Table Of ContentDOL 10.10021anie.200((will be filled in by the editorial staff))
The Origin of Primitive Cells, Nutrient Intake, and Non-
Enzymatic Elongation of Encapsulated Nucleotides **
Uwe J Meierhenrich,* Jean-Jacques Filippi, Cornelia Me inert, Pierre Vierling, and
Jason P..Dworkin
Fatty acids and fatty alcohols are commonly found in Consequently; F'fT reactions have been performed under
experiments simulating the prebiotic 'soup'. These amphiphiles can temperature and pressure control in the laboratory, mimicking
be synthesized under prebiotic conditions, at least as long as the hydrothermal conditions, Starting with aqueous solutions of either
molecules are chemically relatively simple and do not need to be formic or oxalic acid — used as proxies for CO, CO,, and H2 in
enantiomerically pure.lzl In the context of topical origin-of-life hydrothermai fluids in order to overcome the practical difficulties of
theories, two distinct formation pathways for amphiphiles have been adding these volatile gas components to the high pressure reaction
described; one related to geophysical sites, such as marine vessel — as the carbon and hydrogen sources, the formation of Lipid
hydrotheral systems, and another to extraterrestrial sources, such compounds with carbon lengths between C2 and Cis, including n-
as the proto-solar nebula, which was fed by interplanetary and alkanols and n-aikanoic acids, was observed inside reaction vessels
interstellar nebulae. The chemical analysis of each provides after cooling, extraction, and gas chromatography-mass
individual characteristic challenges. spectrometry analyses. The identification of the reaction products
was confirmed using "C labeled reactants. Both formic and oxalic
acid carbon sources yielded the same lipid classes with essentially
2.1. Aqueous Fischer-Tropsch-Type Formation of the same ranges of compounds. The optimum temperature window
Amphiphilic Molecules for the formation of alkanoic acids was shown to be 300 °C; higher
temperatures reduce the yield due to competing cracking
The Fischer-'Fropsch-type (FTT) reaction has drawn the processcs.l"r Table I presents the relative concentrations and carbon
attention of geochemists as a potential starting point for the number ranges of alkanoic acids obtained by aqueous FTT synthesis
formation of organic molecules, including amphiphiles- The FTT at various temperatures.
reaction is known to occur in different geological settings such as Carbon preference index 4CPI) values vary from 0.95 to 1.15,
volcanoes and igneous rocks. For a long time, it was assumed that showing no carbon number predominance:. CPI values close to one
the FTT process cannot occur in the aqueous phase due to likely indicate that the chain growth of the homologue series is by single
inhibition by water, but recent laboratory experiments by the team carbon units. The aqueous FTT reaction thus proceeds by the
led by Simoneit have proven that the chemical formation, transformation of oxalic acid to C t species such as CO, followed by
accumulation, and selection of amphiphiles is feasible via FT`F the insertion of the CO group at the terminal end of a carboxylic
ri
reactions even in the aqueous phase.ls. Aqueous FTT synthesis is acid functionality to form homologue series of alkanoic acids after
important since mid-ocean-ridge hydrothermal systems are reduction.t`'l This mechanism differs from the classically known
discussed with increasing emphasis as a possible starting place for industrial FTT process, in which the growth of the hydrocarbon
the origin of life on Earth. This is due to the discovery of primitive chain relies on the reaction of vapor phase mixtures of CO or CO,
life forms around hydrothermal vent systems at the bottom of the with H2 proceeding by surface-catalyzed stepwise polymerization of
bl
ocean, where magma (liquid rock) spills through the Earth's c-rust methylene.l' Besides arnphiphilie molecules, straight-chain
and reacts with seawater. alkanols, alkyl formates, alkan.als, alkanones, alkanes, and alkenes
Contemporaneous marine hydrothermal systems however. are were identified, and methylalkanes were found above 250 'C with a
dominated by organic compounds derived from all-pervasive maximum concentration at 350 °C.
biological processes, and therefore hydrothermai vent-simulating
laboratory experiments provide the best opportunity for
confirmation of the potential for organic synthesis in such systems.
Uwe J. Meierhennch studied chemistry at Jean-Jacques Fifippi studied natural
the Phillips-University of Marburg, After product chemistry at the University of
earning his Ph. D. at the University of Corsica. He moved to the University of
Bremen, he identifed amirto acids ire Nice-Sophia Antipolis in 2000, where he
artifcW cornets at the. Max-Planck-institute obtained his Ph. p, in 2005. He .spent a
for Solar System Research in Katlenburg- one year post-doc at the University of
Lindau and C.B.M. in Urlaans preparing the Hohenheim in the team of H. Strasdert in
cometary Rosetta mission. In 2005, he was prebiotic chemistry before being
promoted to full Professor at the University nominated assistant professor in 2006 at
o€Niceµ Sophia Antlpolis. His book, Amino LCNIBA. His cunent scientific interests
Acids and the Asymmetry of Life, was focus on flavours and fragrances and
published in 2008. prebiotic chemistry.
Cornelia Meinert received her diploma in Pierre Vierfing studied chemistry at the
chemistry in 2004 at the university of University of Strasbourg where he gained
Leipzig focussing on organic and his doctoral under the supervision of J. M
environmental chemistry, She is currently Lehr. He joined the CNRS in 1879 at the
completing her Ph. D. studies on preparative University of Nice Sophia Antipolis (UNS)-
capillary GC with Wenter Brack at the He was promoted Research Director in
Helmholtz-Centre and became postdoctoral 1998 and is currently head of the LCMBA.
fellow in the group of Uwe Melertenrich at His present scientific interests are
the University of Nice-Sophia Antipol s Her focussed on gene (DATA) delivery systems
research interests focus on the origin of with a particular interest for highly
biomolecular asymmetry, especially fluorinated systems for artificial viruses-
enarbomerseparation using GCxGC and far the specific delivery of DNA to
techniques targeted cells
Advanced experiments with an encapsulated dye confirmed that chromatography including a combustion system (GC-w1RMS)
the amphiphilic components of the droplets assembled into present new opportunities to better define the origins and formation
membranous vesicles providing well--defined interior spaces. pathways of meteoritic organic compounds. They showed SD and
6"C values verifying an interstellar origin of the amphiphilic
molecules.l'51 Besides linear chain monocarboxylic acids with
2.3. ldendfication of Amphiphiles in Carbonaceous carbon chains up to C^^, a large diversity of nearly randomly
Meteorites substituted branched chain inonocarboxvlic acids was identified.
This complex mixture of branched monocarboxylic acids was
Functional organic molecules have been extracted from the proposed to have originated by exothermic and thermodynamically
carbonaceous Murchison meteorite. Murchison belongs to the CM2- favored interstellar gas phase radical reactions proceeding from 10
type meteorites, known to contain tip to several percent of their mass to 100 K. More than 3o years ago_ comparatively primitive
as organic carbon; it has a complex history and is certainly not of analytical studies identified 18 monocarboxylic acids in the Murray
identical chemical composition to the simulated precometary ices and Murchison CM2 carbonaceous meteorites, identical to the core
presented in chapter 2.2. However, in the case of the Murchison analytes now detected by the Pizzarello group P61
Ja_nj
meteorite, enantioenriched amino wids,lo chiral and achiral In 1989, extracts from the interior of a 90 g sample of
[22-231
diamino acids. i2'1 nucleic bases, and amphiphilic molecules Murchison showed evidence for surface activity involving both the
have been identified. Chloroform-methanol extracts of the meteorite formation of monomolecular films at air-water interfaces and self-
sample showed that vesicular structures appear when a phosphate assembly into membranous vesicles with encapsulated polar
buffer is added to the organic extract. To determine whether the solvents."'I This research presented novel observations there
amphiphilic components can assemble into membranous vesicles amphiphilic molecules extracted from the Murchison meteorite were
with interior spaces, Dworkin et al. added a hydrophilic pyranine chemically identified, and they showed lipid-like behavior and self-
dye via an approved dehydration-rchydration cycle '241 to an extract organized into vesicles, suggesting that extraterrestrial materials
of the Murchison meteorite.lujl As shown in Figure 2. among oil could exhibit a far greater range of chemical properties and
droplets and other morphologies, micrometer-sized vesicular behaviors than previously thonght.' l"I Amphiphilic molecules can be
structures encapsulating the fluorescent pyranine dye in interior delivered to planetary surfaces such as the early Earth, where they
spaces were formed. mix with endogenous compounds synthesized on the planet,lt`"
The relevance of fatty acid vesicles to origin-of-life scenarios is
recognized because they are chemically simple versions of
amphiphilcs, in contrast to phospholipids used in contemporary
biological cells. We conclude that fatty acids and other amphiphilic
compounds present in carbonaceous meteorites can participate in
membrane self assembly processes, as can carboxylic acids
synthesized by aqueous FTT reactions ,l''I
2.4. Designing the First Cell. Self-Assembly of
Amphiphiles into Cell-Like Vesicles
Amphiphilic molecules linking a single saturated hydrocarbon
chain to a polar headgroup will, when dispersed in an aqueous phase,
self-assemble into different phases depending on concentration,
chain length, head group characteristics, and environmental factors
such as temperature, counter ions, and pH. Amphiphiles including
medium- and long-chain monocarboxylic acids, alcohols, amines,
alkyl phosphates, and alkyl sulfate&,lul as well as organic-inorganic
Figure 2. Meteoritic compounds seen in a new light pyranine dye nanoparticle hybrid systems''s_ `a1 typically form spherical micelles
encapsulated in vesicles made from an extract of the Murchison above the Kratft tern peraturel'01 and above the critical micellar
meteorite. Vesicles show interior spaces with sizes in the micrometer
concentration (crnc). They can form bilayers and vesicles at a
range, but ail droplets and inverse emulsions are also visible
critical concentration for vesicle formation ievc, sometimes
Copyright (2001) National Academy of Sciences, U.B.A.
abbreviated cbc for critical bilayer coneentration)'31 '21 in rapid
dynamic equilibrium with single molecules and micelles. We point
out that the cvc is usually much higher than the cmc. Nonmicellized
Because the Murchison meteorite extracts are available only in
limited quantities, the exact composition of the membrane-forming or nonvesiculized amphiphiles are always present together with
nneelles and vesicles_I',I
amphiphiles was not established in this studv.t''11
Lipid vesicles, also called liposomes {Iiposomes arc, strictly
New work of Pizzarello et al_ revealed via solid phase micro-
extraction tSPME) sample preparation that low molecular weight speaking, vesicles made out of lipids), Is -,l or often simply vesicles,"`'
monocarboxylic acids are the most abundant water-soluble organic are quasi-spherical shells composed of lipid bilayers, encapsulating
compounds in the Murchison and many other carbonaceous an aqueous space.i" —'' Unilamellar and multilamellar vesicles are
meteorites.'r -1 More than {) monocarboxylic acids were identified in generally formed upon dispersion of amphiphilies for mixtures
1 i.3 g taken from the inside of the meteorite_ representing quantities thereof) that self-organize in ware€ into lamellar phases Thew
10 to 100 times greater than those of amino acids. Compound- quasi-spherical supramolecular structures are composed of
specific isotopic analyses perforated with isotope ratio gas thousands to millions of individual molecules''' with dianreters
existence of a lamellar region D in the phase diagram, as illustrated open bilayers partially rolled into lipid tubules had been identified
in figure 3. Stich a growth process of cell membrane-like bilayers is during vesicle formation using the elastic bending energy
driven by the rapid equilibrium between individual amphiphiles, approach,1511 Spontaneous formation and growth of vesicles in a
micelles, and bilayers resulting in the uptake of the micellar micelle solution had moreover been studied by snail-angle neutron-
components/micelles into the bilayer structure and the concomitant scattering experiments (SARIS), opening up the possibility for few
dissolution of the micelles. hundred millisecond resolution experiments. These data revealed
In principle, the simplest mechanistic models of carboxylic acid that cylindrical micelles form before their con.€inuous transition into
vesicle growth would be: l) the direct fusion of micelles with vesiclesl521 and that the number of micelles required to produce a
vesicles in a single step, 2) the dissolution of micelles into vesicle is about 25-50Yl' Studies on the phase behavior of the
carboxylic acid followed by incorporation into the preformed reverse vesicle-micelle transition applying the cryo-TEM
membrane, or 3) vesicle-vesicle fusion, 14"1 The direct observation of methodology had revealed, too, that not only spherical micelles but
the growth mechanism of fatty acid vesicles was for a long time also long cylindrical micelles form as intermediate nanostruetuxes
inaccessible due to the lack of an appropriate analytical during the solubilization of phospholipid vesicles by surfaetants.l541
methodology but -- due to scientific advancement in this field - was Vesicle formation was furthermore observed to be mediated by
deciphered recently. minerals. It was shown that montmorillonite clayfzl and different
minerals and surfaces such as quartz, pyrite, and gold
nanostrueturesl5_1 accelerated the conversion of fatty acid micelles
3.1. Vivat, Crescat, Floreat: Vesicle Growth into bilayer membrane vesicles. Even silica particles of 6 mn
diameter, a diameter smaller than the smallest possible vesicle,
Cryotransmission electron microscopy (cryo-TE'M) was applied promoted vesicle formation. Nucleation most likely involved the
in the first pioneering study clearly demonstrating the growth of formation of small patches of membrane that can continue to grow
vesicles after the addition of fatty acid micelles. l"1 Here, the water- at their edges independently of the silica spheres. This type of
soluble protein ferritin, which, due to its dense iron core, can be surface-assisted vesicle formation was observed in real dune,
detected by cryo-TEM, was entrapped_ in the internal aqueous enabling researchers to see the formation of vesicles streaming oft a
volume of preformed vesicles The size distribution of filled microsphere just after micelle addition,' "The authors ar;sumed that
(ferritin-containing) and empty vesicles could be distinguished, and a layer of positively charged cations associated with or adjacent to
the cryo-TEM data -- obtained from frozen vesicle suspensions -, the montmorillonite surface attracts negatively charged micelles or
gave evidence for the growth of vesicles upon the addition of fresh free fatty acid molecules, increasing their concentration locally and
surfactant, as well as evidence of fission processes of larger vesicles thus facilitating their aggregation into a bilayer rnetnbrane.las;
that lead to a large number of small vesicles. unfortunately, this
cryogenic method could not be used to follow membrane vesicle
growth in real time. [471 3.2. Dynamic Properties of Vesicles
Recently, the laboratory of Szostak applied an innovative
methodology based on membrane-localized fluorescence resonance In contrast to micelles, membrane vesicles are described as
energy transfer (FRET) dyes to follow fatty acid vesicle growth to systems not to be at chemical equilibrium. They are
distinguish between vesicle growth by direct micelle-vesicle fusion thermodynamically unstable, requiring energy to form.''" In recent
versus vesicle growth by the incorporation of free molecular fatty years it became more evident that nonequilibrium structures appear
acids. Bence, a membrane-implemented FRET donor-acceptor pair at all levels in biology, and, as Kondepudi and Prigogine stated, "we
allowed for measurement of the increasing vesicle surface area cannot describe ?Mature around us without an appeal to
during controlled vesicle growth by the well-adapted addition of nor equilibrium situations."t561 In this context it was shown that
micelles. The FRET efficiency decreased as the surface density of different size populations of vesicles can coexist for several days in
the FRET dyes decreased when incorporating additional fatty acid. the same solution without a tendency to fuse with each other to
In contrast to former experimental approaches, this methodology search for an energy minimum. The different vesicle sizes
provided the advantage of allowing for a) the quantitative correspond to energy minima, but no tendency for a homogeneous
measurement of the growth of preformed vesicles even when new size distribution was observed after mixing. however, the individual
vesicles were formed simultaneously and b) such measurements to amphiphilic molecules were observed to be in local equilibrium with
be made in real time during the process of controlled membrane the vesicular structure, f uisi's team concluded that two populations
formation.'4?' Kinetic data revealed that none of the three above Of different vesicle sizes can not only coexist but also, due to larger
mentioned mechanistic models of vesicle growth is appropriate, and uptake rates of bigger vesicles for amphiphilic monomers present in
a new pathway involving previously unsuspected internediate the surrounding solution, compete with each other, for example for
aggregates was proposed. The structure of these metastable the uptake of reagents.l's'
intermediates could not be elucidated: candidate structures are Bilayer vesicles arc dynamic systems, and individual molecules
bilayer patches, cup-like membrane structures, and long cylindrical
can easily enter and leave the vesicular structure, Fatty acids in a
tnicelles, The .sizes of heterogeneous intermediate aggregates could bilayer membrane are in rapid exchange with the aqueous
be determined by dynamic Eight scattering to have a hydrodynamic i.nvironunent. AtnphiphOic monomers can excharrgs from two
radius of about 45 wn, much larger tbarr spherical rnicelles.';1 different layers within one vesicle.t'I They were observod to flip
A tune-resoived structural study art the micelle-to-vesicle from the outer shell into the inner shell and vice versa.'f7 This
transition had already shown that intermediate inetastable states behavior will be important for the nutrient intake and the inetabohte
occur, which wcm described cr_5 (: Iindricai worm;ike miceiles that outtakc of cell-like vesicles via bilayer membranes.
finally evol^,e via disks itato vesicles. t Membiage patches and
discs had been reported to be :short-li=ed intermediates in n mil elli-
to-csi ,e Mire ition m a trodd t t'c old ,up-like paorti [isr
3.1 A New Generation of Cells: Controlled Vesicle phenomenon might well have triggered the appcarance of cell-typc
Redivision vesicles on the early Earth.
Recent studies have shown that vesicles made from a decanoic
In the absence of the complex machinery that controls the acid-decanol mixture are capable of encapsulating and retaining a
tp-591
division of tnodenr cells, the redivision of growing vesicles variety of organic macromolecules such as tluorescent dyes
must rely on the intrinsic properties of the vesicle and the physico- (Figure 4). The formation of vesicles in the presence of a dye
chemical forces of the environment.1451 In R&D, where vesicles are resulted in the capture of the dye molecules within the vesicles.
used as model membranes and in phannaeeutical applications cohere Subsequent size exclusion chromatography allowed for the
vesicles are applied as nanoscale containers for drug transport and separation of the vesicles from unencapsulated dye, thus releasing
delivety,`601 the most widely used method to prepare vesicles under dye-enclosing vesicles for further investigations, `311 Not only dyes
controlled conditions in the laboratory is by extrusion of vesicle but also enzymes such as catalase, as well as oligonucleatides, can
suspensions through small pore filters. For "division", a vesicle be encapsulated in fatty acid vesicles using the
enters a membrane pore under pressure, transforms into a sphero- dehydration/rehydration method (Figure 4).124, 131
cylindrical shape, and fragments into smaller vesicles with a
diameter somewhat smaller or larger than the pore diameter,
depending on the ratio between vesicle size and pore diameter.P61
Even though this method is widely applied, the actual mechanis n by
which vesicles break up into smaller vesicles remains unclear.txr`"s'L
Szostak's group distinguishes between two distinct mechanisms
for vesicle division: ! t the parent vesicle can be disrupted into
smaller membrane fragments, subsequently resealing into a new
generation of smaller vesicles, or 2) by the pinching-off of smaller
vesicles resulting in insignificant dilution of the vesicle contents.tast
A fluorescent dye (calcein) was therefore encapsulated into 90. nn)
sized myristoleate vesicles that were grown to a size of 140 nm
through slow micelle addition, then extruded through 100 nm pores
to a final mean size of 88 nm. It was found that during extrusion
55 % of the dye had been lost from the vesicles.14s' The results show
that the myristoleate vesicle division proceeds with only a slightly Figure 4. Decanoic acid-decanol vesicles stained with fluorescent
rhodamine (left). 600mers of DNA encapsulated in vesicles of
greater loss of internal contents than that demanded by the
decanoic acid alone by the dehydrationlrehydration method (right).
geometric constraints of deriving two daughter spheres from one
DNA was stained with 3,6-dimethylaminoacridine (acridine orange), a
larger parent.
nucleic acid-selective stain used to enhance the contrast in the
In advanced studies Szostak et al, repeated cycles of growth and
microscopic image. Image courtesy: David Deamer, UC Santa Cruz.
division by growing a population of extruded myristoleate vesicles
by slow feeding with myristoleate micelles and then dividing, by
extrusion. The amount of encapsulated calcein was followed after As described in chapter 3.1. montmorillonite was observed to
each growth period and each extrusion. As expected, essentially no accelerate the conversion of fatty acid micelles into vesicles. The
dye was lost during any of the five growth phases, whereas 40 % of surface-mediated bilayer membrane organization allowed for the
the dye was test after each extrusion. These experiments constitute a vesicular encapsulation of catalytically active surfaces such as
proof-of-principle demonstration that vesicle growth and division montmorillonitc. By previous loading of the montmorillonite surface
can result from simple physicochemical forces, without any with adsorbed RNA. RNA oligonueleotides were incorporated into
complex biochemical mael inery.l°Sl Furthermore, environmental the Vesicles,t's' The observed encapsulation of mineral particles
shear forces can cause vesicles to divide. l451 In a recent experiment, within vesicles thus brought the catalytic potential of the RNA-
a template replication inside a cell-type model vesicle followed by tagged mineral surfaces into the vesicle,
the observed random segregation of the replicated genetic material Photoactive semiconducting particles, such as titanium dioxide
lead to the formation of daughter protocells (see section 5).1571 particles in the 20-nm size range, were incorporated into vesicles via
the dehydration/rehydration method, retaining photoaetivity by
allowing incident light to drive photoelectrochemical reactions in a
4. Towards Life's Dynamics: Nutrient Uptake
comparable manner to contemporaneous photosynthesis possibly
through bilayer Membranes 6b 1
relevant for the origin of life on Earth
4.1. Encapsulation During Vesicle Formation By 4.2. The Static Solubility-Diffusion Theory
Dehydration/R e h ydra ti o n
Phospholipid rrmubranes of extant biological cells show limited
Successfully integrating functional chemical systems into the permeability to ionic nutrients such as amino acids, nucleotides, and
z;
'interior space of vesicles is a key challenge in hiophysics.t' The phosphate with measured permeability coelTicients P w to cm s
dehydration'rehydration method is among the most efficient la 1 Deamer et al.l:n1 raised the question "how might an early form
encapsulation ^ucthods. By the dehydrationirehydration .method. of c0iolar life grain access to nutrient solutes'?-' We are confronted
nutrients and functional target molecules can be segties€e yed into the with the paradoxical situation that required vesicular tttembranes to
interior ^pace of vesicles at the stage of vesicle tonnation. Such a a i ae leer€neahie tnoii =h to enahle the intake of nntrients and b) ac€
as a bamtr Prohibiting the loss of the ettcapsnlated hrirnitivo
catalytic and genetic system. Without the barrier function, newly vesicle's interior/oxtenDr space (see chapter 3.2.). This phenomenon
synthesized substances would diffuse into the surrounding bulk is most important at the main phase transition temperature of the
phase, and the potential for interactive systems and speciation would bilayer and in the fluid state compared to the gel state.
be For increasing membrane permeability to solutes, one The functional enzyme catalase was encapsulated in decanoic
should use membranes in a fluid (liquid-crystalline) state rather than acid-decanol vesicles, and its substrate, hydrogen peroxide, was
in a gel (crystal) state. Another solution is to reduce the membrane added to the external aqueous environment, The bilayer membrane
thickness. These goals can be achieved by reducing the length of the was shown to be penneabte to hydrogen peroxide, releasing oxygen
lipophilic chains in the membrane-constituting amphiphiles, frn,641 by inside the vesicle by maintaining the catalytic function of catalase
introducing cis-unsaturation or branching in the chains, and/or by and protecting the enzyme in the vesicular internal space against
adding amphiphiles with larger head groups [571 external influences, e.g. catalase-degradading protease." , Similarly,
Various mechanisms have been proposed to describe the uptake polymerase enzymes encapsulated with their substrates in a cell-type
of nutrients through bilayer membranes. The static solubility- vesicle led to polymeric products, which were protected from
diffusion theory interprets the bilayer membrane as a liquid degradation by hydrolytic enzymes present in the external
hydrocarbon phase separating two aqueous phases. Permeating medium. 1631 Walde et al. entrapped PNPase enzymes in oleic
molecules will partition into the hydrophobic region, diffuse across, acid,' leaote vesicles, followed by external addition of ADP. The
and leave by redissolving in the opposite aqueous phase, driven by nutrient ADP that carries three negative charges at pit 4 was
its concentration gradient (this is also known as the passive diffusion observed to permeate across the vesicular bilayer into the interior
mechanism). Permeability coefficients can hence be calculated if space, where PNPase catalyzed the formation of poly(A), a stretch
appropriate partition and diffusion coefficients as well as the of ribonucleic acid that was retained inside the membrane vesicle
membrane thickness are known. The solubility-diffusion theory is (Scheme 1).14'1
applicable for uncharged molecules, because of their relatively high We have seen that under well-defined physicochemical
solubility in the intermediate hydrocarbon phase. This theory also conditions, amphiphilic molecules can form a population of bilayer
explains that uncharged amino acid methyl esters permeate lipid membrane vesicles that "replicate" through processes of growth and
bilayers orders of magnitude faster than their zwimt rionic parent division and the ability to entrap ntaeromolecules while
have:
compounds, the firmer being much more lipophilic than the latter. remaining permeable to srnailler polar sohttes.1'6,4_'1 The dynamic
Transmembrane pH gradients are used for the active and pore and flip-flop mechanisms might have allowed early cells to
quantitative loading into vesicles and are based on concentration have access to functional ionic nutrients from the external
gradients as well l`'21 environment.
4.3. The Dynamic Pore Mechanism N^Z
Discrepancies between predicted and
measured pemreabilities were observed for
I Hz
small ions penetrating thinner bilayer -0 0 {N lzyymailc ca C^H N
membranes. The alternative dynamic pore L' ^C3-P-fl._ F rt' ongation i /.
G 0.
mechanism suggests that the permeation of s PNPs^ f!k
'tons through bilayer membranes occurs uta,
014 t^
through pores or cavities that are hydrated
L1H rp^ _., N
transient defects produced by thermal -^ 1
fluctuations within the bilayer causing
disturbances in the lipid packing arder.issi ADP poly lAt
Small ions can enter into these pores located
in the headgroup region of the amphiphiles and pass through such
hydrated defects, evading the high-energy barrier associated with
Scheme 1. ADP permeates across the vesicular bilayer into the
partitioning into the hydrophobic membrane interior.`641 If
interior space of oleic acidoleate vesicles- Catalyzed by
Membranes are sufficiently thin, pores provide the dominant
polynudeotide phosphorylase (PNPase), intraprotoceflular enzymatic
permeation pathways for ions_ Ionic substrates such as the ADP elongation is shown, resulting In poly(A), a stretch of RNA that is
nucleoside triphosphate ATP were shown to permeate DMPC-based prevented from escaping into the extraceliular medium. !441
vesicular Hayers at the gel-fluid main phase transition temperature
of 23.3 `C, at rates capable of supplying an encapsulated template-
dependent RNA polymerase.l^'i Permeation was observed to be 5. Non-Enzymatic Elongation of Encapsulated
greatest at the phase transition temperature. At 3" `C, the optimal Nucleotides Inside Cell-type Vesicles
temperature for cnzymc catalyzed reactions, permeability decreased
by two orders, of magnitude, Even if the authors proposed the It w°as previously assumed that the encapsulation of mineral
dynamic pore mechanism for ATP permeation, the Hip-flop particles within membrane vesicles ailows for the use of the
mechanism cannot be excfudtd for explaining the observed results. catalytic potential of the mineral surface for the elongation of
Alternatively to the dynamic pore mechanism, charged encapsulated nucleotides.l's'
molecules pan coordinate on the external shell of the vcsicular Recently, the elongation of an encapsulated genetic polymer was
membrane to the polrir h: a&roups of the a€nphiphilic molecules. observed inside cell-like vesicles with neither inneral surfac: nor
Amphiphiles can flip fir-orn the outcriinner shell into the inncrfouter enzymatic support: synthetic single-strand DNA moleculcs with
:hell Eo h, ,,, pabkf of € 3 a5in rho clmr,__d l:to , ^Cuics to the vt nine ascs t, €c trappc l 3rsiclz m mb€tFn :.ie: arctirt as
primers and templates for their own elongation. Activated sequence infennation in the template can be transcribed to a
nucleotides containing the complementary guanusine bases were functional molecule.''ul Recently, aligopeptide synthesis from amino
added to the surrounding medium of the vesicles. The mixture of acid monomers inside vesicles made of fatty acids or phospholipids
molecules composing the vesicle membranes, including carboxylic in a simulated hydratherma€ environment was reported. It was found
acids, their corresponding alcohols, and monoglycerides, was that encapsulation of the glycine monomers enhanced
optimized for maximal permeability to ribose, the sugar component 0tig0meri7ari0n.t671 Amino acid nutrients for polymerase and
of RNA, but minimal permeability to polymers such as DNA.I6"d rephcase architecture are moreover required to cross the membrane
The optimized cell-like vesicles showed an elongation of the barrier to enter the interior space of the cell-like vesicles. Controlled
synthetic DNA primer as guanosine-containing imidazole-activated conditions that not only allow for the passage of charged nucleotides
nucleosides were added one by one to the external medium! In but also the uptake of zwitterionic amino acids while retaining
contrast, in experiments with POPC vesicles that were run in polymerized nucleic acids inside vesicles will hopefully enlighten
parallel, the authors observed no elongation 15'1 The authors assumed OUT understanding of the crucial steps of the origin of life in the near
that permeation of the imidazole-activated and negatively charged future.
nuelcotide across the membrane was driven by the interaction of its Discussions are ongoing regarding whether, when fed with
polar functional group with the amphiphile head group, whereas amphiphiles and precursors for membranes, replicases, RNA
non-polar regions of the nutrient interacted with the hydrophobic synthesis, and membrane vesicles will grow and divide, and whether
chains of the amphiphiles. The atnphiphile-nutrient complex then improved replicascst6A] will evolveJ'151 Szostak et al. pointed out that
Mips from the outer to the inner membrane shell (see Chapter 43) a vesicle carrying an improved €eplicase would itself not have an
carrying the nutrient to the internal space of the vesicle. This improved capacity for survival or reproduction. It would not be
experiment shows that prebiotically plausible membranes composed called "alive"_ For this to happen, an RNA-coded activity is needed
of fatty acids provide surprisingly high permeabilities to charged that imparts an advantage in survival, growth, or replication for the
molecules such as nucleotides, which can thus be incorporated from membrane component providing internal control of cell division. l45)
an external source of nutrients to take part in efficient template A ribozyme that synthesizes amphiphilic lipids and thus enables the
copying in the protocell interior (Scheme 2). %netnbrane to grow would serve as an example. The membrane and
The decoded non-enzymatic elongation of encapsulated the genome would then be coupled, and the `orl-,mism' as a whole
nucleotides inside protocells has far-reaching consequences, could evolve as vesicles with improved ribozymes would have a
suggesting that a hcterotrophic origin of life is feasible and that growth and replication advantage. f451
early living organisms or systems incorporated carbon-containing
nutrients already available in the environment. The authors argue
that early protocells trade of fatty acid membranes could not have S. Summary and Outlook: From Amphiphiles to
been autotrophs because internally generated metabolites would leak Living Cells
out.l5^1 Cellular life might first have sourced energy and nutrients
from the environment, and more complex autotrophic lifestytes Endogenous aqueous FTT-type syntheses and exogenous
appeared at a later stage of evolution.t661 These experimental data delivery via meteorites and comets are potentially important sources
again highlight that fatty acid membrane vesicles seem to be a of prebiotic and biogenic molecules to the early Earth. Both
suitable model for a protocell during early evolution leading to processes provide amphiphilic molecules that, under well-defined
cellular life.!"] physicuchemieal conditions, assemble into membrane vesicles.
Vesicles are assumed to have harbored potential prebiotic catalysis.
With compartmentalization, the encapsulated replicase component is
not only capable of, but also inevitably
0 subject to, variation, natural selection, and
rdrzw-_^... . l
thus Darwinian evolmionP= Based on
experimental studies in the laboratory, we
can assume that cell-like membranous
NIH compartments composed of bilayers
M i N14
ap appeared wherever organic compounds
ched to C, " ^ H.^_ MH; © became concentrated, within which
:eri^^dat^ „H nor molecules were trapped. Life began when
^F-
a c_ one or more of the assemblies found a way
o° .. 0 not only to grow but also to reproduce by
incorporating a cycle involving catalytic
%Ri4
functions and genetic iufomtation. Lipid
sudee£ides 21-phc,phoamidate-linked DNA
vesicles may have served as a physical
container housing informational polymers such as DNA and RNA
Scheme 2. Negatively charged imidazole-activated nucleotides cross
and a metabolic system that chernicatly regulates and regenerates
the vesicular membrane and participate in non-enzymatic copying of
cellular components, [421
an cfig©-dC DNA template, Membrane vesicles were composed of
decanoic acid, decanoi, and decanoic acid glycerol monoester. [sr Some authors have suggested that a iipid world may have
preceded an RNA world ,`^ Nonetheless, at =,otne point in prebiotic
evolution assemblies of lipid-like molccatles likely heron to
Cellular evolution teat=.timed to progress_ A typical protocell is incorporate monom"s of' present-day life, such as nucleotdcs and
assumed to cnc^ipsidatc n(.,! only an i?ttE^rt7tation-b arin^? te3^ plate amino acids. =after o igotntrization, calal .y,,is and iifmplating
=qut a:so a po.yn.trase or of nnunr. Lrcids_ so than capacities vokild be enhanced within dw
10
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12
Entry for the Table of Contents
A primitive cell remodeled: Bilayer
.$_ membrane vesicles provide a multi-
U. J. Meierhenrich,` J.-J. Filippi, C. = g faceted microenvironment in which
Meinert, P. Vierling, protometabolic reactions could have
J. P. Dworkin Page— Page been triggered. Here, we report an the
n....
formation of membrane vesicles via the
self-assembly of amphiphiles. When
The Origin of Primitive Cells, Nutrients + ^^-
nucleotides were added to the extra-
Intake, and Non-Enzymatic Elongation
vesicular medium, the intake of these
of Encapsulated Nucleotides
nutrients' was observed, followed by
their participation in non-enzymatic DNA
primer elongation in the vesicular
interior.
la
