THE EVOLUTION OF MEMBRANES

THE EVOLUTION OF MEMBRANES

Lipid Diversity And The Physical Properties Of Membranes

The modern era of membrane study essentially began with the fluid-mosaic model [1]. One class of questions that has recurred frequently since then concerns the large diversity of lipids found in biological membranes. When one takes into account the different polar head groups and acyl chains, a given membrane may often contain well over 100 unique species. Does each lipid have a specific functional role? Or, is the diversity of lipids in many cell types and the non-random differences in lipid composition between different cell types a manifestation of accidental historical 86 M. Bloom and O.G. Mouritsen development rather than the importance of specific lipid composition? At the present time, “...there is no satisfying explanation for the observed patterns” ([1], page 32).

Some of the systematics are immediately understandable in a qualitative sense in terms of optimization of physical properties. With the appreciation of the importance of membrane fluidity, it soon became clear that many organisms actually adjust their lipid composition in response to changes in environmental parameters such as temperature in order to preserve membrane fluidity ([1], page 173). For example, it is well known that lipids with saturated acyl chains have higher melting temperatures than those with unsaturated chains. The mono-unsaturated lipid POPC has a transition temperature about 50 C lower than the saturated lipid DPPC. Many organisms systematically increase (or decrease) the concentration of unsaturated relative to saturated chains in their lipids when the growth temperature is decreased (or increased). The ability of organisms to make such compositional adjustments is presumably due to the enzymes responsible for the synthesis of unsaturated chains having higher activation energies, equivalent to higher temperature-derivatives of their activation rates, than those of enzymes responsible for the synthesis of saturated chains in lipids. This selection of enzymes driven, not simply on the basis of enzymatic efficiency but also by its temperature-derivative, must have occurred via evolutionary processes.

In a similar vein, we have proposed in section 7 a scenario for the optimization of the physical properties of plasma membranes of eucaryotic cells, these membranes having to satisfy special mechanical considerations because of their relatively large size. In this case, nature seems often to require a preponderance of unsaturated lipids, mono-unsaturated for red blood cells and poly-unsaturated for the central nervous system (see section 8.3). Why mono-unsaturated in one case and poly-unsaturated in the other is not known – it would seem that both satisfy fluidity needs. These plasma membranes always have large concentrations of cholesterol ( 30 to 50 mol%). We suggested in section 7 that this is because sterols extend the fluid range, when alloyed with di-acyl lipids, while eliminating the phase transition completely, reducing the passive permeability of membrane and greatly increasing its mechanical strength.

It is likely that many other examples of lipid diversity can be explained in terms of the optimization, via evolution, of the physical properties of membranes. We close this final section with brief accounts of three explicit cases that are presently being studied in many laboratories. Each of these cases focuses on different ways in which evolutionary forces have manifested themselves in the diversity of lipids. Lipid polymorphism brings us into contact with the role of symmetry and the breaking of symmetry in membranes as well as the influence of membrane curvature. Skin lipids give an example of lipids being modified in situ in order to play slightly different physical roles as they move from deep in the skin to the surface. Brain lipids bring us into contact with an important aspect of human evolution, brain development, and how that was made possible through the availability of new types of materials, essential fatty acids. These three topics are large ones and our treatments are brief and not intended as reviews. We hope that they will encourage further work from an evolutionary perspective on the manner in which specific lipids have been used to optimize physical properties of membranes of a variety of organisms.

a. Lipid Polymorphism

One of the areas of modern physics research closely coupled to membrane biophysics is the study of liquid crystals, especially, that of lyotropic liquid crystals, i.e. liquid crystals whose properties depend strongly on water concentration as well as on temperature. The lipid structure of prime importance to cell biology is the fluid bilayer. Lipids that spontaneously aggregate in a bilayer structure, in combination with proteins under physiological conditions, will usually but not always, form lamellar arrays having long range order when mixed with water. Such an aggregate, when fluid, is called the (lamellar) L phase and is comprised of a periodic arrangement of lipid bilayers of thickness dl alternating with water layers of thickness dw to define a one-dimensional liquid crystal of repeat distance equal to dl + dw.

It has been known for many years that lipid-water mixtures can give rise to a variety of liquid-crystalline structures of different symmetries. Their long range order can be elucidated using standard methods such as X-ray diffraction. In addition to the L phase, two types of hexagonal phases are found, corresponding to two-dimensional arrays of hexagonally coordinated cylinders in which the lipid acyl chains are oriented inside (HI) or outside (HII) the cylinders. As illustrated in fig. 9, the molecular origin of the hexagonal phases can be understood qualitatively [39, 66] in terms of the shapes of the lipid molecules. Not shown in fig. 9, but discussed extensively in the paper from which this figure was taken [39], is the cubic liquid crystal phase consisting of cubically coordinated cylinders, mentioned previously (see section 5) as being potentially relevant to archaebacterial membranes [38]. Cubic phases are found, for some lyotropic liquid crystalline systems, in a narrow range of water concentrations between the low concentrations at which the L phase is stable and the higher concentrations at which the HII phase is stable [67].

Biological membranes are not liquid crystalline in the sense of having long range order so that one does not expect to see hexagonal phases in natural materials. Still, the study of hexagonal liquid crystalline phases has been of great value in focusing on potential structural and transitional roles of local cylindrical geometry in biological function.

As an illustration of a potentially important structural role played by local cylindrical geometry, consider the tight junction, which is crucial to the function of epithelial sheets. To quote from a well-known cell biology text [13]: from page 29, “The epithelial sheet has much the same significance for the evolution of complex multicellular organisms that the membrane has for the evolution of single cells.” And from page 793, “Epithelia have at least one important function in common: they give us selective permeability barriers separating fluids on each side that have different chemical compositions. Tight junctions play a doubly important role in maintaining the selective-barrier function of cell sheets”.

It is clear from reading further in the text of Alberts et al. [13] that, though the structure of the tight junction is not known with any confidence at this time, the authors favour a protein-based structure for the strands that produce the symmetry breaking required to couple the two bilayers and maintain the permeability barrier required of the tight junction. Nevertheless, it has been proposed, on the basis of 88 M. Bloom and O.G. Mouritsen

Fig. 10. a: diagram of a cross-section of a tight junction illustrating the offset disposition of a pair of intramembrane cylinders. b: diagram illustrating the paired offset cylinders at a tight junction strand and the different possible planes of fracture through such a junction. c: proposed organization of the phospholipids at a tight junction strand. d: diagram of phospholipids combined with freeze-fracture micrograph to show how fractures through lipid micelles could produce images characteristic of tight junctions. (From ref. [68]).

Freeze fracture electron microscopy [68], see fig. 10, that a pair of intramembrane

Fig. 9. Structure of different anisotropic liquid crystalline phases that membrane lipids can form. (A) Normal hexagonal (HI ) phase in which the acyl chains are in the interior regions of the hexagonally coordinated cylinders; (B) lamellar (L) phase; and (C) reversed hexagonal (HII) phase in which the acyl chains are in the exterior regions of the hexagonally coordinated cylinders. We do not show the cubic liquid crystal phases discussed extensively in the paper [66] from which this figure is taken. (Fromref. [39].)

Cylinders composed of lipids, i.e. of the type that form the building blocks of the HII phase, may well be the principal structural elements comprising tight junctions. If this proposal is correct, then the structure of tight junctions represents a solution via evolutionary methods to a physical symmetry problem, how to couple lamellar structures to each other in such a way as to maintain good control of permeability between them, while the system participates in biological activity. The proposed solution makes use of the range of possibilities opened up by the variety of molecular shapes of the lipid building blocks of membranes. Note that a protein based solution to this problem would involve the same type of symmetry considerations.

There have been many candidates [39, 66, 67, 69, 70] proposed as probable biological manifestations of lipid polymorphism via transitional states of membranes. As in the case of structural examples such as the one described above, most of these candidates involve a change in local symmetry, transient in this case, which is made possible by local cylindrical symmetry of the boundary between the hydrophilic and hydrophobic regions of the lipid-water interface. The fusion of pairs of liposomes, budding of small vesicles from larger liposomes, endo- and exo-cytosis are all related to each other and are examples of changes in the topology of the membrane surfaces. In the transition from one vesicle to two or vice-versa, say, there must be a transitional state in which some part of the local membrane surface deviates appreciably from lamellar symmetry. The study of such changes of topology and shapes is currently an active and important area of both theoretical and experimental study [67, 69, 70]. Though most of the work underway at the present time is on model systems, we anticipate that these studies will have an impact on our understanding of physiological processes in the near future.

b. Skin Lipids - Modification Of Lipids In Situ To Provide A Water-Resistant Surface

The properties of skin lipids give an example of how nature has evolved a structure to provide protection of large scale biological organisms from potential environmental dangers, as in water loss, microbial invasion, ultraviolet irradiation, mechanical trauma, etc. We discuss here, very briefly one of these features, water loss, to make the point that as the cells making up human skin, for example, move from the inner (dermis) to the outer (epidermis) layers (see fig. 11), systematic modifications of the lipids take place in such a way as to ensure that, among other things, the outer (stratum corneum) layer of the epidermis is relatively water repellent. This can only be done with a significant change in cellular structure as ordinary cells are relatively water permeable. The detailed structure and thermodynamic phase properties of the epidermis and its associated epidermis has yet to be worked out. Those readers wishing to learn about recent work in this field as seen by the specialists in the field should consult the literature [71]. We are indebted to Dr. Neil Kitson for his assistance in providing the following extremely simplified description of a complex system.

The epidermis, though only the outer layer of the skin, is itself a composite, but is composed mainly of cells derived from one type known as keratinocytes. There are other cells but no blood vessels; a complex vascular network is located immediately beneath the epidermis in the layer known as the dermis.

Fig. 11. Schematic diagram of the stratum corneum in relation to the overall geometry of the epidermis. The upper part of the figure shows its location and thickness in relation to the outer layers of the skin, with an expanded sketch indicating the type of network of diffusive paths whereby water is believed to pass through the stratum corneum. The lower part of the figure shows the manner in which terminally differentiated epidermal cells (corneocytes) inthestratum corneum are surrounded by intercellular membranes described as ‘lamellar lipid sheets’. (This figure was provided courtesy of Dr. Russell O. Potts.)

The epidermis operates in a manner somewhat analagous to that of blood. In the formation of blood, a population of cells (‘stem cells’) spends its time making new cells. These new cells differentiate into ‘mature’ cells (red blood cells) which are biologically useful for short periods of time, ⁓ 120 days in humans [13], and are then lost, destroyed or recycled. In the case of the epidermis, the stem cells live at the bottom of the epidermis, next to the dermis, and the maturing cells move up towards the skin surface. It takes them about two weeks to move from the bottom to the top of the epidermis where they are discarded as dust. The equivalent of the red blood cell is the ‘corneocyte’, and the equivalent of the circulating blood is the stratum corneum, the very top layer of the epidermis. Corneocytes are filled with keratins (rather than hemoglobin), are glued together with intercellular connections (called desmosomes), and have between them unusual lipid lamellae that provide the waterproofing.

During the maturing process that occurs between the stem cells on the bottom of the epidermis and the stratum corneum on the top, the keratinocytes make small organelles (‘lamellar bodies’) filled with lipids and lipid metabolizing enzymes. These are eventually pushed out of the cell (exocytosis) and fuse with each other to form myelin-like layers around and between the cells. This intercellular lipid is usually referred to as the ‘stratum corneum lipid’ or the ‘intercellular matrix’ or ‘intercellular domains’. It is technically difficult to analyze the lipid composition at various levels of the epidermis. However, there is general agreement in the field now that, from the time they are synthesized in the lamellar bodies until they form mature inter- cellular membranes, three major classes of lipid are represented in approximately equal concentrations: sphingolipids, phospholipids and sterols. During the maturing process, enzymatic modifications take place so that a recent model membrane study [72] could reasonably be based on the assumption that a better approximation to the lipid composition just before the cells are discarded is a three-component equimolar mixture of free fatty acid, ceramide and cholesterol.

The study of skin lipids may have a special twist with regard to evolution. After nature had evolved soft materials, it had to find a way of modifying them so as to provide the special protective features of the skins of animals while using the soft materials as a starting point. The precise way in which this has been done is still not really known, but should be amenable to study using modern physical methods.

c. Essential Fatty Acids And Brain Lipids

The role of dietary fatty acids in the evolution of the human brain has been reviewed by Crawford [73] and discussed in relation to a broad evolutionary perspective in a book aimed at the general reader [74]. In this section we present a brief account of some of the arguments made in the review. An important feature of this discussion is that brain lipids contain a very large fraction of poly-unsaturated fatty acyl chains, which mammals are unable to synthesize because they lack the enzymes to introduce double bonds at carbon bonds beyond C-9 in the fatty acid chain [75]. In humans the poly-unsaturated lipids that seem to be of importance to the central nervous system are formed via elongation and desaturation from essential fatty acids (EFA), where the word essential means that they must be supplied in the diet and cannot be endogenously synthesized. The EFA’s occur as linoleic (n-6) and  -linoleic (n-3) acids, which are elongated and desaturated from 18-carbon chain lengths with two or three double bonds to 20- and 22-carbon chain lengths with four and six double bonds. Since the human brain evolved quite recently on the evolutionary time scale, it will be interesting to understand the physical role played by these poly-unsaturated fatty acids in brain lipids, and why nature evolved a brain system dependent on material derived from the local environment rather than being under the control of the cellular synthetic apparatus. The book by Crawford and Marsh [74] examines the consequences, in evolutionary ‘theory’, of important steps “being dictated by chemistry and physics responding to the prevailing conditions”. Such considerations would, for example, play a role in the relatively simple case of magnetotactic bacteria described in section 2 of this article, where the size distribution of the bacterial magnets is undoubtedly a reflection of the amount of iron available in the bacterial diet.

As expressed by Crawford [73]: “Brain chemistry is characterized by two unique features. First, the brain maintains a constant flow of ionic and electrical information without which it dies. Its sophisticated communication network is achieved by transmembrane transfer systems with an outstandingly heavy investment in lipid biotechnology: some 60% of its structural material is lipid. Brain lipid is composed of cholesterol and phosphoglycerides that are rich in the preformed fatty acids, primarily arachidonic and docosahexaenoic (DHA), and not the parent EFAs. For instance, in the rat, the 20- and 22-carbon long-chain fatty acids are incorporated into the developing brain ten times more efficiently than are the parent EFAs. Most experts agree that both n-6 and n-3 families of fatty acids are required in the diet. It is also clear that the dietary supply of EFAs is limiting for brain growth”.

Some of the physical implications of the correlations discussed by Crawford [73], deserve serious study from an evolutionary perspective: e.g., correlations between the use of DHA and the emergence of important biological features in human evolution such as the development of a photoreceptor, synaptic membranes and the role of inositol phosphoglyceride in its involvement with calcium transport. Such studies could lead to a better understanding of past and future human evolution. In addition, the inherent limitations in brain development now known to result from maternal malnutrition due to inadequate supplies of EFAs in the diet of babies in some third world countries provides a social motivation for serious study of how EFAs influence the physical aspects of fundamental processes in the central nervous system.

REFERENCE:

CHAPTER 2

The Evolution of Membranes;

M. BLOOM, Canadian Institute for Advanced Research & Department of Physics, University of British Columbia, 6224 Agricultural Road, Vancouver, B.C., Canada V6T 1Z1.

O.G. MOURITSEN, Canadian Institute for Advanced Research & Department of Physical Chemistry, The Technical University of Denmark, DK-2800, Lyngby, Denmark.

1995 Elsevier Science B.V. All rights reserved, Handbook of Biological Physics, Volume 1, edited by R. Lipowsky and E. Sackmann