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Physical Routes to Primitive Cells: An Experimental Model Based on the Spontaneous Entrapment of Enzymes inside Micrometer-Sized Liposomes.

D'Aguanno E, Altamura E, Mavelli F, Fahr A, Stano P, Luisi PL - Life (Basel) (2015)

Bottom Line: We have reported that proteins and ribosomes can be encapsulated very efficiently, against statistical expectations, inside a small number of liposomes.Moreover the transcription-translation mixture, which realistically mimics a sort of minimal metabolic network, can be functionally reconstituted in liposomes owing to a self-concentration mechanism.Here we firstly summarize the recent advancements in this research line, highlighting how these results open a new vista on the phenomena that could have been important for the formation of functional primitive cells.

View Article: PubMed Central - PubMed

Affiliation: Science Department, Roma Tre University, Viale G. Marconi 446, I-00146 Rome, Italy. Alfred.Fahr@uni-jena.de.

ABSTRACT
How did primitive living cells originate? The formation of early cells, which were probably solute-filled vesicles capable of performing a rudimentary metabolism (and possibly self-reproduction), is still one of the big unsolved questions in origin of life. We have recently used lipid vesicles (liposomes) as primitive cell models, aiming at the study of the physical mechanisms for macromolecules encapsulation. We have reported that proteins and ribosomes can be encapsulated very efficiently, against statistical expectations, inside a small number of liposomes. Moreover the transcription-translation mixture, which realistically mimics a sort of minimal metabolic network, can be functionally reconstituted in liposomes owing to a self-concentration mechanism. Here we firstly summarize the recent advancements in this research line, highlighting how these results open a new vista on the phenomena that could have been important for the formation of functional primitive cells. Then, we present new evidences on the non-random entrapment of macromolecules (proteins, dextrans) in phospholipid vesicle, and in particular we show how enzymatic reactions can be accelerated because of the enhancement of their concentration inside liposomes.

No MeSH data available.


Related in: MedlinePlus

Experimental results for the CA + CFDA system. (a) Confocal micrographs showing green fluorescent vesicles that appear in the progress of reaction (t = 0, 10, 20, 30 min). The vesicles appear more fluorescent than the background although the same reaction is also occurring outside vesicles. (b,c) The vesicle fluorescence can be estimated by image analysis (by ImageJ software [45])—here shown as the pixel luminosity of samples taken at 5 and 25 min after mixing. Note that the fluorescence values of each vesicle greatly differ from each other (giving a fluorescence distribution, cf.Figure 8) and are well above the background. Note also that the background fluorescence increases with time, as expected.
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life-05-00969-f011: Experimental results for the CA + CFDA system. (a) Confocal micrographs showing green fluorescent vesicles that appear in the progress of reaction (t = 0, 10, 20, 30 min). The vesicles appear more fluorescent than the background although the same reaction is also occurring outside vesicles. (b,c) The vesicle fluorescence can be estimated by image analysis (by ImageJ software [45])—here shown as the pixel luminosity of samples taken at 5 and 25 min after mixing. Note that the fluorescence values of each vesicle greatly differ from each other (giving a fluorescence distribution, cf.Figure 8) and are well above the background. Note also that the background fluorescence increases with time, as expected.

Mentions: Experimental results are shown in Figure 11. It is possible to follow the time course of CFDA hydrolysis by confocal microscopy, recording the fluorescence increase at different times, both in bulk than inside liposomes. Figure 11a shows typical microscopic images referring to 0, 10, 20 and 30 min after CFDA addition to CA-containing liposomes (note that free, non-entrapped CA was not removed, so that the fluorescence background also increases in time). By measuring the fluorescence of the liposomes and of the background (Figure 11b,c), at each sampled time, it is possible to plot the rate of enzyme reaction in bulk and inside liposomes. Note that because the liposomes freely float in the solution, it was not possible to follow their individual behavior (namely, the fluorescence increase inside a certain liposome). We therefore captured images containing a number of liposomes and average their fluorescence (the average value, however, is affected by a bias because it is difficult to spot out liposomes that are only slightly more fluorescent than the background; this means that most of the pictures generally contain the brightest liposomes of the population). The fluorescence-versus-time profiles (inside liposomes and in bulk) are shown in Figure 12a.


Physical Routes to Primitive Cells: An Experimental Model Based on the Spontaneous Entrapment of Enzymes inside Micrometer-Sized Liposomes.

D'Aguanno E, Altamura E, Mavelli F, Fahr A, Stano P, Luisi PL - Life (Basel) (2015)

Experimental results for the CA + CFDA system. (a) Confocal micrographs showing green fluorescent vesicles that appear in the progress of reaction (t = 0, 10, 20, 30 min). The vesicles appear more fluorescent than the background although the same reaction is also occurring outside vesicles. (b,c) The vesicle fluorescence can be estimated by image analysis (by ImageJ software [45])—here shown as the pixel luminosity of samples taken at 5 and 25 min after mixing. Note that the fluorescence values of each vesicle greatly differ from each other (giving a fluorescence distribution, cf.Figure 8) and are well above the background. Note also that the background fluorescence increases with time, as expected.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4390888&req=5

life-05-00969-f011: Experimental results for the CA + CFDA system. (a) Confocal micrographs showing green fluorescent vesicles that appear in the progress of reaction (t = 0, 10, 20, 30 min). The vesicles appear more fluorescent than the background although the same reaction is also occurring outside vesicles. (b,c) The vesicle fluorescence can be estimated by image analysis (by ImageJ software [45])—here shown as the pixel luminosity of samples taken at 5 and 25 min after mixing. Note that the fluorescence values of each vesicle greatly differ from each other (giving a fluorescence distribution, cf.Figure 8) and are well above the background. Note also that the background fluorescence increases with time, as expected.
Mentions: Experimental results are shown in Figure 11. It is possible to follow the time course of CFDA hydrolysis by confocal microscopy, recording the fluorescence increase at different times, both in bulk than inside liposomes. Figure 11a shows typical microscopic images referring to 0, 10, 20 and 30 min after CFDA addition to CA-containing liposomes (note that free, non-entrapped CA was not removed, so that the fluorescence background also increases in time). By measuring the fluorescence of the liposomes and of the background (Figure 11b,c), at each sampled time, it is possible to plot the rate of enzyme reaction in bulk and inside liposomes. Note that because the liposomes freely float in the solution, it was not possible to follow their individual behavior (namely, the fluorescence increase inside a certain liposome). We therefore captured images containing a number of liposomes and average their fluorescence (the average value, however, is affected by a bias because it is difficult to spot out liposomes that are only slightly more fluorescent than the background; this means that most of the pictures generally contain the brightest liposomes of the population). The fluorescence-versus-time profiles (inside liposomes and in bulk) are shown in Figure 12a.

Bottom Line: We have reported that proteins and ribosomes can be encapsulated very efficiently, against statistical expectations, inside a small number of liposomes.Moreover the transcription-translation mixture, which realistically mimics a sort of minimal metabolic network, can be functionally reconstituted in liposomes owing to a self-concentration mechanism.Here we firstly summarize the recent advancements in this research line, highlighting how these results open a new vista on the phenomena that could have been important for the formation of functional primitive cells.

View Article: PubMed Central - PubMed

Affiliation: Science Department, Roma Tre University, Viale G. Marconi 446, I-00146 Rome, Italy. Alfred.Fahr@uni-jena.de.

ABSTRACT
How did primitive living cells originate? The formation of early cells, which were probably solute-filled vesicles capable of performing a rudimentary metabolism (and possibly self-reproduction), is still one of the big unsolved questions in origin of life. We have recently used lipid vesicles (liposomes) as primitive cell models, aiming at the study of the physical mechanisms for macromolecules encapsulation. We have reported that proteins and ribosomes can be encapsulated very efficiently, against statistical expectations, inside a small number of liposomes. Moreover the transcription-translation mixture, which realistically mimics a sort of minimal metabolic network, can be functionally reconstituted in liposomes owing to a self-concentration mechanism. Here we firstly summarize the recent advancements in this research line, highlighting how these results open a new vista on the phenomena that could have been important for the formation of functional primitive cells. Then, we present new evidences on the non-random entrapment of macromolecules (proteins, dextrans) in phospholipid vesicle, and in particular we show how enzymatic reactions can be accelerated because of the enhancement of their concentration inside liposomes.

No MeSH data available.


Related in: MedlinePlus