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PERSPECTIVES

OPINION

Cell evolution and the problem of membrane topology
Gareth Griffiths

Abstract | Cells somehow evolved from primordial chemistry and their emergence depended on the co-evolution of the cytoplasm, a genetic system and the cell membrane. It is widely believed that the cytoplasm evolved inside a primordial lipid vesicle, but here I argue that the earliest cytoplasm could have co-evolved to high complexity outside a vesicle on the membrane surface. An invagination of the membrane, aided by an early cytoskeletal system, may have formed the first cells — initially within primordial vesicles.
Genomic analysis leaves no doubt that the three main kingdoms of life — eubacteria, archaea and eukaryotes — evolved from a common ancestor1. The idea that all life evolved from one source is perhaps the most profound consequence of the Darwin–Wallace model of evolution. With increasing amounts of DNA sequence information available, the search is on to find the minimum set of genes that existed in the last common ancestor (LCA) — the precursor cell(s) from which all living organisms subsequently evolved. The LCA must have been a rather sophisticated cell because it contained all the cellular machinery that is common to all present living forms, including a DNA-based information system2,3. From sequence analysis, the LCA is suggested to contain 250–600 genes4,5. Mycoplasma genitalium, the simplest known prokaryote cell (which is nevertheless dependent on host-cell parasitism), requires 270 of its 380 genes for normal function, as revealed when all genes were individually knocked out6. So, the minimum conceivable cell needs a lot of genetic information. The Earth is ~4.56 billion years (Gyr) old, and the best current estimates argue that the first cells appeared by 3.0–3.3 Gyr at the latest7. How these first cells emerged is one of the biggest unsolved problems in biology. It seems undeniable that the LCA must have been preceded by a spontaneous generation of cells from abiotic precursor
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molecules. Even Darwin in his theory of evolution was obliged to accept the necessity of some kind of spontaneous generation. He brilliantly speculated (in a letter to Hooker) that in a “warm little pond” a particular chemistry involving “all sorts of
Acidic Glu Asp 1 RNA Mineral surface chemistry Asn Gln 2 3 Polar uncharged Ser Pro Val Ala 4 Gly Cys 5

ammonia and phosphoric salts, light, heat, electricity etc. was present so that a protein compound was chemically formed ready to undergo more complex changes on the path towards life”8. It seems likely that his (unpublished) hypothesis was not far from the mark. Modern theories were initiated by Oparin in 1924 (REF. 9) and by Haldane10, who first discussed the origin of membranes and speculated that an ‘oily film’ on the surface of sea water evolved into the lipid-rich cell membrane. Here, I attempt to build up a plausible scenario for how cells may have evolved — with an emphasis on the more difficult question of the origin of the cell membrane. A brief discussion of the (more actively pursued) question of the evolution of cytoplasm, including the nucleic acids, sets the stage for the main hypothesis.
The evolution of the cytoplasm Cells are made up of nucleic acids, proteins, carbohydrates, lipids and many small molecules suspended in a particular ionic
Basic Phe Tyr Aromatic His Trp

Non-polar hydrophobic Thr Ile Met Leu 7 8 9

Lys Arg 10

6

11

12

13

14 DNA

Evolutionary time Vesicles

RNA-DNA Phospholipids

Self-synthesizing membrane

Figure 1 | Biosynthetic pathways for amino acids, phospholipids and central metabolism. Nature Reviews | Molecular Cell Biology It has been proposed by Davis22,26 and many others that primitive biochemical pathways existed before a genetic-based system. These include the reductive carbon cycle (the equivalent of the tricarboxylic acid (TCA) cycle working in reverse), the reductive pentose pathway and the central trunk (proposed to be a remnant of the formose cycle), which together make up the central biochemical pathway (CBP). Davis proposes that the emergence of genetically encoded amino acids correlates with the number of chemical reactions from the CBP that are required to generate each amino acid (evolutionary steps 1–14). Acidic amino acids (Glu and Asp) are close to the CBP (1–2 reactions), whereas aromatic residues (such as Trp) require up to 14 steps to be synthesized and may have appeared later. The hydrophobic amino acids that could associate with membranes required four steps. The synthesis of phospholipids requires 10 steps and, therefore, self-synthesizing membranes might only have arisen after this point. Davis provides detailed analysis to argue that the evolutionary appearance of the different amino acids correlates well with the emergence of their corresponding triplet codes; this implies co-evolution of biochemistry and the genetic code, an idea extensively championed by Wong60. Davis also identified an 11-amino acid sequence in the FtsZ–tubulin family that he mapped to his evolutionary stage 7.5, which could support a role for this protein in cellularization. Gly, Cys and Pro cannot easily be placed into any of the five categories shown.

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When such lipids were extracted with solvents from the Murchison meteorite.18 rather than the high-salt ocean environment proposed by others10. the simplest bilayer-forming lipids are long-chain (>C9) fatty acids. as discussed below. a | A pure lipid vesicle in contact with a protocytoplasmic milieu. Additional proposed mechanisms that overcome the permeability barrier include osmotic forces. These reactions could synthesize many (but not all) amino acids and other key precursors. bilayer-forming lipids15. as well as interplanetary dust particles. the positively charged mineral surface and the negatively charged. as proposed by Blobel38 and Cavalier-Smith39. b | Two proposed membrane scenarios for cellularization during evolution. they formed bilayered vesicles in aqueous solution16. The early liposome system is postulated to induce cisternae to fuse with themselves to form double-membrane ‘obcells’ (inside-out cells)39. including ions. Deamer proposed that the earliest vesicles contained bilayer membranes made up of single acyl chain lipids that are Nature Reviews | Molecular Cell Biology more permeable to many molecules. The emergence of membranes In a water-based system. such as clay or iron pyrites21–25. the environment where the earliest chemistry that preceded life occurred is likely to have been in fresh water17. environment. Phospholipids are the main bilayerforming lipids in bacteria and eukaryotes (assuming that the ether-lipid-based membranes in archaea are a later adaptation to extreme environments22). The latter is also difficult to reconcile with the need for primordial lipid vesicles. see below). The model necessitates the loss of the outer of the two membranes to release a protocell that has the correct topology (with the luminal domains of the membrane proteins facing outwards). The evolution of the cytoplasm is often proposed to have occurred in parallel with the emergence of a liposome system. As the system evolved to use the more complex two acyl chain phospholipids. In the ‘inside-out’ or ‘leaky liposome’ model (left) the cytoplasm evolved within the vesicle and the topology of the membrane (outer leaflet outside) remains in place during the evolutionary process. if so. how were sufficient amounts of the necessary starting materials generated? Following the pioneering experiments of Miller9. 14). d | The obcell model of the ‘outside-in’ hypothesis of cellularization. In the ‘cytoplasm outside’ model. c | The ‘cytoplasm inside the vesicle’ hypothesis requires that the molecular precursors of life must have found a way to pass selectively through the liposome barrier.19. many lipids can self-assemble into bilayer-containing vesicles. A gel-like filamentous material may prevent the diffusion of protocytoplasmic components36. bring an extraordinary selection and amount of chemicals and biochemical precursors of life from space. However.PERSPECTIVES Protocytoplasm Lipid vesicle P – + P – + P P – +– + + P – + P – Obcell Protocell Mineral surface Mineral + surface a b Cytoplasm inside vesicle Gel Cytoplasm outside vesicle d –P + + c Unstable permeable bilayer Transient breaks in the membrane RNA Less permeable bilayer ‘Stable’ bilayer Liposome Ribosome Protein Autonomous cell divides Pore/channel Single acyl chain lipids • Monoglycerides • Fatty acids • Carboxylic acids Two acyl chain phospholipids Impermeable membrane • Electric potential • Proton gradient Figure 2 | Surface interactions and the membrane problem. neutral pH and an ionic composition that is rich in K+. the membrane became more impermeable. transient breaks in the membrane caused by polymerized amino acids such as polyleucine. hydrophobic lipid vesicle. and transient openings in the bilayer caused by temperature changes or freeze-thaw cycles20. the cytoplasm co-evolves with the membrane by associating with the outer membrane surface (which will later become the cytoplasmic surface). extensive studies described many chemical reactions that plausibly occurred under the presumed conditions of the early Earth.16. NATURE REVIEWS | MOLECULAR CELL BIOLOGY The cytoplasms of most modern cells have a similar chemical composition with a reducing environment. such as the Murchison meteorite that landed in Australia in 1969 (REF. The system provides two catalytic surfaces.20. Deamer and colleagues proposed that vesicles containing lipids made up of single acyl chains formed the first template for the VOLUME 8 | DECEMBER 2007 | 1019 © 2007 Nature Publishing Group . Before cells emerged. which are unstable at high salt concentrations20. carboxylic acids and monoglycerides18. which eventually became the delimiting membrane of the cell. A useful ‘yardstick’ for timing the main events leading to cellularization is a scheme proposed by Davis22.26 (FIG. Cl– and Mg2+ but low in Ca2+ and Na+. and precursors of liposomes such as carboxylic acids11–13. Perhaps this universal composition reflects the environment where the first cells evolved. such as purines. For a protocytoplasm to emerge. The key precursors of life could have been made on Earth or in outer space and carried to Earth via meteorites. including >90 different amino acids (of which 19 are found in living organisms) and. many schemes have been postulated that involve the occurrence of chemical catalysis and autocatalytic reactions on the surface of positively charged minerals. Meteorites. 1.

capture precursor molecules within their gel-like matrix or provide a scaffold for protection as well as an additional catalytic surface for emerging biochemistry. In this microenvironment. Most specialists think that such a system could only have evolved within the primordial vesicle20. Significantly. as conceded even by some of the strongest proponents of this hypothesis18. the purple ball and stick structure represents a membrane-spanning protein. 2c). perhaps ATP. As membrane complexity increases. all bilayers are highly impermeable to ions such as K+ (REFS 18. Many elegant experiments have been conducted to try and reconstitute some aspects of this hypothesis. Such analyses are still a long way from reconstituting life in such vesicles. Given the problem of these key precursors diffusing away from the site of action. the bilayer becomes more impermeable. as in modern cells (FIG. In parts a and c. This could initially be a simple polymer such as polysugars or polyglutamic acid that might later be replaced by a cytoskeletal protein polymer such as actin (see below). 36). investigators trapped enzymes and whole transcription– translation systems within vesicles and found that these became functionally active 20. Given the difficulties with the above hypothesis. Under these conditions. As proposed in the evolutionary schemes presented in FIG. with the ball representing the luminal domain. A cytoplasmic cisternal structure (of unknown origin but suspected to originate from the endoplasmic reticulum) wraps around cytoplasmic components such as a mitochondrion to form a double-membrane vesicle. The Golgi vesicles aggregate on the surface of the nucleus (pink) at the spindle pole body to enclose the spore within two membranes58. For example. A mineral surface could provide an environment on which lipid vesicles become attached. 3. This intracellular enveloped virus is transported along microtubules and fuses with the plasma membrane.nature. a | During the cellular release of vaccinia virus. a sophisticated protocytoplasm www.30–34 (FIG. b | During sporulation in the budding yeast Saccharomyces cerevisiae. there would be three catalytic surfaces — the positively charged mineral surface24. let us consider another scenario in which the cytoplasm evolved to a high degree of complexity outside the vesicles. the local polymerization of actin around the TGN-derived membrane facilitates virus release into the extracellular space (extracellular enveloped virus (EEV))57. their overall permeability is several orders of magnitude lower than phospholipid bilayers. How then could the precursors of the key molecules of life have crossed a bilayer that presents a significant barrier to charged molecules? Although vesicles comprised of single acyl chain © 2007 Nature Publishing Group . In this ‘cytoplasm within the vesicle’ scenario.20). 1). c | The formation of autophagic vacuoles is proposed to occur by the formation of a double-membrane structure59. The outer membrane fuses with a late endocytic compartment. the|trans GolgiCell Biology Nature Reviews Molecular network (TGN) cisternal domain engulfs the intracellular mature virus (IMV)56. cell membrane and that more complex (two acyl chain) phospholipids emerged later20 (FIG. actin and microtubules also interact here with membranes. GTP. RNA and maybe even DNA — could evolve. the emerging ribosomes inside the vesicle would eventually evolve the capacity to insert membrane proteins from the inside with their extracellular domains facing outwards. we face a crucial unresolved issue in understanding the origin of cells and the cell membrane: on which side of the liposomes did the first key reactions occur? The scenario of life within the vesicle.PERSPECTIVES Box 1 | Double membrane compartments in modern cells a Vaccinia virus TGN Golgi complex Plasma membrane IMV (Microtubule) (Actin) EEV b Budding yeast Golgi Spore Spindle pole body Nucleus c Autophagy lipid membranes are more permeable to uncharged and charged molecules. a double-membrane prospore cisterna is formed that originates from post-Golgi vesicles that fuse around the forming (haploid) spore (only one spore is shown but up to four may be made).13.25. it is attractive to consider the emergence of a polymer in the space around the vesicles that can form a hydrophilic gel (REF. Phospholipids can be synthesized without enzymes under plausible abiotic conditions12. or shortly thereafter. The mineral surface could itself catalyse the assembly of lipid vesicles25. RNA and even DNA 1020 | DECEMBER 2007 | VOLUME 8 emerged in contact with a system of liposomes. the surface of the gel36 and the negatively charged lipid vesicle surface — that could provide a rich surface for many reactions37. 2b). The evolution of cells If we assume that an increasingly complex cytoplasm with protein-synthesizing ribosomes. but it seems likely that these lipids became more important when RNA ribozymes or ribosomes and protein enzymes emerged that could synthesize them (FIG.com/reviews/molcellbio Mitochondrion Fusion with lysosome There are at least three examples in modern cells that are topologically similar to the ‘life outside the vesicle’ model (see figure). sandwiched between these surfaces. 2b.33–35 (FIG. The ‘outside the vesicle’ scenario. 2). and these surfaces could provide the environment where some rudimentary components of a complex protocytoplasm — for example. whereas the inner membrane is expected to be lysed by the hydrolytic conditions of the lysosomal lumen.c).27–29. nucleotides. proteins. Such systems of filaments could serve to attach the vesicles to the mineral surface. During fusion with the plasma membrane.

actin microtubule-associated proteins are also implicated in this process. microtubules and polarity47. were shown to be true using lamellipodia). which were first are knocked out in rod-shaped bacteria. When these proteins along actin. the topology is such that the fast-growing end eukaryotic cells. Cavalier-Smith39. It had been long appreciated that hexokinase and chaperones of the heat shock protein-70 (HSP70) family were homologous to actin41. In many fusion processes. textbooks dogmatically stated that only eukaryotes contain actin filaFusion + ments and microtubules. in time. which also assembles into filaments (green). for example. recently. most myosins move towards the plus ends. double-layered vesicle (FIG. whereas dynein transports cargo in the opposite direction. with FtsZ. There are no comprehensive models to predict how FtsZ–membrane in many membrane-dependent processes interactions operate mechanistically in this process but. actin homologues interact with the cell membrane. the actin family is involved cytokinesis (bottom panel). the vesicle would interactions between membrane nucleation taneously into the vesicle lumen and I will have to invert upon itself and fuse to form a mechanisms for actin assembly (N-WASP) revisit this issue below. this idea Microtubule has been overturned with the realization that Phagocytosis the actin. First. In these processes. 2c) has no obvious mechanistic precedents in modern cells. Membranes can filaments in vitro and in bacteria42. myosin motors attached to membrane organelles can walk prokaryote shape44. release the cell. The prokaryotic GTPase FtsZ. the system is rarely cited by origin-of-life specialists sorting nexin-9 (SNX9) was identified as a would evolve the capacity to insert membut has been extensively championed by membrane scaffold protein that stimulates brane proteins that spanned the membrane. only two can be surface. Even less is known about the interactions of microtubules with membranes. described in the intracellular transport of Listeria monocytogenes and other cytoplasmic pathogens. the spheres44. I argue that this (admittedly complex) inversion occurred by numerous steps and depended on the earliest cytoskeletal filaments.45. kinesin transports cargo towards the plus end of the microtubule. phagobeen shown to interact directly with FtsZ46.PERSPECTIVES Box 2 | Interactions of actin and microtubules with membranes in modern cells Filopodia – Myosin + Lamellipodia Cell motility Exocytosis – Comet Endocytosis Cytokinesis Membrane nucleation – of actin + of an ‘obcell’ or ‘inside-out cell’ (FIG. who conceived the idea N-WASP and ARP2/3-dependent actin NATURE REVIEWS | MOLECULAR CELL BIOLOGY © 2007 Nature Publishing Group VOLUME 8 | DECEMBER 2007 | 1021 . actin comets. is mechanistic details of how actin or its homologues in prokaryotes interact with membranes is poorly also involved in bacterial cell division and understood49. Of the different mechanisms and processes shown. As in nucleate the assembly of actin and. top panel) and prokaryotes (bottom panel). thus. interacts with the membrane at the site of cell cytokinesis46 (BOX 2). providing a force that contributes to considered to be well understood. with the has recently been shown to interact directly exception of motor proteins that can be bound to membrane organelles. cell motility. cytokinesis and actin homologues that can form actin-like cell polarity.43. For the most part. FtsZ (tubulin homologue) Actin homologues have been discovered in eubacteria and archaea. Recent data have shown direct molecules may indeed have crossed sponcell-membrane topology.48. whereas the tubulin homologue systems work together to drive bacterial FtsZ. Recently. the MreB and In eukaryotes. 2d). the actin. these filaments polymerize (barbed end or plus end (+)) is localized adjacent to the membrane — this means that the insertion of while being attached to the membrane new monomers occurs at this site. Actin (red) and its prokaryotic homologues have many intimate connections with membranes in eukaryotes (see figure. phagocytosis. Functions of the cytoskeleton Not so long ago. in all known cases. MreB (actin homologue) a homologue of tubulin. 2d). Nature Reviews | Molecular Cell Biology Mb1 subfamilies. A similar lack of understanding pertains to the role of cytosis. however.and microtubule-family proteins Macropinocytosis are universally expressed and interact with + Motor + Motor membranes. Whereas the ‘life within the vesicle’ model (FIG. was shown to form filaments and to interact with the membrane during bacterial cell division40. division (it forms a ring structure at the septum known as the Z ring) and is essential to carry out In eukaryotes. cytokinesis and cell actin in the much more complex process of cytokinesis in eukaryotic cells. the actin homologue FtsA has such as exocytosis. Second. Small inside of the vesicle38. the actin family is involved in many membrane-dependent processes such as exocytosis. The outer of and machinery for non-clathrin-mediated If ribosomes could make hydrophobic the two vesicles would then need to lyse to endocytic vesicle formation and for polypeptides. Below.52. polymerizes on membranes and somehow provides a force to pull membranes together might have emerged that interacted first with their extracellular domains facing the and/or push them or keep them apart49–52 with the outer surface of the vesicles. cell motility (for example.and tubulin-based In prokaryotes. This clever idea by Blobel38 vesicle outer surface and. these would interact with the dorsal cell ruffling. endocytosis. One actin homologue. the cells are converted into transport vesicles such as endocytic vesicles and phagosomes. endocytosis. To develop normal (BOX 2). there are examples from present-day cells in which a cisternal wrapping process occurs that is topologically similar to the ‘cytoplasm outside the vesicle’ model (BOX 1). new FtsA (actin homologue) Cytokinesis members of this family. FtsA. Recently.

In parallel. b | The emergence of the cytoplasmic fusion machinery (orange) allows liposomes to fuse together and bend membranes. These reaction systems are universally involved in the synthesis of all 20 common amino acids used by modern cells. Davis26 identified a conserved 11-residue sequence in the FtsZ–tubulin family that he mapped to his evolutionary stage 7. In the model. 1). its assembly and membrane functions. in this model. who argued that the order in which coded synthesis of the different amino acids and lipids emerged during evolution correlates with the number of reactions needed for their synthesis from an already evolved biochemical system.5bisphosphate (PtdIns(4. Besides its role in cytoplasmic fusion processes (where the cytoplasmic leaflets of membranes first interact). Because microtubules and FtsZ. The inside of the vesicle could then develop a different composition to the outside and might later have become the extracellular space. have intricate interactions with modern membranes. This machinery also allows the protocells to fuse together within the extracellular space. The ATPase actin and the GTPase tubulin–FtsZ protein family may have appeared before the cell became surrounded 1022 | DECEMBER 2007 | VOLUME 8 by a membrane and. membrane-spanning proteins. a stage he classified as occurring before the system could self-synthesize membrane phospholipids (stage 10. The proton and other pumps formed chemical and electrical gradients and synthesized ATP on the cytoplasmic side of the membrane. e | The cytoplasmic fusion machinery evolves into the process of cytokinesis. Thus. The cytoskeleton in cellularization? My model of cellularization starts with interactions of the protocytoplasm with the outer surface of the initially pure lipid liposomes (FIG. if so.or even DNA-based. which included the tricarboxylic acid (TCA) cycle and the pentose pathway22. Actin and/or tubulin ancestors then interacted with the outer surface of the membrane and facilitated membrane bending. were inserted. FIG. These are now independent living forms that are capable of self-replication.26 (FIG. www. Such studies start to resolve the long-elusive molecular links between actin. Shown is a possible evolutionary mechanism by which the outside-in model (FIG. ribosomes for synthesizing proteins and a relatively advanced biochemistry. 3a). Later. I propose a speculative model for the role of their precursors in cellularization. I propose that proteins (made by ribosomes present in the protocytoplasm) evolved hydrophobic domains that allowed them to interact with liposomes.PERSPECTIVES a Pre-cytoplasmic environment Ribosome Lumen Actin Microtubule/FtsZ + + + + + + + + + b c External milieu Extracellular space Protocell Channel e Cytokinesis d Extracellular space Luminal fusion Autonomous cells Cytoplasmic fusion Inverted vesicle Figure 3 | The outside-in model of cellularization. c | The fusion machinery could collaborate with the cytoskeleton to form an inwards-budding vesicle. Thus. SNX9 contains a BAR domain that is known to facilitate membrane bending53. The protocells are eventually released when the outer membrane lyses. 2d) may have occurred. assembly. The system has evolved the capacity to insert membrane proteins such that the future extracellular or luminal domains are inside the liposome.com/reviews/molcellbio © 2007 Nature Publishing Group .and tubulinbased cytoskeleton evolved the capacity to interact with the liposome surface. These suggestions are supported by an analysis by Davis. an actin.5)P2)-enriched membrane domains in vitro. Homologues of actin and tubulin have therefore been identified in all kingdoms of life. even sophisticated membrane functions such as proton and electrochemical gradients emerged before cellularization.nature. actin facilitates both cytoplasmic and luminal fusion events (FIG. 3). a recent study shows that N-WASP-based actin polymerization is also essential for two apposing plasma membrane luminal domains to fuse completely during Drosophila melanogaster myoblast fusion54. which allows protocells to divide in a regulated fashion such that each daughter cell contains everything it needs to metabolize and replicate. especially when it is allowed to oligomerize on phosphatidylinositol-4. a | An evolutionary stage exists in which a complex protocytoplasm has a genetic code that is RNA. could have functioned in cellularization. d | The emergence of luminal fusion mechanisms allows the process of fission out of the parental vesicle to occur. Channels and transporters that allow ions to cross the membrane would be important for maintaining the ionic homeostasis of the protocytoplasm inside the protocells and in the extracellular space.5. and especially actin. which is similar to modern endocytosis but has the opposite topology. allows the genetic material (not shown) and the protocytoplasm to Nature Reviews | Molecular Cell Biology enter into a vesicle within a larger vesicle — the protocell. 1). This budding process. including channels and pumps.

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