The attractiveness of the “bottom-up” approach as a viable route for constructing cell-like systems (Luisi, 2002; Stano, 2019) is clearly evident by the ever increasing number of international projects and initiatives dedicated to this fascinating research (Supplementary Text S1). Such cell-like systems, simply called “synthetic cells” (SCs), “artificial cells” or “protocells” (although with slighly different nuances of meaning) are compartment-based systems (often, but not only, liposomes), capable of mimicking some aspects of cell behavior in a range of manners, and can be variously conceived in terms of materials, designs, and scopes. Even if current SCs are not alive, there is a recognized optimism among practicioners about the contribution of this research to basic and applied science, and there is the bet it will become one of the most important biotechnologies in the near future,—not resembling anything existing before—for example in nanomedicine (Leduc et al., 2007; Krinsky et al., 2018, Ding et al., 2018; Lussier et al., 2021). It seems a useful remark, just after mentioning nanomedicine, recalling that RNA-based anti-COVID vaccines—which actually are RNA-loaded lipid nanoparticles (Pilkington et al., 2021) have been actually developed thanks to decades of research on liposomes and other nanovectors. This suggests a highly relevant and pioneer role that current SC research might have on future ‘smart’ nanomedicine scenarios.

SC research is now well recognized within the “bottom-up” or “in vitro” or “cell-free” or “chemical” domains of synthetic biology. Pioneer research, however, dates back to the early 1990s, mainly referred to the construction of protocellular models of minimal complexity for origins-of-life studies (Walde et al., 1994; Oberholzer et al., 1995; Szostak et al., 2001). In that context a minimalist design is generally applied, which means the use of allegedly primitive materials (e.g., fatty acids, ribozymes, short peptides) (Chen et al., 2005), simple architectures (single, individual compartments), and essential functions (e.g., growth-division driven by basic physico-chemical events). Relevance is given to the verification of capabilities, constraints, and properties which might have ruled the primitive life-like dynamics of compartmentalized chemical systems.

On the other hand, SC research has gradually expanded and has incorporated other approaches that enriched and favored its development. In particular, current studies include systems made of various materials, mainly modern biomacromolecules (following, then, a reconstitution philosophy), but also artificial ones (e.g., block copolymers, ad hoc designed reactive surfactants (Kurihara et al., 2011; Budin and Devaraj, 2012), etc.), and their various combinations, including allegedly primitive materials (Figure 1A). Solute-filled liposomes are largely—but not uniquely—employed for that scope. Indeed, the experimental approaches are inspired by the functional (and relational (Rosen, 1991)) roles of SC components rather than their material embodiment. In a sense, SCs are tools for investigating life as it was, as it is, and as it could be.

Interestingly, in addition to single-compartment design, representing SCs with an architecture of minimal complexity, multicompartment SCs can be constructed, leading to very interesting systems with peculiar features. The term “multicompartment” can refer to a “flanked” (sidewise, Figure 1B) or to a “nested” design (Figure 1C). In the first case, one refers to structures having flanked compartments, attached to each other, to generate 1D, 2D or 3D assemblies (or clusters), often considered models of tissues or multicellular systems (Carrara et al., 2012; Elani et al., 2013). In the second case, the SC architecture would resemble an eukaryotic cell, and the small compartments inside the large one mimic biological intracellular organellae (Bolinger et al., 2004; Elani et al., 2018). In the jargon of liposome technology, the latter structures are called “multivesicular vesicles” (MVVs) or “vesosomes” (Kisak et al., 2004; Giuliano et al., 2021). It should not escape from the attention that early studies on vesosomes were actually motivated by the need of constructing better drug delivery vehicles, for example by providing better protection of loaded drugs against degrading enzymes, or easier modular construction of drug cocktails with differential release (by tuning the membrane properties of individual internal compartments); for a detailed discussion, with examples, please refer to (Giuliano et al., 2021).

In this article we will briefly discuss the nested multicompartment architectures, to highlight their advantages, mainly when coupled to vectorial chemistry. As a casestudy we will highlight systems whereby membrane proteins (MPs) provide the necessary function to generate and/or exploit (electro)chemical gradients, for instance to produce ATP inside SCs.

One of the frontier research line in SC construction directly refers to bioenergetics, and deals with endogenous ATP production. Firstly, this is needed to feed processes necessary for complex SCs. Second, such a production would correspond to the emancipation from the current “windup toy” approaches based on endowing SCs, at time zero, with all required chemical energy to run, and the consequent ceasing when such supply runs out. Moreover, when the two cuncurrent processes of ATP production and usage are coupled, the system nicely constitutes a realization of out-of-equilibrium homeostasis (Pols et al., 2019).

The obvious idea is to engage SCs in an upstream phosphorylation process (ADP + Pi → ATP), operated by ATP synthase, and driven by an (electro)chemical proton gradient. The latter is vectorially generated by a membrane protein (MP) system capable of coupling redox or photoredox reactions with proton pumping. Such a goal, when realized by means of nested multicompartment SCs, combines the three concepts defined in Nested Multicompartments: Construction, Modularization, and Vectorial Chemistry.

Decorating the membranes with MP complexes—the ones that realize vectorial chemistry for ATP production, for instance, requires a precise orientation of all MPs involved in it, to avoid futile cycles. From the simplified drawing of Figure 1E, it is evident that SCs designed as single-compartment or as nested multicompartment require two opposite strategies for MP insertion. This means, in turn, that a complete control of this key process is required at any case: either insertion of pre-formed MP delivered with micelle (Jørgensen et al., 2017; Skrzypek et al., 2018), either insertion of nascent, ribosomally synthesized MP (Kuruma et al., 2009), and for any direction (from the inside or from the outside of the compartment). ¹

Systematic studies about vesicle “decoration” with vectorial MPs that include all above-mentioned possibilities are still lacking, although significant advancements have been recently reported for cell-free synthesis approaches (Sachse et al., 2014; Kuruma and Ueda, 2015; Niwa et al., 2015; Jacobs et al., 2019; Kruyer et al., 2021), demonstrating, for instance, MPs insertion in the lipid membrane can be guided bythe secYEG translocon (Matsubayashi et al., 2014).

In recent reports it has been shown how to reconstitute MPs with a proper orientation by treating GVs with MPs solubilized as micelles which were included in their aqueous lumen or in the external phase (Yanagisawa et al., 2011; Altamura et al., 2017), with high orientation, Figure 1F. These strategies can be adapted both for single-compartment and multi-compartment SC approaches.

The multi-compartment “nested” design leads to more complex SCs, but can be operatively simpler because the internal compartments can be prepared in advance (Biner et al., 2020), and later inserted in the larger one (the “host” vesicle). SCs designed as nested systems appear also more functional, as it is easier to control the SC internal milieu, providing optimal conditions for the operations of internalized small compartments. Modularization by sub-compartmentalization offers the additional advantage of segregating the elements present in the inner vesicle avoiding the mixing and possible noxious interactions with the other SC elements. The nested design is currently at the spotligth of SC research, as it has been employed by several relevant studies, not only for ATP production (Hindley et al., 2019; Belluati et al., 2020). As mentioned, this design is quite valuable for ATP production, being the internal compartments essentially a sort of organellae-like structures with a dedicated function. For example, the ATP-producing synthetic organellae (driven by irradiation) have been produced by detergent-driven reconstitution (Lee et al., 2018) or by directed assembly (Feng et al., 2016), or by direct insertion of the in statu nascendi cell-free synthesized membrane proteins (Berhanu et al., 2019). Alternatively, “prefabricated” and highly efficient organellae have been used, borrowing them from photosynthetic bacteria of the genus Rhodobacter (Altamura et al., 2021) (Figure 1G). Hybrid approaches (particles formed by thylakoid fragments of spinach plus lipids) have been also explored, but not inside SCs (Zheng et al., 2018).

In this Opinion article we have highlight a current trend in SCs research, namely the one moving from simple and isolated SCs to systems made of several compartments. “Flanked” (sidewise) or to a “nested” designs allow moving upward in complexity and favour the achievement of novel functions that will drive near-future directions in the field. MPs will be pivotal too. Think, for example, to G-Protein Coupled Receptors or other receptors as a way to access and exploit the sensorium toolbox also in SCs (May et al., 2013; Hamada et al., 2014; Gessesse et al., 2018). In particular, we have remarked that the nested multicompartment design ideally endows SCs with the energy-producing function and decisively contributes to next advancements.