Biological mechanotransduction enables cells to sense and respond to mechanical forces in their local environment through changes in cell structure and gene expression, resulting in downstream changes in cell function. However, the complexity of living systems obfuscates the mechanisms of mechanotransduction, and hence the study of these processes in vitro has been critical in characterising the function of existing mechanosensitive membrane proteins. Synthetic cells are biomolecular compartments that aim to mimic the organisation, functionality and behaviours of biological systems, and represent the next step in the development of in vitro cell models. In recent years, mechanosensitive channels have been incorporated into synthetic cells to create de novo mechanosensitive signalling pathways. Here, I will discuss these developments, from the molecular parts used to construct existing pathways, the functionality of such systems, and potential future directions in engineering synthetic mechanotransduction. The recapitulation of mechanotransduction in synthetic biology will facilitate an improved understanding of biological signalling through the study of molecular interactions across length scales, whilst simultaneously generating new biotechnologies that can be applied as diagnostics, microreactors and therapeutics.
Biological mechanotransduction
Communication is one of the hallmarks of life, facilitating the interaction of organisms with different stimuli in the local environment, from chemical [1] and thermal fluxes [2] to cell-cell adhesion driven by protein-protein interactions [3]. Upon sensing a signal, cells respond through the activation of signal transduction pathways that translate an initial event (e.g. ligand binding) to a downstream change in cell function through changes in gene regulation, protein expression and protein localisation [4].
To enable cells to respond to physical forces in their local environment (e.g. compression, tensile and shear forces [5]), biology has evolved mechanosensitive protein machinery that can sense these forces at the cell membrane via mechanotransduction mechanisms that transmit signals from the extracellular matrix (ECM) to the nucleus, regulating diverse biological processes, including stem cell fate [6], homeostasis [7], and the development of disease [8]. Key mechanosensors that have been shown to convert mechanical forces into biochemical cues include integrin-based focal adhesions, transcription factors and mechanosensitive ion channels [9].
Integrins are recognised as critical mechanotransducers that connect the ECM to the cellular cytoskeleton [10]. Integrins are single pass transmembrane heterodimers consisting of α and β subunits, with the affinity of the integrin receptor for different ECM components (e.g. fibronectin, laminins, collagens) dependent on the subunit combination [11]. Integrins act as a bridge for bi-directional signalling by binding the ECM through an extracellular domain, whilst through a cytoplasmic tail the integrin can form multiprotein complexes capable of binding the cellular actin cytoskeleton [12].
Integrins transduce changes in ECM structure and mechanics into chemical signals sensed by the cell in a process known as outside-in signalling. This can occur via multiple mechanisms, for example through integrin clustering to form focal adhesion complexes that direct downstream signalling processes through a series of effector proteins described as the intergrin adhesome [13]. Key integrin-activated pathways include RhoA/ROCK, which regulates the actin cytoskeleton and its force response [14], as well as the assembly of myosin II filaments and its ability to exert contractile force on actin [15]. Integrins can also communicate via inside-out signalling, where changes in the cytoplasmic tail conformation can drive affinity changes of the integrin dimer for extracellular ligands, enabling the cell to exert forces on its local environment [16].
Beyond integrins, mechanosensitive transcriptional regulators such as Yes-associated protein (YAP) have been shown to transmit signals to the nucleus in response to mechanical cues such as ECM rigidity [17]. The external force results in stretching of the nucleus, increasing the import rate of YAP through nuclear pores and leading to transcription of YAP-associated genes. Finally, mechanosensitive ion channels can respond to changes in membrane mechanics such as increases in membrane tension, gating in response to conduct ions or small molecules across the cell membrane (Figure 1A) [18]. Such channels play critical biological roles for both prokaryotes and eukaryotes and have found application in synthetic biology research as tools to control molecular transport. As such mechanosensitive ion channels will form the primary focus of the rest of this review.
A comparison of mechanosensitive channel-mediated mechanotransduction in biological and synthetic cells.
(A) In biological mechanotransduction, mechanosensitive proteins can respond to the application of an external mechanical force. This includes mechanosensitive channels such as Piezo1, a Ca2+ channel that gates in response to mechanical changes induced in the plasma membrane by tension or shear stress. An increase in cytosolic Ca2+ can initiate various signalling pathways through the activation of calcium-dependent enzymes such as phospholipase C or calcineurin phosphatase. This results in downstream changes in gene regulation and ultimately cell function. (B) In synthetic mechanotransduction, through individual reconstituted mechanosensitive proteins e.g. mechanosensitive ion channels, a synthetic cell can respond to mechanical forces such as increases in membrane tension to enable the influx of diverse signalling molecules (limited by the pore size and selectivity of the channel). This influx can in turn activate protein machinery, for example the activation of transcription factors that lead to expression of gene circuits. As the synthetic cell can be constructed from the bottom-up, additional components of the cell can be designed to interact with the signalling pathway, for example phospholipases that act on internal membranous compartments, or through dynamic condensate formation and mRNA sequestration. Each of these increases the tuneability of downstream synthetic cell function. Created with BioRender.com.
(A) In biological mechanotransduction, mechanosensitive proteins can respond to the application of an external mechanical force. This includes mechanosensitive channels such as Piezo1, a Ca2+ channel that gates in response to mechanical changes induced in the plasma membrane by tension or shear stress. An increase in cytosolic Ca2+ can initiate various signalling pathways through the activation of calcium-dependent enzymes such as phospholipase C or calcineurin phosphatase. This results in downstream changes in gene regulation and ultimately cell function. (B) In synthetic mechanotransduction, through individual reconstituted mechanosensitive proteins e.g. mechanosensitive ion channels, a synthetic cell can respond to mechanical forces such as increases in membrane tension to enable the influx of diverse signalling molecules (limited by the pore size and selectivity of the channel). This influx can in turn activate protein machinery, for example the activation of transcription factors that lead to expression of gene circuits. As the synthetic cell can be constructed from the bottom-up, additional components of the cell can be designed to interact with the signalling pathway, for example phospholipases that act on internal membranous compartments, or through dynamic condensate formation and mRNA sequestration. Each of these increases the tuneability of downstream synthetic cell function. Created with BioRender.com.
Synthetic cell engineering
With the rise of synthetic biology, two main approaches have arisen towards cellular engineering. The first approach — top-down — involves the genetic engineering of living cells [19] with the aim of controlling specific pathways and outputs, for example using metabolic engineering to drive the efficient biosynthesis of drugs and fine chemicals [20], or the production of engineered cell surface receptors to create powerful immunotherapies (e.g. chimeric antigen receptor T cells) [21]. An alternative approach, known as bottom-up synthetic biology, aims to construct cells from their constitutive molecular parts (e.g. protein, lipids, nucleic acids). This approach has led to the field of synthetic cell engineering, where much simpler compartmentalised systems have been designed that mimic the organisation, functions, and behaviours of biology [22]. Synthetic cells have the potential to act as new tools for the study of fundamental biology [23], enabling the reconstitution of isolated processes that can take effect across length scales (e.g. nanoscale protein activation coupled with micro or milliscale diffusional processes). Beyond this, by engineering nano and microsystems with specific functions such as sensing [24], triggered release [25] and biosynthesis [26], synthetic cells could be applied as new diagnostics, therapeutics and microreactors.
Whether engineering living cells or trying to produce them from compartmentalised non-living chemical networks, the development of new signalling machinery and pathways is essential to enable the cell to (a) sense different aspects of its local environment and (b) respond to this through the activation of a downstream process. This is especially important in the development of synthetic cells, which possess far fewer molecular ‘parts’ than living systems, significantly limiting their interaction with their local environment. To overcome this limitation, there has been efforts made in developing new signalling pathways in synthetic cells. These include the development of synthetic membrane-embedded small molecules that can be transported from one face of a lipid membrane to the other (acting as transducers) [27], nucleic acid nanotechnology that can be activated in response to the presence of oligonucleotides with complementary sequences [28], and the reconstitution of protein channels within membranes that facilitate the transport of content in and out of the synthetic cell [29].
One area of recent development in synthetic cell engineering is the construction of synthetic mechanosensitive signalling pathways (Figure 1B). Such a pathway enables the synthetic cell to sense forces in their local environment, enabling them to act as mechanosensors. The simplicity of synthetic cells relative to their living counterparts make them an ideal testing ground for the engineering of mechanosensitive signalling systems, avoiding the cross-talk of different pathways present in living cells that obfuscates the mechanism of specific processes. Furthermore, as mechanosensitive processes in biology are key in regulating cellular homeostasis [7], genetic engineering methods are challenging to employ in this context without affecting cell viability.
In this review, I will discuss the development of synthetic signalling pathways utilising mechanosensitive membrane proteins, with a focus on the mechanosensitive channel of large conductance (MscL). The versatility of MscL to sense diverse molecular stimuli has led to the design of synthetic cells utilising these channels in different compartments, activated by various stimuli, and in each case leading to a change in the function of the synthetic cell. Finally, I will discuss challenges in constructing biological mechanotransduction pathways in synthetic cells, and how different technologies could be used to build future de novo mechanosensitive signalling pathways.
Mechanosensitive membrane proteins
Mechanosensitive membrane proteins have been found in all genera of life, from bacteria to humans [18]. These proteins are responsible for critical cellular and organismal functions, for example acting as osmotic ‘release valves’ that allow bacteria to survive situations of osmotic stress [30] and underpinning the sense of touch in higher animals [31]. One important class of mechanosensitive proteins discovered to-date are mechanosensitive ion channels (MSCs) that can gate in response to increased membrane tension [18]. However, the function of voltage-gated ion channels can be modulated by mechanical stimulation [32–34], and there are recent examples of G-protein coupled receptors that can respond to mechanical forces [35], indicating that mechanosensitive properties may be more common in membrane proteins than previously thought.
MSCs act as primary transducers of mechanical force, converting the force to molecular (ion) flux across a membrane. Such channels can be found in both prokaryotic and eukaryotic organisms and vary significantly in their number of transmembrane helices as well as oligomerisation states [18]. Of these, the homopentameric bacterial MscL channel (∼80 kDa) remains the best studied example [36,37] (Figure 2A). MscL acts as a ‘release valve’ for bacteria in situations of severe hypoosmotic stress [40]. In vivo, MscL responds to high membrane tension by undergoing a large conformational change to form a large non-specific pore ∼3 nm [41] in diameter, through which the efflux of osmolytes and small proteins occurs [42,43]. During this gating transition, MscL is predicted to decrease its thickness in the membrane whilst increasing its surface area, coupling with decreases in bilayer thickness upon tension application [44].
Synthetic mechanosensitive signalling pathways incorporating MscL.
(A) The structure of MscL. MscL is a homopentameric integral membrane protein with two transmembrane domains (TMDs) in each monomer of the channel. The N-terminus of each monomer is an amphipathic α-helix which sits in the polar headgroups of the bilayer on the cytosolic face of the membrane, acting as a tension sensor. TMD1 then connects to TMD2 via a periplasmic loop, before a cytoplasmic helical bundle at the C-terminal. The channel gates through a large-scale conformational change, increasing the occupied surface area of the channel and opening like the iris of a traditional camera. Closed MscL side and periplasmic structure obtained from PDB (2OAR): whilst the open state is a predicted structure of the MscL TMDs from reference 28 (1KYM) visualised using JSmol. (B) The lateral pressure profile. The activation of mechanosensitive proteins that function via FFL mechanisms can be understood through the concept of the lateral pressure profile. In the absence of an external pressure, the sum of the compressive and repulsive forces acting on a membrane total to 0. However, if these forces are considered locally, this is not the case, and very large forces are present in different regions of the bilayer. The key forces are the repulsion between polar lipid headgroups as well as by the hydrocarbon tails of the phospholipids, opposed by a strong compressive force from surface tension acting at the interface between the headgroups and hydrophobic core of the bilayer. In a symmetric membrane, these forces are mirrored in each leaflet of the bilayer, but the introduction of membrane asymmetry results in the application of asymmetrical local forces to membrane proteins present in the bilayer. In the case of MscL, this asymmetric perturbation of the lateral pressure profile results in channel gating. (C) MscL-mediated gene expression in synthetic cells. In a study by Garamella et al. [76] gene circuits were encapsulated within a lipid vesicle-based synthetic cell. Briefly, MscL was constitutively expressed within the cell under control by T7 RNA polymerase. This results in the expression and integration of MscL into the membrane of the synthetic cell. Additionally, three plasmids were encapsulated that enables the induction of the bacterial cytoskeleton protein MReB (expressed as a fusion with the Venus fluorescent protein) only when IPTG binds to the LacI repressor protein. In the absence of IPTG, LacI prevents expression of the σ28 RNA polymerase (and hence expression of MReB). (D) Synthetic mechanotransduction for cytoskeleton synthesis. Synthetic cells as composed in (C) can respond to an initial hypoosmotic shock by enabling the influx of IPTG in the external solution. This results in activation of the inducible gene circuit as LacI dissociates upon IPTG binding, leading to the expression of σ28 RNA polymerase. This in turn drives the expression of the Venus-MreB fusion protein, which self-assembles at the inner face of the membrane forming a new cytoskeleton in the synthetic cell. (E) sPLA2-membrane-MscL communication. In the absence of asymmetry (and membrane tension) MscL is closed, preventing transport across the membrane. If sPLA2 is present in the external solution, it will bind to the membrane. Its catalytic activity produces single tailed lysophopholipids in the external leaflet of the bilayer (yellow), generating a compositional asymmetry which perturbs the lateral pressure profile of the membrane. MscL responds to this through gating, facilitating molecular transport across the membrane. This interaction can be viewed as an interaction between sPLA2 and MscL that takes places through the medium of the lipid membrane. (F) Synthetic mechanosensitive signalling pathways in multicompartment synthetic cells. sPLA2-M-MscL communication highlighted in (E) was used by Hindley et al. [78] in a multicompartment lipid vesicle-based synthetic cell, where nanoscale vesicle compartments containing MscL were co-encapsulated within cell size vesicles alongside the sPLA2 enzyme. The sPLA2-M-MscL interaction was controlled by exploiting the calcium-dependent nature of sPLA2 activity, controlling the concentration of free Ca2+ present in the synthetic cell lumen through co-encapsulating an excess of the Ca2+-chelator EDTA. The signalling pathway was then completed through the addition of αHL protein pores to the outer membrane of the synthetic cell. This enabled the influx of Ca2+ to the cell, which at a critical concentration could activate sPLA2. The activity of sPLA2 on the internal compartments of the cell results in gating of MscL, leading to inducible content exchange between the ‘organelles’ of the synthetic cell and its main compartment. Created with BioRender.com.
(A) The structure of MscL. MscL is a homopentameric integral membrane protein with two transmembrane domains (TMDs) in each monomer of the channel. The N-terminus of each monomer is an amphipathic α-helix which sits in the polar headgroups of the bilayer on the cytosolic face of the membrane, acting as a tension sensor. TMD1 then connects to TMD2 via a periplasmic loop, before a cytoplasmic helical bundle at the C-terminal. The channel gates through a large-scale conformational change, increasing the occupied surface area of the channel and opening like the iris of a traditional camera. Closed MscL side and periplasmic structure obtained from PDB (2OAR): whilst the open state is a predicted structure of the MscL TMDs from reference 28 (1KYM) visualised using JSmol. (B) The lateral pressure profile. The activation of mechanosensitive proteins that function via FFL mechanisms can be understood through the concept of the lateral pressure profile. In the absence of an external pressure, the sum of the compressive and repulsive forces acting on a membrane total to 0. However, if these forces are considered locally, this is not the case, and very large forces are present in different regions of the bilayer. The key forces are the repulsion between polar lipid headgroups as well as by the hydrocarbon tails of the phospholipids, opposed by a strong compressive force from surface tension acting at the interface between the headgroups and hydrophobic core of the bilayer. In a symmetric membrane, these forces are mirrored in each leaflet of the bilayer, but the introduction of membrane asymmetry results in the application of asymmetrical local forces to membrane proteins present in the bilayer. In the case of MscL, this asymmetric perturbation of the lateral pressure profile results in channel gating. (C) MscL-mediated gene expression in synthetic cells. In a study by Garamella et al. [76] gene circuits were encapsulated within a lipid vesicle-based synthetic cell. Briefly, MscL was constitutively expressed within the cell under control by T7 RNA polymerase. This results in the expression and integration of MscL into the membrane of the synthetic cell. Additionally, three plasmids were encapsulated that enables the induction of the bacterial cytoskeleton protein MReB (expressed as a fusion with the Venus fluorescent protein) only when IPTG binds to the LacI repressor protein. In the absence of IPTG, LacI prevents expression of the σ28 RNA polymerase (and hence expression of MReB). (D) Synthetic mechanotransduction for cytoskeleton synthesis. Synthetic cells as composed in (C) can respond to an initial hypoosmotic shock by enabling the influx of IPTG in the external solution. This results in activation of the inducible gene circuit as LacI dissociates upon IPTG binding, leading to the expression of σ28 RNA polymerase. This in turn drives the expression of the Venus-MreB fusion protein, which self-assembles at the inner face of the membrane forming a new cytoskeleton in the synthetic cell. (E) sPLA2-membrane-MscL communication. In the absence of asymmetry (and membrane tension) MscL is closed, preventing transport across the membrane. If sPLA2 is present in the external solution, it will bind to the membrane. Its catalytic activity produces single tailed lysophopholipids in the external leaflet of the bilayer (yellow), generating a compositional asymmetry which perturbs the lateral pressure profile of the membrane. MscL responds to this through gating, facilitating molecular transport across the membrane. This interaction can be viewed as an interaction between sPLA2 and MscL that takes places through the medium of the lipid membrane. (F) Synthetic mechanosensitive signalling pathways in multicompartment synthetic cells. sPLA2-M-MscL communication highlighted in (E) was used by Hindley et al. [78] in a multicompartment lipid vesicle-based synthetic cell, where nanoscale vesicle compartments containing MscL were co-encapsulated within cell size vesicles alongside the sPLA2 enzyme. The sPLA2-M-MscL interaction was controlled by exploiting the calcium-dependent nature of sPLA2 activity, controlling the concentration of free Ca2+ present in the synthetic cell lumen through co-encapsulating an excess of the Ca2+-chelator EDTA. The signalling pathway was then completed through the addition of αHL protein pores to the outer membrane of the synthetic cell. This enabled the influx of Ca2+ to the cell, which at a critical concentration could activate sPLA2. The activity of sPLA2 on the internal compartments of the cell results in gating of MscL, leading to inducible content exchange between the ‘organelles’ of the synthetic cell and its main compartment. Created with BioRender.com.
MscL (as well as other bacterial MSCs such as the mechanosensitive channel of small conductance) can respond exclusively to the forces transmitted from the surrounding lipid membrane, which is now known as the force-from-lipid (FFL) principle [45]. Whilst forces from the membrane total to 0 for a tension-free membrane at equilibrium [46], the local lateral forces exerted in a lipid membrane can exceed several hundred atmospheres of pressure [47]. This lateral pressure profile includes a strong compressive force (interfacial tension) that acts at the headgroup/hydrocarbon tail interface, minimising exposure of the hydrocarbon lipid tails because of the hydrophobic effect. Opposing this are repulsive forces between hydrocarbon tails within the membrane (with contributions from sterics and conformational entropy costs associated with hydrocarbon chain ordering [48] dominating attractive dispersive forces), as well as smaller repulsive steric interactions between head groups (Figure 2B). In a symmetric membrane, these forces are mirrored in each leaflet of the bilayer, but the introduction of membrane asymmetry results in the application of asymmetrical local forces to membrane proteins present in the bilayer. This helps to rationalise the activation of mechanosensitive proteins via FFL, for example the gating of MscL via asymmetric addition of lysophospholipids to the bilayer [49]. Molecular dynamics simulations indicate that asymmetric incorporation of lysophospholipids results in lateral contraction in one leaflet of the membrane and expansion in the other, resulting in gating of the MscL channel [50].
Other mechanosensitive channels have been shown to function solely via the FFL principle, including Piezo1, a eukaryotic cation transporter that gates in response to increases in membrane tension [51,52]. Piezo1 forms a large, triskelion-shaped trimer (∼900 kDa) with bent arms projecting from the central axis, and in vesicles has been shown to locally curve the membrane into a dome structure when in the closed state [53]. By using high-speed atomic force microscopy, Lin et al. [54] controllably applied mechanical force to supported lipid bilayers containing Piezo1, observing a circular expansion of the channel with increasing membrane tension, flattening the local bilayer curvature.
For MscL and Piezo1, their ability to respond solely to forces from the lipid bilayer was established in vitro through isolation of the protein and reconstitution into an artificial membrane environment. In contrast, there are mechanosensitive proteins which have been shown to be linked to cytoskeletal components, and respond to forces exerted by these, known as the force-from-filament (FFF) principle [55]. The FFF relies on the presence of a cytoskeleton, which many synthetic cells currently lack, and the incorporation of a cytoskeleton could allow synthetic cells to act as better model systems for the study of biological mechanotransduction.
The characterisation of mechanosensitive membrane proteins in vitro illustrates the utility of such cell-free approaches for studying biomolecular function. In this light, the design and study of synthetic signalling pathways acts as a logical next step. The feasibility of producing such molecular interaction networks is driven by advances in biomembrane engineering that have taken place over the last 20 years. This includes the development of multiple methods to create giant, cell-sized vesicles capable of encapsulating complex biomolecular solutions [56–58], the construction of multicompartment model systems of varying architecture and compartment number [25,26,59–61], and the development of microfluidic production methods that facilitate the rapid production of giant vesicles with control over their size [38,62,63]. By combining biomembrane engineering with traditional membrane protein reconstitution methods [39,64,65], as well as cell-free protein synthesis systems [66], a variety of signalling pathways have now been designed incorporating mechanosensitive channels as a key component.
Synthetic mechanosensitive signalling pathways
To date, MscL has been the sole mechanosensitive membrane channel incorporated within synthetic cells. Its use can be attributed to its well-characterised structure and function [37], ease of expression (both in-cell [67] and cell-free [68]), large pore size (which facilitates the transport of a variety of molecular cargo) [43], and critically for applications in synthetic cells, its highly versatile gating mechanisms. MscL has been shown to gate in response to multiple stimuli, including membrane tension [36], membrane asymmetry [49], drug binding to the channel [69] and membrane (including allosteric modulation [70]), and through covalent chemical labelling, electrostatics [71] and light [72]. To access these novel gating modalities, mutagenesis of MscL has been used to (i) tune the inherent mechanosensitivity of the channel through changing the hydrophobicity of the pore lumen [73,74] and (ii) install a reactive cysteine residue within the channel lumen (e.g. G22C in Escherichia coli MscL [71]). This enables the modification of the channel with small molecule thiosulfonate labels that have been used to electrostatically gate the pore [71], or enable the light-responsive gating of the pore through photoisomerisation of the label [72].
Membrane tension has been used by Liu and Noireaux as a trigger to activate the function of GUV-based synthetic cells via MscL gating [75,76]. Key to these studies was the cell-free expression of the channel from E. coli lysate within the lumen of the synthetic cell. Through this expression, the synthetic cell gains the ability to respond to osmotic stress. By co-expressing a calcium-responsive protein, a calcium biosensor was constructed that serves as a reporter for osmotic stress events [75]. Building on this, synthetic cells were designed that could respond to osmotic stress via transporting signalling molecules (e.g. IPTG) that enable transcription of co-encapsulated genes (Figure 2C). Using this method, synthetic cells were produced that produce reporter proteins (e.g. eGFP) or structural proteins (e.g. the bacterial cytoskeletal protein MreB) in response to osmotic stress [76]. Through such mechanotransduction an external osmotic stress could be sensed and converted to a structural change in the synthetic cell through the formation of a MreB cytoskeleton (Figure 2D). This highlights the potential of using mechanosensitive channels to control gene signalling programmes inside synthetic cells.
Membrane asymmetry — an important feature in the organisation of biological membranes — has also been exploited as a component in the design of synthetic mechanosensitive signalling pathways. One key example is in the development of membrane-mediated protein communication networks by Charalambous et al. [77]. In this initial work, communication was established between secretory phospholipase A2 (sPLA2) enzymes and MscL through the lipid bilayer containing the MscL channel. Here, the asymmetric production of single-tailed lysophosphocholine (LPC) lipids by sPLA2 results in the generation of an LPC membrane asymmetry, which results in an asymmetric perturbation of the lateral pressure profile (Figure 2E). MscL can sense this asymmetry, and in response the gating of the channel occurs. This approach was used to trigger the efflux of a fluorescent dye, calcein, through MscL pores reconstituted in large unilamellar vesicles ∼100 nm in diameter and serves as proof of concept for the gating of MscL channels through dynamic changes in membrane composition.
Building on this, a synthetic signalling pathway was developed in multicompartment synthetic cells, utilising sPLA2-Membrane-MscL interactions as an activatable module contained within the cell lumen (Figure 2F) [78]. This pathway exploited the calcium-dependent nature of sPLA2 catalysis to link onset of the sPLA2-Membrane-MscL interaction (and subsequent gating of MscL) to an increase in the calcium concentration in the cell. This was achieved through the co-encapsulation of the calcium chelator EDTA alongside MscL vesicles and sPLA2. The pathway was then completed by functionalising the external membrane of the synthetic cell with alpha haemolysin [79], a pore-forming protein that enabled influx of Ca2+. Synthetic cells were designed that could trigger after saturating the encapsulated EDTA concentration, leading to activation of sPLA2 activity, induction of sPLA2-Membrane-MscL communication and the release of calcein dye into the main compartment of the synthetic cell through MscL. This pathway shares similarities with biological signal transduction, including the ∼20 000-fold increase in calcium concentration from nanomolar to millimolar in the main compartment of the synthetic cell upon pathway activation [80], and changes in cell properties driven by sequential activation of protein machinery. This de novo pathway could be used (and extended) to control a variety of downstream synthetic cell functions including protein expression and drug release.
Gating MscL via membrane asymmetry has been further explored in multicompartment droplet-based synthetic cells. In this exciting study by Li et al. [81], a gain-of-function G22S mutant of MscL was incorporated into aqueous droplets within a larger oil droplet, which functions as a multicompartment cell model. In such an environment, droplet interface bilayers (DIBs) can be formed where two droplets (each stabilised by a lipid monolayer) touch [82,83]. Asymmetric dioleoylphosphatidylcholine (DOPC) and diphytanoylphosphatidylcholine (DPhPC) DIBs were created by bringing DOPC and DPhPC droplets into contact, and gating of reconstituted MscL G22S was achieved in this DIB system purely from this membrane asymmetry, monitored by calcium translocation through the channel across the DIB. This highlights how actuation of signalling can be driven by tuning the mechanosensitivity of the MscL channel to match the magnitude of the perturbation from its host lipid bilayer (in this case the designed asymmetry). In the same work, user-controlled actuation of signalling was obtained through magnetic manipulation. Here, acoustic levitation was used to trap the cell. In this state, acoustic forces are dissipated through rotation of the synthetic cell. However, by encapsulating magnetic particles within one compartment of the cell, application of an external magnetic field resulted in locking the synthetic cell, and in this locked state, the acoustic forces induce membrane tension, resulting in the gating of the MscL channel. This system represents a highly novel example of using combined magnetic and acoustic manipulation of synthetic cells to actuate internal processes and highlights the ability of such systems to incorporate non-biological components in pathway design.
Further development of synthetic signalling pathways
Recent work has established that mechanosensitive channels such as MscL can be used as mechanosensors to drive the design of new bioinspired signalling systems. However, this represents only one molecular transducer to convert mechanical force into a (bio)chemical response. Other mechanosensitive membrane proteins could be used to transduce mechanical stress into chemical changes. Alternative MSCs represent obvious tools for integration into synthetic cell designs, for example the eukaryotic Piezo1 MSC. Piezo1 governs the touch sense in mammals and is a mechanosensitive calcium channel that as with MscL, can respond solely to forces in the lipid bilayer [84]. Piezo1 could help to regulate calcium transport within synthetic cells and tissues, leading to the controlled activation of Ca2+-dependent signalling pathways. Piezo1 has been successfully overexpressed, purified, and reconstituted into proteoliposomes [52], further indicating the promise of utilising this channel as a synthetic cell part. Reconstitution of these alternative channels could result in greater control over synthetic cell pathway activation in comparison with MscL, as channel gating results in ion influx into the synthetic cell without loss of contents to the environment, in addition to enabling synthetic cells to respond to differing magnitudes of applied force.
Many biological mechanotransduction pathways transmit force from the external environment through structural elements such as the ECM and the cell cytoskeleton. For such mechanisms to be reconstituted within synthetic cells, it is vital that the synthetic cell possesses a dynamic cytoskeleton that can interface with other encapsulated signalling machinery. Cytoskeletons have been reconstituted into synthetic cells using a variety of molecular structures, including nucleic acids [85–87], hybrid peptide nucleic acids [88], organic molecules [89] and the reconstitution of actin and other biological cytoskeleton components [90–94]. However, the design of synthetic cells that can respond to external forces through changes in cytoskeleton or downstream cell properties is still unrealised. This will require the integration of mechanosensitive components that can activate in response to an external force capable of modulating the state of the cytoskeleton, as well as downstream pathways that respond to changes in cytoskeleton structure. Such pathways would facilitate the design of synthetic cells with dynamic mechanical robustness and enable study of the FFF principle outside the biological cell.
Additionally, just as the cytoskeleton acts to support the integrity of biological membranes under different forces, synthetic cytoskeletons have been shown to increase the mechanical strength of synthetic cell membranes and protect them against external forces such as osmotic pressure [89,95]. Integrating mechanosensitive ion channels alongside the cytoskeleton in synthetic cells would act as a start point for studying more nuanced biological mechanotransduction mechanisms where multiple cell components act together to sense and transduce mechanical forces into downstream changes in cell behaviour. This represents an example of the challenges in integrating different molecular ‘parts’ within the synthetic cell. Addressing this challenge will be essential for the engineering of more advanced cell-like systems and will require the creation of multidisciplinary research teams and community building activities such as the Build-a-Cell project [96]. In the context of mechanosensitive pathways, the development of new molecular and systems level modelling approaches [97] will accelerate the integration of various mechanosensors alongside other sensing and information processing systems within new synthetic cell models.
Cell-free protein expression has been established as a key tool to study the mechanisms of biological transcription and translation as well as act as a method to rapidly prototype protein parts and networks outside their cellular context. Such tools have already been used to express MscL in synthetic cells (as discussed above) and have furthermore been used to investigate the role of membrane mechanics on protein integration [98]. Cell-free expression systems could be used to prototype entire biological mechanotransduction signalling pathways, exploiting the development of sequential gene expression [99] as well as transcriptional and translational regulatory systems that allow the user to control the onset of gene expression, such as riboswitches which have been utilised in multiple synthetic cell designs [100–102].
Combining cell-free protein expression alongside advances in compartmentalisation (enabling localised protein expression) will facilitate the rapid prototyping of synthetic mechanosensitive signalling pathways of increased complexity. Such synthetic cells will act as suitable model systems that can recapitulate key aspects of biological signal transduction, including (a) acting as a framework to study molecular interactions across length scales, from nanoscale protein activation to diffusion-mediated processes and (b) coupling processes that occur across timescales, from the millisecond activation of mechanosensitive ion channels [18] to the minutes to hours timespan necessary for downstream changes in gene expression.
Whilst most multicompartment systems have focused on the production of multiple membrane-containing compartments, recent work has looked to combine membrane-bound compartments with chemical systems capable of undergoing aqueous phase separation [103]. This includes the design of pH-triggered coacervation inside vesicle-based synthetic cells to concentrate enzymes and substrates within the vesicle and modify reaction rates [104], whilst more recently, protein-based coacervates were produced inside synthetic cells to partition mRNA (reducing the rates of expression for subsequent proteins) [105]. Varied compartmentalisation approaches could be integrated alongside mechanosensitive components to further tune the function and dynamics of mechanotransduction in synthetic cells.
Finally, synthetic mechanosensitive signalling pathways could be used to create synthetic cells suited for biomedical application. As recently reviewed by Hsu et al. [106], mechanosensitive synthetic cells could be used in a variety of applications, including activation of the synthetic cell in the bloodstream in areas of high shear stress which could be used for the localised release of drugs at obstructed blood vessels. Mechanosensitive synthetic cells could also be used in bioengineering, for example to help regulate the growth and activity of co-cultured biological cells under in the presence of external forces. The recent development of mechanoresponsive hydrogels by the Liu laboratory could be utilised in such an application, where hybrid polymer-lipid membranes respond to external force through molecular release into the hydrogel [107].
Conclusions
The recent advances in constructing de novo mechanosensitive signalling pathways have been enabled by significant developments in our understanding and characterisation of MSCs, biomembrane engineering, and cell-free synthetic biology. By combining these, pathways have already been constructed to control gene synthesis programmes as well as transduce signals across multiple compartments in a synthetic cell. Such systems could form the foundation of model systems of biological signal transduction, as well as environment-sensing modules that enable synthetic cells to transduce local forces and survive and function in different environments.
Current challenges involve the development of dynamic cytoskeletons within the synthetic cell that can help to mediate force transduction, as well as the integration of mechanosensitive elements alongside other molecular ‘parts’. Succeeding in these efforts will enable the generation of new mechanotransduction pathways that better mimic eukaryotic mechanotransduction, with spatiotemporal control of designed pathways benefitting from the incorporation of genetic and non-genetic regulation of gene expression alongside different types of sub-compartmentalisation. This will lead to new synthetic cell technologies capable of executing multiple functions, exhibiting feedback, and operating in increasingly complex biological environments, be it as biological cell models or applied biotechnologies.
Perspectives
The importance of the field: Mechanotransduction is a critical biological function that underpins cellular survival mechanisms, developmental processes and sensing in multicellular organisms such as touch and hearing. Recapitulating mechanotransduction in synthetic cells will facilitate an improved understanding of mechanobiology as well as generate new technologies for application in medicine and bioengineering.
Summary of the current thinking: Synthetic mechanosensitive signalling pathways have been constructed through combining advances in biomembrane engineering, the cell-free expression of proteins, and the characterisation of mechanosensitive channel proteins. These engineered pathways function in vesicle-based synthetic cells, enabling the mechanotransduction of external forces into downstream changes in synthetic cell function such as induction of protein expression or transport between compartments of the synthetic cell.
Future directions: The further development of synthetic mechanotransduction pathways could incorporate diverse mechanosensitive channel proteins that can be used for tuneable mechanosensation and transport of different substrates. To study many existing mechanisms of biological mechanotransduction, it will be essential to integrate dynamic cytoskeleton components that can act as structural components as well as transducers of mechanical force. Incorporating channel proteins and cytoskeletal elements alongside increasingly complex gene circuits (and regulatory systems) into diverse multicompartment cell models will enable the design of new mechanotransduction systems that will engender synthetic cells with advanced sense-and-response functionality.
Competing Interests
The author declares that there are no competing interests associated with this manuscript.
Open Access
Open access for this article was enabled by the participation of Imperial College London in an all-inclusive Read & Publish agreement with Portland Press and the Biochemical Society under a transformative agreement with JISC.
Author Contributions
J.W.H. conceptualised and created this work.
Acknowledgements
This work was supported by an EPSRC-funded New Horizons 2021 (EP/X018903/1) grant awarded to J.W.H.