Concluding Remarks

Compartments in general and vesicles in particular, play a fundamental role in the origin of life. We have already seen that, basides providing confined space for the occurrence and persistence of molecular reaction networks, the formation of a compartment establishes the emergence ofindividuality and, when the autopoietic conditions become fulfilled, the emergence of self-maintenance, self-bounding and homeostasis. In addition, confinement of self-organized reaction networks within a vesicle creates clear-cut distinction between the "system" and its environment. Several authors have recognized from the theoretical viewpoint the relevance ofcompartments, even though the experimental approach has only been introduced recently. One of the clearest positions is held by Harold J. Morowitz, who states:1

The formation of closed vesicles was a major event in biogenesis: it represented the origin of supramolecular entities, three-phase systems consisting of a polar interior, a nonpolar membrane core and a polar exte rior— the environment. It is difficult to ove rstress the importance of vesicle closure in cellular genesis. This event established the organism-environment dichotomy in the most general sense. Closure led to aphysicalseparation between outside and inside by a barrier of restricted permeability. Without this barrier, the very idea of a cell is hard to visualize.

It is not known how the first living cell originated, but what is clear is that no life is possible without a compartment, since life is an emergent and collective property derived from the self-organization of a complex molecular system. Life is not present in single molecules, such as DNA or ribozymes; it arises instead from self-generating autocatalytic and coupled processes that follow a circular logic. Autopoieis offers a powerful descriptive view of life, even if it does not explain how cellular life originated on the Earth.

Experiments have shown that several reactions, including one of great biochemical significance, can be reconstructed inside the tiny aqueous space of lipid- and fatty acid-vesicles and that vesicles can grow and divide through the incorporation of a membranogenic precursor. These encouraging observations are driving current re

Figure 12.10. A speculative scenario for the emergence of primitive cells. Functional biopolymers and surfactants self-assemble in cell-like structures, i.e., vesicles. Starting from this initial stage, the system must achieve self-maintenance, self-replication and directed membrane synthesis by stepwise chemical evolution. Many steps of this transition are not known, in particular the question of the origin of the first functional macromolecules.

Figure 12.10. A speculative scenario for the emergence of primitive cells. Functional biopolymers and surfactants self-assemble in cell-like structures, i.e., vesicles. Starting from this initial stage, the system must achieve self-maintenance, self-replication and directed membrane synthesis by stepwise chemical evolution. Many steps of this transition are not known, in particular the question of the origin of the first functional macromolecules.

search toward the realization of semi-synthetic constructs where vesicles enclose more complex biochemical pathways, ranging from the expression of genes to the control of vesicle permeability, to internal regulation and to the biosynthesis of lipids, with the aim of synthesizing models of early cells with such minimal and essential functions that they may be called alive. This research area, known as the "minimal cell" area, is attractive also for other disciplines, such as the constructive approach of synthetic biology. Clearly,

Figure 12.11. An experimental approach to semi-synthetic minimal cells. Enzymes, genes and transcription/translation machineries are entrapped inside vesicles. In situ synthesized enzyme(s) can process suitable precursors to produce the membrane-forming compound S. As a consequence, the vesicles will grow and divide. If the internalized components are not produced, the system will die after a number of divisions owing to an excessive "dilution" of the network elements in some of the vesicles.

Figure 12.11. An experimental approach to semi-synthetic minimal cells. Enzymes, genes and transcription/translation machineries are entrapped inside vesicles. In situ synthesized enzyme(s) can process suitable precursors to produce the membrane-forming compound S. As a consequence, the vesicles will grow and divide. If the internalized components are not produced, the system will die after a number of divisions owing to an excessive "dilution" of the network elements in some of the vesicles.

semi-synthetic minimal cells do not answer the question of the historical origin of living cells. Nevertheless the efforts involved in their study will bring deeper knowledge (and verification) of the principles underlying cellular life.

Going back to the main topic of this chapter, the origin of protocells, we must admit that very little is known about the mechanisms of their formation. We believe that the formation of compartments is an early step and that various beneficial properties ofvesicles such as colocalization of reactants, protection from external molecular parasites and inhibitors, potential surface catalysis, accumulation of hydrophobic pigments in the membrane, maintenance of electroosmotic and chemical gradients, storage of macromolecules, emergence of individuality and ultimately creation of a self-sustained biochemical organized system, all serve to emphasize the significance and need of compartmentation in the transition from the nonliving to living matter. Undoubtedly fatty acid vesicles are currently considered the most coherent model of the early compartments: besides the formation of their fatty acid monomers under plausible prebiotic conditions, the vesicles can form by self-assembly of the monomers, give rise to a semi-permeable boundary, host biochemical reactions, grow and divide spontaneously and therefore self-reproduce.

References

Special Readings

1. Morowitz H. Beginnings of cellular life. Metabolism recapitulates biogenesis. Yale University Press, New Haven, 1992.

• Essay on the origins of cellular life, with emphasis on the compartment approach and key aspects of metabolic organization, complexity, networks and bioenergetics.

2. Luisi PL. The emergence of life. From chemical origins to synthetic biology. Cambridge University Press, Cambridge, 2006.

• The book presents a systematic course discussing several theoretical and experimental issues on origins of life studies, as self-organization, emergence, self-replication, autopoiesis, biophysics of compartments, their self-reproduction and use as cellular models.

3. Luisi PL. Autopoiesis: A review and a reappraisal. Naturwissenschaften 2003; 90:49-59.

Box 12.2. Compartments in Other Origins of Life Scenarios

We have seen how the "compartment approach" has been employed as a paradigm for a series of experimental and theoretical investigations. It is clear, however, that compartments alone do not suffice to develop the first cells. The relevance of compartments has been recognized with different personal emphases and biases, by several authors, whose key contributions go beyond a metabolism-first vs gene-first debate. In this paragraph, we will illustrate briefly how some of these authors have integrated the existence and function of compartments into their origin of life scenarios (see also ref. 6).

Manfred Eigen, representing the gene-first viewpoint, has proposed a stable coexistence of several "cooperating" information carrier molecules (RNA) that self-organize into mutually catalytic cycles known as "hypercycles".33 In this way, several replicators cooperate with, rather than compete against, each other. In its initial formulation, the hypercycle consists ofRNA replicators that are translated into replicator enzymes, but a ribozyme hypercycle also has been proposed. Eigen's hypercycles solve the problem of achieving a rich functional set of molecules by overcoming the trap of replication error threshold (Eigen's paradox). One of the problems of hypercycles is that a hypercycle is not an individual in the sense that a bacterium is. Instead it is an ensemble of interacting molecules that is affected by molecular parasites, short-cuts and mutations. To stabilize hypercycles and improve cycle fitness, it has been suggested, although not very passionately, that the hypercycle might be inserted into compartments,34 creating in this way individuals with inheritable genetic information built on the joint occurrence of hypercycle processes and compartment reproduction. Moreover, the compartment protects the hypercycle from parasites and alternative cycles and brings in the benefits of favorable mutations.35,36 Similarly, compartmentalized hypercycles can generate fruitful competition between protocells, thus giving rise by natural selection to more effective enzymes and consequently also longer genes.6

Freeman Dyson asserts,37 on the other hand, that a network oforganic reactions catalyzed by protein-like molecules preceded the appearance and replication of nucleic acids. His theory has strong roots in the Oparin view of a metabolism-first/coacervates origin. According to Dyson, compartments and protocells containing a metabolic system based on protein-like catalysts are first and essential. Such primitive "cells", mostly inspired by Oparin's coacervates, could grow by absorbing building blocks from the environment and reproduce by statistical division, so that a basic form of compositional (and functional) inheritance become possible. Nucleic acids originated later as by products of such metabolism (or as cell invaders) and were parasites of the metabolism at first, then symbionts and finally fully integrated components of the cell. The compartment in the Dyson model is a necessary element that encloses a specific set of components which give rise to proto-metabolism. What is inherited by the daughter structures from the parent is the system of metabolites, protoenzymes and metabolic paths, namely the organization of the metabolism and the architecture ofthe whole system. Although Dyson's model lacks specific chemical considerations, especially those defining the nature of the bounded particle, it makes clear reference to compartments and compartmentalized reactions as a means to achieve individuality and conceives of inheritance routes without referring to the nucleic acids.

Moreover, it focuses on homeostasis as the essential character of living cells.

Another important theoretical approach was formulated some years ago by Stuart Kauffman.38 It follows the paradigm ofcomplex system self-organization, suggesting that some systems can spontaneously reach a state of "order" despite their initial "disorder" state. In order to accomplish this disorder to order transition, the system must be thermodynamically open, so that it can reduce its entropy at the expense of increased entropy in the environment; this way the system can maintain its far-from-equilibrium state without violating the second law of thermodynamics. The roots of this view stem from the dissipative systems of Ilya Prigogine.39 Kauffman states that the spontaneous emergence of self-organized systems is one of the key factors for the origin oflife. Following this idea, he has developed a model on the onset of an autocatalytic reaction network formed by catalytic, interacting biopolymers. The single molecules cannot self-replicate; the system, i.e., the network of interacting catalysts and reactions, need to reproduce itself as a whole. In this view, collective properties emerge as soon as a critical level of molecular complexity is surpassed. Chemically, the catalytic biopolymers are ribonucleic acids and peptides, but the theory does not involve self-replicating RNAs. Compartments enter into Kauffman's theory—although it is not focused on the compartment approach—in two aspects. The first is the confinement required to let reaction proceed by increasing the local concentration of the biopolymers. The second arises from the need of introducing an evolution/selection pathway. New, occasional mutant polymers may be added to the initial autocatalytic set and this will potentially bring the system to diverge toward different directions. If the autocatalytic set is confined in a sort of protocell (Oparin's coacervates, Fox's microspheres and liposomes were suggested), the division process stochastically brings different polymer sets to two daughter cells, this being especially true for polymers present in low copy number in the parent protocell. This will bring into play the evolution of autocatalytic sets within a population of dividing protocells. Kauffman also suggests that liposomes in particular can take on, in addition to their confinement tasks, a key role in facilitating the formation of biopolymers on the basis of osmotic and entropic effects.38

With the "minority control" approach, Kunihiko Kaneko offers a new look at the two main problems of the gene-first or metabolism-first approaches, namely the lack of stability against parasites in the former and problematic inheritance in the second. In his theory, the existence of a compartment (a protocell) is a foremost requirement; and two levels of reproduction, of molecules as well as protocells, are assumed, with the view that compartmentation is important to the origin of genetic information. The minority molecules are low in concentration on account of their slow synthesis. However, because they participate in the mutual catalysis required for the reproduction of the protocell as a whole, they actually control the dynamics of the protocells. The minority molecules must be preserved; and their number fluctuations, having a strong influence on the division time of the protocell, must be dampened. The detailed concepts of the theory of minority control can be found by the interested reader in Kaneko's monograph.40

• Basic autopoiesis is introduced and discussed, including a historical viewpoint; its implication in understanding the logic of cellular life is clearly explained. Principles of emergence and biological autonomy are also briefly discussed.

4. Monnard PA, Deamer DW. Membrane self-assembly processes: Steps toward the first cellular life. Anatomical Records 2002; 268:196-207.

• A recent review on the role of vesicles (from phopsholipids and fatty acids) in the origin of life. Structure, stability, permeability and their use in experimental investigations are well illustrated.

5. Walde P. Surfactant assemblies and their various possible roles for the origin(s) of life. Orig Life Evol Biosph 2006; 36:109-150.

• In this review, a large collection of data on the various possible roles (not only compartmentation) of surfactant assemblies as micelles, reverse micelles and vesicles is well organized and clearly explained.

Precellular Evolution: Vesicles and Protocells Specific References

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