Freeze-thaw cycles induce content exchange between cell-sized lipid vesicles

Abstract

Early protocells are commonly assumed to consist of an amphiphilic membrane enclosing an RNAbased self-replicating genetic system and a primitive metabolism without protein enzymes. Thus, protocell evolution must have relied on simple physicochemical self-organization processes within and across such vesicular structures. We investigate freeze-thaw (FT) cycling as a potential environmental driver for the necessary content exchange between vesicles. To this end, we developed a conceptually simple yet statistically powerful high-throughput procedure based on nucleic acidcontaining giant unilamellar vesicles (GUVs) as model protocells. GUVs are formed by emulsion transfer in glass bottom microtiter plates and hence can be manipulated and monitored by fluorescence microscopy without additional pipetting and sample handling steps. This new protocol greatly minimizes artefacts, such as unintended GUV rupture or fusion by shear forces. Using DNAencapsulating phospholipid GUVs fabricated by this method, we quantified the extent of content mixing between GUVs under different FT conditions. We found evidence of nucleic acid exchange in all detected vesicles if fast freezing of GUVs at −80°C is followed by slow thawing at room temperature. In contrast, slow freezing and fast thawing both adversely affected content mixing. Surprisingly, and in contrast to previous reports for FT-induced content mixing, we found that the content is not exchanged through vesicle fusion and fission, but that vesicles largely maintain their membrane identity and even large molecules are exchanged via diffusion across the membranes. Our approach supports efficient screening of prebiotically plausible molecules and environmental conditions, to yield universal mechanistic insights into how cellular life may have emerged.

Microfluidic Handling and Analysis of Giant Vesicles for Use as Artificial Cells: A Review

“…[96] So far, the focus has been on developing microfluidic technologies for controllably trapping one [28,30,62,76,78] or even two [87] GUVs at a defined location. [97] Moreover, microfluidic handling of large collections of giant unilamellar vesicles as recently presented by the Robinson lab [43] could be adapted for enhanced manipulation and creation of tissue-like systems based on droplet-droplet interfaces (i.e., DIBs). [46] Future works should take advantage of microfluidic methods to create a specific assembly with a defined number of GUVs which will find its uses in creating tissue-like structures from the bottom-up and also for studying artificial cell-to-cell communication.…”

“…These include energy conversion, [100] metabolism, [101] polarisation, [19] growth, [14,16,102] division, [103] signaling/communication, [97,104] and motility. In the context of using the bottom-up approach for the construction artificial cells, there are a number of functionalities currently being explored.…”

Optimization of the Inverted Emulsion Method for High‐Yield Production of Biomimetic Giant Unilamellar Vesicles

“…Although microfluidic devices do yield GUVs, which are more monodisperse in size and with higher encapsulation efficiencies, their use involves more complex instrumental or time‐consuming setups, which are not always available to researchers . On the contrary, the bulk inverted emulsion‐based method has shown great promise in encapsulating macromolecules, such as polymers, DNA, enzymes, cells, and even micron‐sized particles . The method can also be used to produce GUVs with complex multicomponent lipid mixtures allowing them to be used as biomimetic membrane models …”

“…Not only is this low‐throughput, but in the absence of proper optimization, results in very low vesicle yields. Recently, we presented work in which we demonstrated that microtiter plates are ideal for performing this method as it allows multiple parallel experiments . We take advantage of this setup within this work and perform repeatable parallel experiments to allow optimization.…”

Horizontal transfer between loose compartments stabilizes replication of fragmented ribozymes

“…Recent experiments suggest that the random exchange of contents between compartments is plausible. The freeze-thaw cycles, which enhance the assembly of fragments [7], also induce content exchange between giant unilamellar vesicles through diffusion [19] or through fusion and fission [20]. Also, transient compartmentalization, which involves the occasional complete mixing of contents between comparments, is considered to be relevant to maintain functional replicators [17,[21][22][23][24].…”