Self-reproduction Of Vesicles Research Articles

Spectroscopic and Microscopic Approach to Monitor the Changes in Bilayer Rigidity during Cell Penetrating Peptide Induced Self-Reproduction of Phospholipid Vesicles

Understanding peptide-membrane interaction in view of structural change of lipid bilayer is essential to develop new therapeutic strategies. Though different mechanisms are proposed for cell penetrating peptides (CPPs) penetration in lipid bilayer, the exact mechanism of cellular uptake of CPPs remains a significant unanswered piece of puzzle. Here, we report a novel observation that model phospholipid vesicles self-reproduce to generate unilamellar daughter vesicles in presence of a CPP, nonaarginine (R9). Fluorescence lifetime image analysis (FLIM) exquisitely captures the changes of L-α-phosphatidylcholine (LAPC) bilayer in different times and also quantifies the changes through valuable lifetime distributions obtained. Mean square displacement (MSD) calculations from FLIM images of single vesicle trajectory establish CPP induced self-reproduction of LAPC vesicles. Heterogeneous nature of lipid bilayer after 1 hour of R9 addition is observed from fluorescence correlation spectroscopy (FCS) which supposed to consider as the starting point of “mother vesicles” reproduction. Solvation dynamic study delineates the change in dynamics of the encapsulated water inside vesicles which also states different rigidity of lipid bilayer in different states of vesicle self-reproduction. Fluorescence anisotropy image analysis provides information regarding change in lipid bilayer rigidity by quantifying the rotational motion of the fluorophore in presence of perturbation of CPPs. From these experimental observations, we are able to establish phospholipid vesicles self-reproduction in presence of CPPs which may be considered as a possible route of CPPs action to deliver cargos to cell interior through hydrophobic barriers. Overall, our results provide an experimental underpinning of the role of lipid bilayer structural changes in self-reproduction which can exhibit great potential to interpret the protein-lipid interaction in emergence of life.

A cell-penetrating peptide induces the self-reproduction of phospholipid vesicles: understanding the role of the bilayer rigidity.

Model lipid vesicles self-reproduce to generate unilamellar daughter vesicles in the presence of a cell-penetrating peptide. Fluorescence lifetime and anisotropy image analysis exquisitely capture the change in the lipid bilayer rigidity throughout the process, whereas a solvation dynamic study delineates the change in the dynamics of the encapsulated water inside the vesicles. Overall, our results provide an experimental underpinning of the role of the lipid bilayer structural changes in self-reproduction, which can exhibit great potential for interpreting the protein-membrane interaction in the emergence of life and for developing new therapeutic strategies.

Glass Microsphere-Supported Giant Vesicles for the Observation of Self-Reproduction of Lipid Boundaries.

Growth and division experiments on phospholipid boundaries were carried out using glass microsphere-supported phospholipid (DOPC) giant vesicles (GVs) fed with a fatty acid solution (oleic acid) at two distinct feeding rates. Both fast and slow feeding methods produced daughter GVs. Under slow feeding conditions the membrane growth process (evagination, buds, filaments) was observed in detail by fluorescence microscopy. The density difference between supported mother vesicles and newly formed daughter vesicles allowed their easy separation. Mass spectrometric analysis of the resulting mother and daughter GVs showed that the composition of both vesicle types was a mixture of original supported phospholipids and added fatty acids reflecting the total composition of amphiphiles after the feeding process. Thus, self-reproduction of phospholipid vesicles can take place under preservation of the lipid composition but different aggregate size.

Role of Inverse-Cone-Shape Lipids in Temperature-Controlled Self-Reproduction of Binary Vesicles

We investigate a temperature-driven recursive division of binary giant unilamellar vesicles (GUVs). During the heating step of the heating-cooling cycle, the spherical mother vesicle deforms to a budded limiting shape using up the excess area produced by the chain melting of the lipids and then splits off into two daughter vesicles. Upon cooling, the daughter vesicle opens a pore and recovers the spherical shape of the mother vesicle. Our GUVs are composed of DLPE (1,2-dilauroyl-sn-glycero-3-phosphoethanolamine) and DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine). During each cycle, vesicle deformation is monitored by a fast confocal microscope and the images are analyzed to obtain the time evolution of reduced volume and reduced monolayer area difference as the key geometric parameters that quantify vesicle shape. By interpreting the deformation pathway using the area-difference elasticity theory, we conclude that vesicle division relies on (1) a tiny asymmetric distribution of DLPE within the bilayer, which controls the observed deformation from the sphere to the budded shape; and (2) redistribution of DLPE during the deformation-division stage, which ensures that the process is recursive. The spontaneous coupling between membrane curvature and PE lipid distribution is responsible for the observed recursive division of GUVs. These results shed light on the mechanisms of vesicle self-reproduction.

From vesicles to protocells: the roles of amphiphilic molecules.

It is very challenging to construct protocells from molecular assemblies. An important step in this challenge is the achievement of vesicle dynamics that are relevant to cellular functions, such as membrane trafficking and self-reproduction, using amphiphilic molecules. Soft matter physics will play an important role in the development of vesicles that have these functions. Here, we show that simple binary phospholipid vesicles have the potential to reproduce the relevant functions of adhesion, pore formation and self-reproduction of vesicles, by coupling the lipid geometries (spontaneous curvatures) and the phase separation. This achievement will elucidate the pathway from molecular assembly to cellular life.

Novel applications of physical autocatalysis.

The autocatalytic self-reproduction of micelles and vesicles has been studied for several decades. These systems are vital components of certain protocell models and some models for how life may have begun from mixtures of simple chemicals. Here we discuss our recently described autocatalytic systems where self-reproducing micelles are driven by bond-forming reactions. These systems generate increased complexity on both the molecular level, through covalent bond formation, andthe supramolecular level, through spontaneous self-assembly into functional aggregates. This provides the conceptual basis for novel studies of the potential roles of self-reproducing lipid aggregates in the prebiotic world.

Achievements and open questions in the self-reproduction of vesicles and synthetic minimal cells

Supramolecular chemistry was enriched, about twenty years ago, by the discovery of the self-reproduction of micelles and vesicles. The dynamic aspects and complexity of these systems makes them good models for biological compartments. For example, the self-reproduction of vesicles suggests that the growth in size and number of a vesicle population resembles the pattern of living cells in several aspects, but it take place solely due to physical forces. Several reports demonstrate that reverse micelles, micelles, sub-micrometric and giant vesicles can self-reproduce, generating new particles at the expenses of a suitable precursor. Recently, similar studies are in progress on more complex vesicle-based systems, namely semi-synthetic minimal cells. These are artificial cell-like compartments that are built by filling liposomes with the minimal number of biomolecules, such as DNA, ribosomes, enzymes, etc., in order to construct a living cell in the laboratory. This approach aims to investigate the minimal requirements for molecular systems in order to display some living properties, while it finds relevance in origins of life studies and in synthetic (constructive) biology.

Reproduction of Fatty Acid Vesicles

Compartmentalization is an essential step for the origin of life to allow for the formation of more complex biotic building blocks. Possibly in early life preliminary compartments were formed out of prebiotic molecules, such as fatty acids. These molecules organize in bilayers at neutral pH and they show intriguing behavior with respect to self-reproduction of vesicles [1], in that fast addition of fatty acid at high pH to preexisting seed vesicles at neutral pH results in a fast formation of new vesicles with a size distribution that is closely related to that of the seed vesicles. The mechanism behind this so-called matrix effect is still a puzzle. One possibility would be that the fatty acids insert in the outer monolayer of the vesicle and that an excess of material in the outer leaflet with respect to the inner leaflet is then the driving force for budding and subsequent fission. In order to test this possible mechanism, we varied the rate of addition (insertion) of the new material. Slow addition would give the system time for movement of fatty acids from the outer leaflet to the inner leaflet and thus to remove this imbalance. Our results show that such a decrease of the addition rate indeed leads to growth of the vesicles instead of division, supporting our hypothesis. We also found, by including a fluorescent dye which is self-quenching at high concentrations, that during the fission process the content of the vesicles does not leak to the exterior. These observations agree well with results and predictions from coarse-grained molecular dynamics [2] and provide a plausible mechanism for the matrix effect [1].[1] Luisi et al. J. Phys. Chem. B 2008, 112, 14655-14664 [2] Markvoort et al., in preparation

Insights into the self-reproduction of oleate vesicles

In view of the importance of vesicles as models for early cells, several groups have startedwork looking for conditions under which vesicles can undergo growth and division.Evidence for growth and division has been obtained with the help of ferritin-labelledvesicles; furthermore, it has been shown that in such processes the vesicle size distributionis largely conserved. In both cases, the data suggest that the process under study is mainlycharacterized by vesicle growth and eventually division into daughter vesicles. However,direct evidence for vesicle division has not been obtained. In this paper, mostly basedon freeze-fracture electron microscopy, we describe conditions under which forthe first time division intermediates can be trapped in the form of twin vesicles.This finding, together with supporting dynamic light scattering and fluorescenceinvestigations, permits us to establish some additional points in the mechanism of vesicleself-reproduction.

A possible route to prebiotic vesicle reproduction.

Spherical bounded structures such as those formed by surfactant aggregates (mostly micelles and vesicles), with an inside that is chemically and physically different from the outside medium, can be seen as primitive cell models. As such, they are fundamental structures for the theory of autopoiesis as originally formulated by Varela and Maturana. In particular, since self-reproduction is a very important feature of minimal cellular life, the study of self-reproduction of micelles and vesicles represents a quite challenging bio-mimetic approach. Our laboratory has put much effort in recent years into implementing self-reproduction of vesicles as models for self-reproduction of cellular bounded structures, and this article is a further contribution in this direction. In particular, we deal with the so-called matrix effect of vesicles, related to the fact that when fresh surfactant is added to an aqueous solution containing preformed vesicles of a very narrow size distribution, the newly formed vesicles (instead of being polydisperse, as is usually the case) have dimensions very close to those of the preformed ones. In practice, this corresponds to a mechanism of reproduction of vesicles of the same size. In this article, the matrix effect is re-elaborated in the perspective of the origin of life, and in particular in terms of the prebiotic mechanisms that might permit the growth and reproduction of vesicles. The data are analyzed by dynamic light scattering with a new program that permits the calculation of the number-weighted size distribution. It is shown that, on adding a stoichiometric amount of oleate micelles to preformed oleate vesicles extruded at 50 and 100 nm, the final distribution contains about twice the initial number of particles, centered around 50 and 100 nm. The same holds when oleate is added to preformed phospholipid liposomes. By contrast, when the same amount of oleate is added to an aqueous solution (as a control experiment), a very broad distribution ranging between 20 and 1000 nm is obtained. The data can then be seen as a kind of reproduction of the same size vesicles, and the argument is advanced that this may correspond to a simple prebiotic mechanism of vesicle multiplication in prebiotic times, when only physical forces might be responsible for the basic mechanisms of early protocell growth and division. Preliminary data also show that repeated addition of oleate maintains the same basic initial features, and that surfactants other than oleate also respect the reproductive mode of the matrix effect.

V1E1440化学反応で複製するジャイアントベシクルのリアルタイム観察

V1E1440化学反応で複製するジャイアントベシクルのリアルタイム観察

Autopoietic Self-Reproduction of Chiral Fatty Acid Vesicles

The self-reproduction of vesicles formed by (S)- and (R)-2-methyldodecanoic acid (4) was investigated in order to relate the autocatalytic increase of the vesicle concentration with enantioselectivity. 4(R) and 4(S) were synthesized with an enantiomeric excess greater than 98%. 4 forms vesicles in aqueous solution in the pH region between 8.8 and 7.5. Chiral properties of the vesicles were studied by differential scanning calorimetry (DSC) and circular dichroism (CD). For self-reproduction studies, the hydrolysis of the water-insoluble 2-methyldodecanoic anhydride (8) was investigated in a biphasic system consisting of an aqueous solution and 8. The reaction rates of 8(RR) and 8(SS) catalyzed by 4(R) or 4(S) vesicles were the same within experimental errors, indicating that the chiral vesicles cannot induce significant enantioselectivity. However, a clear effect was observed at 10 °C: racemic vesicles destabilized during hydrolysis, causing phase separation, whereas homochiral vesicles remained stable and continued to self-reproduce.

Self-Reproduction of Chemical Structures and the Question of the Transition to Life

AbstractThe principles which underlay the chemical approach to the origin of life are discussed, beginning with Oparin’s notion of molecular evolution, whereby the minimal living emerged from the non livingviaa natural increase of molecular complexity and organization. The philosophical and methodological difficulties inherent in such a view are briefly examined. The scenario of the origin of life provided by the RNA-world is then reviewed, and the great difficulties inherent in this view are emphasized, particularly the one according to which a RNA family is createdex-novoin an enzymefree world in a way which is capable to self-replicate, mutation is concluded that the view of the RNA world for the origin of life, despite its popularity, is not very realistic at all; however it has a great importance as it has introduced a series of fundamental new concepts into the field of origin of life. Particularly important is the notion of self-reproduction, and in the paper the self-reproduction of vesicles is reviewed, pointing to the fact that it is a thermodynamically driven process based on spontaneously self-assembling macromolecualr aggregates. The possible relevance of these experiments for assessing a prebiotic «pre-RNA» world is discussed.

Autopoietic Self-Reproducing Vesicles: A Simplified Kinetic Model

A simplified kinetic model is presented in order to interpret the kinetics of the self-reproduction of vesicles. These experimental systems have been recently described (Walde P. et al., J. Am. Chem. Soc. 1994, 116, 11649) and consist of a biphasic system in which an insoluble surfactant precursor, e.g., an anhydride of a long-chain fatty acid, is overlaid on a basic aqueous solution and is eventually hydrolyzed into surfactant molecules that then assemble spontaneously into vesicles. Under these conditions, a catalytic effect can be observed due to the presence of the aggregates in the aqueous solution. To elucidate this effect within a theoretical framework, the hydrolysis has been described as an irreversible surface reaction that can take place both at the macroscopic interface and on the surface of vesicles. This reaction has been also assumed to be the limiting step so that all the others processes (e.g., formation of vesicles, absorption of molecules anhydride by vesicles, acid−base reactions, etc....

Light microscopic investigations of the autocatalytic self-reproduction of giant vesicles

Light microscopic investigations of the autocatalytic self-reproduction of giant vesicles

Lipase-catalyzed reactions in vesicles as an approach to vesicle self-reproduction.

As a continuation of our endeavor to find conditions under which bounded aggregate structures are able to self-reproduce, we have investigated the reactivity of lipase, both in free solution and vesicle-entrapped, against mixed oleic acid/oleate/ethyl oleate vesicles. Three types of vesicles have been prepared and characterized: (A) oleic acid/oleate vesicles; (B) oleic acid/oleate/ethyl oleate vesicles; and (C) lipase containing oleic acid/oleate vesicles. Long time stability studies by quasi elastic light scattering show that whereas (B) and (C) vesicles remain stable with a diameter of 110-130 nm and monodisperse for over a period of one month, vesicles (A) separated from an initial single population of 105 nm diameter into two populations, having respectively 70 nm diameter (more than 95% of the particles) and 180-210 nm diameter (less than 5% of the total population). In the case of vesicles (C), it could be shown that the enzyme remains localized inside the vesicles and it does not protrude into the water bulk phase. The enzymatic hydrolysis of ethyl oleate (which is water-insoluble) incorporated in the B-vesicles was studied under two configurations: (I) by adding lipase externally to the B-vesicles; (II) by mixing vesicles (B) and vesicles (C). In both cases, the reaction progressed to 100% hydrolysis. In the first case, the reaction was attended by an increase of the number of vesicles, and since this hydrolysis reaction takes place within the boundary of the parent vesicles, the criteria of autopoietic self-reproduction of vesicles are satisfied. In the case (II) instead, no increase of the population number of particles could be detected. The possible reasons for this difference are discussed.