From Self-Assembled Vesicles to Protocells...From Self-Assembled Vesicles to Protocells Irene A....

2010;2:a002170 originally published online June 2, 2010Cold Spring Harb Perspect Biol Irene A. Chen and Peter Walde From Self-Assembled Vesicles to Protocells

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From Self-Assembled Vesicles to Protocells

Irene A. Chen1 and Peter Walde2

1FAS Center for Systems Biology, Harvard University, Cambridge, Massachusetts 021382Department of Materials, ETH Zurich, CH-8093, Zurich, Switzerland

Correspondence: [email protected]

Self-assembled vesicles are essential components of primitive cells. We review the impor-tance of vesicles during the origins of life, fundamental thermodynamics and kinetics ofself-assembly, and experimental models of simple vesicles, focusing on prebiotically plaus-ible fattyacids and their derivatives. We review recent work on interactions of simple vesicleswith RNA and other studies of the transition from vesicles to protocells. Finally we discusscurrent challenges in understanding the biophysics of protocells, as well as conceptual ques-tions in information transmission and self-replication.

For synthetic biologists, a useful operationaldefinition of life is “a self-sustaining chemi-

cal system capable of Darwinian evolution,”which was adopted by the Exobiology programof NASA (Joyce 1994). In the quest to build asimple living system, much recent interest hasfocused on encapsulating a genetic or metabolicsystem inside membrane vesicles (Deamer andDworkin 2005; Luisi et al. 1999; Morowitzet al. 1988; Ourisson and Nakatani 1994; Szo-stak et al. 2001). Vesicles are supramolecularaggregates containing an aqueous interior thatis separated from the bulk solution by one ormore bilayers of amphiphiles.

Why use vesicles? Although they are notstrictly required in this definition of life, thereare two major reasons why vesicle membranesare thought to be important. The first is thatthe membrane forms a semipermeable barrierthat permits small molecules to pass into the

cellular space and traps modified (e.g., phos-phorylated or polymerized) products. The sec-ond reason is evolutionary: The membraneseparates different genomes from one anotherand reduces the problem of inactive parasites(Szathmary and Demeter 1987; Szostak et al.2001). During the origin of life, physical group-ing is a plausibleway for replicatorenzymes (rep-licases) to interact nonrandomly. For example,replicases encapsulated in growing and dividingmembrane vesicles would tend to be trappedwith sequences related to their own sequence,and thus would preferentially copy those se-quences. Because the vesicles separate differentgenomes from each other, poor replicases wouldnot have access to active replicases, whereasmutants with improved replicase activity wouldbenefit directly themselves, as their descendantsremain in the same vesicle and copy eachother. An occasional parasitic sequence would

Editors: David Deamer and Jack W. Szostak

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be separated from most of the active polymerasesduring vesicle division and could not poison theentire system (the “stochastic corrector” model)(Smith and Szathmary 1995; Szathmary andDemeter 1987). Thus, a higher level of popu-lation organization, the cell, greatly facilitatesthe evolution of more efficient replicases (Cava-lier-Smith 2001; Koch 1984; Matsuura et al.2002; Szathmary and Demeter 1987; Szostaket al. 2001).

Membrane vesicles are not the only way tosegregate different genomes. The attachmentof molecules onto surfaces also creates a het-erogeneous distribution of interactions basedon spatial proximity, a scenario that has been

investigated theoretically using cellular autom-ata models (Szabo et al. 2002). Although theymay not have been the initial means of achievinggenomic segregation during the origin of life,membranes are the dominant means of separat-ing cells today. Membranes presumably assumedthis function very long ago, at least three to fourbillion years ago, at some time before the diversi-fication from the last common ancestor.

Vesicle morphologies and topologies cancover a rich and diverse landscape (Fig. 1, top),although experimentalists tend to prefer unila-mellar vesicles because data can be more easilyinterpreted in this context, and because thesevesicles resemble contemporary cells, which

Vesiclescontaininginternalvesicles

Unilamellar

Oligo- ormultilamellar

Potentiallyprebiotic

Synthetic,non-biological

Frombiomembranes

Vesicle shapechanges

Archaea

Eubacteria andeukaryotes

Polymeric

Non-polymeric

+

+

++

++ +

+

Figure 1. Diversity of morphology and composition of self-assembled vesicles. Top: Schematic representation ofpossible morphologies and shape changes. Vesicles may be multi-, oligo-, or unilamellar. They may also bemultivesicular (containing smaller vesicles inside a large vesicle). Under certain conditions and for certainamphiphiles, vesicle shape changes can be induced (e.g., leading to vesicle budding and fission). Vesicles mayalso be nonspherical (e.g., tubular). The diameter of vesicles may vary between about 30 nm and more than100 mm. Bottom: Vesicle formation occurs for a large number of chemically diverse amphiphiles, includingthose naturally occurring in biomembranes as well as completely synthetic amphiphiles. Of particularinterest are single-chain amphiphiles (or mixtures of amphiphiles) that are potentially prebiotic.

I.A. Chen and P. Walde

2 Cite this article as Cold Spring Harb Perspect Biol 2010;2:a002170




use the plasma membrane to separate the cell’sinterior from the outside environment. Theplasma membrane is composed of roughlyequal parts protein and lipid amphiphiles, soone might assume that a protocell membranewas also composed of amphiphilic lipids and/or peptides. However, for simplicity, most exper-imental work thus far has focused on vesiclesmade of only one or two prebiotically plausiblecomponents (e.g., fatty acid). Vesicles can bemade using manydifferent types of amphiphiles,either naturally occurring or synthetic (Fig. 1,bottom). Because of the general robustness ofthe formation of vesicles, this process has beencalled an “archetype of self-assembly” (Anto-nietti and Foerster 2003).

In this article, we first review some funda-mentals of self-assembly and focus on importantfeatures of vesicles made from single chainamphiphiles. For further discussion of vesiclesas model prebiotic experimental systems, werefer the reader to reviews already in the litera-ture (Mansy 2009; Monnard and Deamer 2003;Walde and Ichikawa 2001; Walde et al. 2006)and references contained within these reviews.We then turn to recent results and current chal-lenges in research related tovesicles in the originsof life.

BACKGROUND

Thermodynamics of Self-Assembly ofAmphiphiles

Self-assembled structures of amphiphiles arethe result of a balance of attractive and repulsiveforces. Amphiphiles tend to aggregate becauseof the hydrophobic effect, which stems primar-ily from the strong attraction of water for itself(Tanford 1973). Nonpolar solutes disrupt theisotropic hydrogen bonding of water, causingan entropic loss at the solute-water interface.Therefore nonpolar molecules tend to aggregateto minimize the interface (direct attractive inter-actions, such as van der Waals forces, play a rela-tively small role). In the absence of a repulsiveforce, the hydrophobic effect would lead to bulk-phase separation. However, repulsion amongamphiphiles resulting from sterics (e.g., among

hydrated head groups) or electrostatics favorsthe formation of structured assemblages.

A minimum number of molecules isrequired for effective reduction of the water–hydrophobic surface interface in an aggregaterelative to free solution. This leads to a highlycooperative transition in which the concentra-tion of aggregates increases dramatically near acritical aggregation concentration (cac) andfor micelles, the critical micellization concen-tration (cmc). This process is often modeledas a phase transition; although such modelsare not strictly correct, they often capture theessential behavior of the system within experi-mental error. This approximation is quitegood when aggregates are large (i.e., containing.50 molecules), as is the case for most micellesand vesicles.

Micelles are self-assembled structures witha liquidlike hydrocarbon interior and polarheadgroups on the exterior (Fig. 2A). Severalgeometries are possible, including spherical,cylindrical, and ellipsoidal. The free energy oftransferring an amphiphile from water into amicelle depends linearly on chain length, so thecmc depends exponentially on chain length(Tanford 1973). Vesicles are closed bilayer struc-tures with an aqueous compartment (Fig. 2B).Whether micelles or vesicles are formed canbe explained largely by packing considerations(Israelachvili et al. 1976; Israelachvili et al.1977). The balance of attractive interactionsand repulsive interactions gives an optimal in-terfacial area per molecule. Amphiphiles self-assemble into structures having area exposed tosolvent (a) close to this optimum. Sphericalmicelles have the greatest interfacial area, inwhich v/a ¼ l/3 (where v ¼ volume and l ¼hydrophobic chain length), cylindrical micelleshave v/a ¼ l/2, and vesicles have v/a ¼ l. To afirst approximation, the presence of two chainson an amphiphile doubles v whereas the optimala and l are constant, so a consequence of theseconsiderations is that single-chain amphiphilestend to form micelles whereas double-chainamphiphiles tend to form cylindrical micellesor vesicles. Vesicle formation is also favored byfactors that reduce the optimal interfacial area.For example, fatty acids (prebiotically plausible

Self-Assembled Vesicles to Protocells

Cite this article as Cold Spring Harb Perspect Biol 2010;2:a002170 3




single-chain amphiphiles) form micelles at highpH when their head groups are negativelycharged, but they form vesicles at lower pHwhen their head groups interact more favorably(Gebicki and Hicks 1973).

Packing constraints also imply a minimumchain length required to form vesicles; for fattyacids (a single chain amphiphile), this length isaround eight carbons (Monnard and Deamer2003). Organic extracts of the Murchison mete-orite (Lawless and Yuen 1979) show that abun-dances of fatty acids decreased as chain lengthincreases, with a three-carbon increase corre-sponding to a �10-fold drop in abundance.Roughly speaking, this drop in abundance issomewhat compensated by the decrease of cacas chain length increases (a three-carbon incre-ase corresponds to a �10-fold decrease in cac)(Chen et al. 2006). Although the details of theserelationships depend on the synthetic pathwayand buffer conditions, this general pattern sug-gests that, during prebiotic synthesis, each fattyacid would reach its cac at around the sametime, leading to a relatively sharp transition toa vesicle world.

Kinetics of Self-Assembly of Amphiphiles

The kinetics of nucleation and growth of mi-celles are generally quite fast. Nucleation occurson the order of microseconds to milliseconds,

whereas exchange of monomers through theaqueous phase occurs on the order of nanosec-onds to microseconds. These timescales dependon the association and dissociation rates ofamphiphiles. As chain length increases, dissoci-ation rates decrease (for alkyl sulphates, roughly10-fold per three-carbon increase), whereasassociation rates decrease only slightly becauseof slower diffusion (within one order of magni-tude from C6 to C14) (Aniansson et al. 1976;Hunter 2001).

Vesicle formation from a solution of mi-celles appears to have two relaxation times, arapid mixing or aggregation of micelles,followed by slower growth and closure to formvesicles. For a mixture of anionic and zwitter-ionic surfactant micelles, the micelles mixwithin a few milliseconds and then coalesceinto disclike micelles that grow and close intovesicles within a second (Weiss et al. 2005).For vesicles formed by increasing the pH of asolution of fatty-acid micelles, aggregation ofmicelles occurs very quickly (,12 ms) whereasrelaxation into vesicles takes seconds to minutes(Bloechliger et al. 1998; Chen and Szostak2004a). The kinetics of fatty-acid vesicle forma-tion are particularly interesting with regard tothe origins of life for two reasons: (1) the reac-tion can be autocatalytic, i.e., the presence ofvesicles accelerates the formation of morevesicles (Walde et al. 1994b), and (2) vesicle

O

OH

HO

O

OH

O

POH

HO

OH

N

HO

O

A

E

B C

F

D

HO

O

P

Figure 2. Structures of prebiotically plausible single chain amphiphiles and a commonly used buffer. (A) micelle;(B) vesicle; (C) myristoleic acid; (D) bicine; (E) geranylgeranyl phosphoric acid; (F) n-decylphosphonic acid.






formation is catalyzed by many types of surfaces(Hanczyc et al. 2003; Hanczyc et al. 2007a),including montmorillonite, which also catalyzesRNA polymerization from activated monomers(Ferris and Ertem 1992). Once formed, closedvesicles can be fairly stable as supramolecularstructures, surviving for several days or longer,even if their components exchange relativelyrapidly.

Amphiphiles for Protocells

The primary components of modern cells aredouble-chain amphiphiles, particularly phos-pholipids, which consist of a headgroup andtwo acyl chains. Because of their geometry,double-chain amphiphiles form stable vesiclesvery readily. However, they pose several funda-mental problems for a protocell, which resultfrom slow dynamics. Phospholipids are rela-tively impermeable to charged compounds(e.g., Kþ permeability coefficient: 10210 to10212 cm/s (Paula et al. 1996)), possibly becausethe low mobility of amphiphiles between theleaflets prevents facilitation of transport. Slowflip-flop would also limit vesicle growth byincorporation of new lipid (which requires redis-tribution of lipid from the outer to inner leaflet),inhibiting a key feature of a self-reproducingsystem. Modern cells treat their membranes asrelatively static barriers, using flipases and per-meases to mediate flip-flop and transport,respectively.

On the other hand, single-chain amphi-philes appear to be excellent candidates for pre-biotic protocells for several reasons. They haverelatively high permeability to ions and smallmolecules (e.g., Kþ permeability coefficient:1026 cm/s (Chen and Szostak 2004b)), pre-sumably due at least in part to increased mo-bility of amphiphiles flipping between theleaflets, facilitating ion passage and circumvent-ing the need for specific transporters. Fast flip-flop also permits vesicle growth through addi-tion of an amphiphile feedstock to the vesicleexterior (Berclaz et al. 2001; Chen and Szostak2004a). Single-chain amphiphiles also havehigh dissociation rates and relatively high watersolubility, so the membrane components can be

exchanged among vesicles rapidly, allowingredistribution to “growing” protocells (Chenet al. 2004). The cac is higher for single-chainlipids compared with double-chain lipids ofthe same chain length and similar head groupstructure, yielding a greater reservoir of mono-mers in solution. A limitation of single-chainamphiphiles is that they often form micelles,depending on the molecular geometries andbuffer conditions, so the amphiphiles consid-ered for prebiotic vesicles are constrained tohave relatively small headgroups comparedwith double-chain amphiphiles.

Although we do not know which type ofamphiphiles constituted the membranes ofprotocells, much attention has focused on fattyacids (Fig. 2c) and mixtures of fatty acids withtheir derivatives (e.g., fatty alcohols, amines,and monoacylglycerols). Fatty acids can be syn-thesized abiotically in several ways. For example,Miller-Urey-type electrical discharge reactionsin a solution of ammonia under a nitrogenand methane atmosphere yield fatty acids witha chain length up to C12 (Allen and Ponnam-peruma 1967; Yuen et al. 1981). Abiotic synthe-ses generally yield decreasing amounts of fattyacids of longer chain lengths. A synthesis simu-lating hydrothermal vents yielded fatty acids upto C33, from an aqueous Fischer-Tropsch-typesynthesis using a heated solution of oxalicacid (which disproportionates into H2, CO2,and CO) (McCollom et al. 1999; Rushdi andSimoneit 2001).

Direct evidence for the abiotic presence offatty acids comes from the detection of fattyacids in the interior of the Murray and Mur-chison carbonaceous chondrite meteorites fromAustralia (up to C8), as well as an Asuka carbo-naceous chondrite meteorite (A-881458) fromAntarctica (up to C12) (Lawless and Yuen1979; Naraoka et al. 1999; Yuen et al. 1984;Yuen and Kvenvolden 1973). Fatty acids arerelatively abundant in these meteorites, being20 times more abundant than amino acids inthe organic extract of A-881458. Indeed, organicextracts from the Murchison meteorite formboundarymembraneswhenrehydrated(Deamer1985; Deamer and Pashley 1989). The presenceof fatty acids is particularly suggestive because






the chemical composition of these meteorites isbelieved to resemble that of the early solarsystem.

Depending on the solution pH, fatty acidsself-assemble into different structures (Cistolaet al. 1988; Small 1986). At low pH, the mole-cules are protonated and uncharged, resultingin an oil phase. At high pH, the molecules aredeprotonated and negatively charged, resultingin the formation of micelles with a hydrophobiccore and surface-exposed carboxylates that repelone another. Although the pKa of a carboxylicacid is typically 4–5, the self-assembly of fattyacids leads to a cooperative effect that increasesthe pKa. For example, oleic acid (C18:1) mono-mers have a pKa of 4.5, but oleic acid assem-bled into a bilayer membrane in bicine buffer(Fig. 2D) has a pKa of 8.5. Medium- and long-chain fatty acids incorporated into membraneshave pKas in the general range of 7–9 (Cistolaet al. 1988). When the solution pH is nearthe pKa, fatty acids assemble into bilayer mem-brane vesicles that are capable of entrappingsolutes (Apel et al. 2001; Walde et al. 1994a).These vesicles have a net negative charge, witha formal surface charge density close to half ofthe molecular density of the membrane. As aresult, cations are also associated with thesurface, forming an electrical double layer(Grahame 1947; Hunter 2001). Several articlesdescribe in detail the characteristics and limita-tions of fatty-acid systems (Deamer and Dwor-kin 2005; Mansy 2009; Monnard et al. 2002;Morigaki and Walde 2007; Walde et al. 2006).

Another category of single-chain amphi-philes that may be promising as a model for pre-biotic vesicles uses a phosphate headgroup, suchas the polyprenyl phosphates (e.g., geranyl-geranyl phosphate; Fig. 2E). These amphi-philes can self-assemble into vesicles (Ourissonand Nakatani 1994; Pozzi et al. 1996). In aninteresting parallel to fatty-acid membranes,which can be stabilized to pH changes by acylalcohols (Monnard et al. 2002), polyprenylphosphate membranes are stabilized by addi-tion of polyprenyl alcohols (Streiff et al. 2007).Other phosphate-based systems under inves-tigation include mono-n-alkyl phosphatesand phosphonates (Walde et al. 1997); alkyl

phosphonates in particular have been observedin organic extracts from the Murchison meteor-ite (e.g., decylphosphonate; Fig. 2F) (Cooperet al. 1992).

RECENT RESULTS

Membranes and Nucleic Acids

Vesicles could play a simple role as compart-ment boundaries for ribozymes (Chen et al.2005), but they may contribute to an early cellin other ways as well. Because of the importanceof the RNA world during origins of life, severalgroups are studying direct or indirect interac-tions between vesicle membranes and RNA.One mode of direct interaction between RNAand cationic vesicles is electrostatic attractionto form nucleic acid-lipid complexes (Thomasand Luisi 2005). Such complexes may also beformed with neutral or zwitterionic vesiclesunder the right buffer conditions (e.g., highdivalent cation concentration). For anionicamphiphiles, the electrostatic repulsion betweenRNA (poly-U) and the vesicles can be overcomeby covalently decorating the phopholipid head-group with adenosine (Milani et al. 2007), anal-ogous to the annealing of two complementary,negatively charged nucleic acid strands. Con-versely, the nucleic acid can be conjugated to ahydrophobic moiety, such as cholesterol, whichinserts into the membrane and brings the nucleicacid into the proximity of the vesicle (Banchelliet al. 2008). RNA aptamers for membraneshave also been found by in vitro selection forRNAs that bound to phospholipid vesicles. Thesesequences appear to bind the membrane asaggregates (Janas and Yarus 2003). One suchsequence was modified to include an RNA motiffor tryptophan binding, and together thesemembrane-binding RNAs had a small butmeasurable effect on the rate of tryptophan per-meation through the membrane (Janas et al.2004).

Membrane vesicles can also have surprisingconsequences for the evolution of the RNAworld. Membranes can accelerate the polymer-ization of RNA mononucleotides or aminoacids during drying and wetting cycles (Deamer






et al. 2006; Rajamani et al. 2008; Zepik et al.2007). This effect may be driven by an increasein local concentration of monomers, throughtrapping of monomers between lamellae duringdrying and mass action from dehydration.Although the mechanism is unknown, the liq-uid crystalline structure of the dried lamellaeis believed to be important for this effect.

On the theoretical side, fresh efforts havebeen made in the analysis of polymerizationdynamics relevant to nucleic acids (“prelife”).This mathematical analysis describes the distri-bution of sequences with and without replica-tion and the occurrence of a phase transitionamong these regimes (Manapat et al. 2009;Nowak and Ohtsuki 2008). One interestingdirection for this research would be considera-tion in the context of a protocell, to determinehow small numbers and competition amongprotocells might affect the prelife dynamics.

Systems-Level Properties of Protocells

How did interactions arise between the mem-brane and its encapsulated contents? Two inter-actions have been described based solely on thephysicochemical properties of the system. Thegrowth of vesicles consists of the incorporationof fatty-acid molecules into the outer leafletof the membrane, followed by the flip-flop ofabout half of these molecules into the inner leaf-let. Fatty acid flip-flop into the inner leaflet willalso transport protons (Hamilton 1998; Kampand Hamilton 1992), so growth transduces theenergy from the micelle-vesicle transition intoa pH gradient across the membrane (Chenand Szostak 2004b). However, proton gradientsacross fatty-acid membranes only last as long ascations are not transported in the oppositedirection, so a cell would need to use the energystored in the proton gradient quickly before itdecayed (Kamp and Hamilton 1992; Zenget al. 1998). Regardless, the capture of energyis one way the growth of one component, themembrane, could confer an advantage toanother component (encapsulated contents).

The emergence of the cell can also beapproached from the perspective of the encap-sulated genome. A transmembrane gradient

of any impermeable solute (Dc) exerts osmoticpressure on a semipermeable membrane (DP ¼nDcRT for small solutes in dilute solution,where n ¼ number of particles into which thesolute dissociates). A charged solute generatesmore osmotic pressure, and the osmotic pres-sure of a charged polymer, such as RNA, willbe due primarily to the counterions associatedwith it, in accordance with the Gibbs-Donnanequilibrium (Bartlett and Kromhout 1952;Tinoco et al. 2002). The replication of agenomic polymer would increase the osmoticpressure in a vesicle (assuming that the mono-mers equilibrate across the membrane). Theresulting areal strain favors membrane growth,which relieves the strain by increasing the totalarea of the membrane. For amphiphiles withhigh rates of monomer exchange and flip-flop,like fatty acids, this leads to a competitionamong vesicles for amphiphiles, translating thefitness of the encapsulated replicase into thefitness of the whole cell (Chen et al. 2004).Darwinian competition between “cells” couldhave emerged simply on encapsulation of rep-licases or metabolic cycles inside vesicles; emptyvesicles and vesicles encapsulating less efficientsystems would shrink by losing membrane.

This competition has interesting implica-tions for the natural selection of chemical traitsin the genome and membrane. Because coun-terions cause most of the change in osmoticpressure after polymerization, protocells con-taining charged polymers as the genetic mate-rial, such as RNA or DNA, would outcompetethose with neutral polymers (e.g., peptidenucleic acid). With respect to the membrane,the protocellular competition might favor theevolution of an enzyme that stabilized the mem-brane against loss of amphiphiles, such as bycondensing two fatty-acid molecules together.In turn, the reduced permeability and growthdynamics of stabilized membranes might drivethe coevolution of transporters and moreenzymes. Thus the competition arising fromthe simple physical properties of an encapsu-lated replicase could result in a “snowball” effecton the evolution of cellular complexity. Suchpredictions may suggest further experimentalexploration.






Computer Simulation ofProtocell-Like Systems

In an era of increasingly powerful and accessiblecomputation, molecular dynamics (MD) simu-lation is a promising technique for exploringour understanding of the behavior of relativelylarge chemical systems like vesicles. In generalthese simulations use a combination of empiri-cal and ab initio information to describe energypotentials for molecular conformations andintermolecular forces (dispersion and electro-static forces) and solve the Newtonian equationsof motion numerically. The size of a vesicle sys-tem is roughly hundreds of thousands to a fewmillions of atoms (e.g., a single fatty acid isroughly 50 atoms, and thousands of fatty-acidmolecules would compose a small vesicle, andwater would also compose a substantial molefraction of the system). Currently an MD simu-lation of this size could simulate about 100nanoseconds, which would be enough to studythe fast kinetics of self-assembly. An atomisticsimulation of dipalmitoyl phosphatidylcholine(DPPC) in water recapitulated vesicle self-assembly (de Vries et al. 2004). In addition totesting our understanding of the system, ideallythese simulations could be useful to experimen-talists for building biophysical intuition andemphasizing features to be further explored.For example, the simulation of a DPPC vesicleindicated a large asymmetry in mass and order-ing between the inner and outer bilayers, sug-gesting a potential mechanism for asymmetricinsertion of molecules into the membrane.

Slower dynamics or larger systems could bemodeled using coarse-grained rather thanatomistic simulations (Klein and Shinoda2008). One interesting but poorly understoodphenomenon that might benefit from MD sim-ulation is the recent observation that dried lipidmembranes accelerate the polymerization ofamino acids and nucleotide monophosphates,perhaps by preorganizing the reactants intothe reactive geometry (Rajamani et al. 2008).

Another style of simulation of protocellsfocuses on the reaction (or reaction-diffusion)kinetics of the system without modeling themolecular details. Although much work, such

as Manfred Eigen’s classic model of hypercycles(Eigen 1971), dealt with metabolic cycles with-out explicit reference to a membrane, recentlythe potential for interactions between the mem-brane and encapsulated metabolic reactions hascome under closer scrutiny (Mavelli and Ruiz-Mirazo 2007). Stochastic simulations are of par-ticular interest because the volume of a smallvesicle could be less than one attoliter, suchthat micromolar concentrations would give onlya handful of molecules per vesicle. Althoughcare is always required when dealing with as-sumptions in a model, simulations can providea testing ground for our understanding, andmay become a useful tool for understandingexperimental results and generating interesting,experimentally testable predictions in protocell-sized systems.

CHALLENGES AND FUTURE DIRECTIONS

Physical Forces and Protocells

How would protocells interact with their envi-ronment? Although some interest has focusedon interactions at a molecular level, such asthe permeability and thermostability of vesicles(Mansy et al. 2008; Mansy and Szostak 2008),less work has been performed to relate colloidalprocesses to protocells. For example, transportphenomena might give us clues about protocellmovement through a heterogeneous envir-onment or even initially homogeneous env-ironment. Hanczyc et al. showed how oleicanhydride oil droplets added to an alkaline sol-ution spontaneously move, as exothermichydrolysis of the anhydride sets up a convectivecurrent within the droplet; the directionof motion appeared to be the outcome ofsymmetry-breaking fluctuation (Hanczyc et al.2007b). The theory of osmophoresis suggeststhat vesicles in a solute gradient will move inthe direction of lower solute concentration(Anderson 1983; Nardi et al. 1999). Such effects,if experimentally relevant, could inform ourunderstanding of how a protocell would movewithin its environment as a consequence ofcolloidal forces, without motor machinery.






Entropy might play a very interesting role inprotocells. For example, theory predicts thattwo chromosomes in a cell tend to segregatefrom each other to maximize conformationalentropy (Jun and Mulder 2006). This findingsuggests that protocell genomes might notrequire extra machinery to segregate its geneticmaterial. Along similar lines, cell division ma-chinery (in particular, a contractile ring) wasrecently discovered to be unnecessary for repro-duction and division of Bacillus subtilis (Leaveret al. 2009). The observed mode of division isreminiscent of a physical phenomenon, theRayleigh instability (also known as “pearling”in tubular liposomes), lending credence to theidea that protocells would not require divisionmachinery (Chen 2009). One might also expectprotocells to interact with one another throughentropic effects, such as the depletion effect,in which the entropy of the aqueous solutionis maximized when large particles are closetogether, resulting in an effective attractionbetween the particles. Such effects could beinteresting as leading to assembly of protocellsinto larger structures. Investigation of the roleof physical forces in protocells may presentmany opportunities for collaboration betweentraditional soft condensed matter physicistsand origins of life researchers.

Vesicles from Mixtures

Studies on vesicles formed from potentiallyprebiotic amphiphiles have so far been limited tosystems containing one or two types of amphi-philes. This is in contrast to the output of simula-ted prebiotic chemical reactions, which typicallyproduce very heterogeneous mixtures of com-pounds. In addition, little experimental workhas been performed to integrate peptides and lip-ids. (A notable exceptionto this biastoward work-ing with one or two component systems was astudy using the heterogeneous organic extractfrom a carbonaceous chondrite meteorite toform vesicles [Deamer 1985].) The reason forthis bias is mainly the avoidance of analytical dif-ficulties, but clearly it is not very realistic.

What properties might vary with mem-brane composition? Several features characterize

a particular vesicle system and differentiate itfrom others, including (1) lamellarity, (2) averagemembrane thickness and its fluctuations, (3)amphiphiledynamicsinthemembrane,(4)mem-brane permeability, i.e., the ability to keep solutescaptured inside the vesicles and to stabilize con-centration gradients across the membrane, (5)chemical stability of the amphiphiles, (6) mem-brane surface charge, (7) membrane fluidity, (8)possibility of membrane budding and fission(leading to vesicle divisions), (9) vesicle aggrega-tion or vesicle adsorption to surfaces, (10) vesiclefusion, (11) colloidal stabilityat different temper-atures and bulk solution compositions. All theseproperties ultimately depend on the chemicalstructure of the amphiphiles, the environmentalconditions, and the process of vesicle formation.The process of vesicle formation is relevantbecause most vesicles are kinetically trapped sys-tems (i.e., not at true thermodynamic equi-librium). Lamellarity and size depend stronglyon how the vesicles are prepared, but the otherfeaturesdependprimarilyonthevesiclecomposi-tion and buffer conditions.

There are challenges to using vesicles basedon fatty acids as protocell models (Monnardand Deamer 2002). One limitation for practicalstudies is the failure in preparing vesicles fromlong-chain fatty acids at room temperature,for example from hexadecanoic acid, becauseof the high melting temperature of saturatedlinear hydrocarbon chains. According to thephase diagram, a lamellar phase exists at 708C(Skurtveit et al. 1989). However, recently it wasshown that vesicles form from long-chain fattyacids at room temperature if guanidine hydro-chloride is added (Douliez et al. submitted forpublication). This illustrates how vesicles ofmixed composition can actually have un-anticipated properties beneficial to a protocell,such as robustness to ionic strength, loweredcac, or even propagation of information (as dis-cussed later) (Monnard and Deamer 2003).One of the challenges for the future will beto further study these mixtures to understandthe chemical basis for their effects on vesiclecharacteristics. Other mixtures to explore in-clude mixtures of fatty acids with small mole-cule components, mixtures of different types






of fatty acids, and mixtures of fatty acids withpeptides.

Self-Assembled Structures and Information

The storage and propagation of information is akey feature of biological systems. Nucleic acidsequences fulfill this requirement in a straight-forward fashion, particularly in the case of ribo-zymes, where the informational material itselfhas chemical activity. However, in a very broadsense, molecules other than nucleic acids carryinformation about their reactivity and physico-chemical properties (Graham et al. 2004). Self-assembling molecules contain the informationfor building supramolecular structures, suchas vesicles and micelles. For example, potassiumdecanoate, CH3(CH2)6COO2Kþ, carries infor-mation on the thermodynamics of its self-assembly behavior. When dissolved in water ata concentration of 29.1 g/L (150 mM), micellaraggregates form (Namani and Walde 2005).Each micelle contains a hydrophobic core thatallows some solubilization of molecules thatare not soluble in water, so the self-assemblyof the amphiphilic decanoate molecules confersnew physicochemical properties to the aqueoussolution (Luisi 2002). Could self-assembled,noncovalent systems also propagate their “in-formation”?

An interesting model addressing this ques-tion was proposed recently (Segre et al. 2000).In a solution containing a mixture of amphi-philes, if amphiphiles of a certain type favoredthe incorporation of other amphiphiles in a selec-tive fashion, an assemblage would reach a partic-ular steady state whose composition containssome information. In simulations they observedthat abrupt transitions could occur among differ-ent steadystates, suggesting that different compo-sitions could coexist and thereby providevariation on which natural selection might act.An experimental system corresponding to thismodel would require determining the on- andoff- rates of different amphiphiles in vesiclesspanning a range of compositions. A particularlyinteresting technical challenge associated withthis theoretical problem is the identification ofthe composition of an individual vesicle.

Self-assembly as a Quantitative Trait

Although one working definition of life (a self-sustaining chemical reaction capable of Dar-winian evolution) was given in the introductionto this article, alternative definitions also exist,often in the form of several criteria. The difficul-ties encountered in defining life suggest thatlife is a quantitative, not qualitative, trait, whichcould be measured by an appropriate metric.Chirikjian and coworkers developed metricsfor the degree of self-replication and task com-plexity in a framework of self-replicating robots(Lee et al. 2008). For example, some robots copythemselves by joining two already complexbuilding blocks (low self-replication) in a highlystructured environment with many landmarks(low task complexity), whereas other robotscopy themselves by joining several simplebuilding blocks (high self-replication) in anunstructured environment (high task complex-ity). These calculations require knowledge ofthe components, their interconnections, andthe environment. Application of this quantita-tive approach to chemical systems and to otherproperties (i.e., evolvability and life) poses aninteresting intellectual challenge that might alsohelp integrate our understanding of differentmodel systems of protocells.

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