S U P R A M O L E C U L A R    C H E M I S T R Y
 
Liposomes as models for biological cells - the minimal cell project
 
An important question in the field of the origin of life and evolution concerns the structure of the early cells. Clearly they must have been much simpler than the modern ones, which consist of more than 500 genes. This bears to the question of the minimal cell, the cell having the minimal and sufficient structural complexity to display life. The construction of such a cell (or cells) is the long goal of our project, to be pursued with conventional liposomes,  with giant vesicles (see infra) as well as with reverse micelles(see infra).

Let us consider first the work with conventional liposomes.
Our first attempt in this direction was with lecithin liposomes, containing the enzymes that would construct lecithin from the inside starting from G3P, glycerol-3-phosphate (Schmidli et al 1991). It was difficult to obtain clear results due to the fact that the commercially available enzymes were not pure and not enough active. Presently, this work has been taken up again by a graduate student, Paola Luci. She is now preparing  the first two enzymes (those that produce phosphatidic acid starting from G3P) by recombinant DNA techniques. The isolation and characterization of these enzymes, which are not well known in the literature, has shown to be rather difficult.
In our lab, the entrapment of enzymes and nucleic acids inside liposomes, in order to obtain microreactors for molecular biology, has progressed over the years. Such  reactions were: (i) the polynucleotide phosphorylase (PNPase) reaction, that was able to polymerize internalized ADP into poly(A). This was done with oleate vesicles that were simultaneously self-reproducing (Walde et al 1994); (ii) PCR (polymerase chain reaction) taking place in POPC liposomes (Oberholzer et al 1995a); (iii) the  Q-beta replicase reaction in liposomes (Oberholzer et al 1995b). The enzyme Q-beta replicase, acting upon a RNA template, can make copies of this template in the presence of the nucleotides. Again, this was accomplished in oleate vesicles that were self-reproducing in the process, thus achieving a system in which both the compartment and the RNA were multiplying themselves (although uncoupled). This is illustrated below: 
 

(iv) protein biosynthesis in liposomes: work is now in progress to realize the entire ribosomal machinery inside conventional POPC liposomes. Until now, only a simple synthesis of a polypeptide (Poly-phe)has been obtained using Poly(U) as the m-RNA (Oberholzer et al 1999). We are trying to express a more complex protein, such as the green fluorescent protein (GFP). This appears to be much more difficult. Preliminary results have been obtained by using a novel method of encapsulation of the biological material (Oberholzer and Luisi, in press, in Proceedings of the XII Biophysical International Symposium in Tokyo, 2001). 
This part of the molecular biology inside liposomes, as well as inside giant vesicles see infra - is carried out under the supervision of Dr. Thomas Oberholzer.
This kind of work with biopolymers in conventional liposomes requires still  basic  studies in the general field of enzymes in liposomes, in particular about the transport efficiency of substrate in and out of the lipid bilayer. Prof. Peter Walde is particularly interested in this kind of work. Recently, he investigated together with Prof. Sosaku Ichikawa, who worked as a postdoc in the group, proteinase-containing lipid vesicles. The focus of the work is in the quantitative analysis of experimental kinetic data obtained for the entrapped enzymes, focusing particularly on the substrate permeability properties of the bilayer membranes in these enzyme-containing nanoreactors. In an extension of the work, enzyme-containing vesicles are also used as analytical tool to investigate vesicle-vesicle fusions caused by the external addition of phospholipase D.
As already mentioned, the work towards the minimal cell is also pursued with reverse micelles. In the past, we were able to incorporate DNA, and entire ribosomes inside reverse micelles (Imre & Luisi, 1982 ; Palazzo & Luisi, 1992). Although reverse micelles are less interesting as model for biological cells, they offer the advantage of a better compartimentation of water soluble biopolymers and biomolecules. Presently, a graduate student, Adriana Pietrini, is working in this project, and she was able to extend the work of the incorporation of DNA and plasmids (BBA, in press).
All this work with the minimal cell is carried out in our group with enzymes and DNA-namely we are dealing with  DNA/protein minimal cells. The case of a minimal RNA cell has been considered from a theoretical perspective together with our collaborators in Harvard Prof. Jack Szostak and Prof. D. Bartel (Whitehead Institute for Biomedical Research in Cambridge), and has brought to the perspective that a minimal self replicating cell is possible with only two genes ribozymes (Walde, 2000), as illustrated below. However this minimal cell, even if it could be realized (the ribozymes are not yet available) cannot produce proteins.
 

 
Work with giant vesicles (GV)
 
Mostly Dr. Thomas Oberholzer, Prof. Peter Walde, and Dr. Kenichi Morigaki are presently working in the group with GV. This work with GV has been in our group for many years,  as it was initiated during  the dissertation work of the graduate student Roger Wick, who got his doctor title in 1996. He worked with GV that spontaneously formed from a mixture of oleic acid and oleate (Wick et al,1995), as well as with GV from POPC obtained by electroformation (Wick et al,1996), in collaboration with Dr. Miglena Angelova.  He was also able to inject phospholipase A2 in GV and to study the reaction of this enzyme with the GV lipid membrane (Wick & Luisi,1996). 
 

Later on Aline Fischer, (who graduated in 2000), together with visiting students from the University of Bologna, Alessandra Frazzoli and Andrea Franco, mostly under the supervision of Dr. Thomas Oberholzer,  were able to give a series of additional interesting contribution to the area. Particularly the work by Aline Fischer (Fischer et al,2000) shed a new light on the physical properties of GV, particularly concerning the docking and the permeability of GV towards small proteins. In all these works, we have emphasized the differences in behaviour between GV and conventional liposomes and made the point that GV, because of the large curvature radius, are closer model to biological membranes. 
 
 

Presently, the work with GV is progressing in the direction of using them as model compartments for minimal cells. It has been possible to inject the ribosome system as well as plasmid inside GV, and preliminary data for the expression of proteins inside GV have been collected (in the example of GFP). Reproducibility problems are however still present.