A Survey of Biological Building Blocks for Synthetic Molecular Communication Systems
Christian A. Söldner, Eileen Socher, Vahid Jamali, Wayan Wicke, Arman Ahmadzadeh, Hans-Georg Breitinger, Andreas Burkovski, Kathrin Castiglione, Robert Schober, Heinrich Sticht
IIEEE COMMUNICATIONS SURVEYS & TUTORIALS 1
A Survey of Biological Building Blocks forSynthetic Molecular Communication Systems
Christian A. S¨oldner, Eileen Socher, Vahid Jamali, Wayan Wicke,Arman Ahmadzadeh, Hans-Georg Breitinger, Andreas Burkovski,Kathrin Castiglione, Robert Schober, and Heinrich Sticht
Abstract —Synthetic molecular communication (MC) is a newcommunication engineering paradigm which is expected to enablerevolutionary applications such as smart drug delivery andreal-time health monitoring. The design and implementationof synthetic MC systems (MCSs) at nano- and microscale isvery challenging. This is particularly true for synthetic MCSsemploying biological components as transmitters and receiversor as interfaces with natural biological MCSs. Nevertheless,since such biological components have been optimized by natureover billions of years, using them in synthetic MCSs is highlypromising. This paper provides a survey of biological componentsthat can potentially serve as the main building blocks, i.e.,transmitter, receiver, and signaling particles, for the designand implementation of synthetic MCSs. Nature uses a largevariety of signaling particles of different sizes and with vastlydifferent properties for communication among biological entities.Here, we focus on three important classes of signaling particles:cations (specifically protons and calcium ions), neurotransmit-ters (specifically acetylcholine, dopamine, and serotonin), andphosphopeptides. These three classes have unique and distinctfeatures such as their large diffusion coefficients, their specificity,and/or their uniqueness of signaling that make them suitablecandidates for signaling particles in synthetic MCSs. For eachof these candidate signaling particles, we present several specifictransmitter and receiver structures mainly built upon proteinsthat are capable of performing the distinct physiological func-tionalities required from the transmitters and receivers of MCSs.Moreover, we present options for both microscale implementationof MCSs as well as the micro-to-macroscale interfaces needed for
This work was supported in part by the German Research Foundation underProjects SCHO 831/7-1 and SCHO 831/9-1, in part by the Friedrich-AlexanderUniversity Erlangen-N¨urnberg under the Emerging Fields Initiative, and inpart by the STAEDTLER Foundation.Christian A. S¨oldner and Heinrich Sticht are with the Division ofBioinformatics, Institute of Biochemistry, Friedrich-Alexander-Universit¨atErlangen-N¨urnberg (FAU), Fahrstr. 17, 91054 Erlangen, Germany. (email: { christian.soeldner, heinrich.sticht } @fau.de)Eileen Socher is with the Institute of Biochemistry and the Institute ofAnatomy, Friedrich-Alexander-Universit¨at Erlangen-N¨urnberg (FAU), 91054Erlangen, Germany. (email: [email protected])Vahid Jamali, Wayan Wicke, Arman Ahmadzadeh, and Robert Schoberare with the Institute for Digital Communications, Department of Elec-trical, Electronics, and Communication Engineering (EEI), Friedrich-Alexander-Universit¨at Erlangen-N¨urnberg (FAU), Cauerstr. 7, 91058 Er-langen, Germany. (email: { vahid.jamali, wayan.wicke, arman.ahmadzadeh,robert.schober } @fau.de)Hans-Georg Breitinger is with the Department of Biochemistry, Faculty ofPharmacy and Biotechnology, German University in Cairo (GUC), New Cairo11835, Egypt. (email: [email protected])Andreas Burkovski is with the Division of Microbiology, Department ofBiology, Friedrich-Alexander-Universit¨at Erlangen-N¨urnberg (FAU), Staudtstr.5, 91058 Erlangen, Germany. (email: [email protected])Kathrin Castiglione is with the Institute of Bioprocess Engineering, De-partment of Chemical and Bioengineering, Friedrich-Alexander-Universit¨atErlangen-N¨urnberg (FAU), Paul-Gordanstr. 3, 91052 Erlangen, Germany.(email: [email protected]) experimental evaluation of MCSs. One of the main advantages ofemploying proteins for signal emission and detection is that theycan be modified with tools from synthetic biology and be tailoredto a wide range of application needs. We discuss the properties,limitations, and applications of the proposed biological buildingblocks for synthetic MCSs in detail. Furthermore, we outline newresearch directions for the implementation and the theoreticaldesign and analysis of the proposed transmitter and receiverarchitectures. Index Terms —Molecular communications, transmitter andreceiver architecture, signaling particles, synthetic biology, andtest-bed implementation.
I. I
NTRODUCTION
The development of nanomachines for medical applicationssuch as real-time health monitoring and targeted drug deliveryis a focus area of current nanotechnology research [1]–[3].In order to realize the full potential of such applications,it is necessary that the nanomachines be able to efficientlycommunicate with each other [4]–[7]. In particular, it isenvisioned that a network of communicating nanomachinescan help realize the concept of the Internet of Bio-NanoThingswhich is expected to enable nanomachines to perform complextasks [8], [9]. For instance, a group of nanomachines maydetect a metabolic condition and communicate this obser-vation to another nanomachine which is then responsiblefor triggering the release of a drug into the body. Sinceconventional communication techniques are not well suited forcommunication at nano- and microscale, especially in liquidmedia, molecular communication (MC), where molecules areused as information carriers, has been proposed as a promisingbio-inspired mechanism for enabling communication amongnanomachines [4], [5].The general structure of a (synthetic) MC system (MCS)is depicted in Fig. 1. In response to a certain input signal,which may be artificial (e.g. a light impulse or an electricalstimulation) or biological (e.g. a nerve signal), the transmitterreleases a pattern of signaling particles , which represents theinformation to be conveyed. Depending on how sophisticatedthe transmitter is, it may also apply advanced encoding andmodulation techniques for efficient representation of the databefore releasing the corresponding signaling particles intothe channel. The signaling particles propagate through thechannel, e.g. via free diffusion where the propagation may be Throughout this paper we use the terms molecules and particles interchange-ably, although the latter term is broader as not all particles are molecules. a r X i v : . [ c s . ET ] J u l EEE COMMUNICATIONS SURVEYS & TUTORIALS 2
Control/Computing UnitControl/Computing Unit
ReleaseMechanismReceptionMechanism SignalingParticlesEncoding,Modulation, ...Detection,Decoding, ...
ReceiverTransmitter
Information Source
Natural/SyntheticStimulus, ...
Information Sink
New Action,Readout, ...
Focus of This Survey
Physical Channel
Fig. 1. General block diagram of an MCS. This paper surveys suitable biological building blocks for implementation of the release and reception mechanismsfor several classes of signaling particles. The color code used to represent transmitter, channel, and receiver will be applied throughout the paper. further accelerated by advection [10]. The receiver observes thesignaling particles and recovers the data by applying suitabledemodulation and decoding techniques. Thereby, the data mayeither be read out using an artificial mechanism (e.g. via alight emission or an electrical current) or trigger a biologicalprocess (e.g. a nerve signal).
A. Motivation and Scope
Although synthetic MC has received considerable interestfrom the research community over the past decade, the researcharea is still in its infancy. In particular, the design, analysis,and implementation of microscale biological MCSs requireinherently a multidisciplinary approach with contributionsfrom different engineering disciplines, including electrical,biological, and chemical engineering, and different branches ofscience, including biology, chemistry, physics, and medicine.Particularly, the field of synthetic biology is expected to play acrucial role in the fabrication and implementation of the maincomponents of future synthetic MCSs, i.e., the transmitter,receiver, and signaling particles.In this paper, we review biological components suitablefor implementation of MCSs. In order to define the scopeof this survey paper and to facilitate the classification of thedifferent research directions in the field of synthetic MC, wepresent a roadmap for the development of synthetic biologicalMCSs from the basic biological building blocks to commercialapplications, see Fig. 2. • Stage 1 – Enabling Basic Biological Hardware:
Thefundamental feature of MCSs is that signaling particlesare employed as information carriers [11]. Therefore, inits most basic form, an MCS consists of a transmitterthat is able to release signaling particles into the channeland a receiver which is able to detect the presence of thesignaling particles. The primary focus of this survey isthe compilation of various biological options for realizingthe release mechanism at the transmitter and the receptionmechanism at the receiver for several different types ofsignaling particles. • Stage 2 – Communication-Theoretical Modeling andDesign:
The next step needed for the design of an MCS is the development of communication-theoretical modelsfor the release, propagation, and reception of the signalingparticles that account for the features and constraints ofthe adopted biological building blocks [12]–[17]. Basedon these models, the basic functionalities of MCSs such aschannel coding [18], [19], modulation [20], [21], detection[21]–[23], decoding [19], [24], synchronization [25], [26],and estimation [27], [28] can be developed and theirperformance can be analyzed. • Stage 3 – Control and Computing Modules:
Theimplementation of the communication-theoretical conceptsdeveloped in Stage 2 depends on where the correspond-ing operations are to be performed. For instance, forhealth monitoring applications where the observationscan be collected and accessed from outside the MCenvironment, a personal computer may be responsiblefor part of the processing. For other applications, such astargeted drug delivery, sophisticated nano-transmitters andnano-receivers may have to process the data themselves.Various options have been proposed for realizing con-trol/computing units at nano- and microscale for biologicaltransmitters/receivers including molecular circuits (i.e.,cascaded networks of chemical reactions) [24], [29], [30]and genetic circuits [31]–[33]. • Stage 4 – Experimental Verification:
The concepts anddesigns developed in Stages 1-3 have to be verified vialaboratory experiments [34]–[36]. Stage 4 is challengingdue to the fact that controlling an MCS at microscaleis difficult. Therefore, in addition to the development ofmicroscale MCSs, it is advantageous to also develop micro-to-macroscale interfaces that enable their observationand test. The role of this stage in the development ofMCSs is analogous to employing spectrum analyzers,channel sounders, and other measurement devices to testand analyze the components of wireless communicationsystems [37]. Therefore, this paper does not only surveyoptions for the implementation of MCSs but also theinterfaces required for their experimental evaluation. • Stage 5 – Prototyping for Commercial Applications:
Depending on the application, suitable building blocks
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Stage 1
Basic Biological Hardware
ReleaseMechanism SignalingParticles ReceptionMechanism
Stage 2
Communication-Theoretical Modeling and Design
TransmitterDesign ChannelModeling ReceiverDesign
Stage 3
Control and Computing Modules
MolecularCircuits GeneticCircuits
Stage 5
Prototyping for Commercial Applications
Fig. 2. This figure illustrates a roadmap for the development of syntheticbiological MCSs towards commercial applications. The focus of this surveyis on Stage 1, where we present different biological options for realizingthe release mechanism at the transmitter and the reception mechanism atthe receiver for several different types of signaling particles. The biologicalbuilding blocks proposed in this paper may help theoreticians to develop modelsand communication-theoretical system designs that account for the relevantbiological constraints and features imposed by the proposed building blocks (inStage 2). Moreover, we propose architectures suitable for implementation ofboth microscale MCSs as well as the micro-to-macroscale interfaces requiredfor experimental evaluation of synthetic MCSs (in Stage 4). from Stages 1-3 (that have also been experimentallyverified in Stage 4) are chosen to develop first-orderprototypes and ensure that these blocks successfully worktogether. At this level, there are numerous applications forMCSs including smart drug delivery, health monitoring,and even the realization of the Internet of Bio-NanoThings[8], [9], [38], [39].So far, the main focus of the MC literature has been onStages 2 and 3 assuming often quite abstract and simple modelsfor the underlying biological building blocks in Stage 1. Inaddition, as a proof of concept, MCSs have been demonstratedat macroscale [12], [40]–[44] and at microscale [34]–[36],[45]–[49] (i.e., Stage 4). The latter systems employ biologicalcomponents as transmitter and/or receiver but were eitherdemonstrated only for single pulse transmission or offer verylow data rates on the order of one symbol per hour. However,for nano- and microscale MC to become practical, continuoustransmission at much higher data rates is needed. Both thedesign and the implementation of such systems require a soundunderstanding of the biological building blocks that can be usedto construct them. This paper surveys candidates for signaling molecules, release mechanisms, and reception mechanismsneeded in Stage 1 as well as the micro-to-macroscale interfacesneeded for their experimental evaluation in Stage 4.Several survey and tutorial papers focusing on differentaspects of MC have been published over the past few years[5]–[7], [10], [50]–[67], see Table I for a brief summary ofthese survey and tutorial papers. In particular, the authors of[5]–[7], [56] provide general overviews of the field of MC, itsfuture applications, and related challenges. In [7], [50], [51],[55], networking aspects of MCSs are discussed and potentialnetwork layer architectures for MCSs are proposed. A generalsurvey on synthetic MCSs, including aspects such as particletransport, communication engineering aspects, testbeds, andapplications, is presented in [52]. The theoretical aspects ofMC are surveyed in detail from the perspectives of informationtheory in [58], [59], physical-layer channel modeling in [10],and transmitter and receiver design in [60]. The design ofgenetic circuits is surveyed in [62] and the mutual impactof connected biological components is discussed in [63].Potential medical applications of synthetic MCSs are surveyedin [53], [67]. Drug delivery applications are discussed in[55] and applications of mobile MCSs are presented in [61].In addition, the authors of [64] survey research works witha particular focus on MCs in the synaptic cleft and [57]surveys tools from bioinformatics for the analysis of protein-protein interactions. Finally, the authors of [65] proposeelectrochemical methods to interface with biological MCSswhereas [66] studies optogenomic interfaces for controllinggenes and their interactions in the cell nucleus. These surveypapers are either general overviews (e.g., [5]–[7], [52], [56]) orfocus mainly on Stage 2 (e.g., [10], [58]–[61]) and Stage 3 (e.g.,[62], [63]) of the development roadmap illustrated in Fig. 2,and although most of them also consider nanomachines ascomponents of MCSs, the corresponding survey of the relatedliterature is very brief. A comprehensive survey of potentialbiological building blocks of MCSs (in Stage 1) and theirmicro-to-macroscale interfaces (in Stage 4) is not available inthe literature, yet.While biological building blocks of MCSs have receivedlittle attention in the MC literature, in the field of syntheticbiology, there is a vast body of literature on biological systemsthat can potentially be used as components of MCSs. However,for researchers not well versed in synthetic biology, it canbe challenging to find the relevant literature and to relateit to MCS design. Therefore, in this paper, we provide acomprehensive survey of biological building blocks that canpotentially be engineered to serve as components of nano- andmicroscale synthetic MCSs operating in aqueous environments.Since, unlike what is often assumed in the MC literature,biological systems are very specific, the signaling particles,transmitters, and receivers have to be carefully matched toeach other. In particular, the design of the transmitter andreceiver in MCSs crucially depends on the adopted signalingparticles. Hence, in this survey, we adopt a signaling particlecentric approach and first present several candidate signalingparticles for synthetic MCSs. Then, for each of the consideredsignaling particles, we provide several candidate transmitterand receiver structures. We believe that this survey is useful
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TABLE IS
UMMARY OF SURVEY AND TUTORIAL PAPERS ON
MCS S . M OST OF THE PAPERS LISTED IN THIS TABLE PROVIDE AN OVERVIEW OF
MCS
S AND CONTAINCONTENT RELATED TO ALL DEVELOPMENT STAGES SHOWN IN F IG . 2. F OR CLARITY , THE MAIN FOCUS OF EACH SURVEY / TUTORIAL PAPER IS REPORTED INTHE FOURTH COLUMN OF THIS TABLE . Reference Year Type Main Focus More Detailed Notes
Akyildiz et al. [5] 2008 Survey Overview Nanonetworks, architectural aspects, and expected featuresDressler et al. [6] 2010 Survey Overview Bio-inspired networking approachesNakano et al. [7] 2012 Survey Stage 2 Physical and network layers of MCSsDarchini et al. [50] 2013 Survey Stage 2 MCSs via microtubules and physical contactNakano et al. [51] 2014 Survey Stage 2 Layered architecture of MCSsFarsad et al. [52] 2016 Survey Overview/Stage 2 Underlying physical principles of MCSs, communication engineering aspects, andsimulation toolsFelicetti et al. [53] 2016 Survey Overview Medical applications of MCSs, diagnostic and treatment applications, andimplementation interfacesChahibi et al. [54] 2017 Survey Overview Targeted drug delivery, component and modeling approachesOkonkwo et al. [55] 2017 Survey Stages 1, 2,and 4 Targeted drug delivery, application concepts, propagation channel modeling, andsystem designWang et al. [56] 2017 Survey Overview Diffusive MCSs, communication theoretical designs, and cooperative relay-basednetworksJamali et al. [10] 2019 Tutorial Stage 2 Channel modeling of diffusive MCSs, physical principles, communication theoretical,simulation-based, and data-driven models, and model derivation methodologiesAkyildiz et al. [58] 2019 Tutorial Stage 2 MC theory and models for functional blocks of MCSs based on chemical kinetics andstatistical mechanicsRose et al. [59] 2019 Tutorial Stage 2 Capacity of point-to-point MCSsKuscu et al. [60] 2019 Survey Stage 2 Transmitter and receiver architectures of MCSs, modulation, coding, and detectiontechniquesNakano et al. [61] 2019 Survey Stage 2 Mobile MCSs, modeling approaches, and networkingNguyen et al. [62] 2019 Tutorial Stage 3 Asynchronous genetic circuitsMcBride et al. [63] 2019 Tutorial Stage 3 Synthetic biomolecular circuitsVeleti´c et al. [64] 2019 Survey Stage 2 Synaptic communication engineering and brain–machine interfaceKim et al. [65] 2019 Survey Overview Reduction/oxidation (redox) reactionsJornet et al. [66] 2019 Survey Overview Optogenomic interfacesQadri et al. [67] 2020 Survey Stage 2 Internet of Nano-Things for healthcare applicationsS¨oldner et al. (this paper) – Survey Stages 1 and 4 Potential biological building blocks of MCSs, microscale implementations, andmacroscale interfaces to both theoreticians and experimentalists. For researchersworking on the theoretical aspects of MCS design, taking intoaccount the specific properties of the underlying biologicalbuilding blocks, which are reviewed here and described indetail in the provided references from synthetic biology, willallow them to develop more realistic communication-theoreticalmodels and designs of MCSs. For researchers interested indeveloping MC testbeds and experiments, the survey outlinesthe advantages and disadvantages of potential design choicesand the provided references contain the detailed informationneeded for implementation. In the following subsections, wefirst explain some basic biological concepts and components.Then, we provide a brief overview of the considered signalingparticles and matching biological components, which can beused to construct transmitters and receivers.
B. Some Important Basic Biological Concepts and Components
In the following, to assist readers that do not have abackground in biology, we explain some essential basicbiological concepts and components used throughout this article.A summary of additional biological concepts and terminologyappearing in the text is provided in Appendix A. • Vesicles:
A vesicle is a small, round or oval-shapedcontainer whose wall consists of a lipid bilayer membrane which encloses a liquid substance [68]. Being muchsmaller than a cell, natural vesicles are formed by defor-mation and subsequent budding from the cell membraneor the membrane of cellular organelles such as the Golgiapparatus or the endoplasmic reticulum. They are usedfor transport purposes, e.g. in the context of secretion, forthe storage of certain biomolecules, and as compartmentswith particular reaction conditions. Different proteins maybe embedded in their lipid membrane which may facilitatefor instance transmembrane transport. Moreover, vesicleswith transmembrane proteins can be created artificiallyusing biochemistry and molecular biology tools. Suchartificially generated lipid vesicles are called liposomes. • Ion channels:
An ion channel is a special type of trans-membrane protein with a pore that becomes permeable forspecific types of ions under certain circumstances [69]. Ionchannels only allow passive transport which means thatthey merely facilitate diffusion along an already existingconcentration gradient. Ion channels may be subdividedinto different groups based on the conditions that lead tochannel opening (“gating”). For instance, there are voltage-gated (a certain transmembrane potential is required),ligand-gated (a certain molecule has to bind from theoutside), and mechanosensitive (stretching of/pressure on
EEE COMMUNICATIONS SURVEYS & TUTORIALS 5 the membrane is required) ion channels [69]. • Carriers:
Another kind of protein involved in the move-ment of ions or small molecules across membranes arecarriers [70]. The transported particles are also referred toas substrate in this context. So called uniporters transportonly one specific type of substrate. Upon stimulation, theyundergo a conformational change whereby the particle iscarried through the membrane to be released at the otherside. As other secondary carriers (see below) they areable to accumulate their substrate against a concentrationgradient [70]. However, there are carriers which do nottransport one type of substrate alone, but two or moredifferent types of substrates (e.g. particles A and B) simul-taneously, either both in the same direction (symporter) orin opposite directions (antiporter). This principle, whichis called secondary active transport, allows to transportsubstrate A against its concentration gradient if there isa sufficiently high concentration or charge gradient forsubstrate B that can be used as a source of energy for thetransport process. Often, the concentration gradient forsubstrate B is actively maintained by use of an ion pump[70]. • Ion pumps:
Similar to ion channels and carriers, ionpumps are transmembrane proteins which can transportions across a membrane. In contrast to ion channels, ionpumps use an external source of energy such as lightor adenosine triphosphate (ATP) to facilitate an activetransport which also works against the concentrationgradient of the respective ion [71]. This type of transportis also called primary active transport. • Voltage-clamp method:
The voltage-clamp technique is amethod where a microelectrode is placed inside a vesicleor a cell to manipulate or measure the current acrossthe vesicle membrane at a certain voltage [72]. Thereby,changes in membrane potential can be induced, e.g. inorder to open a voltage-gated ion channel. Moreover, thetwo-electrode voltage clamp technique can be used tomeasure the transmembrane current that arises when ionchannels are opened. The two-electrode voltage clamptechnique allows adjustment of the transmembrane poten-tial and recording of currents through separate electrodes[72], and is mostly used for measurements on oocytes orvery large vesicles/cells ( > • Reversibility:
In this article, we refer to transmitters as“reversible” if the signaling particles are recycled after theirrelease so that they can be used repeatedly. For example,a simple vesicle-based transmitter may eventually getexhausted over time having released all signaling particlesthat were stored inside at the beginning. In contrast, a“reversible” transmitter is able to regenerate its content, e.g.by pumping the signaling particles back inside. In addition,reversibility will require that the vesicle exhibits a highstability and remains intact during multiple regenerationcycles of the transmitter.
C. Signaling Particles
Nature uses a vast number of different molecules forinformation exchange between different entities. For concrete- ness, in this survey, we focus on three important types ofsignaling particles, namely cations, neurotransmitters (NTs),and phosphopeptides as a representative class of modifiedproteins, see Fig. 3. These classes of signaling particles areattractive for use in synthetic MCSs as they allow the designof simple transmitter and receiver structures employing only asmall number of protein components. Furthermore, as will beexplained in the following, the considered classes of signalingparticles differ substantially in their behavior and properties,such that they are collectively suitable for a wide class ofdifferent MCSs.
Cations:
Cations are small positively charged ions thathave the advantage of fast diffusion [74], [75]. In addition,protons, as a specific ion, can jump from one water moleculeto the next through the formation and concomitant cleavage ofcovalent bonds, the so-called Grotthuss mechanism [76], [77],which makes them appear to move even faster than they wouldalready be by classic diffusion due to their small size. Sinceions interact with a plethora of proteins in organisms [78]–[80], there exists a large set of possible biological structuresthat can be used as components of transmitters and receivers.Although many types of cations and anions may be appropriatefor MCSs, the present article focuses on protons due to theirunique speed of diffusion, and on calcium ions, because theyplay an important role as messengers in cellular signaling[81]. For instance, calcium ions are involved in the process ofapoptosis (programmed cell death of damaged cells) [82] aswell as in the coupling between the electrical excitation andthe consecutive contraction of muscle fibers [83]. Neurotransmitters:
As an alternative class of particles forMCSs, we consider NTs. NTs are bigger than cations andtherefore diffuse more slowly. On the other hand, they aremore specific and are therefore more likely to avoid unintendedsignal interference from and to different natural processes e.g.in the human body. The class of NTs comprises several distinctmolecules (e.g. acetylcholine, dopamine, and serotonin) that allfunction in a similar fashion in signal transduction. This hasthe general advantage that similar building blocks can be usedfor the construction of transmitters and receivers for differentNTs. In nature, NTs are used to trigger or suppress neuronalfiring (the so-called action potentials) between neurons or froma neuron to a muscle fiber [84]. Thus, NTs are particularlyinteresting because they could be used to directly interact withneurons [7], e.g. in the context of nerve lesions which needto be bridged, the control of a prosthesis, and the treatmentof neurological diseases. One possible application would betargeted drug delivery. For example, Parkinson’s disease whichis caused by a selective loss of dopamine producing neurons ina specific region of the brain, the so-called substantia nigra , isnowadays treated by a systemic oral administration of levodopa,a precursor of dopamine synthesis [85]. In this context, adirect delivery of dopamine via MC to the region where it isrequired would greatly enhance the treatment effectiveness ofParkinson’s disease.
Modified proteins:
The final class of signaling particles In a similar manner, anions (i.e., small negatively charged ions) can beused for signaling in MCSs.
EEE COMMUNICATIONS SURVEYS & TUTORIALS 6 discussed in this article are proteins that become signaling par-ticles by post-translational modification (PTM). PTM describesa covalent enzymatic modification of proteins that occurs afterprotein biosynthesis. There are different types of PTMs (e.g.phosphorylation, methylation, acetylation, and ubiquitination),which differ in their functional groups, their size, and the waythey are linked to the protein [86]. Protein phosphorylation,one of the most frequent types of PTM, is an importantcellular regulatory mechanism as many proteins (e.g. enzymes,receptors) are activated/deactivated by phosphorylation anddephosphorylation events. More than two-thirds of the 21,000proteins encoded by the human genome were shown to bephosphorylated [87] indicating that phosphorylation plays arole in almost every physiological process. Abnormal pro-tein phosphorylation may lead to severe diseases includingAlzheimer’s disease, Parkinson’s disease, and cancer. From acommunication-theoretic perspective, the protein moiety isalways present in the channel but it is only activated forsignaling if it carries a phosphate group (orange phosphorouswith red oxygen atoms in Fig. 3) that is transferred by a kinase[88], a special type of enzyme. We consider phosphorylationsbecause of their versatility due to the large number of differentsignaling particles that can be generated by variation of theprotein sequence. In addition, phosphorylated proteins may beminiaturized to generate smaller signaling particles (henceforthtermed ‘phosphopeptides’) that have the advantage of fasterdiffusion due to their smaller size compared to proteins.Fig. 3 provides an overview of the properties of the differentsignaling particles that we survey for their applicability inMCSs. From top to bottom, they are ordered according to theirmolecular weights (MWs). As mentioned before, one commonproperty of all three considered classes of signaling particlesis that their respective transmitters and receivers require onlya few biological components which makes them attractive forapplication in synthetic MCSs. In addition, they are intrinsicallyrather homogeneous groups of signaling particles, whichfacilitates the design of transmitter and receiver structuresas well as communication protocols that can be applied tomultiple members of these classes.
Remark 1:
We refrained from considering hormones assignaling particles in this survey, because they are verydivergent as far as their size (molecular weights from about150 g/mol up to insulin which is a protein with 51 aminoacids and a molecular weight of 5800 g/mol), the involvedreceptors (transmembrane, intracellular, and nuclear), and alsothe effect that they have on their target cell are concerned[91]. Therefore, it is not possible to develop a general MCSarchitecture valid for the entire group of hormones. We also donot consider complex biological communication systems suchas chemotaxis or bacterial quorum sensing, which would requirethe challenging implementation of the signaling cascadesinvolved [92], [93]. Quorum sensing, for example, depends onthe conditional activation of gene transcription [93] and wouldthus only be possible if complete bacterial cells were used astransmitters/receivers. In this paper, we focus on the designof simple transmitters and receivers consisting of only a fewprotein components.
Remark 2:
In the MC literature, there are several works that have analyzed the performance and the design of MCSsfor the signaling particles introduced above. For example, theuse of calcium/proton ions as signaling particles in MCSs isconsidered in [36], [94]–[100]. Furthermore, several theoreticalworks investigate the design and analysis of neuronal MCSsemploying NTs as information particles, see e.g. [101]–[106].However, these existing theoretical works either consideredabstract models for the transmitter and receiver or focusedon transmitter and/or receiver structures of natural
MCSs. Inthis paper, unlike the existing works, we investigate differenttransmitter and/or receiver structures for each of the consideredsignaling particles with the design of synthetic MCSs in mind.Furthermore, the proposed transmitter and receiver structuresare not limited to those existing in nature.
D. Biological Components of Transmitter and Receiver
The signaling particles described in Section I-C are compat-ible with several biomolecules that can be used to constructtransmitters and receivers for MCSs. An overview of somerelevant classes of such biomolecules is given in Fig. 4. Onerelevant protein family are channels that allow ions to passthrough a membrane via a channel pore in response to anexternal stimulus (e.g. voltage, pH, ligands, or mechanicalstress) (Figs. 4a, d, e, f). In contrast to such ion channels,which only allow diffusion of ions along a concentrationgradient, pumps (Fig. 4b) and symporters (Fig. 4g) can movemolecules against a concentration gradient at the expense ofenergy consumption. G-protein coupled receptors (Fig. 4h)detect ligands outside the cell and convert the correspondingsignal to intracellular responses. These biomolecules can beintegrated into the membrane of cells or artificial vesiclesusing the tools of synthetic biology [117]–[121]. In additionto these membrane-bound components, there exist also solubleproteins with properties relevant for MCSs. For instance,kinases (Fig. 4j) can convert peptides into signaling particlesand enzymes (Fig. 4i) allow the degradation of signalingparticles.Fig. 5 presents different classifications of the biologicaltransmitter and receiver architectures discussed in this paper.As mentioned in Section I-A, experimental verification of theconcepts conceived in Stages 1-3 of the proposed roadmapfor the development of MCSs is in general difficult sincecontrolling an MCS at microscale is a challenging task.Therefore, for each class of signaling particles, we presentoptions for both microscale implementation of the MCSs andmicroscale-to-macroscale interfaces that can be used for theirexperimental evaluation (i.e., in Stage 4). For instance, light-driven ion-pumps can be inserted into the membrane of thetransmitting cell to enable the release of ions (as signalingparticles) in response to an external light stimulus which is easyto control in laboratory experiments. For practical microscaleMCSs, other stimuli may be preferred such as other molecules(originating potentially from other synthetic or natural sources)which open corresponding ligand-gated channels to release thesignaling particles.Conceptually, transmitter architectures can be also cate-gorized based on whether the particle release is induced
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Fig. 3. Overview of the considered signaling particles, their molecular weight (MW), diffusion coefficient (D), and chemical structure. Carbon atoms arecolored in black, hydrogen atoms in white, oxygen atoms in red, nitrogen atoms in blue, and phosphorous atoms in orange. Diffusion coefficients were takenfrom [74] (protons, acetylcholine), [75] (calcium ions), and [89] (dopamine, serotonin). The structure images were created using UCSF Chimera [90]. manually (e.g., via a pipette in a laboratory experiment);initiated by binding other molecules to ligand-gated channelson the transmitter surface; or triggered by an electrical, optical,or chemical stimulus. Similarly, receiver architectures can beclassified based on whether the signaling particles bind toreceptors on the receiver surface and trigger another secondarysignal inside the receiver or the reception process causesan electrically, optically, or chemically detectable signal. Inaddition to design principles for synthetic transmitters andreceivers, we also discuss natural mechanisms in the humanbody for the release and detection of the considered signalingparticles. These mechanisms can be interpreted as naturaltransmitters and receivers or natural sources of interference.
E. Organization of the Paper
In the following, we outline how the remainder of thepaper and the individual sections are organized. In particular,in Sections II and III, we discuss transmitter and receivermechanisms for protons and calcium ions, respectively. InSection IV, transmitter and receiver structures for NT signaling particles are presented. Section V introduces transmitters thatuse phosphorylation as a mechanism for controlling the releaseof peptide particles as well as different receivers that can detectphosphorylated peptides. Several extensions of the proposedtransmitter/receiver structures to more complex architectures,capable of performing more elaborate processing tasks, are alsoprovided. In Sections II-V, the respective different synthetictransmitter and/or receiver architectures are presented inorder of increasing sophistication and complexity required forintegrating the necessary components into the transmitter andreceiver. Comparatively simple building blocks, which requireexternal devices or the use of detergents, are rather intended for in vitro purposes such as testbeds or benchmarking experiments.They could be used for an initial proof of concept whereasmore complex cell- or vesicle-based systems may be bettersuited for in vivo applications. We note that there is no one-to-one mapping between the orders in which the transmittersand the receivers are presented. In other words, depending onthe specific application, the proposed transmitter and receiverstructures can be flexibly combined. Moreover, in Section VI,
EEE COMMUNICATIONS SURVEYS & TUTORIALS 8 a ) Voltage-gated channel: Ca v [107]). ( b ) Light-driven pump: Bacteriorhodopsin (PDB code [108]). ( c ) Fluorescent protein: Green fluorescent protein (GFP; PDBcode [109]). ( d ) pH-dependent channel: Acid-sensing ion channel (ASIC; PDB code [110]). ( e ) Ligand-gated ion channel: Serotonin receptorsubtype 5-HT3A (PDB code [111]). ( f ) Mechanosensitive ion channel: Transient receptor potential vanilloid 4 (TRPV4; PDB code [112]). ( g ) NTsymporter: Dopamine transporter (DAT; PDB code [113]). ( h ) G-protein coupled receptor (GPCR): M3 muscarinic receptor (PDB code [114]). ( i )NT degrading enzyme: Acetylcholinesterase (PDB code [115]). ( j ) Kinase: Mitogen-activated protein (MAP) kinase 1 (MEK1; PDB code [116]).Structure images were created using UCSF Chimera [90]. EEE COMMUNICATIONS SURVEYS & TUTORIALS 9 P r opo s ed B i o l og i c a l M C B u il d i ng B l o cks S i gna li ng P a r t i c l e s R e c ep t i on M e c han i s m R e l ea s e M e c han i s m I on s N eu r o t r an s m i tt e r P r o t e i n M od i fic a t i on s P r o t on s C a l c i u m I on s A c e t y l c ho li ne D opa m i ne S e r o t on i n P ho s pho - pep t i de s T y pe L i gand - ga t ed C he m i c a l E l e c t r i c a l N a t u r a l M anua l O p t i c a l S c a l e M i c r o sc a l e I m p l e m en t a t i on M a c r o sc a l e I n t e r f a c e T y pe L i gand - ga t ed C he m i c a l E l e c t r i c a l N a t u r a l O p t i c a l S c a l e M i c r o sc a l e I m p l e m en t a t i on M a c r o sc a l e I n t e r f a c e E x a m p l e T , F i g . F i g . / T ab l e I V T , F i g . T , T ab l e II T , F i g . T / T , F i g . T / T , T ab l e II T , F i g . T , F i g . T , F i g . T / T , F i g . T / T , T ab l e II T / T / T , F i g . T , F i g . T , T ab l e II T , F i g . E x a m p l e R , F i g . R / R , F i g . R / R / R , F i g . R / R , T ab l e II R , F i g . R , F i g . R , F i g . R / R , F i g . R / R , T ab l e II R , F i g . , F i g . Fig. 5. Classification of the biological building blocks for the MCSs presented in this paper.
EEE COMMUNICATIONS SURVEYS & TUTORIALS 10 we compare the different signaling particles studied in thispaper, discuss potential medical applications of the consideredMCSs, and present several practical considerations for theimplementation of these MCSs including relevant biological mechanisms for inter-symbol interference (ISI) mitigation andbottlenecks for the achievable data rates. Furthermore, inSection VII, we outline possible future research directionsincluding the new research problems that should be tackledfor the modeling and design of MCSs based on the proposedbiological building blocks as well as challenges that need tobe addressed for the implementations of such MCSs. Finally,the conclusions of the paper are drawn in Section VIII.II. P
ROTONS AS S IGNALING P ARTICLES
Protons (represented by the symbol H + ) are the first type ofsignaling particles we consider for MCSs. The correspondingoptions for transmitter systems are depicted in Fig. 6, those forreceiver systems are presented in Fig. 7. They are describedin detail in the following. A. Transmitters
We consider six different transmitter structures (Fig. 6,T1-T6). While the first transmitter (T1) is quite simple (forlaboratory experiment purposes), transmitters T2-T5 are morecomplex and consist of a vesicle containing an acidic solutionand employ different mechanisms for the controlled releaseand/or reuptake of protons. Finally, we also briefly considerproton emitters (T6) which may naturally occur in the humanbody.
T1 (Pipette):
The simplest transmitter for protons is a pipetteby which an acidic solution is released dropwise into thechannel (Fig. 6, T1). This transmitter may be suitable forcontrolling the release of protons at macroscale.
T2 (Degenerating vesicle):
As second and more complextransmitter system for protons, we propose the usage ofvesicles that contain an acidic solution (Fig. 6, T2). In thesimplest case, the release of the vesicle content could betriggered by adding a detergent destroying the membraneor by means of electroporation [122] where the membranepermeability is increased due to an externally applied electricfield. Both approaches are very effective; however, they alsodestroy the entire vesicle and thus release the entire contentat once. In order to transport only partial quantities of thesignaling particles from the interior of the vesicle to theoutside, different transporter proteins can be incorporated intothe vesicle membrane as will be explained in the following.
T3 (Ion channels):
For the third transmitter model, we proposeto use ion channels (e.g. voltage-gated proton channels) for acontrolled release of the signaling particles from the vesicle(Fig. 6, T3). Voltage-gated ion channels are transmembraneproteins (Fig. 4a), which undergo conformational rearrange-ments due to changes in the electrical membrane potentialnear the channel. Such changes of membrane potential canbe induced artificially, e.g. by the voltage-clamp technique[72]. The channel opens through these conformational changesand ions can leave the vesicle. Voltage-gated proton channelsexhibit a high selectivity allowing only protons to leave the vesicle [123], which makes them perfect candidates for outwardtransportation.
T4 (Ion pumps):
Alternatively, for the fourth transmittermodel, light-driven or ATP-driven pumps are exploited forthe proton transmitter system (Fig. 6, T4). While voltage-gated ion channels conduct cations or anions in a passivemanner, ion pumps need an external source of energy andfunction as active transporters, which allows them to build upa proton concentration gradient [124]. As light-driven outwardproton pump, for instance, bacteriorhodopsin [125] may beused (Fig. 4b), which can be embedded in the membrane ofproton containing vesicles. In particular, there are several knownvariants of bacteriorhodopsin which differ in the wavelengthrequired for activation [126]. Recently, an experimental testbedemploying bacteriorhodopsin has been reported in [36]. As anATP-driven pump, V-ATPases or P-ATPases [127] could beused and activated by addition of ATP in the vicinity of thevesicle.
T5 (Inward ion pumps):
The previous two proposed trans-mitter mechanisms (Fig. 6, T3 and T4) enable the controlledrelease of signaling particle; however, they lack reversibility,i.e., the recycling of the signaling particles by the transmitter.By recycling the signaling particles, the transmitter can harvestsome of the previously released protons for future releases.Moreover, when transmitter and receiver are placed close toeach other, as e.g. in the synaptic cleft, recycling the signalingparticles aids in clearing the channel for future releases, andthereby, reducing ISI. Reversibility can be achieved by addinga second biomolecule that transports the signaling particle backinto the vesicle after signal detection. As inward transporter,light-driven inward proton pumps can be additionally integratedinto the vesicle [124]. These inward pumps are able to transportprotons from the surrounding solution into the interior of thevesicle (Fig. 6, T5). In case that light-driven outward protonpumps are used for signaling particle release (Fig. 6, T4), itis important to choose outward and inward proton pumps thatare activated by different wavelengths in order to avoid thesimultaneous operation of both proton pumps.
Remark 3:
In Fig. 6, light-driven pumps are labelled by hν because the energy of the photon used as a driving force maybe calculated as E = hν , where h denotes the Planck constantand ν is the frequency of the light [128]. In T5, the inwardlight driven pump is referred to as hν (cid:48) , indicating the fact thata different activation frequency/wavelength is needed comparedto the outward pump. T6 (Natural transmitters):
Besides these designed syn-thetic transmitter systems (T1-T5), there are also some pro-cesses in the human body that may be interpreted as protonemission. For example, some tissue has a higher protonconcentration (lower pH value) than other tissue (Fig. 6, T6).Lower pH values can be observed e.g. in inflamed tissue butalso the extracellular pH of tumors can be heterogeneous andacidic [129], which may be used for detection of cancer cells.
B. Receivers
Protons as signaling particles lead to an increase of theproton concentration at the receiver or equivalently a reduction
EEE COMMUNICATIONS SURVEYS & TUTORIALS 11
T2:
Vesicle with acid, destruction of vesicle (detergents, electroporation)
T1:
Pipette with acid (protons )
T3:
Vesicle with acid and voltage-gated proton channel h ν T4:
Vesicle with acid and light-driven proton pump (e.g. bacteriorhodopsin) h ν h ν ' T5:
Vesicle with acid and light-driven proton pump; reuptake of protons with an inward light-driven proton pump
T6:
Tissue with decreased extracellular pH (e.g. tumor tissue, in fl ammation) light-driven pumpght-drivpump tumortissuevoltage-gated hannel light-driven pump Fig. 6. Transmitters for protons as signaling particles. The suggested building blocks for different transmitter systems are presented in order of increasingcomplexity and include designed artificial systems and physiological emitters, which may naturally occur in the human body. of the solution pH. We discuss five possible synthetic receiverarchitectures (R1-R5) for protons and two natural processes inthe human body (R6, R7) that are triggered by changes in theproton concentration.
R1 and R2 (pH sensors):
The simplest approaches to detecta change in the proton concentration of a solution are pH-measuring instruments (Fig. 7, R1) and pH-sensitive dyes (pHindicators) (Fig. 7, R2) as they are routinely used in chemicaland biological laboratories. The changes of dye colors can forexample be detected by photometry in a second step.
R3 (Fluorescence proteins):
In biological experiments, pH-sensitive fluorescence proteins are also often used as pHindicators and they are good candidates for use in nanoscaleMCSs. One example for such a fluorescent protein is the greenfluorescent protein (GFP) (Fig. 7, R3) with its characteristic β -barrel structure and the fluorescent chromophore (fluorophore)in the center (Fig. 4c). The fluorescence is dependent on theprotonation of the fluorophore and in the last years many different variants were developed, which possess an alteredpH-sensitive excitation spectrum. For some of these variants, aresponse time of less than 20 ms could be demonstrated [130].Besides GFP variants with disappearing fluorescence throughdecreasing pH values, also GFP variants which emit light ofdifferent color at different pH values were reported [131]. Forexample, for excitation at 388 nm, the GFP from the sea cactus Cavernularia obesa has a blue fluorescence at pH 5 and below,whereas it shows a green fluorescence at pH 7 and above[132]. In case of long-term fluorescent measurements, the light-induced bleaching of the fluorophores has to be considered.Photobleaching results in a decreasing signal that is independentfrom pH variations. Here, ratiometric pH-sensitive GFP variants,such as pHluorin2, have a big advantage since the pH is notestimated from simple changes in the fluorescence level butfrom the ratio of the fluorescence intensities at two differentexcitation wavelengths [133]. Thus, photobleaching will notaffect the correctness of the pH measurement, but only the
EEE COMMUNICATIONS SURVEYS & TUTORIALS 12
Fig. 7. Receivers for protons as signaling particles. The suggested building blocks for different receiver systems are presented in order of increasing complexityand include designed artificial systems and physiological receivers, which naturally occur in the human body. Protons are indicated as blue dots. lifetime of the receiver.
R4 (FRET):
Another possibility for a receiver system is theusage of biomolecules, which undergo large conformationalchanges upon a decrease in the surrounding pH value. Oneexample are peptides consisting of polyglutamate, whichform α -helices in acidic solution while being disordered atintermediate or high pH (Fig. 7, R4) [134]. This conformationalrearrangement of the structure can be visualized and madedetectable with a coupled FRET (F¨orster resonance energytransfer or fluorescence resonance energy transfer) experiment.The underlying mechanism is an energy transfer betweentwo light-sensitive molecules (chromophores). A donor chro- mophore, which is excited by irradiation with light of a certainwavelength, transfers energy to an acceptor chromophore. Theshorter the distance between the two chromophores, the moreenergy can be transferred from the donor to the acceptorchromophore. Hence, FRET is very sensitive to small changesin distance between the chromophores. This effect can be readout by monitoring the ratio of the respective light intensitiesemitted by the donor and the acceptor at different wavelengths.Alternatively, the intensity of the donor can be comparedin the presence and absence of the acceptor [135]. We We note that the underlying FRET mechanism as an option for communi-cation at nanoscale has been theoretically investigated in [136]–[140].
EEE COMMUNICATIONS SURVEYS & TUTORIALS 13 propose the construction of a FRET-based receiver employinga fusion protein with two FRET partners at the termini and apolyglutamate sequence in between as described in [134]. Uponhelix formation in acidic pH, the distance between the two endsdecreases. In biological experiments, a common donor-acceptorpair is the cyan fluorescent protein (CFP) combined with theyellow fluorescent protein (YFP) [131], [141] which are bothcolor variants of GFP (Fig. 4c).
R5 (Proton-gated channels):
As a further option for a pH-dependent receiver system, we propose the usage of proton-gated channels, like acid-sensing ion channels (ASICs, Fig. 4d)[142], [143] or proton-gated proton channels (e.g. the viralp7 protein) [144] embedded in a vesicle (Fig. 7, R5). Theseligand-gated ion channels are permeable for certain types ofcations after activation by high proton concentration at theirextracellular side [142], [143]. The cations can flow via thechannel pore in the vesicle membrane into the interior of thevesicle and increase the concentration of cations in the vesicle,which can be detected by measuring the transmembrane currentby methods such as the two-electrode voltage clamp method[73]. For reversibility, an additional light-driven outward pump(e.g. KR2 of the marine bacterium
Krokinobacter eikastus [145] for sodium ions or bacteriorhodopsin for protons) can beembedded into the vesicle membrane. With such light-drivenpumps, it would be possible to pump the cations from theinterior of the vesicle back into the surrounding solution.
R6 (Proton-triggered protein dissociation):
In nature, thereare numerous examples where a protein complex dissociatesin response to decreasing pH values (Fig. 7, R6). One suchexample is described for the periplasmic protein HdeA, whichforms a well-folded homodimer at neutral pH. Under acidicconditions, HdeA unfolds and exhibits an enhanced tendency todissociate into monomers [146], [147]. Another example is thehuman Hsp47 protein, which is a collagen-specific molecularchaperone and indispensable for molecular maturation ofcollagen [148]. Hsp47 transiently binds to procollagen in theendoplasmic reticulum (ER, neutral pH) and dissociates from itin a pH-dependent manner once this complex is transported to acompartment with lower pH (e.g. the cis -Golgi or the ER-Golgiintermediate compartment) [148]. These examples demonstratethat various physiological processes may be triggered bymodulating proton concentrations.
R7 (Proton-modulated “acidic-metabolic” vasodilatation):
In the human body, decreasing pH values during hy-poxia/ischaemia can also lead to a physiological mechanismknown as “acidic-metabolic” vasodilatation [149] (Fig. 7, R7),which improves blood flow and oxygen supply. Vasodilatornitric oxide (NO) is generated through a non-enzymaticreduction of inorganic nitrite (NO − ) to NO, a reaction that takesplace predominantly during acidic/reducing conditions [149].Thus, “acidic-metabolic” vasodilatation represents anotherphysiological process that may be modulated by proton-basedsynthetic MCSs.III. C ALCIUM I ONS AS S IGNALING P ARTICLES
Besides protons, other types of ions may be suitable forsynthetic MCSs as well. In many cases, there exist building blocks similar to those presented in Section II. Due to theirparticular role as second messengers in the human body, weillustrate in this section how the concepts proposed for protonsmay be transferred to calcium ions (represented by the symbolCa ). Table II summarizes the proposed building blocks andunderlines the high similarity of the underlying componentsfor both types of signaling particles. A. Transmitters
Transmitter
T1 (pipette) as well as the simple vesicle-basedtransmitter
T2 (degenerating vesicle) , which does not containspecific membrane proteins, may be adapted for calcium ionssimply by replacing the acid with a solution containing acalcium salt such as CaCl . Compared to protons, there aresome additional degenerating carriers triggered by light ortemperature which may be used as an alternative .Biological transmitters T3-T5 can be constructed by replac-ing the described proton channels and proton pumps in Fig. 6by calcium-specific proteins with an analogous function. Somesuggestions for how this could be realized in detail are givenbelow. T3 (Ion channels):
As proposed for protons, the outwardtransport of calcium ions may be accomplished with ionchannels as well. There exist some voltage-gated channelsfor calcium ions such as Ca v neuron. Thiswould allow to use the input from a nerve fiber for activationof the transmitter. This general principle can also be appliedto other pairs of ligand-gated channels and their physiologicalligand, of course. This allows for the direct coupling of abiological process and a synthetic MCS.Another interesting option are mechanosensitive calciumchannels, which are opened in response to mechanical stress Intracellular chemical substance whose concentration changes upon aprimary signal, e.g. a ligand binding to a transmembrane receptor [150]. One type of such carriers may be so-called light-sensitive caged compounds[160], [161], which are already commercially available. Like in the vesicle,the calcium ions are shielded at the beginning and thus biologically inactive,which is due to a bound photoswitchable molecule. Upon irradiation with lightof a certain wavelength, the shielding agent gets cleaved and the calcium ionsare released from their cage [160], [161]. The same general concept, but withheat instead of light as a trigger mechanism, could also be realized by the useof thermosensitive microcapsules as described in [162]. Neuron/synapse which produces serotonin or uses serotonin as an NT.
EEE COMMUNICATIONS SURVEYS & TUTORIALS 14
TABLE IIT
RANSFERABILITY OF THE BUILDING BLOCKS SUGGESTED FOR PROTONS (F IGS . 6
AND TO MCS
S USING C ALCIUM IONS AS SIGNALING PARTICLE .C OMPARISON OF POSSIBLE TRANSMITTERS AND RECEIVERS . Component Protons (H + ) Calcium ions (Ca )T1 (Pipette) Pipette with acidic solution Pipette with solution containing calcium salt (e.g. CaCl ) T2 (Degenerating carriers)
Vesicle with acidic solution Vesicle with calcium ions, alternatively light-sensitivecaged calcium or thermosensitive microcapsules
T3 (Ion channels)
Vesicle with acid and passive transport via voltage-gatedproton channel [123] Vesicle with calcium ions and passive transport via calciumchannel, different gating mechanisms: • Voltage-gated (e.g. Ca v • Ligand-gated (e.g. 5-HT-3A + serotonin [152]) • Mechanosensitive (e.g. TRPV4 [153])
T4/T5 (Ion pumps)
Vesicle with acid and active transport via proton pump,different energy sources: • Light-driven (e.g. bacteriorhodopsin [125]) • ATP-driven (e.g. V-ATPases, P-ATPases [127]) Vesicle with calcium ions and active transport via calciumpump, different energy sources: • Light-driven (e.g. engineered pump from [154]) • ATP-driven (e.g. Ca -ATPase [155]) R1 (Measuring instrument) pH-meter Conductivity measuring instrument
R2 (Dye) pH-sensitive dye/pH indicator Calcium-sensitive fluorescent dye (e.g. Fura-2, Indol-1,Fluo-3, Fluo-4, Calcium Green-1 [156], [157])
R3 (Fluorescence proteins)
GFP (different variants available) [130], [132] Fusion constructs of GFP variants and calmodulin (e.g.GCaMP [158])
R4 (FRET) α -helix stabilization due to protonation, fluorescenceproteins at the termini forming a FRET pair [134] Calcium indicators containing troponin C and a FRET pair(e.g. TN-XL (CFP+YFP) [159]) [163]. One possible approach could be the insertion of thevesicles between the fibers of the extracellular matrix at somelocation in the body. Upon mechanic shear or pressure onthe respective tissue, a calcium release would be triggered.This principle could be useful in the context of targeted drugdelivery, e.g. in order to facilitate the local administrationof an anesthetic. One example for such a mechanosensitivecalcium channel involved in nociception, i.e., the encoding andprocessing of pain stimuli in the human body, is the transientreceptor potential vanilloid 4 (TRPV4) [153] which is depictedin Fig. 4f. T4/T5 (Ion pumps):
Besides passive channels, similar toprotons, calcium ions can actively be transported using either alight-driven pump, that has been artificially created [154], or anATP-driven Ca -ATPase [155]. If two light-driven pumps areintended to be used in the same vesicle to allow for transmitterregeneration (reversibility), the second pump would need to beengineered to work at a different wavelength as has alreadybeen reported for some bacteriorhodopsin mutants [126]. B. Receivers
Regarding possible receivers for calcium ions, buildingblocks which are similar to R1-R4 presented for protons canbe employed (see Table II for a comparison).
R1 (Conductivity measurement):
In response to the releaseof calcium ions, the conductivity of the medium surroundingthe transmitter would increase. Analogous to the usage of a pH-meter in case of protons, the easiest way to construct a receiverfor calcium ions is thus an instrument, which can measure theconductivity of the solution in the channel. While this approachmight be suitable for testbeds and applications outside thebody, it is challenging for biological systems because the high background concentration of other ions in the environmentrequires the detection of rather small changes of the total ionicstrength.
R2 (Fluorescent dye):
Similar to a pH indicator, a calcium-sensitive fluorescent dye could be used to detect changes in thecalcium ion concentration. When such a dye is illuminated withlight of a certain wavelength, fluorescence occurs if calciumions are present. Examples for such dyes are Fura-2, Indol-1,Fluo-3, Fluo-4, and Calcium Green-1 [156], [157], which allhave a high specificity for calcium ions in common.
R3 (Fluorescence proteins) : For the third receiver structure,we propose the use of calcium-sensitive fluorescent proteins.They are generally fusion constructs of GFP (Fig. 4c) or one ofits variants, and the calcium-binding protein calmodulin. Oneexample is GCaMP [158] which shows only feeble activity ifcalcium ions are absent, but undergoes a conformational changeupon calcium binding leading to a pronounced fluorescence.
R4 (FRET):
Alternatively, as described for protons, calciumions may be detected via receivers relying on the FRETmechanism [159], [164]. There exist calcium ion indicatorswhich are based on a FRET pair of two different fluorescentproteins, connected via the calcium-binding protein troponinC. One example, TN-XL [159], consists of CFP and YFP.If no calcium ions are present, the protein has an extendedconformation where the two fluorescent subunits are distantfrom each other. If CFP is activated by illumination with acertain wavelength, only cyan fluorescence occurs. As soon as acalcium ion binds to troponin C, the conformation of the fusionprotein changes, such that the two fluorescent building blocksget into mutual vicinity. Upon illumination and activation ofCFP, the energy is partly transferred to YFP via FRET so thatyellow fluorescence can be observed as well.
EEE COMMUNICATIONS SURVEYS & TUTORIALS 15
IV. N
EUROTRANSMITTERS AS S IGNALING P ARTICLES
Another important class of signaling particles well suited forthe design of synthetic MCSs are NTs such as acetylcholine,dopamine, and serotonin. In the human body, NTs are usedto transmit nerve signals (action potentials) at the chemicalsynapses between two neurons (e.g. dopamine, serotonin) orfrom a neuron to a muscle fiber (acetylcholine) [84]. In thissection, we describe some general principles and buildingblocks for the design of synthetic transmitters and receivers forNTs. The general design strategies for the transmitter systemsare depicted in Fig. 8, those for the receiver systems arepresented in Fig. 9. These general design principles can beapplied to all three NTs discussed in this survey. Thus, the mostsuitable NT can be selected depending on the requirementsimposed by the desired application. It is important to note thatthe design of a specific signaling pathway requires the choiceof suitable protein components depending on the type of NTused. Candidate components for acetylcholine, dopamine, andserotonin are summarized in Table III.
A. Transmitters
In the following, we consider six different possible types oftransmitters for NTs. The first two transmitters (T1, T2) aresimple and can be used as macroscale interfaces. TransmittersT3-T5 are vesicle based and employ different biologicalmechanisms for NT release. The final transmitter (T6) is theaxon terminal of a nerve fiber.
T1 (Pipette):
Analogous to cations (Fig. 6, Table II), thesimplest and least sophisticated transmitter is a pipette bywhich a solution containing the NTs can be released dropwiseinto the channel (Fig. 8, T1).
T2 (Caged compounds):
As described for calcium ions,another interesting concept, which is based on shieldingthe NTs until they need to be released, are so called light-sensitive caged compounds (Fig. 8, T2). In this approach, theNTs are enclosed and thus inactivated by a photoswitchablemolecule. Upon irradiation with light of a certain wavelength,the photoswitchable molecule is either cleaved or changes itsconformation and as a consequence, the NTs are releasedfrom their cage. Caged compounds have been developedfor acetylcholine, dopamine, serotonin, and many other NTs[165]–[167]. Some of them are already commercially available.Recently, caged serotonin has been suggested to be used fortargeted drug delivery in the context of neurodegenerativediseases [167]. Alternatively, the NTs could be shielded usingthermosensitive microcapsules [162] which release their contentupon an increase of temperature.
T3 (Degenerating vesicles):
Vesicles containing a solution ofparticular NTs may be used as transmitter (Fig. 8, T3). As asimple option, the vesicle can be destroyed to release its contentas described previously. However, this has the disadvantagethat only a one-time release is possible.
T4 (Symporters):
If a vesicle-based approach is used assuggested in T3, a transmitter which pumps the NTs in amore controlled manner from the inside of the vesicle intothe channel may be a better option than simply destroying thevesicle and releasing its entire content at once. To this end, special proteins for the outward transport of the NTs maybeinserted into the vesicle membrane. One possible outwardtransporter are NT sodium symporters (Fig. 8, T4), whichare driven by a sodium concentration gradient [168]. Thestructure of sodium symporters for the target NT dopamine,i.e., dopamine transporters, is shown in Fig. 4g. In particular,the underlying mechanism of sodium symporters is as follows:When both a sodium ion and an NT bind to the transportersimultaneously, they are carried across the vesicle membrane bya conformational change of the transporter. This means that theNT will be pumped outwards as soon as a sodium concentrationgradient from the inside to the outside is established. Sucha sodium gradient may be realized by a light-driven sodiumpump which transports sodium inside when it is illuminatedby light of a certain wavelength. Thus, the combination of anNT-sodium-symporter and a light-driven sodium-pump can beused for a finely controllable release of NTs from the vesicle.
T5 (Antiporters):
An alternative strategy with the same levelof complexity and controllability as the previous transmitterstructure (T4) is to use vesicles with NT antiporters instead ofsymporters [169] (Fig. 8, T5). In contrast to sodium symporters,in this case, the driving force for transportation of NTs acrossthe vesicle is a proton gradient. In particular, if a protonbinds to the antiporter from the outside of the vesicle and anNT simultaneously from the inside, a conformational changeoccurs by which the proton is transported inside and the NTis transported outside. To avoid a constitutive NT release, theinside of the vesicle has to be more acidic than the surroundingchannel when the transmitter is inactive. Coupled with alight-driven proton pump such as bacteriorhodopsin, one canthen remove protons from the vesicle upon illumination witha certain wavelength and thereby induce a proton gradienttowards the inside which triggers a controlled release of theNTs.
T6 (Natural transmitters):
Besides the vesicle-based trans-mitters (T3-T5), a main advantage of NTs is the possibility todirectly use a physiological transmitter, i.e., the axon terminal(presynaptic part) of a nerve fiber in the human body (Fig. 8,T6). Upon excitation of a nerve fiber, an electrical signal (actionpotential) is generated, moves along the axon terminal, andtriggers the release of the NTs, which are stored in vesicles[169]. This natural biological transmitter is inexhaustiblebecause the vesicles are regenerated by the neuron. Possibleapplications of such a direct interface to a neuron [7] arethe bridging of nerve lesions, the release of drugs in certainconditions (e.g. an analgesic combined with a neuron involvedin pain reception), and the movement of a prosthesis.
B. Receivers
In this subsection, we discuss two synthetic receivers forNTs which employ NT receptors embedded into the membraneof a vesicle. Such NT receptors exist in the human body,e.g. in postsynaptic cells, and there are different types of NTreceptors. Here, we consider ligand-gated sodium channelssuch as the serotonin 5-HT3A receptor (R1, Fig. 4e) and G-protein coupled receptors (R2, Fig. 4h). In addition, we alsoconsider one natural receiver for NT which can serve as an
EEE COMMUNICATIONS SURVEYS & TUTORIALS 16
Fig. 8. Transmitters for NTs as signaling particles. The suggested building blocks for different transmitter systems are presented in order of increasingcomplexity and include designed artificial systems and physiological emitters, which naturally occur in the human body.
EEE COMMUNICATIONS SURVEYS & TUTORIALS 17
R2:
Cell with G-protein coupled receptor (GPCR),detection with FRET-experiment+ light-driven pump for ion reversibility
R1:
Vesicle with ligand-gated sodium channels and measurement of the transmembrane current
R3:
Postsynaptic terminal time A ligand-gated channel h ν GPCR G-proteinFRETassaylight-driven pump
Fig. 9. Receivers for NTs as signaling particles. The suggested building blocks for different receiver systems are presented in order of increasing complexityand include designed artificial systems and physiological receivers, which naturally occur in the human body. Neurotransmitters and sodium ions are indicatedas orange and green dots, respectively. interface for the control of biological systems (R3).
R1 (Ligand-gated ion channels):
For ligand-gated sodiumchannels, upon binding of the target NT, a pore in the receptor isopened which allows sodium ions to pass through the membrane[180]. Based on the concentration gradient, sodium ions movefrom the outside to the inside of the cell, which leads to theformation of a transmembrane current that can be measuredby methods such as the two-electrode voltage clamp method(Fig. 9, R1, upper panel) [73]. For proper functionality of thevesicle, the concentration of sodium ions inside the vesiclehas to be lower than the concentration outside. To ensure thatafter detection the required sodium ion gradient is restored toenable future receptions, reversibility has to be integrated in thevesicle. This can be accomplished by further integrating light-driven sodium-pumps into the membrane of the vesicle (Fig. 9,R1, lower panel) [181]. Then, the light-driven sodium pumpscan operate e.g. in the time interval between two consecutive transmissions to pump out the sodium ions so that the receiveris replenished.
R2 (GPCRs):
Besides ligand-gated ion channels, G-proteincoupled receptors (GPCRs) are another class of receptor forNTs [180]. The structure of one example GPCR, namely theM3 muscarinic receptor, is shown in Fig. 4h. When an NTbinds to a GPCR, this leads to a conformational change ofthe receptor. This conformational change is conveyed to theinside of the cell via an intracellular binding protein such as aG-protein or arrestin, where it may activate or inhibit a varietyof second messenger molecules and thereby have an impact onthe cell metabolism [182] rendering it suitable for microscaleapplications. However, a GPCR could also be used as receptionmechanism for a macroscale interface (Fig. 9, R2). Such asetup will most probably require an intact cell, which has sucha receptor in its membrane, because it would be extremelydifficult to synthetically reconstruct the corresponding complex
EEE COMMUNICATIONS SURVEYS & TUTORIALS 18
TABLE IIIC
OMPARISON OF DIFFERENT NT S AS SIGNALING PARTICLES . E
XEMPLARY OPTIONS FOR DIFFERENT COMPONENTS . Component Acetylcholine Dopamine SerotoninT3-T5 Vesicle yes yes yes
T4 Symporter(sodium-driven) — dopamine transporter (DAT) [114] serotonin transporter (SERT) [170]
T5 Antiporter(proton-driven) vesicular acetylcholine transporter(VAChT) [171] vesicular monoamine transporter 2(VMAT2) [172] vesicular monoamine transporter 2(VMAT2) [172]
T6 Physiological transmitter axon terminal at neuromuscularjunction [173] axon terminal in the centralnervous system (CNS); regulationof executive functions, motorcontrol, motivation, arousal,reinforcement, and reward [174] axon terminal in the CNS;regulation of mood, emotion,memory processing, sleep,cognition [174]
R1 Ligand-gated channel nicotinic acetylcholine receptor(nAChR) [175] — serotonin receptor subtype 5-HT [176] R2 G-protein coupledreceptor (GPCR) muscarinic acetylcholine receptors(mAChR) M − [177] dopamine receptors DRD1-DRD5[178] serotonin receptor subtypes5-HT , , − [179] R3 Physiological receiver trigger contraction of muscle fiber[173] trigger nerve impulses in the CNS[174] trigger nerve impulses in the CNS[174] signaling cascade in a vesicle. There are several commerciallyavailable kits which use FRET experiments allowing opticaldetection of the level of GPCR activation.
R3 (Natural receivers):
NTs can be used to directly interactwith biological systems. For example, if the NTs are released inproximity of a postsynaptic terminal or a muscle fiber, they maystimulate a nerve or induce a muscle contraction (Fig. 9, R3).This stimulation occurs by binding of an NT to a receptor, e.g. aligand-gated ion channel, on the membrane of the postsynapticterminal. The resulting ion influx leads to a depolarizationof the cell membrane which propagates then as a new actionpotential along the cell. This principle can be exploited inmedical applications of MC [7] for bridging of nerve lesionsor targeted intervention into a deregulated neuronal circuitry,e.g. in the context of neurodegenerative diseases.V. P
HOSPHOPEPTIDES AS S IGNALING P ARTICLES
As outlined in Section I-C, protein modifications representa widespread principle of signal transduction in nature. Phos-phorylation, where a phosphoryl group is added to a peptide,represents one of the most frequent modifications in cellularsignaling, and will be considered in the following in more detail.To exploit this principle for the design of MCSs, it appearsadvisable to reduce the size of the respective phosphoproteinsto the vicinity of the phosphorylation sites. These smaller‘phosphopeptides’ have the advantage of faster diffusion dueto their smaller size compared to intact proteins.Phosphopeptides are complementary to the particles de-scribed in the previous sections as they have very differentproperties. The most important difference is that peptides donot function in isolation, but require attachment to a chemicalfunctional group. This step is mediated by a kinase, a specifictype of enzyme. It is important to note that the peptide unitrepresents more than a mere carrier molecule to transportthe phosphoryl group from the transmitter to the receiver but also plays an important role for the specificity of thesignal transduction process. The proteins discussed as receiversbelow generally do not only recognize the phosphoryl groupitself, but also the physico-chemical properties of the peptidein its vicinity. This allows the design of various types ofphosphopeptides with different signaling specificity.
A. Transmitter
In contrast to the systems described in Sections II-IV, inthe case of peptide modifications, the signaling particles donot need to be stored at the transmitter but can be generatedupon a stimulus (e.g. light or external ligand), see Fig. 10. Thestimulation is provided by kinases that transfer a phosphorylgroup from the chemical energy carrier molecule ATP toa peptide, thereby creating a phosphopeptide, the signalingparticle. In this process, ATP is hydrolyzed to adenosinediphosphate (ADP), which can be regenerated to ATP by othercellular processes.For phosphorylation, several different peptides and corre-sponding kinases are available. The human proteome containsat least 518 different protein kinases [183]. Most proteinkinases phosphorylate either the amino acids serine/threonineor tyrosine (specific types of amino acids). However, there arealso dual-specificity protein kinases that can phosphorylateboth serine/threonine and tyrosine residues. Within thesegroups, kinases additionally differ in their specificity, i.e.,the amino acid sequence in the environment of the potentialphosphorylation site can determine whether an amino acidbecomes phosphorylated by a certain kinase or not. Dueto the large variability of the peptide sequences with thecorresponding kinases, a large number of different signalingparticles is available.An important prerequisite for the use of signaling particlesin MCSs is the controllability of particle generation, i.e., in thiscase, the ability to switch the kinases on and off. Because kinase
EEE COMMUNICATIONS SURVEYS & TUTORIALS 19
Fig. 10. Phosphopeptides as signaling particles. General composition of the communication system, which uses switchable kinases (activation by light, ligands,or pH change) as transmitters. The receiver consists of a phosphopeptide-binding domain, which allows detection of the binding process via changes intryptophan fluorescence or in the mass of the complex. activity has profound effects on cellular processes, proteinkinases are generally highly regulated, i.e., there are manymechanisms for switching them on and off. An overview ofphysiological and engineered mechanisms for kinase regulationis given in Table IV.
B. Receiver
For the detection of phosphorylated peptides, there existsa large set of protein domains in nature that may be used asreceivers in synthetic MCSs. The binding of the phosphopeptideto such domains can for example be detected by a change intryptophan fluorescence that occurs upon binding (Fig. 10).Alternatively, the change in mass upon peptide binding maybe detected via surface plasmon resonance [194].Similar to the versatility on the transmitter side, there existmany different adapter domains that can be used as specificreceivers. Serine/threonine phosphorylated peptides can berecognized by a large number of different domain types,including 14-3-3, BRCT, FF, WW, and FHA domains [86].Tyrosine phosphorylated peptides can be recognized by SH2or PTB domains [195]. The SH2 domain family represents thelargest class of tyrosine phosphopeptide recognition modulesand is found in 111 different human proteins [196]. Inaddition to the phosphorylated tyrosine residue (pTyr) itself,these domains also recognize peptide residues adjacent to thephosphorylation site. For example, the SH2-domains of the SHPprotein preferentially bind to a pTyr-X-X-Leu sequence stretch,i.e., they recognize a leucine (Leu), which is three amino acidsapart from the phosphorylation site (“X” denotes a variableamino acid). In contrast, CRK SH2-domains recognize a pTyr-X-X-Pro sequence, which contains a proline (Pro) insteadof leucine at the respective sequence position [197]. Thisrecognition of additional residues in the peptide ensures ahigh specificity at the receiver side and underscores that thepeptide moiety of the signaling particle is more than a merecarrier, but instead plays an important role for the constructionof specific transmitter-receiver pairs.
C. More Complex Architectures for MC
Compared to cations and NTs, phosphopeptides have amore sophisticated structure which provides more degrees offreedom for system design. In particular, by combining severalkinases with receiver domains of corresponding specificity,various communication concepts can be realized. Here, wediscuss orthogonal channels, diversity, coding, and jamming,see Fig. 11.
P1 (Orthogonal channels):
Orthogonal channels can berealized by using two kinases, which differ in the typeof their activation mechanism and the specificity of theirphosphorylation (Fig. 11a). For example, transmitter 1 (T1)could be a light-activated kinase and T2 a pH-activated kinase,each combined with a specific receiver domain (R1 or R2). Inthis system, changes in irradiation and pH can then be detectedin the same setup based on the signals S1 and S2. This setupallows the interference free multiplexing of signals.
P2 (Diversity):
By selecting suitable signal peptides and re-ceiver domains, two signals cannot only be observed separately,but can also be processed jointly to produce a combined outputsignal. One setup for such a processing is shown in Fig. 11b.Here, it is sufficient if one of the two stimuli is present totrigger the signal at the receiver. The difference to the situationshown in Fig. 11a is that instead of two specific recognitiondomains, a receiver domain (R3) with low specificity is nowused which can bind the phosphorylation sites x and y ofboth peptides. This can be interpreted as a form of diversity.For example, let’s assume that both peptides convey the sameinformation (e.g. both convey information bit “1”). If we furtherassume that diffusion is the main transportation mechanismto bring the phosphorylation sites of the peptides into contactwith the recognition domain at the receiver, then, due to therandom nature of the diffusion process, one of the peptidesmay not arrive at the receiver. Alternatively, one of the peptidesmay not be phosphorylated at all, because the respective kinasewas not activated by a stimulus. However, for the consideredsetup, it is sufficient if one of the peptides carrying one of the EEE COMMUNICATIONS SURVEYS & TUTORIALS 20
TABLE IVS
UMMARY OF THE TYPE OF STIMULI THAT CAN BE USED TO CONTROL THE ACTIVITY OF PROTEIN KINASES . T
HE TABLE DISTINGUISHES BETWEENPHYSIOLOGICAL (p)
PRINCIPLES OF ACTIVATION AND THOSE THAT WERE ACHIEVED BY MOLECULAR ENGINEERING (e). I
N ADDITION TO THE TARGETKINASE AND THE TYPE OF STIMULUS , A BRIEF DESCRIPTION OF THE UNDERLYING MOLECULAR MECHANISM IS GIVEN . Type of stimulus Origin Target kinase Molecular mechanismPhosphorylation p Lck Lck contains two regulatory tyrosyl residues (Tyr394, Tyr505). Phosphorylation ofthese residues controls Lck activity in T cells [184].
Ubiquitination p receptortyrosine kinases(RTKs) RTKs can become modified by ubiquitin, which causes their endocytosis from theplasma membrane and degradation [185]. pH change p egg cortextyrosine kinase This kinase shows significant changes of activity within the physiologically relevant pHrange from 6.8 to 7.3 and may therefore be used as a pH sensitive transmitter [186].
Regulatory protein p cyclindependentkinases (CDKs) The activity of CDKs is modulated by the interaction with specific cyclins that act asregulatory partners [187].
Allosteric ligand e Fyn, Src, Lyn,Yes, PAK1 Through insertion of a modified FK506 binding domain, these kinases were engineeredto allow activation by the allosteric ligand rapamycin [188], [189].
Photoresponsive lig-and e Protein kinase C(PKC) When exposed to light, a photoresponsive small molecule becomes an active inhibitorof PKC. This turning on of enzyme inhibition with light allows to control enzymefunction [190].
Light e receptortyrosine kinases(RTKs) RTKs were engineered to include light-oxygen-voltage (LOV)-sensing domains,resulting in kinases that can be activated by light [191].
Light e Tropomyosin-related kinase(Trk) Trk was engineered to include the photolyase homology region of cryptochrome 2 (ablue-light photoreceptor) resulting in a light-controllable kinase [192].
Light e Raf1, MEK1,MEK2, CDK5 A photodissociable dimeric protein (Dronpa) was engineered that dissociates in cyanlight and re-associates in violet light. Insertion of Dronpa into protein kinases allowedto create photo-switchable kinases [193]. phosphorylation sites arrives at the receiver, which implies adiversity gain.
P3 (Coding):
For the architecture shown in Fig. 11c, bothstimuli (e.g. light and pH change) must be present so that asignal can be detected at the receiver. The carrier moleculeused is a peptide that has two distinct phosphorylation sitesfor kinases T1 and T2. This requires a receiver with tworecognition sites for phosphoryl groups, each of which on itsown binds the phosphoryl groups too weakly to trigger thesignal. The simultaneous binding of two phosphoryl groupsresults in a significantly stronger binding, which triggers adetectable signal at the receiver. This may be seen as a formof repetition coding as a signal is generated only if bothphosphoryl groups are observed at the receiver. This principleis used in nature, for example, by the ZAP70 adapter protein,which has two SH2 domains. The simultaneous binding of bothSH2-domains causes a > P4 (Jamming):
For the architecture shown in Fig. 11d, thetransmitter and the signal peptides are similar to those forthe coding scheme shown in Fig. 11c. However, the receiver(R4) has different properties compared to R3. If a secondphosphorylation is added at position y , this leads to a weakeningof the binding due to unfavorable interactions with the receiver, such that no signal is detected. In a communication context,this may be interpreted as a jamming of the signal. In particular,if the intended message is encoded via phosphorylation site x ,adding the second phosphorylation y jams the received signal.An example of such a receiver in nature is a 14-3-3 protein thatspecifically recognizes a Cdc25B signal protein phosphorylatedat the serine 323 position. If a second phosphorylation is addedat the adjacent serine 321, the interaction with the receiver isdisrupted [199].The principles for switchable interactions described in Fig. 11are widely used in nature. The ELM.switches database [200]provides an overview of known switchable systems, whichmight be exploited for MCS design. As a long-term goal, aquantitative understanding of these signaling processes mayguide the design of signaling particles that interfere with cellularsignal transduction processes in a desired fashion, e.g. bycounter-balancing impaired signaling originating from disease. Remark 4:
We emphasize that cations and NTs also allow therealization of some of the above complex architectures albeitto a lesser degree. For instance, the number of NT-receptorpairs that allow realization of orthogonal channels is muchsmaller compared to what can be realized by phosphopeptides.In fact, the sophisticated structure of phosphopeptides allowsthe system designer to engineer multiple types of signaling
EEE COMMUNICATIONS SURVEYS & TUTORIALS 21 T P x P x S T P y S y a P x P y T T P x P y S P x P y T T SR P T T S P x P y P x P y R cbd Fig. 11. More complex architectures for MC based on phosphopeptides. (a) Simultaneous orthogonal transmission of two different signals (S1, S2). MCSsrealizing (b) diversity, (c) coding, and (d) jamming. Transmitters (T) and receivers (R) of different types are labelled with small subscript numbers. x and y denote two distinct phosphorylation sites either in two different peptides (a, b) or within the same peptide (c, d). molecules whose release and detection can be controlled eitherseparately or jointly, depending on the desired application.Such high level of flexibility does not exist for cations andNTs.VI. C OMPARISON , A
PPLICATIONS , AND P RACTICAL C ONSIDERATIONS
In this section, we first compare the properties, advantages,and disadvantages of the signaling particle classes studied inthis paper. Subsequently, we present several medical applica-tions of the proposed biological MCSs and corresponding de-sign options for the transmitter, receiver, and signaling particles.Furthermore, we discuss some practical communication-relatedconsiderations of the considered MCSs, namely ISI mitigationand the speed of communication.
A. Comparison of the Considered Signaling Particles
In Sections II-V, we have described three different classesof candidate signaling particles for synthetic MCSs. Each ofthese classes has certain advantages and disadvantages for implementation. For example, ions exhibit a high particlestability and speed of diffusion, but different ions may generatethe same signal at the receiver. This may cause considerableinterference in physiological environments where syntheticand natural MCSs employ different ions that interact with thesame receiver. NTs provide a highly unique and specific signal,but the reversibility of their use still represents a bottleneckin the designs proposed here. In contrast to the other classes,phosphopeptide-based communication does not require vesiclesand is highly versatile , but the particle stability (susceptibilityto proteases and phosphatases) may constitute a limitation. Theproperties of the considered signaling particles are summarizedin Table V and will be discussed more in detail in the followingwith respect to their medical applications, options for mitigationof ISI, and the speed of communication. Phosphopeptides are versatile signaling particles because they can be usedwith large variations in the setup, i.e., different transmitters (different kinases)and receivers (different recognition domains).
EEE COMMUNICATIONS SURVEYS & TUTORIALS 22
TABLE VP
ROPERTIES AND ADVANTAGES OF THE DIFFERENT CLASSES OF SIGNALING PARTICLES . Properties Ions NTs PhosphopeptidesTransmitter control light, voltage, mechanical stress,ligand indirect via ion channel (light,voltage) light, ligand
Membrane vesicles required yes yes no
Regeneration possible(Reversibility) yes, by returning signaling particlesto the vesicles difficult: in the presented settingsonly with pipette or axon terminal yes, kinase is not destroyed (ATP isregenerated in living cells)
Energy source(if reversible) light by using light-driven pumps irreversible chemical (ATP)
Receiver output signal optical (fluorescence), ion flow(conductivity), ligand release fromprotein ion flow (conductivity), biological(GPCR signal) optical (fluorescence), surfaceplasmon resonance
Signal removal external addition of chemicals: base(OH − ), chelation (EDTA) enzymatic degradation:acetylcholinesterase (AChE),monoamine oxidases (MAO) enzymatic dephosphorylation withphosphatases Underlying physiologicalcommunication type intercellular intercellular intracellular
System complexity moderate high moderate
Signaling particle stability very high high moderate
Speed (diffusion) very fast fast moderate
Uniqueness of signal moderate very good good
Versatility high moderate very high
B. Potential Medical Applications
In the following, we present some practical examples forpotential medical applications of the proposed building blocksof synthetic MCSs. The signaling particles presented in thisarticle were chosen because changes in their concentration areeither causative for diseases or because they may be used asimportant diagnostic markers to detect pathological conditions.Consequently, a combination of appropriate transmitters andreceivers could allow for the construction of medical nanoma-chines that would be able to recognize specific regions of thebody or abnormal tissue such as inflammations or tumors dueto an altered concentration of certain signaling particles. Anoverview of possible medical applications covering a broadrange of diseases such as bacterial or viral infection, cancer,inflammation, and neurodegeneration or circulatory diseases isprovided in Table VI. This table lists the respective signalingparticles and proposes building blocks which could be used todesign interfering nanomachines.Which combination of transmitter and receiver is mostappropriate critically depends on the application of interest,of course. In the following, we focus on one of the medicalapplications listed in Table VI, namely the design of a bridgingdevice for a damaged neuron, cf. Fig. 12, in order to illustratethe design principles for choosing suitable biological buildingblocks for the desired MCS. In the physiologic state (Fig. 12,upper panel), a nerve signal is transduced consecutivelyfrom one neuron to the next via the release of NTs at thechemical synapse between adjacent neurons. If one of theneurons within this chain is damaged due to an injury ora neurodegenerative disease (Fig. 12, middle panel), signal transduction is interrupted. Depending on the type of nerve,this could result in a paralysis or a loss of sensory perception.One conceivable option to repair this damage is the insertionof a bridging device in order to replace the damaged neuron(Fig. 12, lower panel). Such an artificial cell or micelle couldbe fully integrated within physiological signaling. Theoretically,it would consist of two MCSs, one between the healthy neuronon the left-hand side and the bridging device (MCS1) andone between the bridging device and the healthy neuron onthe right-hand side (MCS2). In MCS1, the healthy neuronon the left side would serve as a physiological transmitter(building block NT, T6). As soon as a nerve signal occurs,NTs are released within the synaptic cleft. On the surface ofthe bridging device, ligand-gated Na + channels (building blockNT, R1) could be expressed, which upon binding of the NTwould transport Na + ions to the inside. This would lead toan enhanced concentration of Na + inside the vesicle. Thereby,MCS2 on the right-hand side of the bridging device would beactivated. Na + -NT symporters (building block NT, T4) wouldexport NTs into the second synaptic cleft from where theywould be detected by the healthy neuron on the right-hand side(building block NT, R3). Consequently, the interruption withinthe transmission of the nerve signal would be repaired. Thisexemplary MCS may be further optimized: Reversibility couldbe ensured by adding transporters for a reuptake of the NT tothe left-hand side of the second synaptic cleft. Such a bridgingdevice is well suited for in vivo application as it is based on abiocompatible vesicle or cell and no external equipment suchas light sources or devices for the measurement of membranepotentials are needed. EEE COMMUNICATIONS SURVEYS & TUTORIALS 23
TABLE VIE
XAMPLES FOR MEDICAL in vivo
APPLICATIONS OF
MCS
S INCLUDING THE RESPECTIVE MEDICAL CATEGORY AND THE INVOLVED SIGNALING PARTICLES .B UILDING BLOCKS , WHICH ARE SUITABLE FOR THE DESIGN OF THE CORRESPONDING NANOMACHINES , MAY BE REALIZED USING THE TRANSMITTERSAND RECEIVERS PROPOSED IN S ECTIONS II TO V. Medicalcategory Exemplary disease mechanism and signaling particle Proposed building blocksBacterialinfection
Some bacterial proteins interfere with physiological signaling pathwaysbased on phosphopeptides or phosphoproteins [201]. An example isSpvC from
Salmonella
Typhimurium, which can inactivate MAP kinasesthat are involved in the activation of the immune response [202]. The depleted kinases could be replaced by a synthetictransmitter in order to restore the physiological immuneresponse (e.g., transmitters in Fig. 10 and Table IV).
Viral infection
Some viral proteins interfere with physiological signaling pathwaysbased on signal peptides [203]. One example is HIV Vpu [204] whichcontains phosphorylated residues that are detected by the receiver proteinh- β TrCP within cells of the human immune system. This interactionleads to a degradation of the immune receptor CD4 which is requiredfor pathogen recognition [205]. One possibility to interfere with this pathological signalingpathway would be to design an artificial receiver that bindsand sequesters Vpu in a more affine manner than h- β TrCP(e.g., receiver in Fig. 10).
Cancer orinflammation
Proton concentrations are elevated in inflamed tissue [206] or cancer[207], [129] due to an enhanced/altered metabolism. MCSs with receivers for protons could be used to detectaffected tissue, which would be useful for targeted drugdelivery (e.g., R5 in Fig. 7).
Depressivedisorders orParkinson’sdisease
Dysregulations within the dopaminergic system resulting in a lack ofthe NT dopamine play a role for the development of the hedonicdeficits of patients suffering from depression [208]. A selective lossof dopaminergic neurons within the
Substantia nigra is causative forParkinson’s disease [85]. Receivers which are activated by dopamine could be used todetect dopaminergic synapses. Then, transmitters releasingdopamine could be used to potentiate the signal (e.g., T4 inFig. 8 and R1 in Fig. 9).
Dysregulatedsignalingpathways
Many physiological signaling pathways are based on phosphopeptidesor phosphoproteins [209], [210]. Mutations of the respective phospho-rylation sites which occur in various diseases can thus have deleteriouseffects [211]. One example are several mutations in the protein c-Mycthat lead to the loss of a phosphorylation site. As a result, c-Myc isinefficiently degraded which may lead to tumor formation (Burkitt’slymphoma) [212]. Synthetic receivers for mutant c-Myc could be used in orderto bind and thus inactivate the signaling pathway that leads totumor formation (e.g., receiver in Fig. 10).
Hypertension
One approach to treat hypertension is the administration of drugs whichlead to a widening of blood vessels and can thereby reduce the vascularresistance [213]. An increased proton concentration results in “acidic-metabolic” vasodilatation [149]. Transmitters for protons could be used to increase the protonconcentration in peripheral blood vessels in order to treathypertension (e.g., T3 in Fig. 6).
Nerve lesions
NTs such as acetylcholine, dopamine, or serotonin are missing becausethe physiological transmitter, i.e., the releasing neuron is damaged.Therefore, the transmission of nerve signals is interrupted [214]. The damaged nerve could be repaired using a bridging deviceconsisting of two MCSs, cf. Fig. 12 (e.g., T4 in Fig. 8 and R1in Fig. 9).
Remark 5:
We note that for the exemplary applicationscenarios in Table VI, the end-to-end MCS may be in generalpartially synthetic and partially natural . For example, in Fig. 12,the receiver in MCS 1 is a synthetic cell whereas the transmitteris a natural cell (the nerve cell on the left-hand side in Fig. 12).On the other hand, for MCS 2, the transmitter is a synthetic cellwhereas the receiver is a natural cell (the nerve cell on the right-hand side in Fig. 12). More advanced applications of MCSsmay necessitate the use of both partially and fully synthetic end-to-end MCSs. For instance, in targeted drug delivery for cancertherapy, on the one hand, the drug-carrying nanomachines mayneed to communicate with each other in order to collaborativelyfind cancerous tissue [38], [55]. Here, the end-to-end MCS issynthetic, i.e., transmitter and receiver are nanomachines andthe signaling particles have to be chosen by the system designerby considering the specific characteristics of the application.On the other hand, after the targeted cancerous tissue has beenlocalized, the drug-carrying nanomachines that are close to thetissue have to release their payload drug for cancer therapy[2], [39]. This communication system can be interpreted as a partially synthetic MCS whereby the transmitter is a syntheticnanomachine, the particles are the drug molecules, and thereceiver is the cancerous tissue.
C. ISI Mitigation via Signal Removal
MCSs are impaired by ISI which is caused by the dispersivenature of the diffusion process. In particular, when the trans-mitter emits consecutive symbols, the signaling particles ofpreviously transmitted symbols may be observed at the receiverin the current symbol interval. Since ISI is a common problemalso in conventional wireline and wireless communicationsystems, many approaches for ISI mitigation exist [215].In conventional communication systems, equalization at thereceiver is employed to combat ISI. Although, equalizationhas also been proposed as an option for MCSs [22], it may bechallenging to implement even simple schemes, such as linearequalization, at nano-scale. Alternatively, in natural MCSs, ISIis usually mitigated by removing the signaling particles fromthe channel. This biological approach also seems to be a viableoption for ISI mitigation in synthetic MCSs. In the following,
EEE COMMUNICATIONS SURVEYS & TUTORIALS 24 nerve signalPhysiologic statePathologic state/Nerve lesionnerve signalnerve signalBridging device damaged neuron ligand-gated Na+ channel Na+-NT symporter no signalNT, T6 NT, R1MCS 1 NT, T4 NT, R3MCS 2
Fig. 12. Bridging device for nerve lesions as a potential medical application for MCSs. The upper panel shows the physiological state with three intact neurons.The middle panel illustrates a nerve lesion in which the second neuron is damaged so that the signal is not transmitted. In the lower panel, a potential vesicle-or cell-based bridging device is shown that could be used to repair the damaged nerve. The building blocks used in the MCS are described in Figs. 8 and 9. we will discuss mechanisms for particle removal for each ofthe considered signaling particles. • Protons : If protons are used as signaling particles, thenadding a basic solution capable of reversing the pH changecaused by the transmitter system is the simplest optionfor removal of the signaling particles. Like the acidicsolution (Fig. 6, T1), this basic solution can be releasedinto the channel e.g. by an electrically controlled pipetteafter symbol detection. In a physiological environment,where such a pipette is not available, raising the pH valuecan be achieved for example by decarboxylation reactionsof amino acids or urea, which consume protons [216]. Inthe presence of the enzyme urease, urea is cleaved intoCO and NH . The latter acts as a base and can thuslead to a neutralization of the acidic liquid in the channel.This cleavage of urea occurs also in nature, e.g. as amechanism to remove acidic carbohydrate fermentationproducts in human saliva [217]. Moreover, pathogenic Helicobacter pylori , which evokes gastritis and gastriccancer in humans, uses urease to buffer the highly acidicgastric liquid [218]. • Calcium ions : Calcium ions can be chemically shieldedwith a chelating agent such as ethylenediaminetetraacetate(EDTA) [219] or ethylene glycol-bis( β -aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA). In particular, these substancescan bind to calcium ions and prevent them from fur-ther interaction with their environment. By releasing anEDTA/EGTA solution into the channel as soon as asignal has been detected, the calcium ions released in the current symbol interval are neutralized and cannotcause interference in future symbol intervals. Hence, ISIis mitigated. • NTs : For NTs, in nature, signal removal is either achievedby symporters, which pump the NTs into separatedcompartments [168], or by enzymes, which degrade theNTs [84]. The latter principle has the advantage thatit can be easier to implement in synthetic MCSs asfewer biological building blocks are needed. A theoreticalstudy of NT removal via enzyme has been providedfor acetylcholine in [220], which can analogically toa physiological process at neuromuscular junctions beeliminated by the enzyme acetylcholinesterase (Fig. 4i).In an analogous manner, dopamine and serotonin can bedegraded by monoamine oxidase-A. • Phosphopeptides : For modified peptides as signalingparticles, the phosphorylation can be removed by phos-phatases to avoid interference with subsequent signals.One option for regulating such phosphatases are light-controlled inhibitors, which are reversibly activated by ir-radiation with UV light [221]. Alternatively, phosphataseswith pH-dependent activity were developed [222]. Amodular protein assembly approach was used to designa prototype that can detect tyrosine phosphorylation andimmediately activate phosphatase without requiring furtherexternal stimuli [223].
D. Speed of Communication
The time scales at which the different processes neededfor signal transmission occur at transmitter and receiver as
EEE COMMUNICATIONS SURVEYS & TUTORIALS 25 well as in the channel are crucial for MCS design as theyultimately limit the achievable data rate. The quantity, which isthe most straightforward to measure, is the speed of diffusion(Fig. 3). However, in order to estimate the speed of the entirecommunication process, the speed of the proteins involvedas well as the time for building up concentration gradientssufficient for detection need to be taken into account as well. Acomprehensive review of these processes is beyond the scopeof this article. We will therefore only give a broad overviewof the respective time scales; a comprehensive overview oftime ranges of biological processes with many useful modelcalculations and the corresponding database are given in [224]and [74], respectively. One approach that may be exploitedto speed up synthetic MCSs is flow. In particular, flow isalso used in nature as a biological process that facilitates thetransportation of chemical species across the human body. • Transport in channel:
Individual molecular events, suchas an ion flowing through an ion channel or an enzymecatalyzing a reaction, are typically very fast. For example,one bacteriorhodopsin molecule can pump about 188protons per second [74]. However, depending on thedensity of bacteriorhodopsin in the vesicle, the protongradient needed to activate the receiver, and the distancebetween transmitter and receiver, it may take considerablylonger to achieve a desired macroscopic effect. Somemodel calculations for such macroscopic processes forbacteriorhodopsin have been performed in [125]. In an ex-perimental study,
E. coli cells were used to synthetize withblue-absorbing and green-absorbing proteorhodopsins,which are two different types of proton pumps closelyrelated to bacteriorhodopsin. A change of − nM inproton concentration of the cell suspension was achievedafter about 60 s with an approximately linear evolutionover time [225]. • Ion channels:
For ion channels, a good estimate forthe number of ions that can be transported through asingle channel is on average about 10 per second [74]with a range from about 10 s − to 10 s − . Someexemplary permeability rates (number of ions that flowthrough the channel in a certain time span) are summarizedin Table VII. This compilation includes a subset ofthe channels suggested in this article for which suchinformation was available in the literature. Although thechannels are rather fast on the level of a single molecule,the time needed to detect an effect at the cellular level(for example in Xenopus oocytes) is in the range of afew seconds [226]. However, if artificial vesicles withion channels are created, the required time span will behighly dependent on the density of the ion channels inthe membrane. • Transporters:
Transporters are in most cases much slowerthan ion channels with a typical transport rate of ≈ − [224]. Nevertheless, if a cell has e.g.10,000 transporters of a certain type in its membrane,10 molecules can be transported in total per second. Forglucose transporters in certain cell types, it has beenestimated that they make up about 2 % of the total membrane surface [224]. How many molecules can beexported per time unit will critically depend on how easilythe corresponding membrane protein can be inserted intoa vesicle membrane and what density can be achieved. • Receptors:
For GPCRs, ligand binding happens on amicrosecond time scale [234] and the conformationalrearrangements required for receptor activation take placeon a micro- to millisecond range [235]. However, currentFRET experiments to readout the receptor activation atthe cellular level would then take multiple tens of secondsor even minutes [236]. • Enzymatic reactions:
For some of our proposed buildingblocks, such as the degradation of NTs and the addi-tion and removal of peptide modifications, enzymes arerequired. The speed at which an enzyme catalyzes itsreaction is dependent on the concentration of the enzymeitself and the substrate (target molecule) the enzymebinds to. The general form of an enzymatic reaction is asfollows: E + S k (cid:10) k − ES k cat (cid:42) E + P (1)where enzyme E forms with its substrate S an enzyme-substrate complex ES with a certain rate constant k .This complex can either dissociate, with the reaction rateconstant k − , and form E and S, or react, with the reactionrate constant k cat , and form an E molecule and a productmolecule P [237]. Note that the enzyme itself is notconsumed in this process. The speed v of this reactionmay be approximated (apart from additional factors suchas cooperative binding that can affect v ) by use of theMichaelis-Menten equations [237] as follows v = k cat · [ E ] t · [ S ] K M + [ S ] = v max · [ S ] K M + [ S ] (2)where square brackets denote the concentrations of therespective molecules, [ E ] t is the total concentration ofthe enzyme (free+substrate-bound), v max is the maximumreaction speed at the given enzyme concentration, and K M is the Michaelis constant. Thus, knowing k cat , K M ,and the concentrations of enzyme and substrate, the speedof the reaction can be calculated. In particular, in (2), k cat stands, in simple terms, for the number of reactions anenzyme makes per unit time and K M for the substrateconcentration at which 50 % of the maximum reactionspeed is reached [224]. Remark 6:
Typical values of k cat vary between 10 − s − and 10 s − with the median approximately at 14 s − .Moreover, the range of the values of K M varies approxi-mately from 10 − mM up to 10 mM with a median valueof 0.130 mM [224], [246]. Please note that these valuesare not only enzyme-specific, but they also depend on therespective substrate and environmental conditions such aspH or ionic strength. For illustration, we provide concretevalues of k cat and K M for some selected exemplaryenzyme-substrate combinations in Table VIII. Specificvalues for many more enzymes and substrates are availablein the BioNumbers data bank [74]. EEE COMMUNICATIONS SURVEYS & TUTORIALS 26
TABLE VIIP
ERMEABILITY RATES FOR SELECTED ION CHANNELS . C
HANNEL IS AN EXAMPLE FOR A VOLTAGE - GATED PROTON CHANNEL (P ROTONS , T3),
CHANNEL IS A VOLTAGE - GATED CALCIUM CHANNEL (C ALCIUM IONS , T3). C
HANNEL CAN BE USED AS A RECEIVER FOR ACETYLCHOLINE (NT S , R1). C HANNELS
4, 5, 6,
AND ARE ADDITIONAL EXAMPLES DEMONSTRATING THE TYPICAL RANGE OF ION TRANSPORT RATES . Ion channel Permeability rate [s − ] References1 Voltage-gated proton channel H v . · [227], [228] v ≈ [81] . · [229] ≈ [230] v ≈ [231] . · [232] . · [233]TABLE VIIIK INETIC CONSTANTS FOR SELECTED ENZYME - SUBSTRATE COMBINATIONS . E
NZYMES AND CAN BE USED FOR NT DEGRADATION , ENZYME IS ANEXEMPLARY ENZYME FOR PROTEIN PHOSPHORYLATION AND ENZYME FOR PROTEIN DEPHOSPHORYLATION (T HEIR INDICATED KINETIC VALUES WEREDETERMINED FOR A SAMPLE PEPTIDE .). E
NZYMES
5, 6,
AND ARE ADDITIONAL EXAMPLES DEMONSTRATING THE ACTIVITY RANGE OF DIFFERENTENZYMES . Enzyme Substrate k cat [ s − ] K M [ mM ] References1 Acetylcholinesterase acetylcholine . · dopamine 1.83 0.23 [240]serotonin 1.80 0.40 peptide 0.7 0.013 [241] α peptide 0.3 0.09 [242] H O . · glyceraldehyde3-phosphate . · N-acetyl-Val-OMe . · −
88 [239], [245]
VII. F
UTURE R ESEARCH D IRECTIONS
In this section, we outline some future research directionsincluding open research problems that should be tackled inStage 2 of the roadmap in Fig. 2 by theoreticians for modelingand designing the biological building blocks proposed in thispaper as well as the challenges that should be addressed byexperimentalists for implementation of these building blocksin Stage 4 of the roadmap. A summary of the proposed futureresearch directions are provided in Fig. 13.
A. Directions and Challenges for Theoretical Research
The design of reliable and efficient synthetic MCSs cruciallydepends on the accuracy and simplicity of the correspondingtransmitter, channel, and receiver models. Simplicity is desir-able since complicated models typically do not allow for thederivation of insightful design guidelines which are requiredfor efficient system design while a certain level of accuracyis also needed to ensure the relevance of the results and thecorresponding design. Although there exists a rich literature inbiology that analyzes the proposed biological building blocksfor MCSs, the corresponding models are too complex to bereadily applicable to communication system design. In fact,for many of the transmitter and receiver architectures proposed in this paper, communication-theoretical models have not beendeveloped by the MC community, yet. In the following, wehighlight some of the related open research problems: • Ion pumps vs. ion channels:
While ion channels allowpassive diffusion of the signaling particles across themembrane and rely on a concentration gradient, ion pumpsenable the transport of the signaling particles across themembrane even against a concentration gradient at thecost of energy. Communication-theoretical channel modelshave been developed for ion pumps and ion channelsin [125] and [247], respectively. However, these resultsare preliminary and contain no comparative study of ionpumps and ion channels. To quantitatively compare theseoptions, the impact of the energy consumption of ionpumps and the concentration gradient of ion channelshave to be modeled and analyzed. This has not been fullyaddressed in the existing MC literature, yet. • Reuptake:
The particle reuptake mechanism enablesa reduction of ISI and the possibility of “harvesting”signaling particles for re-transmission. However, severalaspects affect the performance of the reuptake processincluding the position of the reuptake pumps on the cellsurface, their number/density, and the required energy
EEE COMMUNICATIONS SURVEYS & TUTORIALS 27
Future Research Directions
Theoretical Research Experimental Research
Ion Pumps vs.Ion Channels ReuptakeProcess
Phosphopeptides asSignaling Particles
End-to-endChannel Model Impact ofInterfaces RecombinantExpressionof Proteins ProteinEngineering MembraneProteinReconstitutionMembranePermeability OpticalStimulation PolymersomesArtificial Vesiclesvs. Entire Cells
Fig. 13. Summary of potential future research directions. consumption. Related preliminary results by the MCcommunity can be found in [248]–[250], where the authorsproposed the transmission of signaling molecules by onenode and harvesting them by inward reuptake pumps bythe same or another node. • Phosphopeptides:
As discussed in Section V-C, phospho-peptides are quite flexible which allows the constructionof complex system architectures. In addition, unlike othertypes of signaling particles, peptide molecules can bealways present in the channel and become phosphorylatedto create the signaling molecules, namely phosphopeptides,upon an external stimulus (e.g. light or external ligand).This interesting class of signaling particles has not beeninvestigated in the MC literature, yet. Hence, there existmany open research problems ranging from the modelingof the synthesis and reception of phosphopeptides to thedesign of new network architectures addressing the needsof different applications. • End-to-end channel model:
In communication theory,we are often interested in an end-to-end channel modelwhich relates the received signal to the transmit signal. Inthe context of MCs, the number of signaling moleculesreleased by the transmitter is typically considered as thetransmit signal and the number of signaling moleculescounted at the receiver constitute the received signal.However, the release and reception mechanisms in MCSsmay include multiple stages which should be carefullytaken into account for end-to-end channel modeling. Forinstance, for the NT receiver R1 in Fig. 9, the NTsactivate ligand-gated sodium channels which allow thesodium ions to enter the receiving cell and potentiallytrigger another action. Therefore, the number of sodiumions entering the receiver should be considered as theactual received signal rather than the number of activatedsodium channels. Although these two quantities are related,the latter accounts for the available concentration ofsodium ions outside the receiver, which may considerablyaffect the performance. For example, although signalingparticles (i.e., NTs) may activate many ligand-gatedsodium channels, a strong signal cannot be generated inside the receiver if the concentration of sodium ionsoutside the receiver is low. • Impact of interfaces:
We presented various micro-to-macroscale interfaces to facilitate the experimental eval-uation of the proposed biological building blocks forMCSs. For the experimental verification in Stage 4 of theproposed development roadmap in Fig. 2, the relativeimpact of the microscale MCSs and the interface onthe received signal have to be carefully investigated. Forinstance, the impairments (noises and nonlinearities) thatdiffusion, reactions, and interference from natural cellscause to the received signal are different from those thata light source, a pH meter, or a current meter cause.These two different categories of impairments have to beseparately and quantitatively analyzed.
B. Implementation Challenges
In order to realize the biological building blocks proposedin this article, some technical challenges have to be met, whichare discussed in the following. • Recombinant expression of proteins:
To generate suffi-cient amounts of the required proteins (e.g. those shown inFig. 4), they have to be expressed recombinantly in cellsfrom either prokaryotic (e.g.
E. coli ) or eukaryotic origin(e.g. yeast, insect or mammalian cells). Therefore, a DNAcoding for the respective protein has to be inserted, suchthat the native cellular machinery of protein biosynthesiscan be utilized [251]. The fact that crystal structures formost of the proteins shown in Fig. 4 exist indicates thatprotocols for the recombinant expression and subsequentpurification of larger amounts of these proteins are alreadyavailable. • Additional engineering:
The vast majority of the proteinsproposed as biological transmitter and receiver compo-nents were directly adapted from functional biologicalsystems and do not require further modification, i.e., theycan be readily obtained by recombinant expression or byusing them directly in the context of the native biologicalsystem. However, protein engineering [252], [253] offersthe possibility to modify protein function in order to make
EEE COMMUNICATIONS SURVEYS & TUTORIALS 28 them more suitable for application in MCSs. The simpleststrategy is the introduction of point mutations, i.e., theexchange of single amino acids within the protein. Thisstrategy has for example been applied to engineer a light-driven calcium pump [154], bacteriorhodopsin mutantswith activity at different wavelengths [126], and GFPvariants with different pH optima [130]. This strategymay also be used to design light-driven calcium pumpswhich operate at different wavelengths (prerequisite forreversibility, Table II, T5). Such components have not yetbeen reported, but can presumably be constructed in asimilar manner as the already available bacteriorhodopsinvariants (Fig. 6, T5) by introducing point mutations.Besides point mutations, another strategy for proteinengineering is the fusion to unrelated protein domains ofsynergistic function , which can be considered as part of amodular toolbox [253]. For instance, a fusion construct ofGFP (Fig. 4c) and the calcium-binding protein calmodulinhas led to the development of the calcium sensor GCaMPwhich shows a pronounced fluorescence only upon thepresence of calcium ions [158]. Moreover, light-inducibleprotein kinases have been developed using a similarapproach [191]–[193]. Another interesting applicationwould be a fusion to domains that allow for a fixationat certain carrier materials (e.g. magnetic nanoparticles)[254]–[256] to facilitate the design of MCSs with a definedgeometry.Additional engineering will also be required to implementsome of the more complex systems proposed in this survey.For example, membrane proteins have to be forced intothe correct orientation when they are inserted into vesicles(see below). • Membrane protein reconstitution:
Some of the pro-posed systems require the reconstitution of membraneproteins into vesicles, which is a challenging task. How-ever, the artificial construction of such proteoliposomeshas been widely reported [117]–[121], [257], and dueto the fast progress in the field of synthetic biology,there is a continuous development of corresponding newmethods [257]. As vesicles containing only physiologicaltypes of lipids are sometimes not sufficiently stable, so-called polymersomes which have membranes consistingof non-lipid polymers, or hybrid vesicles containing bothpolymers and lipids may be used as an alternative [117]. • Polymersomes:
The significantly higher mechanical sta-bility is not the only advantage that polymersomes haveover liposomes in the context of MC. The higher stabilitygoes hand in hand with a lower intrinsic permeability,since both aspects correlate with membrane thickness[258]. While lipid membranes are usually 3 – 5 nm thick[259], polymersomes with up to 40 nm thick membraneshave been reported [260]. This leads to permeabilitycoefficients that are several orders of magnitude lower thanthe corresponding values determined for liposomes [261].As a consequence, the uncontrolled passive diffusion of Cooperative function of two or more agents which allows for fasterachievement of a common goal. substances over the compartment boundaries and, thus, thebackground noise in MCS is much lower. The excellentstability and encapsulant retention are also reasons whypolymersomes have attracted significant attention asvehicles for (targeted) drug delivery [262]. In addition, thechemical versatility of the amphiphilic polymer monomersfrom which the polymersomes are formed enables themembrane properties to be tailored to the intended applica-tion. For some polymers, such as the ABA-type triblock co-polymer poly(2-methyloxazoline)-poly(dimethylsiloxane)-poly(2-methyloxazoline) (PMOXA-PDMS-PMOXA) ithas been shown that they do not cause cytotoxic effectsor inflammatory responses in biological systems [263],[264]. PMOXA-PDMS-PMOXA also allows the functionalintegration of diverse transmembrane proteins, as reviewedrecently [265]. With regard to the communication speed,the number of proteins per vesicle is decisive. In poly-mersomes with a mean diameter of 100 nm, a maximumnumber of 120 transmembrane proteins per vesicle hasbeen inserted so far [266]. This number corresponds to acoverage of 3% of the available surface area. However,due to the rapid advances in the field of preparativepurification of membrane proteins and their functionalreconstitution in artificial membranes, it is expected thatthis value will increase in the future [267]. A systematicstudy of membrane protein reconstitution in liposomesand polymersomes revealed that similar amounts ofproteins can be incorporated in both types of vesicle,with a tendency to slightly higher values in liposomes.Interestingly, the specific activity of reconstituted protonpumps was higher in polymer membranes, which pointsto the possibility that the same biological effect can beachieved with a smaller number of membrane proteins[268]. • Membrane permeability:
The permeability coefficientsof lipid membranes for charged molecules are generallylow. As a result, pure lipid membranes are often referred toas “impermeable” for ions if there is no protein-mediatedtransport. However, whether or not a certain membranecan be considered (im)permeable also depends on theconsidered time frame, since any molecule will permeateat some point due to passive diffusion if a concentrationgradient across the membrane is applied. This leads toan uncontrolled mass transport over the compartmentboundaries of vesicles, which increases the backgroundnoise in MCS. This aspect is of special importance forproton-based transmitters involving lipid vesicles (T2-T5,Fig. 6) since the permeability coefficients of protons ( − cm s − ) are several orders of magnitude higher than thecorresponding values for other ions, such as calcium ( . x − cm s − ) [269]. As a consequence, the intrinsicmembrane permeability may not be negligible for certainMCSs. • Optical stimulation:
Some of the suggested buildingblocks require external optical stimulation. Although itwould be preferable to use internal stimuli for in vivo applications because they are less invasive, light is stillconsidered as a unique stimulus since it is easy to control
EEE COMMUNICATIONS SURVEYS & TUTORIALS 29 and does not interfere with other physiological stimuli.For research purposes, it is already nowadays the stateof the art to use optical systems in animals (e.g, livingrats) in order to stimulate light-dependent proteins [270].For instance, optogenetics employs light to stimulategenetically engineered neurons, which express light-drivenion channels [271]–[274]. A further miniaturization of therespective equipment could eventually allow for a medicaluse in humans. The example of deep brain stimulation,which is widely applied for therapy of Parkinson’s disease,dystonia, and obsessive compulsive disorders [275], showsthat such interfaces between technical devices and thehuman body are feasible. • Artificial vesicles vs. entire cells:
Instead of employingartificial vesicles, most of the presented building blockscould be realized using entire cells in which the respectivemembrane proteins are recombinantly expressed. Whilethis may be easier from a technical point of view andfacilitate the implementation of reversibility, it would alsorequire higher concentrations of signaling particles due tothe much bigger size of the transmitter/receiver systems.Moreover, it would most certainly lead to a high level ofnoise because of the high complexity and the plethora ofnatural transmembrane proteins and signaling cascadespresent in cells which could interfere with the desiredsignaling processes. Finally, in the context of applicationswithin the human body (e.g. targeted drug delivery), theusage of entire cells may be problematic because of thehigh amount of surface proteins which could trigger animmune reaction.VIII. C
ONCLUSIONS
In this paper, we provided a comprehensive survey of thebiological components that can serve as building blocks forthe transmitter and the receiver and as signaling particlesfor synthetic MCSs. Adopting a signaling particle centricpresentation, we argue that cations, NTs, and phosphopeptidesrepresent promising candidate signaling particles. They can beused to interact with natural physiological processes and asinformation carriers in synthetic MCS employing engineeredprotein systems as transmitters and receivers. The engineeredtransmitter/receiver systems considered in this survey mainlyrely on physiological protein components of well-characterizedfunctionality. However, the engineering of functional trans-mitter/receiver systems based on these isolated componentsstill remains a challenging task, particularly when insertion ofproteins into vesicle membranes with defined inside-outsidegeometry is required. For each of the presented syntheticMCSs architectures, the ensuing advantages and limitations areoutlined and references to the relevant literature in syntheticbiology are provided. This survey will help both theoreticiansand experimentalists to develop a better understanding ofthe options available for the design and implementation ofbiological synthetic MCSs and interfaces for natural MCSs.Some of the related open research problems for communication-theoretical modeling/design and technical implementation ofthe proposed MCS building blocks were outlined. A
PPENDIX AG LOSSARY OF B IOLOGICAL C ONCEPTS AND T ERMS
For convenience, in Table IX, we explain the most importantbiological concepts and terms appearing in this article.R
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EEE COMMUNICATIONS SURVEYS & TUTORIALS 36
TABLE IXE
XPLANATION OF RELEVANT BIOLOGICAL AND CHEMICAL TERMS . F
OR MORE IN - DEPTH DISCUSSION , WE RECOMMEND REFERENCES [276]–[278].
Term Description
From amino acids to proteins
Amino acid
Organic compound containing an amine (NH ) group, a carboxyl (COOH) group, and a specific side chain.Building block of a protein. Peptide
Short chain of amino acids (smaller than a protein) linked by peptide bonds.
Protein
Large biomolecule, consisting of one or more long chains of amino acids.
Crystal structure
Three-dimensional model of the structure of a molecule (e.g. a protein) resolved by X-ray crystallography.
Residue
Single amino acid within a peptide or protein.
Proteome
Entire set of proteins expressed by a certain organism, tissue or cell.
Protein folding and modifications
Chaperone
Protein that assists the folding process of other proteins or the assembly of macromolecular structures.
Molecular maturation
Collective term for additional modifications taking place after protein biosynthesis, such as cleavage,posttranslational modifications and oligomerization.
Posttranslational modification ofproteins/peptides
Covalent modification of a protein or a peptide after protein biosynthesis.
Methylation/Acetylation/Ubiquitination
Examples for posttranslational modifications: addition of a methyl (CH ) / acetyl (CH CO) / ubiquitin (asmall regulatory protein) group.
Different oligomerization states
Monomer
Single molecule which can undergo oligomerization and thereby contribute a constitutional unit to amacromolecule.
Dimer
Chemical structure formed from two subunits.
Oligomer
Macromolecule formed from a few subunits.
Homo-/Hetero-
Consisting of identical/different sub-molecules.
Protein domains
Protein domain
Part of a protein which can evolve, fold, and function independently from the rest of the protein chain.
Adapter domain
Domain mediating specific interactions with a binding partner.
Homologous domain
Domain which is similar to another one due to a common evolutionary origin.
Enzymes – Biological catalysts
Kinase
Transfers a phosphate group from ATP to a protein.
ATPase
Catalyzes cleavage of ATP to ADP and a phosphate group, uses released energy to drive a chemical reactionthat would not occur otherwise.
Protease
Cleaves a protein by hydrolysis of peptide bonds.
Phosphatase
Removes a phosphate group from a phosphorylated protein.
Allosteric regulation
Regulation of an enzyme by binding an effector molecule at a site other than the enzyme’s active site.
Overview of chemical terms
Moiety
Chemical group (e.g. methyl moiety, acetyl moiety, peptide moiety)
Acidic conditions
Environmental conditions characterized by a low pH value, i.e., a high proton concentration.
Protonation
Covalent addition of a proton to a molecule, formation of the conjugate acid.
Hydrolysis
Cleavage of a biomolecule accompanied by the consumption of a water molecule.
Chelating agent
Chemical agent that binds certain metal ions by forming two or more coordinate bonds. May be used toremove or chemically shield the metal ions.
Miscellaneous
Surface-plasmon resonance
Biophysical method to detect biomolecular interactions.
Recombinant DNA/protein
DNA/protein sequence engineered by laboratory methods of genetic recombination such as molecular cloning.
EEE COMMUNICATIONS SURVEYS & TUTORIALS 37
Christian A. S¨oldner received the B. Sc. and M.Sc. degree in molecular medicine from the FriedrichAlexander University Erlangen-N¨urnberg, Germany,in 2014 and 2016, respectively. During his Ph.D. inthe subject of bioinformatics, he focused his researchinterest on the analysis of receptor-ligand interactions.He studied GPCRs and the macrophage surfacereceptor Mincle by means of molecular dynamicssimulations and developed a metadynamics-basedprotocol for the determination of ligand bindingmodes. After his Ph.D. degree, which he receivedin 2020, he joined Siemens Healthineers and is currently working as anapplication specialist and sequence developer for magnetic resonance imaging.
Eileen Socher received the B.Sc. and M.Sc. degreesin Molecular Medicine (with a focus on pathol-ogy and bioinformatics) from Friedrich-Alexander-University Erlangen-N¨urnberg (FAU), Erlangen, Ger-many, in 2011 and 2012, respectively. In 2017, shereceived the doctoral degree in the field of bioin-formatics from the Faculty of Sciences, Friedrich-Alexander-University Erlangen-N¨urnberg (FAU). Cur-rently, she is a postdoctoral researcher at the FAUFaculty of Medicine in Erlangen, Germany. Herresearch interests include structural bioinformatics ingeneral and the molecular modelling of proteins as well as their investigationby using molecular dynamics simulations in particular. Within this field, sheworks, for instance, on the computer-based analysis of pH-induced effectson protein structure or protein-protein interactions between viral and humanproteins.
Vahid Jamali (S’12, M’20) received the B.Sc. andM.Sc. degrees (honors) in electrical engineeringfrom the K. N. Toosi University of Technology,Tehran, Iran, in 2010 and 2012, respectively, and thePh.D. degree (with distinctions) from the Friedrich-Alexander-University (FAU) of Erlangen-N¨urnberg,Erlangen, Germany, in 2019. In 2017, he was aVisiting Research Scholar with Stanford University,CA, USA. He is currently a Postdoctoral Researcherwith the Institute for Digital Communication, FAU.His research interests include wireless and molecularcommunications, Bayesian inference and learning, and multiuser informa-tion theory.Dr. Jamali has served as a member of the Technical Program Committeefor several IEEE conferences and he is currently an Associate Editor ofthe IEEE C
OMMUNICATIONS L ETTERS and IEEE O
PEN J OURNAL OF THE C OMMUNICATIONS S OCIETY . He received several awards for his publicationsand research work including the Best Paper Award from the IEEE InternationalConference on Communications in 2016, the Doctoral Research Grant fromthe German Academic Exchange Service (DAAD) in 2017, the GoldenerIgel Publication Award from the Telecommunications Laboratory (LNT),FAU, in 2018, the Best Ph.D. Thesis Presentation Award from the IEEEWireless Communications and Networking Conference in 2018, the Best PaperAward from the ACM International Conference on Nanoscale Computing andCommunication in 2019, and the Postdoctoral Research Fellowship by theGerman Research Foundation (DFG) in 2020.
Wayan Wicke (S’17) was born in Nuremberg,Germany, in 1991. He received the B.Sc. and M.Sc.degrees in electrical engineering from the Friedrich-Alexander University Erlangen-N¨urnberg (FAU), Er-langen, Germany, in 2014 and 2017, respectively,where he is currently pursuing the Ph.D. degree. Hisresearch interests include statistical signal processingand digital communications with a focus on molecularcommunication.
Arman Ahmadzadeh (S’14) received the B.Sc.degree in electrical engineering from the FerdowsiUniversity of Mashhad, Mashhad, Iran, in 2010, andthe M.Sc. degree in communications and multimediaengineering from the Friedrich-Alexander-Universit¨at(FAU) Erlangen-N¨urnberg, Erlangen, Germany, in2013, where he is currently working toward the Ph.D.degree in electrical engineering at the Institute forDigital Communications. His current research inter-ests include physical layer molecular communications.Mr. Ahmadzadeh has served as a member of theTechnical Program Committee of the Communication Theory Symposiumfor the IEEE International Conference on Communications (ICC) 2017-2020.He received several awards including the Best Paper Award from the IEEEICC in 2016, IEEE ICC in 2020, and the Student Travel Grants for attendingthe Global Communications Conference (GLOBECOM) in 2017. He wasrecognized as an Exemplary Reviewer of IEEE C
OMMUNICATIONS L ETTERS in 2016.
Hans-Georg Breitinger received the Ph.D. in Or-ganic Chemistry from Heinrich Heine University(HHU), D¨usseldorf, Germany, in 1993. He was a post-doctoral researcher at Cornell University from 1993-1997, and completed the Habilitation (Universityteaching certificate) for Biochemistry at Friedtrich-Alexander University, Erlangen, in 2003. In 2004 hejoined the German University in Cairo as Full Pro-fessor and Head of the Department of Biochemistry.His research focuses on neuronal ion channels andviroporins, the biochemistry of membrane receptors,antimicrobial peptides and isolation of new pharmaceuticals from naturalresources. His postdoctoral stay at Cornell University was supported by afellowship of the Swiss Federation of Sciences.
Andreas Burkovski received his diploma in Biologyin 1989 and his doctoral degree in 1993, both at theUniversity of Osnabr¨uck. After postdoc positionsat the University of Osnabr¨uck and the ResearchCenter J¨ulich, he became group leader at the Uni-versity of Cologne in 1997. In 2005 he received aprofessorship in Microbiology at the University ofErlangen-Nuremberg. His research interests focus onthe analysis of host-pathogen-interactions, regulatorynetworks and synthetic biology.
EEE COMMUNICATIONS SURVEYS & TUTORIALS 38
Kathrin Castiglione received the B.Sc. and M.Sc.degrees in Molecular Biotechnology from the Tech-nical University of Munich (TUM). After her PhDat the Institute of Biochemical Engineering of TUMin 2009, she moved to the Toyama PrefecturalUniversity in Japan as a postdoctoral fellow of theJapanese Society of the Promotion of Science. Atthe end of 2010, she returned to TUM and becamehead of the biocatalysis group at the Institute ofBiochemical Engineering. Since 2012 she has beenthe leader of an independent junior research groupworking on artificial reaction compartments based on functionalized polymervesicles. Since 2018 Kathrin Castiglione has headed the Institute of BioprocessEngineering of the Friedrich-Alexander University Erlangen-N¨urnberg (FAU).Among other things, she works on the design of functionalized polymersomesfor molecular communication.
Robert Schober (S’98, M’01, SM’08, F’10) receivedthe Diplom (Univ.) and the Ph.D. degrees in electricalengineering from the Friedrich-Alexander University(FAU) Erlangen-N¨urnberg, Erlangen, Germany, in1997 and 2000, respectively. From 2002 to 2011, hewas a Professor and Canada Research Chair at theUniversity of British Columbia (UBC), Vancouver,Canada. Since January 2012, he is an Alexandervon Humboldt Professor and the Chair for DigitalCommunication at FAU. His research interests fallinto the broad areas of Communication Theory,Wireless Communications, and Statistical Signal Processing.Robert received several awards for his work including the 2002 HeinzMaier-Leibnitz Award of the German Science Foundation (DFG), the 2004Innovations Award of the Vodafone Foundation for Research in MobileCommunications, a 2006 UBC Killam Research Prize, a 2007 WilhelmFriedrich Bessel Research Award of the Alexander von Humboldt Foundation,the 2008 Charles McDowell Award for Excellence in Research from UBC, a2011 Alexander von Humboldt Professorship, a 2012 NSERC E.W.R. StacieFellowship, and a 2017 Wireless Communications Recognition Award bythe IEEE Wireless Communications Technical Committee. He is listed as a2017 Highly Cited Researcher by the Web of Science and a DistinguishedLecturer of the IEEE Communications Society (ComSoc). Robert is a Fellowof the Canadian Academy of Engineering and a Fellow of the EngineeringInstitute of Canada. From 2012 to 2015, he served as Editor-in-Chief of theIEEE T
RANSACTIONS ON C OMMUNICATIONS . Currently, he is the Chairof the Steering Committee of the IEEE T
RANSACTIONS ON M OLECULAR ,B IOLOGICAL AND M ULTI -S CALE C OMMUNICATIONS , a Member of theEditorial Board of the Proceedings of the IEEE, a Member at Large of theBoard of Governors of ComSoc, and the ComSoc Director of Journals.