New Results

Encoding extracellular modification of artificial cell membranes using engineered self-translocating proteins

Alexander Harjung, Alessandro Fracassi, Neal Devaraj
doi: https://doi.org/10.1101/2023.10.06.561148

Abstract

A common method of generating artificial cells is to encapsulate protein expression systems within lipid vesicles. However, to communicate with the external environment, protein translocation across lipid membranes must take place. In living cells, protein transport across membranes is achieved with the aid of complex translocase systems which are difficult to reconstitute into artificial cells. Thus, there is need for simple mechanisms by which proteins can be encoded and expressed inside synthetic compartments yet still be externally displayed. Here we present a genetically encodable membrane functionalization system based on mutants of pore-forming proteins. We show that the membrane translocating loop of α-hemolysin can be engineered to translocate functional peptides up to 52 amino acids across lipid membranes. Engineered hemolysins can be used for genetically programming artificial cells to display interacting peptide pairs, enabling their assembly into artificial tissue-like structures capable of signal transduction.

Main

Living cells are compartmentalized by phospholipid membranes, which separate the contents of a cell from the extracellular environment and provide a barrier that is crucial for cell survival and identity. However, lipid barriers also limit interaction and communication of cells with the extracellular environment since cellular membranes are generally not permeable to most biological molecules.1 To resolve this issue, cells have evolved many complex mechanisms that enable modification and functionalization of their membranes. This functionalization is often achieved through the insertion of transmembrane proteins which serve as a connection between the intracellular and extracellular environment and facilitate nutrient uptake, signaling, and tissue formation.2 Membrane proteins, due to their amphipathic nature, rarely spontaneously insert into membranes. Instead, these proteins achieve proper topology and folding within the membrane with the aid of highly evolved cellular translocase systems. An important example of such a mechanism is the Sec61-dependent protein translocation pathway in which several proteins form a translocation channel enabling protein insertion into a membrane.3

Recently there has been increasing interest in developing artificial cells as models that mimic the structure and function of living cells. Perhaps the most common method for generating artificial cells is to encapsulate proteins within membrane-bound vesicles consisting of phospholipid membranes. Advances over several decades have enabled researchers to routinely form membrane-bound vesicles and even encapsulate the necessary biological components for cell free protein synthesis.4 However, like in living cells, the lipid membranes of artificial cells constitute a barrier that prevents interaction of internally expressed proteins with other cells or the external environment.

Given the importance of membrane proteins in living cells, there exists a major need for simplified mechanisms by which proteins internally expressed within vesicles can embed themselves into membranes such that the protein translocates through the membrane. If a significant part of the protein is exposed on the outer leaflet, interactions with the external environment of the artificial cell would then become possible.

While advances have been made in reconstituting the natural protein insertion machinery in protocells, this approach remains highly complex and has limited efficiency.5 Alternatively, researchers have tackled this problem through several alternative mechanisms. For instance, one approach involves functionalizing artificial membranes by doping the membrane with an affinity ligand. This ligand can then serve as a handle by which the membrane can be chemically functionalized using proteins bearing appropriate ligand binding groups.6 However, in natural cells membrane modification is self-encoded as part of the genome of the cell.

One strategy that could enable external membrane modification of individual artificial cells using genetically encoded proteins is to take advantage of soluble proteins that are able to self-translocate across lipid membranes. There are some rare examples of proteins, such as pore forming toxins (PFTs) which can self-insert into biological membranes, independent of any insertion machinery.7,8, 9,10,11,12 A well-studied example of a PFT is the bacterial toxin α-hemolysin (αHL). Pore formation occurs when αHL binds to the membrane, first as a soluble monomer, and then subsequently forming a heptameric complex, which spontaneously translocates across the lipid membrane as a barrel-like structure.13 αHL has been extensively studied due to its biological role in infection and its utility in nanopore sequencing. Due to its self-insertion and pore-forming ability, αHL has also been used in artificial cell systems to make lipid membranes permeable to small molecules, which can promote internal biochemical reactions like transcription and translation.14 Multiple seminal studies have demonstrated that αHL can be mutated in several positions without losing its pore forming activity.15

While αHL assembly leads to spontaneous formation of a transmembrane protein, only a very small region of each monomer, the loop formed by amino acids 128-131, fully translocates across the lipid membrane upon pore formation (Fig. 1a).16 If amino acids 128-131 were modified to contain peptide tags or other small proteins, while still allowing for αHL pore assembly and insertion, artificial cells internally expressing engineered hemolysins would externally display peptide tags that could subsequently interact with the external environment (Fig. 1b). Our envisioned system would constitute a simple way of achieving self-encoded extracellular membrane functionalization in artificial cells.

Fig. 1:
Fig. 1: Scheme of α-hemolysin facilitated membrane translocation of peptides.

a, αHL translocates peptide inserts in loop128-131 across a lipid membrane. If engineered α-hemolysins remain functional, then pore formation and insertion would cause the peptide insert (red) to be translocated across the membrane b, Intracellular expression of αHL with a peptide insert in loop128-131 would enable self-encoded extracellular membrane functionalization in artificial cells.

Several studies have reported mutating single amino acids in αHL loop128-131. In particular the G130C-mutant has been shown to be fully active, with the cysteine translocating across the membrane during pore formation.17,18,19,20,21 However, to our knowledge, there are only a few literature reports about making larger modifications to loop128-131.22,23,24,25 It was shown for instance that substituting amino acids 130-134 with 5 histidines does not disrupt the pore forming ability of αHL and endows the pores with the ability to bind zinc ions, which blocks transport.22,23 Another study replaced amino acid 129 with a cysteine flanked by two flexible linkers, representing an addition of 10 amino acids to the loop.24 However, it was not investigated if the resulting protein can assemble into functional pores on its own. Instead, hetero-heptamers were formed in which 6 wild-type αHL monomers were mixed with one monomer that had the 10 amino acid insert in the loop.

Here we demonstrate that functional peptides up to 52 amino acids in length can be inserted into loop128-131 without disrupting αHL’s membrane insertion and pore formation ability. We screened peptides of different sizes to determine the types of inserts that are tolerated. αHL proteins encountering one side of a synthetic membrane can spontaneously translocate peptide epitopes across the membrane and interact with peptide binding antibodies on the other side. By genetically encoding modified αHL that can be expressed within giant unilamellar vesicles (GUVs), we create artificial cells that can self-display peptides on their extracellular membrane. By mixing populations of artificial cells that display peptides that interact with one another, we demonstrate self-encoded formation of tissue-like structures. Finally, we show that self-encoded artificial tissue formation is modular and can be combined with other functional systems as demonstrated by implementing a simple artificial cell-cell signaling pathway.

Results and discussion

Loop128-131 of α-hemolysin tolerates inserts up to around 50 amino acids. We initially tested insertion of a 6XHis-tag between D128 and K131 of the membrane translocating loop of αHL, with the inserted peptide replacing T129 and G130. We also hypothesized that the addition of flexible linkers might make the 6XHis-tag more accessible by increasing the distance between the affinity ligand and the membrane, reducing undesirable steric interactions. A C-terminal GFP fusion was added to enable visualization of membrane binding by the αHL mutant. We initially used a simple GUV binding/leakage assay to determine if αHL mutants retained functionality. GUVs encapsulating Cy5 were formed by the inverse emulsion method26 using a mixture of DOPC and cholesterol. Upon treatment of Cy5-encapsulating GUVs with αHL containing the 16-mer L-6XHis-L peptide insert, GFP localization to the membrane was observed as well as leakage of internalized Cy5 dye molecules (Fig. 2), suggesting the formation of a fully functional pore despite the insertion of a 16-mer peptide in the loop128-131 region. We next increased the flanking linker to (GGGGS)2 and (GGGGS)3. Surprisingly, despite adding even a 36-mer peptide insert in the loop, GUV binding and leakage assays indicated that the protein was still able to form functional pores.

Fig. 2:
Fig. 2: α-hemolysin with a peptide inserted in loop128-131 forms functional pores which induce leakage of Cy5 from vesicles.

a, General scheme of αHL membrane binding and leakage assay. GUVs encapsulating Cy5 (purple stars) are treated with αHL mutants that contain peptide inserts (red) in the loop region of αHL. The αHL is also fused to GFP (green star) to enable visualization of membrane binding. Pore formation in the GUV membrane leads to the leakage of Cy5. b, Confocal microscopy images of the leakage assay using αHL containing a 16-mer peptide insert (6XHis-tag flanked by two linkers). Green channel shows membrane binding of the αHL-GFP fusion protein. Leakage of Cy5 (purple channel) can be observed over time. Scale bar: 10 μm

Encouraged by our results, we next investigated if αHL could tolerate alternative functional peptide inserts. For instance, we were interested if cyclic peptides could be inserted into the loop region, as cyclic peptides have found widespread use in medicinal chemistry.27 We inserted the sequence for somatostatin-1428 flanked by two flexible linkers into loop128-131. We found that αHL containing a somatostatin-14 insert was also able to induce leakage of Cy5 from GUVs., suggesting that cyclic peptides are tolerated for membrane translocation using αHL loop mutants.

To determine the size limitation of the peptide inserts that can be tolerated in the loop region of αHL, we tested a series of additional inserts (Supplementary Table 1 and 2). We tested a 52 amino acid peptide by inserting a GLP1 peptide hormone29 flanked by a (GGGGS)2 linker. The GLP1 containing αHL was also able to bind to GUVs and induce Cy5 leakage. However, we found that larger peptide inserts (75-248 amino acids, Supplementary Table 2) generated αHL mutants that were unable to bind to lipid membranes and do not form pores and induce Cy5 leakage from GUVs. Thus, with respect to inserting simple peptides, we estimate that the cutoff length for retaining αHL functionality is approximately 50 amino acids, though undoubtedly the nature of the insert with respect to charge, hydrophobicity, and structure likely have important effects on the assembly of the pore and its interaction with lipid membranes.

Peptide inserts in αHL loop128-131 can interact with large biomolecules after membrane translocation

We wanted to confirm that the peptide inserts in the loop get translocated across the membrane and are accessible from the other side. One way to test translocation is to determine if large macromolecules encapsulated in GUVs, like monoclonal antibodies, can bind and recognize the translocated peptide if the αHL loop mutant is delivered from the outside of the GUV. We generated GUVs encapsulating Cy5-conjugated antibodies directed against the various peptides (6XHis, somatostatin, GLP-1) that were shown to insert into αHL without disrupting pore formation. We individually encapsulated the fluorescent antibodies into GUVs. GUVs were then treated with the αHL loop mutant containing the appropriate peptide insert. If the αHL loop mutants fully translocate across the membrane and the loop is fully accessible on the other side, then antibody recruitment to the membrane would take place and this would be visualized by Cy5 fluorescence at the membrane (Fig. 3a). Because monoclonal antibodies have a high molecular weight, they themselves cannot diffuse through the αHL pores, and therefore any observed membrane staining should be due to internal binding.14 As an initial test, we treated GUVs encapsulating an anti-6XHis antibody conjugated to Cy5 with αHL-GFP containing the L-6XHis-L insert. Using fluorescence microscopy, we observed that, after treatment with the αHL fusion protein, antibodies became localized to the GUV membrane (Fig. 3b). Incubation at room temperature for one hour resulted in nearly all GUVs showing membrane localization of the Cy5 anti-6XHis antibodies (Fig. 3c). In comparison, untreated GUVs encapsulating Cy5-conjugated anti-6XHis antibodies do not show membrane localization of the antibody even after incubation for several hours (Supplementary Fig. 1a). Similarly, GUVs encapsulating Cy5 anti-6XHis-tag antibody show no membrane localization of Cy5-antibody after 4 h treatment with αHL L2-GLP1-L2 (Supplementary Fig. 1b). By using different monoclonal antibodies, similar results were obtained for all the peptide inserts we had previously tested, suggesting that our approach is a general method for translocating peptides across lipid membranes such that they are accessible to binding ligands (Fig. 3d).

Fig. 3:
Fig. 3: Peptide inserts in loop128-131 are capable of interacting with encapsulated antibodies after membrane translocation.

a, General scheme for testing translocation of peptides by GUV antibody binding assay. A Cy5-conjugated monoclonal antibody specific for given peptide insert is encapsulated into GUVs. Upon external treatment of GUVs with αHL-GFP fusion proteins containing the peptide insert, the antibody will localize to the membrane, indicating that the peptide insert has fully translocated across the membrane and can be bound by the internal antibody. b, GUVs encapsulated with Cy5 modified anti-6XHis-tag antibodies before and after treatment with αHL-GFP containing the L-6XHis-L loop insert. The GUV membrane shows increased Cy5 (antibody) signal after treatment. c, A larger population of GUVs encapsulating the Cy5 anti-6XHis-tag antibody, all showing increased membrane fluorescence after treatment with αHL-GFP containing the L-6XHis-L loop insert. d, Cy5 antibody assays with all the peptide inserts that were shown to form functional pores. Scale bar: 10 μm.

Cryo-EM reveals that the peptide inserts in loop128-131 of αHL do not interfere with formation of the heptameric pore structure

To further verify that inserts in loop128-131 of αHL do not interfere with pore formation and membrane insertion, we collected structural data using cryo-EM. We chose to investigate αHL mutants with the L2-GLP1-L2 loop insert because it was the largest insert that showed pore formation and peptide translocation based on our previously discussed GUV experiments. Due to its size, the effects of the L2-GLP1-L2 insert on αHL protein structure and functionality should be the largest out of all the inserts we tested. Adapting a previously published protocol,30 we prepared small unilamellar vesicles (SUVs) through hydration of a lipid film (DOPC 60%, cholesterol 40%) followed by extrusion through a 100 nm membrane, and then treated these vesicles with αHL containing the L2-GLP1-L2 insert, which was expressed using the PURExpress® system. Cryo-EM images revealed the formation of numerous αHL pores embedded in the membranes of the SUVs (Fig. 4a). It is possible to see the cap of the αHL pores both from a side-view (red arrow) and from a top-down-view (yellow arrow). In addition, we were also able to generate 2D class averages showing a top-view of the heptameric αHL pore as well as a low-resolution side-view of the cap in the membrane (Fig. 4b). By inducing pore formation through the addition of sodium deoxycholate micelles, we were able to generate 2D class averages with a higher resolution (Supplementary Fig. 2). These results further confirm that the loop128-131-region of αHL tolerates large peptide inserts without losing its pore forming or membrane insertion abilities.

Fig. 4:
Fig. 4: Cryo-EM data and lipid bilayer channel recordings of αHL with peptide inserts in loop128-131.

a, Cryo-EM image of a representative lipid vesicle after treatment with αHL containing the L2-GLP1-L2 loop insert. Red arrow: Side-view of αHL cap. Yellow arrow: Top view of αHL cap. Scale bar: 10 nm. b, 2D class averages of cryo-EM images of αHL containing the L2-GLP1-L2 loop insert in a lipid bilayer. Top view shows heptameric pore structure. c, Lipid bilayer channel recordings of αHL containing the L-6XHis-L loop insert showing pore blockage events. d, Lipid bilayer channel recording of αHL containing the L2-GLP1-L2 loop insert. Increasing the length of the flexible linker increases the frequency of pore blockage events. Scale bar 20 ms.

In addition, we also looked at the effect of peptide inserts on the conductance of the αHL pore in lipid bilayer channel recordings. For the L-6XHis-L insert (Fig. 4c) we could observe pore blockage events, even in the absence of molecules such as PEG that have been shown to be translocated through the pore.31,32 We hypothesize that this is due to the flexible nature of the glycine serine linkers, which might allow the insert to flip in and out of the pore leading to these pore blocking events. Previous work has shown that flexible inserts in the cap region can trigger similar pore blockage events during conductance measurements.33 Consistent with this hypothesis, pores formed from αHL containing longer inserts, like the L2-GLP1-L2 insert (Fig. d), showed an increase in the frequency of these pore blocking events. Despite this high current variability, we were able to observe an approximate channel conductance between 0.1 nA and 0.2 nA, in line with our previous experiments demonstrating that loop inserts do not inhibit the ability of αHL to form pores that can induce the leakage of small molecules across lipid membranes.

α-Hemolysin-peptide fusions enable self-encoded extracellular membrane functionalization of artificial cells and the formation of artificial tissue-like structures

To display peptides through engineered αHL on artificial cells, we encapsulated an in vitro transcription-translation system in a GUV, adapting previously published protocols.34 Each artificial cell would then be able to functionalize its own extracellular membrane through internal expression of a gene coding for αHL with a functional peptide insert in loop128-131. Upon expression of the protein inside the membrane-bound artificial cell, the soluble monomer would be expected to assemble into heptameric pores on the lipid membrane, which would lead to spontaneous translocation of 7 copies of the peptide insert across the membrane onto the extracellular membrane side (Fig. 1b).

While several possible applications could be imagined for external display self-encoded peptides, we decided to explore whether we could use αHL loop mutants to encode for the spontaneous generation of synthetic tissues. While engineering artificial cells has been a long-standing goal of bottom-up synthetic biology, self-organization and self-assembly of individual artificial cells into multi-cellular structures may enable significantly expanded functionality and complexity.35,36,37 Examples of current ways of implementing synthetic cell-cell adhesion are the use of affinity ligands (e.g. streptavidin and biotin),38 electrostatic interactions,39 or the reconstitution of natural cellular proteins like claudin.40 However, one significant difference between all these methods compared to natural cells is that they require the researcher to manually modify the artificial cell membrane externally, for instance by doping the membrane with an affinity ligand. In contrast, natural cells can functionalize their own outer membranes through the self-encoded internal expression and subsequent insertion of membrane proteins which in turn can induce cell-cell adhesion.41 We envisioned that our described αHL peptide translocation system could be used in artificial cells to overcome this limitation and create a self-encoded cell-cell adhesion system.

To genetically encode self-assembly into artificial cells we selected two peptide inserts expected to bind to one another. A simple strategy is to design peptide inserts that can associate due to electrostatic interactions (Fig. 5a). We created two αHL mutants with peptide loop inserts K3 and E3 (each 17 amino acids in length, sequences given in Fig. 5a) which would be predicted to interact with each other by forming salt-bridges between the positively charged lysine and the negatively charged glutamate residues.42,43,44 We tested the ability of αHL to tolerate each insert by performing a membrane binding and leakage assay as previously described (Supplementary Fig. 3). Each heptameric complex can translocate 21 positive or negative charges across an artificial cell membrane. Complimentary artificial cells bearing opposite charges can interact, eventually leading to cell-cell binding and aggregation of multiple cells into artificial tissue-like structures (Fig. 5b).

Fig. 5:
Fig. 5: Self-encoded membrane functionalization system enables artificial tissue formation.

a, αHL pores with the K3 peptide insert can interact with pores containing the E3 peptide insert due to electrostatic interactions. b, General scheme of the tissue formation experiment. Two populations of GUVs express αHL with one of two mutually interacting peptide inserts, K3 (positively charged) or E3 (negatively charged). After internal protein expression, αHL oligomerizes on the artificial membrane and forms pores, while at the same time translocating the K3 or E3 peptide insert across the membrane for extracellular display. After translocation, the K3 expressing GUVs can interact with E3 expressing GUVs leading to GUV self-assembly into tissue-like structures. c, Confocal microscopy image of tissue-like structures formed through mixing K3-αHL and E3-αHL expressing GUVs. Specific interactions between K3-αHL and E3-αHL expressing vesicles were observed and controls (see supporting information) did not result in the formation of assemblies. Scale bar: 10 μm.

We encapsulated the PURExpress® system into GUVs for expression of αHL with either the K3 or E3 loop insert using the T7p14 vector (myTXTL®) as an expression system. The GUVs were formed through the inverse emulsion method with a lipid composition of 60% DOPC and 40% cholesterol. To distinguish GUVs expressing the K3 insert from GUVs expressing the E3 insert, we marked the GUVs by also encapsulating pre-expressed mCherry (K3 insert) or CFP (E3 insert). The two GUV populations were mixed at equal concentrations and protein expression was induced at 37 °C. After one hour of protein expression, the GUVs started to aggregate. The two populations of GUVs showed strong GUV-GUV interactions which ultimately led to the formation of a tissue-like vesicular network structure that had alternating contacts between vesicles displaying the K3 and E3 peptide insert (Fig. 5c). GUV-GUV-interactions were specific to the K3 and E3 inserts.

With insufficient protein expression, we observed no formation of aggregates (Supplementary Fig. 4). Control experiments with GUVs only expressing the K3 insert or only expressing the E3 insert also did not lead to the formation of GUV networks (Supplementary Fig. 5 and 6).

Fig. 6:
Fig. 6: Hydrogen peroxide signaling in self-encoded artificial tissue-like structures.

a, General scheme of an artificial cell signaling pathway using hydrogen peroxide. Glucose oxidase (GO) containing sender artificial cells (marked with mCherry) create hydrogen peroxide in the presence of glucose. Hydrogen peroxide can enter receiver artificial cells which contain Hyper7 as a peroxide reporter. Catalase (CA) is added to the extracellular solution to prevent peroxide signaling outside of the artificial tissue-like structures. b, Confocal microscopy image of hydrogen peroxide signaling in a large tissue-like assembly. Hyper7 containing artificial cells inside the vesicular assembly show increased fluorescence in the green channel. Hyper 7 containing artificial cells outside of tissue (marked with white boxes) show much weaker fluorescence signal. Scale bar: 25 μm c, Quantification of Hyper7 fluorescence intensity. 1: Control experiment. Mean fluorescence of artificial cells in tissue-like structure in the absence of glucose (Supplementary Fig. 7). 2: Mean fluorescence of artificial cells outside of tissue-like structure in the presence of glucose. 3: Mean fluorescence of artificial cells in the tissue-like structure in the presence of glucose.

Formation of self-encoded artificial tissue-like structures facilitates a simple signaling pathway

Apart from enabling cell-cell adhesion, self-encoding artificial tissue formation with αHL mutants can provide further functionality due to the formation of pores in the membranes. αHL with the K3 and E3 insert forms functional pores that allow small molecules to leak across membranes (Supplementary Fig. 3). This in turn also means that vesicles that are part of the artificial tissue are permeable to small molecules. Improved transport of small molecules through nanopores could help facilitate artificial cell-cell signaling within the tissue. While individual artificial cells within a population often act independently of one another, interconnected artificial tissues may be designed to perform specialized actions based on cell-cell communication and interaction. As such, artificial tissues could exceed simple independent and non-interacting artificial cells because communication enables collective action and the formation of spatially ordered tissues, which would be a pre-requisite for the creation of life-like structures reminiscent of natural tissues.45

As a proof of concept, we envisioned engineering a simple signal transduction pathway, where one population of GUVs can generate and send a signaling molecule to a second population of GUVs, which are able to recognize the signaling molecule and produce a detectable readout. We decided on using hydrogen peroxide as the signaling molecule. Hydrogen peroxide has been shown to readily diffuse across lipid membranes46 and, by adding catalase to the outer solution, hydrogen peroxide that diffuses outside of the artificial cells can be rapidly quenched (Fig. 6a). As a readout, we used the protein Hyper7, a hydrogen peroxide responsive fluorescent protein.47 To generate hydrogen peroxide we used glucose oxidase.48,49,50 One population of GUVs, marked with pre-expressed mCherry, expresses αHL with the K3 loop insert and also contains glucose oxidase (sender cells). A second population of GUVs expresses αHL with the E3 loop insert and contains Hyper7 (receiver cells). Signaling is initiated through the addition of glucose. Expression of the αHL mutants enables tissue formation based on the interaction of the E3 and K3 insert and makes the GUVs permeable to glucose, which can traverse the αHL pore. Catalase was added to the outer solution to quench hydrogen peroxide in the extracellular environment. Due to its much higher molecular weight compared to glucose, catalase would not be able to enter the GUVs through the formed αHL pores. Under these experimental conditions we could observe a fluorescence increase for Hyper7 containing GUVs that had assembled with glucose oxidase containing GUVs (Fig. 6b). Unaggregated GUVs (marked with a white box) displayed significantly lower Hyper7 fluorescence, indicating that there is proximity and aggregation-based exchange of hydrogen peroxide. A negative control without glucose showed lower fluorescence intensity (Fig. 6c).

In conclusion, we have demonstrated a straightforward method for the self-encoded extracellular functionalization of artificial cells using internally expressed proteins. Our method relies on the insertion of functional peptides into loop128-131 of the self-translocating pore toxin αHL. We show for the first time that lengthy peptides, up to approximately 50 amino acids, can be inserted into the αHL loop without affecting its membrane binding or translocation function. We also validate by cryo-EM that the inserts do not affect formation of the heptameric pore. The ability of individual artificial cells to encode their own extracellular membrane modification opens new ways for programming interactions between artificial cells and their environment. We demonstrate this by programming the formation of functional synthetic tissues using GUVs that self-encode the surface display of interacting peptides.

Our work may also have implications beyond providing a platform for the programmable functionalization of artificial cells. For instance, at the origin of life, it is unlikely there were highly sophisticated translocase systems, and our work may shed light on the mechanisms by which protocells functionalized their membranes and communicated with their environment. Furthermore, a better understanding of membrane translocation could lead to the development of tools for the non-endocytic delivery of macromolecular therapeutics into living cells.

Methods

Protein expression and purification

Gene constructs were cloned into the pTNT™ vector (Promega). BL21(DE3) Competent E. coli cells (NEB) were transformed with the respective plasmid. 300 ml of LB-medium with carbenicillin (100 μg/ml) was inoculated with a single colony and incubated on a shaker at 37 °C overnight. Cells were collected through centrifugation for 5 minutes at 5000 g. The resulting cell pellet was resuspended in 3 ml lysis buffer (HEPES 20 mM pH = 7.5, NaCl 500 mM, imidazole 10 mM, PMSF 1 mM). Cells were lysed by sonication on ice (3 s on, 3 s off, 10 min, 50% amplitude). The lysate was centrifuged (15.000 x g, 30 min, 4 °C) and filtered through a 0.45 μm syringe filter. The resulting cleared solution was applied to a gravity Ni-NTA column, loaded with 1 ml HisPur™ Ni-NTA Resin that had been pre-equilibrated with lysis buffer. After incubation with the lysate on an overhead spinner at 4 °C for 30 min, the column was washed 4 times with wash buffer (HEPES 20 mM pH = 7.5, NaCl 500 mM, imidazole 30 mM; 1 ml each wash). Protein was eluted into 4 volumes of elution buffer (HEPES 20 mM pH = 7.5, NaCl 500 mM, imidazole 300 mM; 0.5 ml each elution fraction). Protein fractions were identified by SDS–PAGE and the relevant fractions pooled and dialyzed against storage buffer (HEPES 20 mM pH = 7.5, NaCl 500 mM). Proteins were stored at 4 °C for several days or at -80 °C for long-term storage.

Expression of Hyper7

Hyper744 was expressed using the myTXTL® T7 Expression Kit. Expression was set up according to the manufacturer’s instructions. After 18 h of protein expression at 29 °C, TCEP was added to a final concentration of 50 mM. Following incubation at RT for 30 min, the solution was centrifuged at 80.000 x g, 30 min, 4 °C. Excess TCEP was removed from the supernatant using a PD SpinTrap G-25 column. The resulting reduced protein was concentrated on an Amicon ultra spin-concentrator (10 kDa cut-off) and was used without further purification.

GUV formation

GUVs were formed through the inverse emulsion method.26 Briefly, a 1 mM lipid stock (DOPC 60 mol%, cholesterol 40 mol%) in mineral oil was prepared. To 100 μl of this lipid solution was added 10 μl of encapsulation solution, containing all the components to be encapsulated into GUVs. This mixture was emulsified by vortexing. The resulting emulsion was layered on top of lower buffer solution. If not specified otherwise, this lower buffer solution comprised of 20 mM HEPES pH = 7.0, NaCl 500 mM. This two-layer system was subjected to centrifugation at 10.000 x g for 10 minutes. The oil layer was removed and the GUV pellet was resuspended in 20 μl lower buffer. For forming GUVs encapsulating a PURExpress® expression system, this GUV formation protocol was scaled down by half: 50 μl lipid solution, 5 μl encapsulation solution, 50 μl lower buffer solution.

Vesicle leakage assay

GUVs were formed as described with encapsulation solution containing Cy5 (1 μM) and 3.5% (wt/vol) Ficoll® 400 in buffer (20 mM HEPES pH = 7.0, NaCl 500 mM). The resulting GUVs were treated with protein to a final protein concentration of 10 μM. After incubation on an overhead spinner at room temperature for 2 h, the GUVs were imaged on a confocal microscope.

Antibody assay

GUVs were formed as described with the encapsulation solution containing a solution of Cy5-conjugated antibody (1:500 dilution) and 3.5% (wt/vol) Ficoll® 400 in buffer (20 mM HEPES pH = 7.0, NaCl 500 mM). The resulting GUVs were treated with protein to a final protein concentration of 10 μM. After incubation at room temperature for 1 h, the GUVs were imaged on a confocal microscope.

Cryo-electron microscopy of αHL pores in lipid bilayers

A 2.5 mM vesicle solution (DOPC 60 mol%, cholesterol 40 mol%) was prepared through hydration of a lipid film with buffer. (Tris 20 mM pH = 7.0, NaCl = 250 mM). The resulting vesicles were extruded ten times through a 100 nm polycarbonate membrane using a commercial mini-extruder (Avanti Polar Lipids).

αHL monomer with the L2-GLP1-L2 insert was prepared through in-vitro expression with the PURExpress® system. A 25 μl reaction was incubated at 37 °C for 4 hours and then purified using reverse His-tag purification according to the manufacturer’s instructions. To the resulting protein solution was added the solution of extruded vesicles described above to a final lipid concentration of 1 mM. After incubation at room temperature for 1 hour, cryo-EM grids were prepared.

Cryo-electron microscopy grids (Lacey Carbon Film, Electron Microscopy Sciences #LC300-Cu) were glow-discharged (Emitech K350 unit at 20 mA for 30 s), deposited with 3.0 μL of the protein-treated vesicles, blotted for 4 seconds, and then plunged into liquid ethane using a Vitrobot (Mark IV, Thermo Fisher Scientific). Images were acquired on a Talos Arctica (FEI) operated at 200 kV equipped with a Falcon 4i Direct Electron Detector (Thermo Fisher) and collected with a total dose of 40 e/Å2 at 0.95 Å/pixel and at -3 μm nominal defocus. 2837 individual exposures were collected automatically using EPU (Thermo Fisher). Exposures were analyzed in cryoSPARC. Top View 2d class average: Initial particle picks were obtained using cryoSPARC Live’s blob picker (100 – 200 Å circular blobs). The resulting 2247 particles were used for the generation of 2d class averages (20 classes in total). Side View 2d class average: Initial particle picks were obtained using cryoSPARC’s manual picker (256 px box size). The resulting 355 particles were used for the generation of 2d class averages (20 classes in total).

Cryo-electron microscopy of detergent-stabilized αHL pores

α-hemolyisn monomer with the L2-GLP1-L2 insert was prepared through in-vitro expression with the PURExpress® system. 5 reactions (25 μl each) were incubated at 37 °C for 4 hours and then purified using reverse His-tag purification according to the manufacturer’s instructions. The resulting protein solution was concentrated on an Amicon ultra spin-concentrator (10 kDa molecular weight cut-off). To induce pore-formation, sodium deoxycholate was added to a concentration of 6.25 mM. After incubation at RT for 30 min, the solution was diluted with buffer (Tris 20 mM pH = 7.0, NaCl = 250 mM) to a sodium deoxycholate concentration of 1 mM. The resulting dilute solution of αHL pores was concentrated using an Amicon ultra spin-concentrator (100 kDa molecular weight cut-off).

All samples were prepared on UltraAuFoil 1.2/1.3, 300 mesh grids that had been freshly plasma-cleaned using a Gatan Solarus II plasma cleaner (10 s, 15 Watts, 75% Ar/25% O2 atmosphere), deposited with 3.0 μL of the protein solution, blotted for 4 seconds, and then plunged into liquid ethane using a Vitrobot (Mark IV, Thermo Fisher Scientific). Images were acquired on a Titan Krios G4 (Thermo Fisher) operated at 300 kV and equipped with a Selectris X energy filter and a Falcon 4 Direct Electron Detector. Micrographs were collected with a total dose of 55 e/Å2 at 0.935 Å/pixel and at -3 to -1 μm nominal defocus range. 5357 individual exposures were collected automatically using EPU (Thermo Fisher). Exposures were analyzed in cryoSPARC. Initial particle picks were obtained using cryoSPARC Live’s blob picker (100 – 200 Å circular blobs), which was used to generate templates for one round of template picking. The resulting 36740 particles were used for the generation of 2d class averages (30 classes in total).

Single-channel recordings

Recordings were taken on an Orbit Mini instrument (Nanion Technologies) using MECA 4 chips with a 50 μm microcavity. The signal was filtered as such: Range: 2 nA; Sampling frequency (SR): 5 kHz; Final Bandwidth: SR/8. The Orbit Mini was used as described in the manufacturer’s instructions. In short: Recording buffer (3 M KCl, 20 mM Tris pH = 7.0, 150 μl) was added to the chip. Lipid membranes were painted from a DOPC solution in n-octane (5 M). 20 μl of a PURExpress expression solution of the respective protein was added to the chip. Measurements were taken at + 50 mV.

Artificial tissue formation

GUVs expressing either αHL with the K3 insert or the E3 insert were formed as described with the encapsulation solution containing a PURExpress® expression mix prepared according to the manufacturer’s instructions. Two different batches of GUVs were prepared. For GUVs expressing αHL with the K3 insert, to 5 μl PURExpress® mix was added mCherry to a final concentration of 1 μM, plasmid coding for αHL with the K3 insert (75 ng), and 3.5% (wt/vol) Ficoll® 400. For GUVs expressing αHL with the E3 insert, to 5 μl PURExpress® mix was added CFP to a final concentration of 1 μM, plasmid coding for αHL with the E3 insert (75 ng), and 3.5% (wt/vol) Ficoll® 400. To account for the high protein concentration in the PURExpress® mix, the lower buffer solution was changed to 50 mM Tris pH = 7.0, alanine 100 mM, BSA (66 μM). The GUV pellet was resuspended in expression buffer (PURExpress® solution A (2.0 μl), H2O (3.0 μl)).

GUVs expressing αHL with the K3 insert and GUVs expressing αHL with the E3 insert were mixed at a ratio of 1:1 and incubated on an overhead rotator at 37 °C for 2 hours to induce protein expression and subsequently aggregation into tissue-like structures. The resulting tissue-like structures were imaged by confocal microscopy.

Hydrogen peroxide signaling in artificial tissues

For the formation of sender and receiver cells the above protocol for artificial tissue formation was modified slightly: For sender GUVs, to 5 μl PURExpress® mix was added mCherry to a final concentration of 1 μM, plasmid coding for αHL with the K3 insert (75 ng), glucose oxidase (1 U/ml) and 3.5% (wt/vol) Ficoll® 400. For receiver GUVs, to 5 μl PURExpress® mix was added plasmid coding for αHL with the E3 insert (75 ng), 3.5% (wt/vol) Ficoll® 400 and Hyper7 to a final concentration of 2 μM. A 1:1 mixture of sender and receiver GUVs were supplemented with 1% (wt/vol) glucose and catalase to a final concentration of 3 U/ml. The resulting mixture was incubated on an overhead rotator at 37 °C for 2 hours to induce protein expression, aggregation into tissue-like structures and hydrogen peroxide signaling. The formed tissue-like structures were imaged by confocal microscopy.

Statistics and reproducibility

Statistically significant differences in Fig. 6c are indicated based on an independent t-test (two-tailed): ***P < 0.001; **P < 0.01; NS, not significant. Specifically, 1 (GUVs in artificial tissue in the absence of glucose) vs 2 (GUVs outside of artificial tissues in the presence of glucose) P = 0.0830. 1 (GUVs in artificial tissue in the absence of glucose) vs 3 (GUVs in artificial tissue in the presence of glucose) P = 0.00351. 2 (GUVs outside of artificial tissues in the presence of glucose) vs 3 (GUVs in artificial tissue in the presence of glucose) P = 0.000210.

Acknowledgements

This material is based upon work supported by the Department of Defense (Army Research Office) through the Vannevar Bush Faculty Fellowship (Award N00014-22-1-2800). The authors acknowledge the facilities along with the scientific and technical assistance of the staff of the cryo-EM facility at UC San Diego, in particular Dr. Mariusz Matyszewski and Dr. Inga Kuschnerus.

References

  1. 1.↵
    Edidin, M. Lipids on the frontier: a century of cell-membrane bilayers. Nat. Rev. Mol. Cell. Biol. 4, 414–418 (2003).
    OpenUrlCrossRefPubMedWeb of Science
  2. 2.↵
    Cymer, F., von Heijne, G. & White, S. H. Mechanisms of integral membrane protein insertion and folding. J. Mol. Biol. 427, 999–1022 (2015).
    OpenUrlCrossRefPubMed
  3. 3.↵
    Osborne, A. R., Rapoport, T. A. & van den Berg, B. Protein translocation by the Sec61/Secy channel. Annu. Rev. Cell Dev. 21, 529–550 (2005).
    OpenUrlCrossRefPubMedWeb of Science
  4. 4.↵
    Guindani, C., da Silva, L. C., Cao, S., Ivanov, T. & Landfester, K. Synthetic cells: From simple bio-inspired modules to sophisticated integrated systems. Angew. Chem. Int. Ed. 61, e202110855 (2022).
    OpenUrl
  5. 5.↵
    Matsubayashi, H., Kuruma, Y. & Ueda, T. In vitro synthesis of the E. coli Sec translocon from DNA. Angew. Chem. Int. Ed. 53, 7535–7538 (2014).
    OpenUrl
  6. 6.↵
    Noba, K. et al. Bottom-up creation of an artificial cell covered with the adhesive bacterionanofiber protein AtaA. J. Am. Chem. Soc. 141, 19058–19066 (2019).
    OpenUrl
  7. 7.↵
    Yabal, M. et al. Translocation of the C terminus of a tail-anchored protein across the endoplasmic reticulum membrane in yeast mutants defective in signal peptide-driven translocation. J. Biol. Chem. 278, 3489–3496 (2003).
    OpenUrlAbstract/FREE Full Text
  8. 8.↵
    Brambillasca, S., Yabal, M., Makarow, M. & Borgese, N. Unassisted translocation of large polypeptide domains across phospholipid bilayers. J. Cell Biol. 175, 767–777 (2006).
    OpenUrlAbstract/FREE Full Text
  9. 9.↵
    Corcoran, J. A. et al. Myristoylation, a protruding loop, and structural plasticity are essential features of a nonenveloped virus fusion peptide motif. J. Biol. Chem. 279, 51386–51394 (2004).
    OpenUrlAbstract/FREE Full Text
  10. 10.↵
    Chang, D. K., Cheng, S. F. & Chien, W. J. The amino-terminal fusion domain peptide of human immunodeficiency virus type 1 gp41 inserts into the sodium dodecyl sulfate micelle primarily as a helix with a conserved glycine at the micelle-water interface. J. Virol. 71, 6593– 6602 (1997).
    OpenUrlAbstract/FREE Full Text
  11. 11.↵
    Islam, Md. Z., Sharmin, S., Moniruzzaman, Md. & Yamazaki, M. Elementary processes for the entry of cell-penetrating peptides into lipid bilayer vesicles and bacterial cells. Appl. Microbiol. Biotechnol. 102, 3879–3892 (2018).
    OpenUrlCrossRef
  12. 12.↵
    Gilbert, R. J. C. Pore-forming toxins. Cell. Mol. Life Sci. 59, 832–844 (2002).
    OpenUrlCrossRefPubMedWeb of Science
  13. 13.↵
    Dinges, M. M., Orwin, P. M. & Schlievert, P. M. Exotoxins of Staphylococcus aureus. Clin. Microbiol. Rev. 13, 16–34 (2000).
    OpenUrlAbstract/FREE Full Text
  14. 14.↵
    Noireaux, V. & Libchaber, A. A vesicle bioreactor as a step toward an artificial cell assembly. Proc. Natl. Acad. Sci. USA 101, 17669–17674 (2004).
    OpenUrlAbstract/FREE Full Text
  15. 15.↵
    Deamer, D., Akeson, M. & Branton, D. Three decades of nanopore sequencing. Nat. Biotechnol. 34, 518–524 (2016).
    OpenUrlCrossRef
  16. 16.↵
    Walker, B. & Bayley, H. Key residues for membrane binding, oligomerization, and pore forming activity of Staphylococcal α-hemolysin identified by cysteine scanning mutagenesis and targeted chemical modification. J. Biol. Chem. 270, 23065–23071 (1995).
    OpenUrlAbstract/FREE Full Text
  17. 17.↵
    Palmer, M. et al. Staphylococcus aureus alpha-toxin. Production of functionally intact, site-specifically modifiable protein by introduction of cysteine at positions 69, 130, and 186. J. Biol. Chem. 268, 11959–11962 (1993).
    OpenUrlAbstract/FREE Full Text
  18. 18.↵
    Valeva, A. et al. 18 Transmembrane β-barrel of staphylococcal α-toxin forms in sensitive but not in resistant cells. Proc. Natl. Acad. Sci. USA 94, 11607–11611 (1997).
    OpenUrlAbstract/FREE Full Text
  19. 19.↵
    Ward, R. J., Palmer, M., Leonard, K. & Bhakdi, S. Identification of a putative membrane-inserted segment in the alpha-toxin of Staphylococcus aureus. Biochemistry 33, 7477–7484 (1994).
    OpenUrlCrossRefPubMedWeb of Science
  20. 20.↵
    Krasilnikov, O. V., Capistrano, M.-F. P., Yuldasheva, L. N. & Nogueira, R. A. Influence of cys-130 S. aureus alpha-toxin on planar lipid bilayer and erythrocyte membranes. J. Membr. Biol. 156, 157–172 (1997).
    OpenUrlCrossRefPubMedWeb of Science
  21. 21.↵
    Krishnasastry, M., Walker, B., Braha, O. & Bayley, H. Surface labeling of key residues during assembly of the transmembrane pore formed by staphylococcal α-hemolysin. FEBS Lett. 356, 66–71 (1994).
    OpenUrlCrossRefPubMedWeb of Science
  22. 22.↵
    Walker, B., Kasianowicz, J., Krishnasastry, M. & Bayley, H. A pore-forming protein with a metal-actuated switch. Protein Eng. Des. Sel. 7, 655–662 (1994).
    OpenUrlCrossRefPubMedWeb of Science
  23. 23.↵
    Russo, M. J., Bayley, H. & Toner, M. Reversible permeabilization of plasma membranes with an engineered switchable pore. Nat. Biotechnol. 15, 278–282 (1997).
    OpenUrlCrossRefPubMed
  24. 24.↵
    Hall, A. R. et al. Hybrid pore formation by directed insertion of α-haemolysin into solid-state nanopores. Nat. Nanotechnol. 5, 874–877 (2010).
    OpenUrlCrossRefPubMedWeb of Science
  25. 25.↵
    Valeva, A. et al. Membrane insertion of the heptameric staphylococcal alpha-toxin pore. A domino-like structural transition that is allosterically modulated by the target cell membrane. J. Biol. Chem. 276, 14835–14841 (2001).
    OpenUrlAbstract/FREE Full Text
  26. 26.↵
    Pautot, S., Frisken, B. J. & Weitz, D. A. Production of unilamellar vesicles using an inverted emulsion. Langmuir 19, 2870–2879 (2003).
    OpenUrlCrossRefWeb of Science
  27. 27.↵
    Zorzi, A., Deyle, K. & Heinis, C. Cyclic peptide therapeutics: past, present and future. Curr. Opin. Chem. Biol. 38, 24–29 (2017).
    OpenUrlCrossRefPubMed
  28. 28.↵
    de Herder, W. W. & Lamberts, S. W. J. Somatostatin and somatostatin analogues: diagnostic and therapeutic uses. Curr. Opin. Oncol. 14, 53 (2002).
    OpenUrlCrossRefPubMedWeb of Science
  29. 29.↵
    Müller, T. D. et al. Glucagon-like peptide 1 (GLP-1). Mol. Metab. 30, 72–130 (2019).
    OpenUrl
  30. 30.↵
    Pang, S. S. et al. The cryo-EM structure of the acid activatable pore-forming immune effector macrophage-expressed gene 1. Nat. Commun. 10, 4288 (2019).
    OpenUrl
  31. 31.↵
    Aksoyoglu, M. A. et al. Size-dependent forced PEG partitioning into channels: VDAC, OmpC, and α-hemolysin. Proc. Natl. Acad. Sci. USA 113, 9003–9008 (2016).
    OpenUrlAbstract/FREE Full Text
  32. 32.↵
    C. Machado, D. et al. Effects of alkali and ammonium ions in the detection of poly(ethyleneglycol) by alpha-hemolysin nanopore sensor. RSC Adv. 6, 56647–56655 (2016).
    OpenUrl
  33. 33.↵
    Jung, Y., Cheley, S., Braha, O. & Bayley, H. The internal cavity of the Staphylococcal α-hemolysin pore accommodates ∼175 exogenous amino acid residues. Biochemistry 44, 8919– 8929 (2005).
    OpenUrlCrossRefPubMedWeb of Science
  34. 34.↵
    Nishimura, K. et al. Cell-free protein synthesis inside giant unilamellar vesicles analyzed by flow cytometry. Langmuir 28, 8426–8432 (2012).
    OpenUrlCrossRefPubMedWeb of Science
  35. 35.↵
    Villar, G., Graham, A. D. & Bayley, H. A tissue-like printed material. Science 340, 48–52 (2013).
    OpenUrlAbstract/FREE Full Text
  36. 36.↵
    Bayley, H., Cazimoglu, I. & Hoskin, C. E. G. Synthetic tissues. Emerg. top. life sci. 3, 615– 622 (2019).
    OpenUrl
  37. 37.↵
    Booth, M. J., Schild, V. R., Graham, A. D., Olof, S. N. & Bayley, H. Light-activated communication in synthetic tissues. Sci. Adv. 2, e1600056 (2016).
    OpenUrlFREE Full Text
  38. 38.↵
    Chiruvolu, S. et al. Higher order self-assembly of vesicles by site-specific binding. Science 264, 1753–1756 (1994).
    OpenUrlAbstract/FREE Full Text
  39. 39.↵
    Carrara, P., Stano, P. & Luisi, P. L. Giant vesicles “colonies”: A model for primitive cell communities. ChemBioChem 13, 1497–1502 (2012).
    OpenUrlCrossRefPubMed
  40. 40.↵
    Belardi, B. et al. Claudin-4 reconstituted in unilamellar vesicles is sufficient to form tight interfaces that partition membrane proteins. J. Cell Sci. 132, jcs221556 (2018).
    OpenUrl
  41. 41.↵
    van Roy, F. & Berx, G. The cell-cell adhesion molecule E-cadherin. Cell. Mol. Life Sci. 65, 3756–3788 (2008).
    OpenUrlCrossRefPubMedWeb of Science
  42. 42.↵
    Wörsdörfer, B., Woycechowsky, K. J. & Hilvert, D. Directed evolution of a protein container. Science 331, 589–592 (2011).
    OpenUrlAbstract/FREE Full Text
  43. 43.↵
    Beck, T., Tetter, S., Künzle, M. & Hilvert, D. Construction of Matryoshka-type structures from supercharged protein nanocages. Angewandte Chemie International Edition 54, 937–940 (2015).
    OpenUrl
  44. 44.↵
    Sun, H. et al. Self-assembly of cricoid proteins induced by “soft nanoparticles”: An approach to design multienzyme-cooperative antioxidative systems. ACS Nano 9, 5461–5469 (2015).
    OpenUrl
  45. 45.↵
    Lin, A. J., Sihorwala, A. Z. & Belardi, B. Engineering tissue-scale properties with synthetic cells: Forging one from many. ACS Synth. Biol. 12, 1889–1907 (2023).
    OpenUrl
  46. 46.↵
    Abuin, E., Lissi, E. & Ahumada, M. Diffusion of hydrogen peroxide across DPPC large unilamellar liposomes. Chem. Phys. Lipids 165, 656–661 (2012).
    OpenUrlPubMed
  47. 47.↵
    Pak, V. V. et al. Ultrasensitive genetically encoded indicator for hydrogen peroxide identifies roles for the oxidant in cell migration and mitochondrial function. Cell Metab. 31, 642–653.e6 (2020).
    OpenUrlCrossRefPubMed
  48. 48.↵
    Wang, X. et al. Chemical information exchange in organized protocells and natural cell assemblies with controllable spatial positions. Small 16, 1906394 (2020).
    OpenUrl
  49. 49.↵
    Li, S., Wang, X., Mu, W. & Han, X. Chemical signal communication between two protoorganelles in a lipid-based artificial cell. Anal. Chem. 91, 6859–6864 (2019).
    OpenUrl
  50. 50.↵
    Yang, B., Li, S., Mu, W., Wang, Z. & Han, X. Light-harvesting artificial cells containing cyanobacteria for CO2 fixation and further metabolism mimicking. Small 19, 2201305 (2023).
    OpenUrl
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Supplementary Material

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Encoding extracellular modification of artificial cell membranes using engineered self-translocating proteins
Alexander Harjung, Alessandro Fracassi, Neal Devaraj
bioRxiv 2023.10.06.561148; doi: https://doi.org/10.1101/2023.10.06.561148
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