What are protocells and their applications?

By Jhonata Lam

The use of protocells in modern research is able to assist in a range of subjects, from studying the origins of life to biotechnology.¹ Involving the generation of synthetic cells, protocell research is therefore increasing in popularity. A benefit of its implementation is that it provides a significant degree of control to cell biology researchers with respect to membrane compositions and a cell’s internal compartments. In contrast, natural cells (such as our own) are usually too complex to manipulate without off-target responses.

In the past, there has been some difficulty in reproducing a synthetic biological membrane using human phospholipids – owing in part to the thermodynamic restrictions present in their chemistry.² This is normally compensated for with the protein abundance in naturally derived cells. Yet simpler single fatty acid chains – with an alcohol or carboxylate group on one end – have seen greater success in generating representative membranes.

Protocells are, fundamentally, imitations of natural cells, acting as synthetic membrane encapsulations.³ In the laboratory, diversifying their generation allows a variety of molecular components to be explored in creating these membranes. We could opt for a conventional approach utilising lipid bilayers, for example, the same phenomenon that we observe in our cells. In another experiment, we might also produce protocells from coacervates, aqueous solutions that are rich in polyelectronic macromolecules like proteins. The benefits of this method lie in the breadth of amino acids employed to make protocell membranes, making their function changeable according to the researcher’s requirements.

Whilst lipid protocells could be produced through simpler methods, protein-based coacervate (or “proteinosome”) formation requires greater experimental manipulation. In vitro synthesis has been demonstrated by implementing BSA-NH₂/PNIPAAm (a chemically modified form of bovine serum albumin, i.e. BSA) and ethyl-1-hexanol mixtures.^4,5 Covalent linkage of the modified BSA protein to one another is then facilitated through the addition of cross-linking agents. These react with the BSA-NH₂ amine functionalities to generate proteinosomes, creating an encapsulated environment in the protocell. This environment is manipulated by including, as one example, cell-free gene expression kits before cross-linking – in turn isolating gene expression in these compartments. Unlike lipid-based methods, the compartment is isolated by covalent linkages, potentially indicating its resistance to drastic alterations in hydrophobicity.

The complexity of protocells can be increased through charge-driven liquid-liquid phase separation techniques.⁶ The results of this can be seen under a confocal microscope, in which coacervate compartments form within a larger coacervate compartment. Fascinatingly, researchers have noted that these resemble nucleoli. The implications of which mean that researchers may be able to further store biomolecules in multi-compartmental organisations. This is required for higher-order functions in human cells. To mediate respiration, for instance, glycolytic enzymes exist in the cytoplasm of cells, whereas enzymes of the TCA cycle exist in the mitochondrial matrix.

With some expertise in genetics and protocell engineering, one can also organise protocell populations to perform complex Boolean (i.e., “false” or “true”) operations. Researchers have demonstrated, with a “displacement circuit”, that AND gates may be engineered for the detection of inputs in a biological context.⁷

Additionally, this was shown with encapsulated DNA strands, originally bound to two inhibitory DNA strands that each partially cover a stretch of their respective sequence. An input – here a high-affinity nucleic acid – displaces the inhibitory strand in the protocell population. Another input strand must displace the other inhibitory strand for a fluorescent response to then be produced. The result is a circuit that requires two inputs for a single output. Thus, responsive genetic circuits may be synthesised in these cells, which could have a variety of therapeutic applications – such as in self-healing materials.¹

From another perspective, exciting developments have occurred by interfacing protocells with “natural” cells. Here, researchers formulated water-in-oil droplets that either encased E. coli bacteria or cell free expression systems⁸ – the former case acting as the “natural” component held in the protocell. These protocells were precisely engineered to encode genes that form a response to inducer molecules like IPTG and the quorum-sensing AHL, alongside an inducible lux operon ahead of a green fluorescent protein (GFP) gene. Referring back to AND gates, only in the presence of IPTG and AHL can the amplification of inducer molecules occur. The researchers oriented these cells one after the other in a capillary and added the inducer AHL from one end. Subsequently, they demonstrated green GFP fluorescence increased sequentially as a function of time – much like a propagating electrical current in a wire. It is suggested that these function similarly to division-inducing molecule (i.e., morphogen) gradients, a well-studied concept in the early differentiation of cells.⁸ New avenues to artificially study developmental processes are thus opened by the field.

The significance of self-assembling droplets in organising chemical reactions has been postulated even in the earliest forms of life.⁹ With this in mind, some researchers have put forward the usage of protocells in studying prebiotic membrane compartmentalisation. Consequently, they may advance contemporary knowledge on the origin of life.

Modern cell division requires cytoskeletal machinery like microtubules and energy in the form of ATP to drive membrane separation. Comparatively, it has been described that Ostwald ripening (the name given to the growth of droplets) can be attenuated if kept away from thermodynamic equilibrium by certain chemical reactions.⁹ Occasionally, this can lead to the division of these droplets, possibly providing insight into the origin of cell division.

In other instances, we may find support for the RNA world hypothesis, which states that primitive RNA molecules could stably exist without proteins or DNA. RNA is particularly substantial in that it can sometimes serve both information-storing and catalytic roles, with RNA strands that can mediate the latter often referred to as ribozymes. Studies have presented the viability of coacervate structures in housing active catalytic ribozymes.¹⁰ With lower levels of RNA and its building blocks on early Earth, primordial forms of life would have had to overcome this by some means. Especially notable are the researchers proving that coacervates were able to concentrate oligonucleotides without additional energy input – lending credence to the RNA world theory.

In all, protocells appear to have an optimistic future in the life sciences industry. They may also play critical roles in understanding remarkable problems such as how life began and the mechanisms by which this may have occurred prebiotically.

References:

1.         Elani Y. Interfacing Living and Synthetic Cells as an Emerging Frontier in Synthetic Biology. Angewandte Chemie International Edition. 2021;60(11):5602–11.

2.         Toparlak OD, Mansy SS. Progress in synthesizing protocells. Exp Biol Med (Maywood). 2019 Mar 1;244(4):304–13.

3.         Abbas M, P. Lipiński W, Wang J, Spruijt E. Peptide-based coacervates as biomimetic protocells. Chemical Society Reviews. 2021;50(6):3690–705.

4.         Huang X, Li M, Green DC, Williams DS, Patil AJ, Mann S. Interfacial assembly of protein–polymer nano-conjugates into stimulus-responsive biomimetic protocells. Nat Commun. 2013 Jul 30;4(1):2239.

5.         Huang X, Patil AJ, Li M, Mann S. Design and Construction of Higher-Order Structure and Function in Proteinosome-Based Protocells. J Am Chem Soc. 2014 Jun 25;136(25):9225–34.

6.         Lu T, Spruijt E. Multiphase Complex Coacervate Droplets. J Am Chem Soc. 2020 Feb 12;142(6):2905–14.

7.         Joesaar A, Yang S, Bögels B, van der Linden A, Pieters P, Kumar BVVSP, et al. DNA-based communication in populations of synthetic protocells. Nat Nanotechnol. 2019 Apr;14(4):369–78.

8.         Schwarz-Schilling M, Aufinger L, Mückl A, Simmel FC. Chemical communication between bacteria and cell-free gene expression systems within linear chains of emulsion droplets. Integr Biol. 2016 Apr 18;8(4):564–70.

9.         Zwicker D, Seyboldt R, Weber CA, Hyman AA, Jülicher F. Growth and division of active droplets provides a model for protocells. Nature Phys. 2017 Apr;13(4):408–13.

10.      Drobot B, Iglesias-Artola JM, Le Vay K, Mayr V, Kar M, Kreysing M, et al. Compartmentalised RNA catalysis in membrane-free coacervate protocells. Nat Commun. 2018 Sep 7;9(1):3643.