenance of not necessarily in maintaining the basic structures of enzymatic activities12,21,60-63. membranes. They appear to be more important in There is a cost for not maintaining appro- providing non-membrane protein attachment sites, priate cell membrane asymmetry, and it is not just scaffolding, tethering or membrane -supporting the appropriate display of enzymes, receptors and structures, membrane curvature-preserving www.discoveriesjournals.org/discoveries 4 Updated Fluid-Mosaic Model of cellular membranes components, and attachment points for soluble actin-containing cytoskeletal components are enzymes and signaling molecules. involved in moving or restraining trans-membrane Peripheral (or extrinsic) membrane proteins integral membrane proteins through intermediate were originally operationally defined as proteins that peripheral membrane proteins and other could be removed from membranes without components9. destroying basic membrane microstructure1,13. This Even by 1972 there were examples of was an operational not an exact definition to help restriction of mobility of integral membrane explain the roles of different membrane proteins in components and involvement of the cytoskeleton in defining basic membrane micro-structures. translocation of membrane components. For Peripheral membrane proteins do not have to be example, during antigen capping certain initially strictly globular in structure, and certainly not mobile cell surface antigens, even when present in amphipathic, and by definition, they include any small mobile clusters, were found to be trapped into Robinson proteins with extensive β-sheet structures large, relatively immobile trans-membrane that can bind to membranes mainly by ionic and complexes in a temperature- and energy-dependent other interactions3,5. process involving cytoskeletal elements65-67. This Soon after the F-MMM proposal1, it became process eventually resulted in cytoskeletal-mediated apparent that another class of membrane proteins endocytosis of some but not all of the large was needed. This new class of membrane-associated macromolecular complexes (receptor-rich domains proteins was proposed, even thought they are not or “receptor patches”)68. We now know in lymphoid strictly membrane proteins9. They are cytoskeletal cells that antigen clustering, domain formation, and associated signaling proteins at the inner internalization, acidification of the resulting membrane surface and certain glycoproteins and endosomes, degradation, and membrane recycling linked glycosaminoglycans at the outer membrane are all part of the normal lymphocyte activation surface (see Figure 1 of reference9). These process68. membrane-associated components are thought to be The organizational structures that mediate involved in stabilizing membranes (and thus cells) trans-membrane linkages between clusters of and immobilizing membrane components to the integral membrane receptors and the cytoskeleton extracellular matrix or to cytoskeletal networks were ultimately found to be much more complex inside cells. There they can function as parts of than the cartoons of the day9. They are now thought adhesion structures or cell motility traction points. to involve multiple membrane peripheral proteins, Thus, these protein components are membrane- lipid-protein-receptor domains, and enzymes that associated but not directly involved in the integral assemble into a submembrane plaque or microstructure of cell membranes, and membrane supramolecular structure that secures the membrane structure is not dependent on their presence. to a complex system of cytoskeletal elements69-71. However, they are important in maintaining The mobility of integral membrane membrane function and dynamics, and they are components can also be restricted by cell-cell and especially important in various cellular activities, cell-matrix interactions. When cells are bound to such as cell adhesion and its stabilization, cell other cells or to extracellular matrix, at least some of motility and spreading, endocytosis, exocytosis and their membrane receptors are immobilized in the many other important actions9. process72-74. Cell adhesion complexes that are immobilized Cytoskeletal and Extracellular Matrix Membrane by matrix interactions are capable in some cells of Interactions communicating signals that are transmitted through a Cytoskeletal and extracellular matrix membrane- dynamically assembled actin-containing cyto- associated interactions can alter cell membrane skeleton to generate mechanical forces that can macrostructure by causing reductions or restrictions move cells or resist exterior mechanical stresses75,76. in freedom of movement or mobility and by causing This serial system of highly specialized global movements of membrane glycoprotein and glycoproteins and proteins (extracellular matrix, lipid domains by tethering these complexes to integral membrane proteins, peripheral membrane cellular or extracellular structures9,10. This later proteins, adaptor proteins, cytoskeletal elements, situation can occur when cell membrane-associated among other components) may have evolved to www.discoveriesjournals.org/discoveries 5 Updated Fluid-Mosaic Model of cellular membranes convert biochemical signals into mechanical forces membrane protein and glycoprotein complexes are that are important in cellular behavior. Along with not made up of single proteins or glycoproteins88-90. better-known biochemical signaling pathways, these Membrane integral protein-protein interac- molecular-mechanical pathways have been proposed tions, which can be driven by ligand binding, are to be key regulators of cell function76. involved in the dynamic formation of trans- Early proposals on the role of membrane- membrane signaling complexes. Eventually the associated cytoskeletal elements and their influence complexes can become activated for recruitment of on membrane structure and dynamics have proven to additional peripheral proteins at the inner cell be relatively simplistic9. Even now this complex membrane surface to form supramolecular trans- system is being carefully dissected and its multiple membrane structures that are competent for cellular subcomponents identified71,77-82. For example, we signaling76,90-92. For example, upon binding of cell now know that certain membrane-interacting surface trans-membrane integrin receptors to their components, such as septins, GTPases, and other ligand, the integrin heterodimers are thought to components, can form higher-order bundles, undergo a ‘bending’ conformational change that filaments and even ring structures that bind to actin allows recruitment of submembrane plaque proteins filaments and microtubules83-85. Although membrane that, in turn, directly or indirectly bind to actin peripheral proteins have been identified in complexes linked to the cytoskeleton78. cytoskeletal interactions86, membrane lipids are also Subsequently a potentially larger group of other important in such interactions. Indeed, specialized signaling molecules and enzymes can be bound to phospholipids may regulate interactions between the submembrane supramolecular complexes, certain membrane lipid domains, such as lipid rafts leading to the formation of stable, trans-membrane (specialized lipid domains), using super-complexes78,92. phosphatidylinositol isoform-binding proteins87. There are a variety of cell membrane Thus, cells can be considered completely glycoprotein-protein oligomeric complexes that are integrated mechanostructures, and cell membranes involved in cell-cell interactions and the formation are not autonomous and separate from other of specialized super-structures between adjacent intracellular membranes and organelles. They are cells in tissues. These will not be discussed here, but continuously interacting with other cellular the reader can find additional examples elsewhere 10. structures-receiving signals, directing contacts, sending instructions, maintaining cellular polarity Membrane Lipid-Lipid Interactions and mechanical properties, while undergoing As discussed above, membrane lipids are constant turnover of their constituent components. asymmetrically arranged in cell membranes (reviewed in20,21,54-56,58,60). They are also unevenly Membrane Protein-Protein Interactions distributed in the membrane plane (reviewed The majority of membrane proteins and in30,44,62,93-96). Certain lipids change the fluidity, glycoproteins are not likely to be isolated compo- dynamics and lateral structures of cell membranes, nents or complexes floating freely in a fluid lipid such as cholesterol, which as the only sterol present environment, as in Figure 11. Functionally they are and the single most abundant lipid in animal cell often assembled into macromolecular complexes, for membranes is particularly important in the formation example, to initiate signaling process that are of membrane lipid domains21,62,95-97. Lipid-lipid important in a variety of cellular functions. As their interaction studies using mixtures of membrane cellular and biochemical functions have been phospholipids, cholesterol and sphingomyelin have elucidated over the years, it has become much shown the importance of domain structures in model clearer how super-complexes of membrane proteins lipid membranes62,98,99. and glycoproteins (domains) perform a variety of Cholesterol is particularly important in cell cellular functions. membrane lipid organization62,96-99. This is thought To demonstrate their functional activities to be due, in part, to cholesterol’s “schizophrenic” membrane proteins have been cloned, sequenced and affinity for fluid and solid phases of membrane lipid functionally expressed15-17. In such studies, single bilayers96. Cholesterol partitions into liquid ordered membrane components can be involved, but usually and disordered phases to roughly the same extent

www.discoveriesjournals.org/discoveries 6 Updated Fluid-Mosaic Model of cellular membranes and changes the properties of different lipid phases and a spectrum of submicro- or nano-domains may or lipid domains95. exist that contain different lipid and protein In plasma membranes sphingolipids are also compositions, physical characteristics and important in the formation of more-ordered functions106. There may be a limited number of membrane lipid domains35,94. Sphingomyelins and allowed lipid compositions or combinations that can phosphatidylcholines constitute more than one-half form lipid domains, and these domains are not of plasma membrane phospholipids and form the randomly distributed; they tend to adopt a main partners for cholesterol interactions94,100. superlattice structure in membranes110. Sphingomyelins and cholesterol are critically important in the separation and formation of ordered Membrane Lipid-Protein Interactions lipid domains (lipid rafts) that are generally As mentioned in the section above, integral surrounded by liquid phase lipids20,94,101. membrane proteins can interact with different The formation of more ordered lipid phases in membrane domains, but they must also interact with plasma membranes is generally important in membrane lipids, in order to produce an intact membrane domain formation and, in particular, the plasma membrane. Specifically, portions of their lipid raft hypothesis20,35,44,45. The formation of structures must directly interact with the acyl chain membrane lipid domains or rafts is now thought to portions of membrane phospholipids or the be a dynamic and reversible process that confers hydrophobic portions of other membrane lipids. functional change at the outer surface of plasma This is accomplished by hydrophobic matching membranes30,35,45,102-104. Lipid domain formation between the hydrophobic lipid bilayer acyl core and appears to be driven by multiple forces, such as a stretch or combination of hydrophobic amino hydrogen bonding, hydrophobic entropic forces, acids15,16,21,43,48,111. charge pairing and van der Waals forces62,101. The concept of hydrophobic matching Lipid domains or rafts in plasma membranes between the hydrophobic core of the lipid bilayer contain specific lipids, integral proteins and and hydrophobic stretches of amino acids in integral peripheral proteins, and they can be platforms for membrane proteins was essential for understanding signal transduction and other cellular functions35, 104- the formation of a stable membrane structure96,111-114. 106. Plasma membrane lipid rafts are now thought to If the hydrophobic portions of this structure are constitute functional, dynamic nano-sized domains mismatched, an elastic distortion of the lipid matrix of diameter <300 nm (most ~10-200 nm) that are around the integral membrane protein occurs48,96,114. characterized by enrichments of cholesterol and In order to produce an appropriate structure sphingolipids31,37,45,107. However, cell membrane hydrophobic matching of particular lipids adjacent to lipid rafts were found to be much smaller than the membrane proteins (boundary lipids) must take lipid domains found in artificial membrane bilayers, place, or there will be an energy penalty that results and their boundary lipids were found to exchange in an elastic distortion of the lipid matrix rapidly (every 10-100 nsec) with lipids in the bulk immediately around the integral protein96,111-114. If membrane. They also tended to exclude unsaturated the penalty is large enough, the integral protein may phospholipids and cholesterol from their undergo a conformational change, and this could boundaries108. potentially cause effects on protein function. It Integral and peripheral membrane proteins could also affect protein-protein interactions and may be sequestered into plasma membrane lipid result in integral protein aggregation in the rafts30,104,106,109. Neuman et al.106 have discussed the membrane plane114. properties of lipid rafts (and other membrane There exists a range of protein interactions domains) that make them biologically important, with lipids in cellular membranes, and these are such as their involvement in cellular processes: apparently controlled by the coherence lengths of the endocytosis, signal transduction, cell death, among interactions between proteins and their boundary other events. Lipid rafts are dynamic structures, and membrane lipids. If very large, this could result in the lipids in these domains can quickly exchange capillary-condensation phenomena and wetting with lipids in the bulk fluid membrane as well as around the integral protein43,115. Other interactions, other rafts. Neuman et al. speculated that there may such as electrostatic interactions between charged be different turnover rates for each raft constituent, amino acids and phospholipids, can complicate this www.discoveriesjournals.org/discoveries 7 Updated Fluid-Mosaic Model of cellular membranes picture, and Mouritsen114 anticipated that under stress53. This property has some similarities to certain circumstances electrostatic interactions might hydrophobic matching, and the binding of integral even overcome hydrophobic matching. Lipid proteins to particular lipids could change the preference for certain integral proteins is proposed to conformation of nearby integral proteins, for result in capillary condensation, and if this occurs example, to open or close membrane channels113. around two or more integral membrane proteins, it Alternatively, the binding of peripheral membrane can result in wetting and the formation of a capillary proteins directly to the lipid head groups could condensate between adjacent integral proteins. This decrease or promote lipid curvature as the lipids can produce a lipid-mediated force that drives the conform to the protein shape53. formation and stabilization of integral protein In addition to hydrophobic matching of complexes96,114,115. proteins and lipids in cellular membranes, there are The hydrophobic matching principle may also additional physical considerations, such as lateral be an essential property in the formation of pressure forces, lateral lipid composition and phases, specialized lipid domains or rafts (see previous membrane curvature, ionic interactions, among section), and it could also be an important others, that must be taken into account to produce an mechanism for selective partitioning of integral overall tensionless membrane structure43,96,112,113,115. proteins into specialized membrane lipid domains. To be sequestered into a lipid domain an integral Membrane Restrictions on Lateral Mobility protein’s hydrophobic structure must match up with We now know that integral membrane proteins are the hydrophobic thickness of the domain115. If the not necessarily completely free to laterally move in a hydrophobic portions of this structure are fluid lipid matrix, as originally proposed1. In fact, mismatched, there will be an elastic distortion of the there are subtle restrictions on the lateral movements boundary lipid matrix around the integral membrane of most integral membrane proteins, and at least protein. Without hydrophobic matching of particular some lipids, in cellular membranes (reviewed boundary lipids immediately adjacent to particular in9,10,26,27). Restrictions on the lateral movements of membrane proteins, there will be an energy penalty integral membrane proteins have been linked to: (a) that causes an elastic distortion of the boundary lipid extracellular restrictions, such as binding to matrix96,114. If the energy penalty is large enough, the extracellular matrix, (b) the formation of specialized integral protein may undergo a conformational membrane domains, such as lipid rafts, (c) the change, and this could potentially cause effects on formation of large, supramolecular protein protein function. It can also result in the exclusion complexes and (d) the formation of peripheral of certain lipids, such as cholesterol, from the membrane barriers at the inner membrane surface, boundary lipid layer due to unfavorable membrane such as membrane-associated corrals or skeletal protein hydrophobic matching96,114. fence works10,22,23,26-28,41,69. As mentioned above, lipid boundary effects Reviewing the results from various optical can also cause changes in protein-protein methods that have been used to follow the dynamics interactions that could result in membrane integral of cell surface integral proteins, Jacobson et al.23 protein aggregation in the membrane plane. By have placed the lateral movements of membrane examining the rotational diffusion rates of rhodopsin proteins into four main categories: (a) transient in reconstituted bilayer membranes, Kusumi and confinement by obstacle protein clusters (fenceposts Hyde108 were able to relate specific phospholipid or pickets) (Figure 2, A); (b) transient confinement acyl chain-lengths to the state of rhodopsin by the cytoskeletal meshwork into defined domains aggregation. They found that hydrophobic mismatch or corrals (Figure 3, B); (c) directed motion by direct between proteins and lipids is unfavorable or indirect attachment to the cytoskeleton (Figure 5, energetically, but the mismatching can be minimized C); and (d) free, random diffusion in the fluid by the transient formation of protein-protein membrane (Figure 5, D). A slightly different list complexes. was produced by He and Marguet35, who Another property important in lipid-protein characterized the categories of lateral movement as: interactions is the tendency of certain lipids to (a) free diffusion; (b) movement limited by induce curvature stress and the ability of certain meshwork barriers (such as fences or corrals); and membrane peripheral proteins to overcome this (c) movement limited by traps and domains (such as www.discoveriesjournals.org/discoveries 8 Updated Fluid-Mosaic Model of cellular membranes lipid rafts). Thus, the original description of integral membrane protein diffusion (reviews:9.66.117). More membrane proteins freely diffusing in the membrane recently the impedance of mobility of membrane plane (Figure 1) pertains to only one of these components can now be directly related in many cell modes. types to cytoskeletal fencing and the formation of It is now well known that a substantial portion cytoskeletal corrals (see Figure 2)23,26-28. of integral membrane proteins are confined, at least The partitioning of plasma membranes in transiently, to small membrane domains by lattices order to limit the dynamics of their integral and corrals, and they are thus not freely diffusing in membrane protein components, at least part of the the membrane plane (reviewed in10,26,27). Integral time, to fenced corrals, or tethering them directly or proteins can escape from one domain to an adjacent indirectly to membrane-associated cytoskeletal domain, and even escape such domains altogether, elements, creates relatively stable membrane zones and this may be related to the sizes of their (domains) of enhanced receptor densities. Also, cytoplasmic structures, their cytoskeletal and extracellular networks or lattices can potentially extracellular interactions, and their abilities to partition the plasma membrane into stabilized dyamically undergo protein complex formation26,27. domains9,34. Moreover, the trapping of mobile The approximate areas of plasma membrane integral membrane proteins inside a corral receptor domains have been estimated from 0.04 to constructed of cytoskeletal fencing (or extracellular 0.24 μm2, and the approximate transit times of lattices) may be dependent on the state of protein membrane receptors in these membrane domains can complex formation. Some integral protein range from 3 to 30 sec27-29. Overall, cell membrane monomers have been found to escape from corrals, domains can range in diameter from 2-300 nm. For but not their oligomeric complexes, or at least they example, actin-cytoskeletal fence domains have been cannot escape at the same rates28. Therefore, found in the range of 40-300 nm, lipid raft domains membrane corrals (or extracellular lattices) may in the range of 2-20 nm, and dynamic integral selectively limit free diffusion in the membrane membrane protein complexes in domains of 3-10 plane, and at the same time they present enhanced nm27. Cells must process many types of signal receptor densities in specific membrane domains27. mechanisms, and the use of different types of cell Cell membrane dynamic compartmentalization membrane domains may allow another level of or the enhanced presentation of specific components signal selection and complexity. in specialized domains may be important in signal transduction, cell activation, cell differentiation, Membrane Hierarchical Organization identification, and other complex membrane events. Cells possess dynamic, multi-dimensional plasma This can occur by changing the range of movements, membrane architectures so that they can quickly distributions and collision rates of various cellular respond to intracellular and extracellular signals, and receptors, and thus affecting their display and ability environmental events. Kusumi and colleagues26,27 to associate into higher order complexes. Kusumi et have proposed that plasma membranes are organized al.27 have proposed that plasma membranes possess into dynamic hierarchical structures. Within these hierarchical architecture consisting of various hierarchical structures membrane components are membrane domains or compartments, such as: limited in their diffusion rates from 5- to 50-times cytoskeletal limited domains or corrals formed by slower than when the same components are cytoskeletal fences, fenceposts or pickets; lipid raft reconstituted into artificial membranes. Conversely, domains; and dynamic, oligomeric integral the macroscopic diffusion rates in cell membranes membrane protein domains that may or may not be can also be increased 20-fold through disruption of linked to the cytoskeleton, among other possibilities. membrane-associated cytoskeletal networks27. However, the basic nano-scale membrane unit would The notion that membrane-associated still be a fluid-mosaic membrane based on the 1972 cytoskeletal networks can slow and limit the model26,27. mobility of trans-membrane integral proteins compared to free lateral diffusion is not a new An Updated Fluid—Mosaic Membrane Model concept and was discussed previously9,22,23,41,116. Some forty years after the original F-MMM Indeed, cytoskeletal-disrupting drugs have been proposal1, one would expect that significant and known for some time to change the rates of integral extensive revisions would be necessary24,25. www.discoveriesjournals.org/discoveries 9 Updated Fluid-Mosaic Model of cellular membranes

Figure 3. An updated Fluid-Mosaic Membrane Model representation that contains membrane domain structures, membrane - associated cytoskeletal and extracellular structures. The cell membrane has been pealed back to the left to reveal the bottom membrane surface and membrane-associated cytoskeletal elements that form barriers (corrals) that limit the lateral motions of some of the integral membrane proteins. In addition, membrane-associated cytoskeletal structures are directly interacting with integral membrane proteins at the inner membrane surface along with matrix components at the outer surface. Although this diagram presents possible mechanisms of integral membrane protein mobility restraint, it does not accurately represent the sizes and structures of integral membrane proteins, lipid domains or membrane-associated cytoskeletal structures.

However, the basic nano-scale Singer-Nicolson Finally, as discussed throughout this brief model1 has been found worthy, even if it is not review, the plasma membrane is not a static, entirely accurate when lipid properties, complex autonomous cellular structure. It is linked in several membranes domains, and higher hierarchical levels ways to the cell cytoplasm through cytoskeletal of mosaic organization are considered10,21,26,27,97. networks, signal transduction systems, transport Although a number of shortcomings of the 1972 systems, and other structural, enzymatic and model have been noted previously19,22,27, the basic communication networks. In tissues, it is also linked nano-scale part of the F-MMM does not require outside the cell to extracellular matrix, other cells, extensive revision beyond the 1976 update published and to interstitial protein structures. Thus, cell previously9. What the F-MMM does require is an membranes are fully integrated structures within update that takes into consideration events and tissues, and they are sensitive and reactive to structures above and below the basic membrane, environmental changes and signals. This is probably along with some of the unforeseen cell membrane why plasma membranes have evolved to become domain and boundary properties absent in the earlier such complex, dynamic structures. They have to models. Thus an updated F-MMM must take into quickly and selectively respond to a number of account contributions that have been made since the disparate signals from within and outside cells. 1970s, such as recent data on boundary lipids, These subtle, dynamic and sensitive cellular membrane lipid and protein domains, cytoskeletal structures will continue to fascinate researchers who corrals, extracellular matrix, and other properties and seek to uncover their secretes. conditions that were almost completely unknown in the 1970s. Obviously many of these criticisms Acknowledgements: cannot be easily addressed in any static cartoon This contribution was supported by donations to the model of the plasma membrane, but most of the Institute for Molecular Medicine. The assistance of important criticisms and newer information have Mr. John Michael is gratefully acknowledged. been incorporated into an undated model (Figure 3). What is difficult to portray in any cartoon is the Conflict of Interest: concept that the components of cell membranes The author has no conflict of interest to disclose. function in various domains as non-uniform, non- random, cooperative, dynamic elements in thermodynamic equilibrium21.

www.discoveriesjournals.org/discoveries 10 Updated Fluid-Mosaic Model of cellular membranes

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