One of the major challenges confronting attempts to synthesize artificial forms of life is understanding how a structurally simple protocell could accomplish the apparently complex biological function of self-replication in the absence of evolved biological machinery.(1
) A self-replicating protocell requires, minimally, two essential components: a self-replicating genome such as an RNA polymerase ribozyme(2
) or a chemically replicating nucleic acid, and a membrane compartment that can grow and divide.3,4
We have recently demonstrated the spontaneous copying of a vesicle-encapsulated genetic template,(5
) and the thermal strand separation of an encapsulated DNA duplex,(6
) suggesting that the spontaneous replication of encapsulated genetic polymers may be possible. Here we describe simple processes that lead to the efficient growth and division of model protocell membranes.
Fatty acid vesicles have long been studied as a model system for protocell membranes,3,5,7−9
as fatty acids and similar membrane-forming amphiphilic molecules have been isolated from meteorites and synthesized under simulated prebiotic conditions.10−16
The physical properties of small (typically 100 nm in diameter) unilamellar fatty acid vesicles have been studied in depth.3,5,6,17−20
For a population of vesicles to grow, fresh lipid molecules must be supplied. Pioneering studies in the laboratory of P. L. Luisi showed that when vesicles in a buffered solution are fed with alkaline fatty acid micelles (which become thermodynamically unstable at lower pH), the lipid molecules can either be incorporated into pre-existing membranes (leading to growth),3,18,21
or self-assemble into new vesicles.21−25
While vesicle growth by feeding with fatty acid micelles can be very efficient,(3
) vesicle division by extrusion through small pores results in the loss of a substantial fraction of the encapsulated vesicle contents to the environment.3,4
Furthermore, it is unlikely for an analogous vesicle extrusion process to occur in a prebiotic scenario on the early Earth, because vesicle extrusion by flowing suspended vesicles through a porous rock would require both the absence of any large pores or channels, and a very high pressure gradient (text S1, Supporting Information
). The possible spontaneous division of small unilamellar vesicles after micelle addition has been discussed,23,26
and electron microscopy has revealed structures that are possible intermediates of growth and division.(27
) However, the inheritance of the contents and membranes of parental vesicles by the newly formed vesicles has not been experimentally confirmed.
In contrast to the small unilamellar vesicles discussed above, fatty acid vesicles that form spontaneously by the rehydration of dry fatty acid films, or by the acidification of concentrated solutions of micelles, tend to be large (several microns in diameter) and multilamellar.3,8
Until recently, we have avoided using multilamellar vesicles for laboratory studies, because populations of such vesicles are so heterogeneous that quantitative studies of growth and division are difficult. To address this problem, we developed a simple procedure for the preparation of large (~4 μm in diameter) monodisperse multilamellar vesicles by large-pore dialysis.(28
) This gentle procedure preserves the original physical properties (e.g., multilamellar structure, volume, and osmolarity) of the large multilamellar vesicles. When we added fatty acid micelles to large monodisperse multilamellar vesicles prepared in this manner, we were able to directly observe a novel and unexpected mode of vesicle growth that allows for efficient division under modest shear forces.