Revised manuscript Directed Growth of Biomimetic 1 Microcompartments 2 3 4 5 Directed Growth of Biomimetic Microcompartments 6 7 8 9 1 Ivan Ivanov1*, Rafael B. Lira2, T-Y. Dora Tang3, Titus Franzmann3, Adam Klosin3, Lucas 10 11 4 3 4 2 12 2 Caire da Silva , Anthony Hyman , Katharina Landfester , Reinhard Lipowsky , Kai 13 14 3 Sundmacher1, Rumiana Dimova2* 15 16 17 4 1Max Planck Institute for Dynamics of Complex Technical Systems, Process Systems Engineering, 18 19 20 5 Sandtorstrasse 1, 39106 Magdeburg, Germany 21 22 2 23 6 Max Planck Institute of Colloids and Interfaces, Theory and Bio-Systems, Science Park Golm, 24 25 7 14424 Potsdam, Germany 26 27 28 8 3Max Planck Institute of Molecular Cell Biology and Genetics, Organization of Cytoplasm & 29 30 31 9 Dynamic Protocellular Systems, Pfotenhauerstrasse 108, 01307 Dresden, Germany 32 33 4 34 10 Max Planck Institute for Polymer Research, Physical Chemistry of Polymers, Ackermannweg 10, 35 36 11 55128 Mainz, Germany 37 38 39 40 12 * Correspondence: 41 42 13 Corresponding Authors 43 44 14 [email protected], [email protected] 45 46 47 48 15 Keywords: growth, vesicles, protein-rich droplets, coacervates, polymersomes, fusion, fatty 49 50 16 acids 51 52 17 53 54 18 Abstract 55 56 57 19 Contemporary biological cells are sophisticated and highly compartmentalized. 58 59 20 Compartmentalization is an essential principle of prebiotic life as well and a key feature in bottom-up 60 21 synthetic biology research. In this review, we discuss the dynamic growth of compartments as an 61 62 63 64 1

65 Directed Growth of Biomimetic 1 Microcompartments 2 3 22 essential prerequisite for enabling self-reproduction as a fundamental life process. We focus on 4 5 23 micron-sized compartments due to their cellular dimensions. Two types of compartments are 6 7 24 considered, membraneless droplets and membrane-bound microcompartments. We review growth 8 9 25 mechanisms of aqueous droplets such as protein (condensates) or macromolecule-rich droplets 10 11 26 (aqueous two phase systems) and coacervates, for which growth occurs via Ostwald ripening or 12 27 coalescence. For membrane-bound compartments, we consider vesicles, composed of fatty acids, 13 14 28 lipids or polymers, where directed growth can occur via fusion or uptake of material from the 15 16 29 surrounding. The development of novel approaches for growth of biomimetic microcompartments 17 18 30 can eventually be utilized to construct new synthetic cells. 19 20 31 21 22 23 24 32 1 Introduction 25 26 27 33 Despite the long and ongoing debate about the essence of life, there is a widespread consensus about 28 29 30 34 some common features of all living systems. While some phenomena, such as metabolism, remain 31 32 35 arguable benchmarks (and place viruses in the twilight zone), it is widely recognized that the 33 34 36 formation of micro- and nanocompartments is an essential ingredient of all forms of life. This spatial 35 36 37 37 constraint serves numerous purposes, including the segregation and protection from the environment 38 39 38 (to allow for individuality and maintenance), the establishment of gradients (to enable and make use 40 41 42 39 of out-of-equilibrium conditions), and the role of two- and three-dimensional confinement for self- 43 44 40 organization phenomena, all of which serve to overcome the overall “dilution problem”. In order to 45 46 47 41 fulfil another cornerstone of life – proliferation – compartments also need to change with time by 48 49 42 fusion, growth, and division. In particular, the dynamic growth of compartments is an essential 50 51 52 43 prerequisite for enabling self-reproduction as a fundamental life process, both in simplistic systems 53 54 44 such as droplets or fatty-acid based vesicles, as well as for lipid vesicle compartments with 55 56 45 membranes that resemble the biomembranes of today’s cells. In this context, we argue that growth 57 58 59 46 deserves more attention, not only because growth precedes division but also because of the difficulty 60 61 47 to realize growth compared to division, especially in the case of lipid vesicles, where budding and 62 63 64 2

65 Directed Growth of Biomimetic 1 Microcompartments 2 3 48 division has been observed in response to various factors This growth aspect has also been 4 5 6 49 recognized in basic theoretical models of living systems such as Ganti’s chemoton [1, 2], for which 7 8 50 the increase of membrane area (referred to as membrane formation) was postulated to be one of three 9 10 11 51 subsystems, characterizing living entities from a chemical viewpoint. The autopoietic theory was also 12 13 52 centered on the membrane but focused on self-maintenance rather than on (self-)reproduction [3, 4]. 14 15 53 However, such a self-maintaining system could also enter a self-reproduction mode, manifested by 16 17 18 54 growth, if homeostatic misbalance leads to excess membrane formation as shown in a thought 19 20 55 experiment by Luisi [3]. 21 22 23 24 56 In this paper, we review the existing approaches for growth of compartments in the context of 25 26 57 bottom-up synthetic biology and protobiology. We consider mainly micron-sized compartments due 27 28 29 58 to their characteristic cellular dimensions; this feature also ensures that the area and curvature of the 30 31 59 interface or the bounding membrane has less influence on the enclosed solution. Microcompartments 32 33 34 60 of various origins and chemistries have been used as protocell models, and many studies have 35 36 61 addressed growth and division simultaneously in an ambitious effort to mimic self-reproduction. 37 38 62 Here, we focus on growth and divide the literature examples into two categories: membraneless and 39 40 41 63 membrane-bound microcompartments, whereby we review aqueous droplets (such as protein- or 42 43 64 macromolecule-rich droplets and coacervates) on the one side, and vesicles, composed of fatty acids, 44 45 46 65 lipids or polymers, on the other. This is by no means a universal and comprehensive classification of 47 48 66 compartment types in nature1, but rather an attempt to classify reported examples. 49 50 51 52 53 54 55 56 57 58 59 60 1 61 We conceptually include examples of fatty acid vesicles as presumable predecessors, as well as vesicles made of 62 synthetic chemicals as prospective successors of today’s cells. 63 64 3

65 Directed Growth of Biomimetic 1 Microcompartments 2 3 67 2 Coacervates and protein-rich droplets – volume and area grow simultaneously 4 5 6 7 68 In the 1920’s Oparin hypothesized that membrane-free liquid-like droplets formed via complex 8 9 69 coacervation could have been precursors to modern cells [5, 6]. Extensive research has demonstrated 10 11 12 70 that small molecules and oppositely charged polymers can phase separate to form structures with 13 14 71 emerging liquid-like properties. More recently, it was discovered that certain types of proteins 15 16 72 undergo intracellular phase separation to form membrane-free/membraneless liquid-like protein-rich 17 18 19 73 droplets. Both of these membraneless microcompartments represent interesting systems to 20 21 74 circumvent the coupling between volume and surface area, and their austerity in comparison to 22 23 24 75 membrane-bound microcompartments has qualified them as useful models to demonstrate 25 26 76 compartment formation and growth. The relevance of membraneless compartments to bottom-up 27 28 29 77 synthetic biology, regardless of their chemical composition, remains undisputed, since they 30 31 78 successfully represent or mimic certain biological aspects. Thus, phase-separated droplets represent 32 33 34 79 useful models of self-organized entities, independent of the natural or man-made origin of the 35 36 80 macromolecules. Form a protobiology perspective, coacervates made of synthetic polymers are 37 38 81 logically excluded as potential ancestors of today’s cells, but such coacervates have played an 39 40 41 82 important role in the history of protocells, starting with the first experiments on gum arabic and 42 43 83 gelatin. 44 45 46 47 84 2.1 Protein-rich droplets 48 49 50 85 Compartmentalization is a hallmark of the eukaryotic cell. It enables cells to spatially separate their 51 52 53 86 complex biochemistry into microreactors. Well-known examples of compartments are the 54 55 87 mitochondrion and the nucleus, both of which are surrounded by lipid bilayers. However, in recent 56 57 58 88 years it has become clear that the eukaryotic cytoplasm is further organized by compartments that 59 60 89 lack membranes. These compartments have been termed biomolecular condensates, also known as 61 62 63 64 4

65 Directed Growth of Biomimetic 1 Microcompartments 2 3 90 membraneless organelles [7]. Intracellular condensates form by the process of liquid-liquid phase 4 5 6 91 separation [8-13]. Phase separation is a highly cooperative and concentration-dependent process: 7 8 92 above a certain saturation concentration, a solution becomes unstable and demixes to form two or 9 10 11 93 more distinct co-existing aqueous phases, a protein-dense and a protein-poor phase that continuously 12 13 94 exchange molecules and maintain a steep concentration gradient across their interfaces. Recent work 14 15 95 suggests, that the formation of biomolecular condensates is primarily driven by RNA and proteins 16 17 18 96 that exhibit a large degree of intrinsic disorder and/or multivalency. Among the growing list of 19 20 97 proteins that undergo liquid-like phase separation are the well-studied P-granule proteins LAF1 and 21 22 23 98 PGL-3 [14, 15], the stress granule proteins FUS and hnRNPA1 [16-18], the centrosome protein SPD- 24 25 99 5 [19], as well as the nucleolar proteins Fib1 and Npm1 that form immiscible phases to organize the 26 27 28100 nucleolus [20]. When expressed in a test tube, these proteins readily phase-separate to form two- 29 30101 phase systems at physiological concentrations. These protein-rich droplets grow by taking up 31 32102 material from the environment (Ostwald ripening) or via coalescence of two smaller droplets to form 33 34 35103 a larger one; see Figure 1. The condensates exhibit liquid-like characteristics, such that they can 36 37104 coalesce, wet surfaces and deform under shear stress. The dynamics of these processes is governed 38 39 40105 by internal viscosity and surface tension [8, 14, 21]. Importantly, condensates display selectivity, and 41 42106 client molecules such as interacting proteins or RNA will partition into the condensates. 43 44 45 46 47 48 49 50 51 52 53 54 55 56107 57 58 59108 Figure 1. (A) Protein-rich droplets can grow by the uptake of material from the surrounding and at 60109 the expense of smaller droplets (Ostwald ripening). (B) Still fluorescence images from a time-lapse 61 62 63 64 5

65 Directed Growth of Biomimetic 1 Microcompartments 2 3110 movie of a Hela cell expressing the YFP-labelled disordered region of the RNA helicase DDX4 4111 (unpublished data). The disordered region forms an intracellular liquid-like droplet. With time, the 5 6112 bigger droplet grows, whereas the smaller droplets shrink and eventually disappear. (C) 7113 Quantification of the bigger droplet area shown in (B) as a function of time (unpublished data). (D) 8114 Growth of liquid-like protein droplets can also occur via coalescence upon encounter of two or more 9115 droplets that merge to form a bigger one. (E) Still bright field images from a time-lapse movie of 10 11116 protein droplets formed from the prion protein Sup35 [22]. The protein droplet was held with an 12117 optical tweezers and brought in the vicinity of other droplets, which coalesce leading to growth with 13118 time. (F) Quantification of the droplet area shown in (E) as a function of time. The step-wise increase 14119 in area is indicative of coalescence. 15 16120 Recent work has identified that the saturation concentration of the FET family of proteins (FUS- 17 18 19121 EWSR1-TAF15 protein family) is quantitatively determined by the associative cation-pi interactions 20 21122 between segregated tyrosine and arginine sticker residues [23]. Other types of interactions, such as 22 23 24123 pi-pi and electrostatics between charged residues, play important roles as well [24]. For instance, the 25 26124 disordered region of the condensate protein helicase DDX4 displays a great degree of segregated 27 28 29125 charges [25]. Importantly, weak multivalent interactions underlie the nucleation process [11]. 30 31 32126 In the context of the cell, these systems are driven away from equilibrium, e.g. by chemical reactions. 33 34 35127 However, in the test tube, droplet growth continues until the thermodynamic equilibrium has been 36 37128 reached. Accordingly, mechanisms must exist that regulate the nucleation as well as the size and the 38 39129 mechanical properties. Liquid-like condensates can also undergo liquid-to-solid transitions [18, 22, 40 41 42130 26, 27]. For FET family proteins, the composition of interspacing residues (so-called spacers) that 43 44131 segregates the stickers plays an important role in modulating the interaction strength and thus the 45 46 47132 liquid-to-solid transition [23]. The liquid-to-solid transition of the centrosome forming protein SPD-5 48 49133 may be mediated by coiled-coil domains to establish a force-resistant meshwork for microtubule- 50 51 52134 mediated chromosome segregation [19]. Coupling phase separation to gelation may provide an ideal 53 54135 size-determining mechanism. Moreover, coupling the phase behavior to biochemical reactions that 55 56136 drive the system away from equilibrium establishes control over nucleation, droplet growth and 57 58 59137 under certain circumstances may even provide a mechanism for droplet division [28, 29]. Phase 60 61138 separation is exquisitely sensitive to environmental changes, including temperature, ionic strength or 62 63 64 6

65 Directed Growth of Biomimetic 1 Microcompartments 2 3139 pH, parameters that also fluctuate within cells [30, 31]. Cells have harnessed the phenomenon of 4 5 6140 phase separation to orchestrate complex processes and evolved precise mechanisms to control 7 8141 nucleation of biomolecular condensates. Taken together, it is now possible to predict the saturation 9 10 11142 concentration, as well as the material constants for minimal proteinaceous systems. Linking synthetic 12 13143 phase separation units with enzyme functions provides an opportunity to organize and polarize 14 15144 biochemical reactions within a synthetic cell. 16 17 18 19145 2.2. Polymer-rich droplets 20 21 22146 Coacervate microdroplets are generated from associative liquid-liquid phase separation 23 24 25147 (coacervation) processes between oppositely charged polymers or small highly charged molecules. 26 27148 They form from synthetic polymers (PDDA, polyethylemine, etc.) [32, 33], as well as from 28 29 30149 biologically relevant molecules including proteins (BSA) [34], polypeptides (polylysine, 31 32150 polyarginine) [35, 36], nucleic acids (DNA, RNA) [33, 37, 38], and co-factors (ATP, FAD, NAD) 33 34 35151 [39]. In comparison, dissociative liquid-liquid phase separation into two aqueous phases can be 36 37152 triggered in solutions of neutral macromolecules such as polyethylene glycol (PEG) and dextran, and 38 39153 more recently, this process was also established in the closed compartment of lipid vesicles [40, 41]. 40 41 42154 It is commonly considered that the general mechanism of phase separation occurs via two steps: an 43 44155 enthalpic contribution to the free energy from the electrostatic interaction between the molecules (in 45 46 47156 the case of charged polymers), which draws the molecules towards each other, and an entropic 48 49157 driving force from the rearrangement of ions and water leading to a lowering of the Gibbs free energy 50 51 52158 and the formation of membraneless, chemically enriched microdroplets. It has been proposed that this 53 54159 mechanism is analogous to liquid-liquid phase separation in biology [42, 43]. The surface tension of 55 56160 coacervates is low, between 1 µN/m – 1 mN/m [44], and even lower for aqueous two-phase systems 57 58 59161 of neutral polymers [45]. The droplets will grow in size over time via coalescence events [46]. The 60 61 62 63 64 7

65 Directed Growth of Biomimetic 1 Microcompartments 2 3162 rate of coalescence and therefore growth can be tuned by the overall charge ratios of the coacervate 4 5 6163 components. 7 8 9164 Studies have shown that polymer-rich microdroplets will selectively partition a range of different 10 11 12165 molecules leading to molecular localization and further chemical enrichment [39, 47-49]. Moreover, 13 14166 enzymes, which partition into the microdroplets maintain activity within the highly charged and 15 16167 crowded interior [50, 51]. Enzymes capable of phosphorylation and dephosphorylation have led to 17 18 19168 cycles of growth and degradation of the coacervate droplets [37, 52]. In addition, growth and decay 20 21169 of coacervate droplets can be instigated by switching the pH of the solution by bubbling carbon 22 23 24170 dioxide and ammonia through the dispersion of coacervate droplets [39]. These results indicate that 25 26171 the physical parameters such as molar ratio of polymers or pH and temperature of coacervate droplets 27 28 29172 can be used to tune and drive droplet growth and disassembly. Additionally, the ability of 30 31173 membraneless coacervate droplets to partition and support enzyme reactions could suggest that these 32 33 34174 systems could be plausible models for predicted growth and division cycles driven by flux of 35 36175 molecules across the interface [29]. 37 38 39176 3 Fatty acid vesicles as primitive protocells – membrane formation due to fast equilibrium 40 41 42 43177 The vesicular (membrane) systems represent another compartment type with the same structural 44 45178 features as living cells. Worth noting with respect to the field of minimal cells, and growth in 46 47 48179 particular, are the pioneering works of Luisi and Szostak [53, 54], which mainly relied on fatty acid 49 50180 micelles and vesicles. The latter have been investigated extensively as protocell models because fatty 51 52 53181 acids have been proposed to be prebiotically relevant due to their structural simplicity compared to 54 55182 phospholipids [55]. Therefore, the spontaneous uptake of fatty acids into preformed vesicles has 56 57 58183 often been considered as a primitive growth mechanism. As far as structural simplicity is concerned, 59 60184 the emphasis on fatty acids is analogous to the RNA hypothesis (single-/double-stranded vs. one or 61 62 63 64 8

65 Directed Growth of Biomimetic 1 Microcompartments 2 3185 two hydrophobic tails) but the quest for the origin of life is intrinsically speculative and we will 4 5 6186 refrain from statements about the evolutionary relevance of these compartments. Regardless of this 7 8187 open question, fatty acid vesicles and fatty acids have served as rewarding models for mimicking life 9 10 11188 processes, including growth. 12 13 14189 We also note that the majority of studies with fatty acids employed small (~30-50 nm) and large 15 16190 (~100-200 nm) unilamellar vesicles – below the limit of optical resolution – which poses the question 17 18 19191 whether those are relevant protocell models from another perspective, namely their small size. A 20 21192 workshop dedicated to defining the minimal compartment size of life, related to possibilities for 22 23 24193 extraterrestrial life and nanobacteria, dealt with this question 20 years ago, see proceedings [56]. The 25 26194 biologists view regarding the threshold size of primitive compartments was set to diameters of 27 28 29195 250±50 nm determined by a requirement for the compartment to accommodate minimum amount of 30 31196 necessary proteins, genes and ribosomes. Others views were more speculative suggesting ⁓50 nm as 32 33 34197 the minimal size required to sustain potential forms of primordial life. The latter notion was 35 36198 supported by reports on enhanced protein expression in 100 nm liposomes [57]. Considered from a 37 38199 simplistic chemoton perspective, smaller size implies higher surface-to-volume ratio, which is 39 40 41200 associated with enhanced inward flux (proportional to the surface) of membrane precursors and other 42 43201 metabolites and thus could be considered as beneficial. However, other factors such as packing, 44 45 46202 curvature, etc., could result in micrometer size (as in modern cells) optimal for a specific functions. 47 48203 In this review, we focus on biomimetic microcompartments, while we do not aim to set the size 49 50 51204 limits of protocells. However, we include reports on nanocompartments as well, due to their 52 53205 historical and mechanistic significance. Furthermore, microcompartments such as giant unilamellar 54 55206 vesicles (GUVs) [58-61] have proven to be a practical system, whose properties, growth and 56 57 58207 response to external factors can be monitored and manipulated directly under a microscope. 59 60 61 62 63 64 9

65 Directed Growth of Biomimetic 1 Microcompartments 2 3208 Compared to phospholipids, fatty acids exhibit a very quick exchange between the membrane leaflets 4 5 6209 as well as faster exchange kinetics between the vesicle and the solution. In addition, stabilizing head 7 8210 group interactions are more important, which endows distinct properties, such as growth and self- 9 10 11211 reproduction of vesicles [62-64]. Due to their single-chain structure, the concentration of monomers 12 13212 in equilibrium with vesicles is significantly higher than for phospholipids, which enables fast flip- 14 15213 flop and exchange of molecules; Figure 2A. These beneficial properties have qualified fatty acids as 16 17 18214 a prominent system for studying protocells, including the demonstration of membrane growth [62]. A 19 20215 notable phenomenon, observed during the growth of oleate vesicles, was the so-called matrix effect, 21 22 23216 which exemplified itself when a seed of preformed vesicles was added to the micellar oleate solution, 24 25217 resulting in a narrow size distribution, corresponding to the size of the seed [65]. The growth of 26 27 28218 oleate vesicles was further investigated in detail, which lead to kinetic [66] and molecular dynamics 29 30219 [67] models. The growth process was also demonstrated under flow [68], whereby filamentous 31 32220 microcompartments were formed, and also employed as a mechanism for ribozyme activation [69] to 33 34 35221 manifest a form of homeostasis. In addition, the filamentous growth of multilamellar oleate vesicles 36 37222 (⁓4 µm) was used to mimic the full proliferation cycle of growth and division [70] and the chemistry 38 39 40223 was extended to other surface-active molecules beyond oleate [71]. 41 42 43 44224 While fatty acids and surfactants are efficiently incorporated into existing membranes due to their 45 46225 optimal physicochemical properties, phospholipids – the membrane constituents of modern living 47 48 49226 cells – form structures, which are stable over a wider range of concentrations (phospholipids exhibit 50 51227 lower critical micelle concentration, CMC, compared to fatty acids). With respect to this distinction, 52 53228 the addition of oleate to preformed phospholipid vesicles was used in other attempts to mimic self- 54 55 56229 reproduction as an intermediate approach. Growth and division has been shown and investigated in 57 58230 detail in several examples of fatty acid incorporation into smaller liposomes [72-74] and the method 59 60 61231 has been later on scaled up to GUVs [75, 76]. In a similar fashion, the addition of detergents and 62 63 64 10

65 Directed Growth of Biomimetic 1 Microcompartments 2 3232 other water-soluble membrane active molecules at low concentration leads to their intercalation into 4 5 6233 the bilayer, which in turn could lead to an increase in the membrane area. In GUVs this increase 7 8234 depends on whether the inserting molecules exhibit fast or slow flip-flop. Fast flip-flop induces a 9 10 11235 large, visible increase in membrane area and fluctuations [75, 77, 78]. Slow flip-flop molecules 12 13236 induce an increase in the membrane spontaneous curvature due to their asymmetric incorporation into 14 15237 the outer leaflet of the membrane, resulting in tubulation [79-81], increased membrane tension and 16 17 18238 eventually rupture [75, 77]. Interestingly, the incorporation of simpler amphiphilic molecules or 19 20239 micelles leads to vesicle growth, often followed by division [70, 82], which is in line with predictions 21 22 23240 for phospholipids [83]. The limitation of detergent-like molecules is that at high concentrations, they 24 25241 lead to membrane permeabilization and eventual solubilization, depending on the membrane phase 26 27 28242 state and the CMC of the surfactant [84, 85]. In general, although this mixed approach provides a 29 30243 tangible system for studies of liposome growth, there is major conceptual drawback in the context of 31 32244 minimal cells – such microcompartments would lose their chemical identity over several generations 33 34 35245 and therefore could not sustain a continuous life cycle. The same drawback applies to another 36 37246 approach, in which blends of phospholipids and fatty acids or cationic surfactants were fused by 38 39 40247 attractive electrostatic interactions [86]. 41 42 43248 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 11

65 Directed Growth of Biomimetic 1 Microcompartments 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24249 25 26 27250 Figure 2. Some approaches for establishing growth of giant vesicles. (A) Growth via uptake of fatty 28251 acid molecules, which remain in dynamic exchange with the external and internal GUV solution and 29 30252 exhibit fast flip-flop across the membrane. (B) Growth via fusion mediated by the formation of 31253 transmembrane metal-ligand complex (left) or fusion protein mimetics (right). (C) Electrofusion 32254 relies on poration of two opposing membranes exposed to strong electric fields. The phase contrast 33255 snapshots on the right illustrate the fusion of two GUVs brought in contact and aligned by means of 34256 AC field, followed by the application of a DC pulse; reproduced from [87]. Copyright (2006) 35 36257 National Academy of Sciences. The white arrow indicates the direction of the field. (D) Growth of 37258 giant vesicles can be established by efficient fusion of small liposomes with the GUV, where the two 38259 membrane types are oppositely charged. Fusion is illustrated with the cartoon (left) and the confocal 39260 images (right) of one negatively charged GUV (green) exposed to a solution of positively charged 40 41261 small liposomes (red). Upon fusion, Förster resonance energy transfer leads to decrease in the red 42262 signal and the GUV area increases significantly [88]. The change occurs within seconds. Reprinted 43263 from [88] Copyright (2019), with permission from Elsevier. Scale bars in (C, D) represent 20 µm. 44 45264 46 47 48 49265 4 Growth of lipid vesicles – a difficult task, solved by fusion 50 51 52266 The system of highest interest for mimicking the proliferation of living cells is the phospholipid 53 54 55267 vesicle, which grows as a result of the incorporation of phospholipids in its membrane and eventually 56 57268 divides. The importance of liposomes arises from the chemical analogy with modern cell membranes. 58 59 60269 The cellular self-reproduction found in nature involves very complex biochemical interactions and 61 62 63 64 12

65 Directed Growth of Biomimetic 1 Microcompartments 2 3270 machineries. According to the reductionist concept, research in the context of protobiology and 4 5 6271 bottom-up synthetic biology aims to establish this phenomenon in a minimal system, based on 7 8272 simpler physicochemical mechanisms in a proof-of-concept for the origin and understanding of life. 9 10 11273 So far, however, growth of pure lipid vesicles has proved to be a challenge. Due to the difficulty of 12 13274 experimental realization of the membrane formation subsystem, however, theoretical studies have 14 15275 preceded practical examples in the case of phospholipids. Different kinetic [89] and thermodynamic 16 17 18276 [83, 90, 91] aspects have been considered and summarized together with other, chemoton-like 19 20277 approaches in a recent review [92]. On the practical side, the self-reproduction of a biomembrane, i.e. 21 22 23278 a phospholipid vesicle building its own membrane, has been addressed several times, but with only 24 25279 modest success so far. 26 27 28 29280 4.1 Growth via uptake of synthesized membrane components 30 31 32281 Efforts for synthesizing membrane components in liposomes date back to the early 90s. In particular, 33 34 35282 Luisi and coworkers assembled a four-enzyme cascade for phosphatidylcholine (PC) synthesis and 36 37283 deduced its incorporation into the membrane based on geometrical considerations (eventually the 38 39284 liposome size decreased, which was ascribed to the higher spontaneous curvature triggered by the 40 41 42285 newly synthesized short-chain PC – a partially undesired outcome with respect to growth) [93]. The 43 44286 authors did not unequivocally determine, whether the enzymes were present in the interior, whereby 45 46 47287 significant dimensional limitations could arise from the small liposome radius (23–26 nm), but they 48 49288 ascertained the enzyme association with the membrane to maintain an active form. In another study, 50 51 52289 the synthesis of phosphatidic acid by acyltransferases, expressed via cell-free methods in vesicles, 53 54290 was demonstrated but no growth was observed [94]. The latter observation was ascribed to the low 55 56291 amount of product due to the limited encapsulation, which will be difficult to overcome unless 57 58 59292 efficient transport mechanisms for the various precursors are established. Recently, this cell-free 60 61293 approach was extended to a more comprehensive pathway, starting from acyl-CoA and glycerol-3- 62 63 64 13

65 Directed Growth of Biomimetic 1 Microcompartments 2 3294 phosphate, and the liposome compartments were equipped with α-hemolysin to facilitate uptake of 4 5 6295 small polar molecules [95]. Although the authors did not specifically follow the vesicle growth, they 7 8296 identified practical barriers causing the low lipid synthesis rate, speculated about transport 9 10 11297 mechanisms for acyl-CoA and discussed relevant crowding and confinement effects. Another 12 13298 example in a similar direction was the biochemical synthesis of palmitate based on a FAS type I 14 15299 enzyme, encapsulated in liposomes [96], where vesicle growth has indeed been observed but the 16 17 18300 incorporation of foreign membrane components remains a conceptual problem. 19 20 21301 To circumvent protein synthesis issues, inherent to cell-free systems, an eight-enzyme cascade for the 22 23 24302 biosynthesis of phospholipids from fatty acids and glycerol 3-phosphate as building blocks has 25 26303 recently been assembled in vitro [97]. In the latter study, the enzymes and precursors were not 27 28 29304 encapsulated and synthesis occurred outside the liposomes. Experiments measuring the dequenching 30 31305 of rhodamine dye showed an approximately 30% membrane expansion, which was subsequently 32 33 34306 limited by the depletion of substrate. The growth was ascribed to the incorporation of oleic acid into 35 36307 the preformed liposomes, while the subsequent biosynthetic conversion to phosphatidic acid did not 37 38308 contribute to further expansion. Despite these limitations, the reconstitution of the biochemical 39 40 41309 membrane formation machinery is undisputedly a landmark in the area of bottom-up synthetic 42 43310 biology. 44 45 46 47311 The continuous search for more realistic growing protocell models has led to the use of phospholipid- 48 49312 like molecules, supplied from outside, instead of internally synthesized. The Sugawara group 50 51 52313 demonstrated growth, division and amplified DNA distribution in daughter vesicles [98]. This was 53 54314 achieved by adding a cationic precursor, which was hydrolyzed into the membrane lipid by an 55 56315 embedded catalyst. Notably, DNA amplification enhanced the efficiency of both growth and 57 58 59316 division. However, the elegant process was ultimately exhausted because it was limited by the 60 61317 consumption of DNA precursors inside the vesicles and the dilution of membrane catalyst and 62 63 64 14

65 Directed Growth of Biomimetic 1 Microcompartments 2 3318 phospholipids. Similar approaches were undertaken by the replacement of the complex biochemical 4 5 6319 lipid synthesis pathway with a self-reproducing autocatalyst, which resulted in triazole phospholipids 7 8320 formation and membrane growth [99]. Both cases seem to function with aggregates and multilamellar 9 10 11321 vesicles, and the exotic chemistry of the membranes might hinder interactions with some biological 12 13322 species (e.g. sensitive membrane proteins), thus potentially limiting other applications in the context 14 15323 of minimal cells. 16 17 18 19324 4.2 Growth via vesicle fusion 20 21 22325 In the case of authentic phospholipids, the only established and practical approach for achieving 23 24 25326 growth until now seems to be via vesicle fusion. Membrane fusion is a ubiquitous process in biology 26 27327 and it is fundamental for a number of cellular processes. It involves merging of two otherwise 28 29 30328 separated membranes, forming a compartment, whose area is the sum of the two fusing bilayers. In 31 32329 other words, increase in membrane area inevitably accompanies fusion, although the former is 33 34 35330 scarcely reported because the vast majority of fusion assays are insensitive to it. Driving forces of 36 37331 various nature have been employed to trigger fusion, some approaches are illustrated in Figure 2. 38 39332 These range from simple physicochemical triggers such as electrostatic interactions [100-102] and 40 41 42333 volume depletion [103] to biochemical approaches, relying on natural protein fusion machinery 43 44334 [104], as well as combinations thereof [105]. Furthermore, fusion methods have been expanded to 45 46 47335 biomimetic strategies [87, 106] in addition to more exotic approaches, involving the use of light (to 48 49336 heat nanoparticles [107] or isomerize azocompounds [108]) or electric fields [87, 109, 110] to perturb 50 51 52337 and porate the bilayer. In some of the examples fusion has led to spontaneous budding, correlating to 53 54338 predictions made by theoretical models [90], which has been put in the context of growth and 55 56339 division cycles for self-reproduction [109]. Presumably, the simplest mechanism to induce membrane 57 58 59340 fusion is based on membrane tension as has been elucidated by molecular simulations [111]. 60 61 62 63 64 15

65 Directed Growth of Biomimetic 1 Microcompartments 2 3341 Membrane fusion of small liposomes has been used for decades [112, 113] and only more recently 4 5 6342 fusion using giant vesicles has been reported [114]. In cells, membrane fusion is mediated by specific 7 8343 interactions between fusion proteins and such fusion reactions have been reconstituted in synthetic 9 10 11344 systems, in which small liposomes fuse with giant vesicles [104, 115, 116]. However, possibly due to 12 13345 the low fusion efficiency of protein-reconstituted systems or the low protein density, vesicle growth 14 15346 has not been detected. Fusion mediated by pH-sensitive lipids has also resulted in undetectable area 16 17 18347 increase [117]. In contrast, when mediated by high charge density, the fusion of positively-charged 19 20348 liposomes induces extensive area increase of the negative GUVs [88, 118], see also Figure 2D. 21 22 23349 Although very efficient, the process is limited by eventual charge neutralization. 24 25 26350 The fusion of two GUVs can be induced and observed also in a more controllable way. This can be 27 28 29351 achieved by bringing a pair of vesicles in contact by manipulation with ultramicroelectrodes [119], 30 31352 by trapping them within microfluidic devices [120], by applying an electric field [87, 110], or by 32 33 34353 direct manipulation using micropipettes [87, 121] or optical tweezers [107, 122]. After initial contact, 35 36354 fusion can be triggered by the application of a strong DC pulse [87, 123], via ligand-mediated ion 37 38355 interactions [87], transmembrane domains of fusogenic proteins [124] or through localized 39 40 41356 nanoparticle-mediated [107] or laser-mediated [122] heating. The common feature of all these studies 42 43357 is that an increase in membrane area is clearly observable. Although GUV fusion may result in 44 45 46358 visible area increase, the resulting area has never been quantified and fusion efficiency has been 47 48359 determined by changes in the measured fluorescence rather than the measured volume changes of the 49 50 51360 vesicle to characterize vesicular growth. Generally, the excess area results in increased membrane 52 53361 fluctuations and formation of membrane folds as a consequence of volume expansion lagging behind 54 55362 the area growth. The GUV-GUV fusion approach in its variations has been used to mix chemical 56 57 58363 precursors for nanoparticle formation inside the vesicular compartment [119, 122, 123], to 59 60 61 62 63 64 16

65 Directed Growth of Biomimetic 1 Microcompartments 2 3364 controllably obtain vesicles with well-defined compositions [121], and to unravel the formation of 4 5 6365 the hemifusion diaphragm [124, 125] and the rapid nature of the fusion neck expansion [87]. 7 8 9366 Importantly, fusion is not limited to pure phospholipid GUVs, but can be also extended to fusion of 10 11 12367 GUVs and cells [126, 127] or GUVs made of synthetic polymers. In contrast to liposomes, fusion of 13 14368 polymersomes is limited by the restricted mobility and high stability of the polymer membranes 15 16369 [128-130] associated with slowed down polymer rearrangement, which is a prerequisite for processes 17 18 19370 such as fusion. Some strategies have been successful in inducing fusion events in polymer GUVs, 20 21371 including exposure of vesicles to osmotic pressure gradients [128], ultrasound treatment [131], 22 23 24372 membrane perturbation by azobenzene photoisomerization [132] and addition of membrane 25 26373 disruptive agents into the dispersing medium [133]. 27 28 29 30374 In relation to bottom-up synthetic biology and protobiology, although fusion has been traditionally 31 32375 considered from a biophysical and biochemical perspective, spontaneous liposome growth could in 33 34 35376 principle occur (or could have occurred in the evolutionary history) by the discrete steps of vesicle 36 37377 fusion [134]. In addition, apart from a mechanism for increasing the membrane area, it has been 38 39378 speculated as a scenario, in which two systems combine their properties and thus increase the degree 40 41 42379 of biocomplexity [135], for instance considering that vesicular trafficking is a major transport 43 44380 mechanism for proteins in natural living cells [136]. This aspect was used to supply feeding 45 46 47381 components and trigger gene expression in the case of fusion of oppositely charged vesicles [137]. 48 49 50382 5 Synthetic chemistry enables new potential approaches for growth 51 52 53 54383 In parallel to the continuing efforts to form and grow new types of membraneless 55 56384 microcompartments, enable efficient fusion via new approaches, and ideally reconstitute the natural 57 58 59385 machinery for phospholipid synthesis, there is another possible pathway, which brings synthetic 60 61386 chemistry in the foreground of bottom-up synthetic biology. This aspect has been already partially 62 63 64 17

65 Directed Growth of Biomimetic 1 Microcompartments 2 3387 addressed by the demonstration of life-like properties of dichloromethane and nitrobenzene oil 4 5 6388 droplets, which grow, divide and partition cargo [138, 139]. The versatility of synthetic reactions was 7 8389 also used to augment phospholipid vesicles and make them grow [98, 99] as noted above. With 9 10 11390 respect to this, advances in the preparation of functional amphiphilic block copolymers provide 12 13391 powerful synthetic tools for the engineering of artificial compartments that could grow upon 14 15392 stimulation by light, temperature or pH. Here the stimuli would induce the build-up of membrane 16 17 18393 stresses that could relax through fusion events. In this way the chemistry of living systems could be 19 20394 extended far beyond natural building blocks, enabling not only the reverse engineering or re- 21 22 23395 engineering, but also the de novo engineering of life. Besides a powerful toolbox, which may enable 24 25396 futuristic applications, the latter concept of synthetic (in its conventional semantics of chemical) has 26 27 28397 resorting implications on the understanding of life as a display of self-organization and will expand 29 30398 our search criteria for other forms of life. 31 32 33 34399 6 Conflict of Interest 35 36400 The authors declare that the research was conducted in the absence of any commercial or financial 37401 relationships that could be construed as a potential conflict of interest. 38 39402 7 Author Contributions 40 41 42403 All authors listed have made a substantial, direct and intellectual contribution to the work, and 43404 approved it for publication. 44 45405 8 Funding 46 47406 This work is part of the MaxSynBio consortium, which is jointly funded by the Federal Ministry of 48 49407 Education and Research of Germany (BMBF) and the Max Planck Society (MPG). 50 51408 9 References 52 53409 [1] T. Ganti, Organization of Chemical-Reactions into Dividing and Metabolizing Units - 54410 Chemotons, Biosystems 1975, 7, 15-21. 55 56411 [2] T. Ganti, Biogenesis itself, Journal of Theoretical Biology 1997, 187, 583-593. 57412 [3] P.L. Luisi, Autopoiesis: a review and a reappraisal, Naturwissenschaften 2003, 90, 49-59. 58413 [4] F.G. Varela, H.R. Maturana, R. Uribe, Autopoiesis: the organization of living systems, its 59414 characterization and a model, Biosystems 1974, 5, 187-196. 60415 [5] A.I. Oparin, Origin of life, Macmillan, New York, 1938. 61 62 63 64 18

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