Comparison of actin- and microtubule-based motility systems for application in functional nanodevices

Salhotra A., Zhu J., Surendiran P., Meinecke C.R., Lyttleton R., Ušaj M., Lindberg F.W., Norrby M., Linke H., Månsson A.

Abstract Significant advancements have been made towards exploitation of naturally available molecular motors and their associated cytoskeletal filaments in nanotechnological applications. For instance, myosin motors and actin filaments from muscle have been used with the aims to establish new approaches in biosensing and network-based biocomputation. The basis for these developments is a version of the in vitro motility assay (IVMA) where surface-adsorbed myosin motors propel the actin filaments along suitably derivatized nano-scale channels on nanostructured chips. These chips are generally assembled into custom-made microfluidic flow cells. For effective applications, particularly in biocomputation, it is important to appreciably prolong function of the biological system. Here, we systematically investigated potentially critical factors necessary to achieve this, such as biocompatibility of different components of the flow cell, the degree of air exposure, assay solution composition and nanofabrication methods. After optimizing these factors we prolonged the function of actin and myosin in nanodevices for biocomputation from <20 min to >60 min. In addition, we demonstrated that further optimizations could increase motility run times to >20 h. Of great importance for the latter development was a switch of glucose oxidase in the chemical oxygen scavenger system (glucose oxidase–glucose–catalase) to pyranose oxidase, combined with the use of blocking actin (non-fluorescent filaments that block dead motors). To allow effective testing of these approaches we adapted commercially available microfluidic channel slides, for the first time demonstrating their usefulness in the IVMA. As part of our study, we also demonstrate that myosin motor fragments can be stored at −80 °C for more than 10 years before use for nanotechnological purposes. This extended shelf-life is important for the sustainability of network-based biocomputation.

Biquet-Bisquert A., Labesse G., Pedaci F., Nord A.L.

The bacterial flagellar motor (BFM) is a rotary molecular motor embedded in the cell membrane of numerous bacteria. It turns a flagellum which acts as a propeller, enabling bacterial motility and chemotaxis. The BFM is rotated by stator units, inner membrane protein complexes that stochastically associate to and dissociate from individual motors at a rate which depends on the mechanical and electrochemical environment. Stator units consume the ion motive force (IMF), the electrochemical gradient across the inner membrane that results from cellular respiration, converting the electrochemical energy of translocated ions into mechanical energy, imparted to the rotor. Here, we review some of the main results that form the base of our current understanding of the relationship between the IMF and the functioning of the flagellar motor. We examine a series of studies that establish a linear proportionality between IMF and motor speed, and we discuss more recent evidence that the stator units sense the IMF, altering their rates of dynamic assembly. This, in turn, raises the question of to what degree the classical dependence of motor speed on IMF is due to stator dynamics vs. the rate of ion flow through the stators. Finally, while long assumed to be static and homogeneous, there is mounting evidence that the IMF is dynamic, and that its fluctuations control important phenomena such as cell-to-cell signaling and mechanotransduction. Within the growing toolbox of single cell bacterial electrophysiology, one of the best tools to probe IMF fluctuations may, ironically, be the motor that consumes it. Perfecting our incomplete understanding of how the BFM employs the energy of ion flow will help decipher the dynamical behavior of the bacterial IMF.

Ruijgrok P.V., Ghosh R.P., Zemsky S., Nakamura M., Gong R., Ning L., Chen R., Vachharajani V.T., Chu A.E., Anand N., Eguchi R.R., Huang P., Lin M.Z., Alushin G.M., Liphardt J.T., et. al.

Precision tools for spatiotemporal control of cytoskeletal motor function are needed to dissect fundamental biological processes ranging from intracellular transport to cell migration and division. Direct optical control of motor speed and direction is one promising approach, but it remains a challenge to engineer controllable motors with desirable properties such as the speed and processivity required for transport applications in living cells. Here, we develop engineered myosin motors that combine large optical modulation depths with high velocities, and create processive myosin motors with optically controllable directionality. We characterize the performance of the motors using in vitro motility assays, single-molecule tracking and live-cell imaging. Bidirectional processive motors move efficiently toward the tips of cellular protrusions in the presence of blue light, and can transport molecular cargo in cells. Robust gearshifting myosins will further enable programmable transport in contexts ranging from in vitro active matter reconstitutions to microfabricated systems that harness molecular propulsion. High-performance engineered myosins robustly change speed or direction in response to an optical signal. In living cells, these motors localize to the tips of protrusions when illuminated and deliver molecular cargos.

Théry M., Blanchoin L.

The stochastic switching between microtubule growth and shrinkage is a fascinating and unique process in the regulation of the cytoskeleton. To understand it, almost all attention has been focused on the microtubule ends. However, recent research has revived the idea that tubulin dimers can also be exchanged in protofilaments along the microtubule shaft, thus repairing the microtubule and protecting it from disassembly. Here, we review the research describing this phenomenon, the mechanisms regulating the removal and insertion of tubulin dimers, as well as the potential implications for key functions of the microtubule network, such as intracellular transport and cell polarization.

Ušaj M., Moretto L., Vemula V., Salhotra A., Månsson A.

Benefits of single molecule studies of biomolecules include the need for minimal amounts of material and the potential to reveal phenomena hidden in ensembles. However, results from recent single molecule studies of fluorescent ATP turnover by myosin are difficult to reconcile with ensemble studies. We found that key reasons are complexities due to dye photophysics and fluorescent contaminants. After eliminating these, through surface cleaning and use of triple state quenchers and redox agents, the distributions of ATP binding dwell times on myosin are best described by 2 to 3 exponential processes, with and without actin, and with and without the inhibitor para-aminoblebbistatin. Two processes are attributable to ATP turnover by myosin and actomyosin respectively, whereas the remaining process (rate constant 0.2–0.5 s−1) is consistent with non-specific ATP binding to myosin, possibly accelerating ATP transport to the active site. Finally, our study of actin-activated myosin ATP turnover without sliding between actin and myosin reveals heterogeneity in the ATP turnover kinetics consistent with models of isometric contraction. With fluorescence based-TIRF microspectroscopy, Ušaj et al. unveil mechanistic details about the ATP turnover rates by myosin and actomyosin with enzymatic reaction pathways that were not possible to obtain from ensemble studies. This study could be important to the field of molecular motors.

Hennell James R., Deme J.C., Kjӕr A., Alcock F., Silale A., Lauber F., Johnson S., Berks B.C., Lea S.M.

Three classes of ion-driven protein motors have been identified to date: ATP synthase, the bacterial flagellar motor and a proton-driven motor that powers gliding motility and the type 9 protein secretion system in Bacteroidetes bacteria. Here, we present cryo-electron microscopy structures of the gliding motility/type 9 protein secretion system motors GldLM from Flavobacterium johnsoniae and PorLM from Porphyromonas gingivalis. The motor is an asymmetric inner membrane protein complex in which the single transmembrane helices of two periplasm-spanning GldM/PorM proteins are positioned inside a ring of five GldL/PorL proteins. Mutagenesis and single-molecule tracking identify protonatable amino acid residues in the transmembrane domain of the complex that are important for motor function. Our data provide evidence for a mechanism in which proton flow results in rotation of the periplasm-spanning GldM/PorM dimer inside the intra-membrane GldL/PorL ring to drive processes at the bacterial outer membrane. Using cryo-electron microscopy, the authors describe the structure and function of a molecular motor powering both the Bacteroidetes type 9 protein secretion system and the associated gliding motility apparatus.

Vemula V., Huber T., Ušaj M., Bugyi B., Månsson A.

Actin is a major intracellular protein with key functions in cellular motility, signaling, and structural rearrangements. Its dynamic behavior, such as polymerization and depolymerization of actin filaments in response to intracellular and extracellular cues, is regulated by an abundance of actin binding proteins. Out of these, gelsolin is one of the most potent for filament severing. However, myosin motor activity also fragments actin filaments through motor-induced forces, suggesting that these two proteins could cooperate to regulate filament dynamics and motility. To test this idea, we used an in vitro motility assay, where actin filaments are propelled by surface-adsorbed heavy meromyosin (HMM) motor fragments. This allows studies of both motility and filament dynamics using isolated proteins. Gelsolin, at both nanomolar and micromolar Ca2+ concentration, appreciably enhanced actin filament severing caused by HMM-induced forces at 1 mM MgATP, an effect that was increased at higher HMM motor density. This finding is consistent with cooperativity between actin filament severing by myosin-induced forces and by gelsolin. We also observed reduced sliding velocity of the HMM-propelled filaments in the presence of gelsolin, providing further support of myosin-gelsolin cooperativity. Total internal reflection fluorescence microscopy–based single molecule studies corroborated that the velocity reduction was a direct effect of gelsolin binding to the filament and revealed different filament severing pattern of stationary and HMM propelled filaments. Overall, the results corroborate cooperative effects between gelsolin-induced alterations in the actin filaments and changes due to myosin motor activity leading to enhanced F-actin severing of possible physiological relevance.

Canty J.T., Yildiz A.

Cytoplasmic dynein is an AAA+ motor that drives the transport of many intracellular cargoes towards the minus end of microtubules (MTs). Previous in vitro studies characterized isolated dynein as an exceptionally weak motor that moves slowly and diffuses on an MT. Recent studies altered this view by demonstrating that dynein remains in an autoinhibited conformation on its own, and processive motility is activated when it forms a ternary complex with dynactin and a cargo adaptor. This complex assembles more efficiently in the presence of Lis1, providing an explanation for why Lis1 is a required cofactor for most cytoplasmic dynein-driven processes in cells. This review describes how dynein motility is activated and regulated by cargo adaptors and accessory proteins.

Robert-Paganin J., Pylypenko O., Kikuti C., Sweeney H.L., Houdusse A.

Generating force and movement is essential for the functions of cells and organisms. A variety of molecular motors that can move on tracks within cells have evolved to serve this role. How these motors interact with their tracks and how that, in turn, leads to the generation of force and movement is key to understanding the cellular roles that these motor-track systems serve. This review is focused on the best understood of these systems, which is the molecular motor myosin that moves on tracks of filamentous (F-) actin. The review highlights both the progress and the limits of our current understanding of how force generation can be controlled by F-actin-myosin interactions. What has emerged are insights they may serve as a framework for understanding the design principles of a number of types of molecular motors and their interactions with their tracks.

Funk J., Merino F., Venkova L., Heydenreich L., Kierfeld J., Vargas P., Raunser S., Piel M., Bieling P.

The actin cytoskeleton drives many essential biological processes, from cell morphogenesis to motility. Assembly of functional actin networks requires control over the speed at which actin filaments grow. How this can be achieved at the high and variable levels of soluble actin subunits found in cells is unclear. Here we reconstitute assembly of mammalian, non-muscle actin filaments from physiological concentrations of profilin-actin. We discover that under these conditions, filament growth is limited by profilin dissociating from the filament end and the speed of elongation becomes insensitive to the concentration of soluble subunits. Profilin release can be directly promoted by formin actin polymerases even at saturating profilin-actin concentrations. We demonstrate that mammalian cells indeed operate at the limit to actin filament growth imposed by profilin and formins. Our results reveal how synergy between profilin and formins generates robust filament growth rates that are resilient to changes in the soluble subunit concentration.

Farhana T.I., Nakagawa T., Ohara S., Shintaku H., Kotera H., Yokokawa R.

Cooperativity of motor proteins is essential for intracellular transport. Although their motion is unidirectional, they often cause bidirectional movement by different types of motors as seen in organelles. However, in vitro assessments of such cellular functions are still inadequate owing to the experimental limitations in precisely patterning multiple motors. Here, we present an approach to immobilize two motor proteins, kinesin-1 and dynein, using the aqueous two-phase system (ATPS) made of poly(ethylene glycol) and dextran polymers. The negligible influence of polymer solutions on the attachment and velocity of motor proteins ensures the compatibility of using ATPS as the patterning technique. The selective fixation of kinesin and dynein was assessed using polarity-marked microtubules (PMMTs). Our experimental results show that on a patterned kinesin surface, 72% of PMMTs display minus-end leading motility, while on a dynein surface, 79% of PMMTs display plus-end leading motility. This work offers a universal and biocompatible method to pattern motor proteins of different classes at the nanoscale, providing a new route to study different cellular functions performed by molecular motors such as the formation of mitotic spindles.

Saper G., Hess H.

Biological molecular motors (or biomolecular motors for short) are nature's solution to the efficient conversion of chemical energy to mechanical movement. In biological systems, these fascinating molecules are responsible for movement of molecules, organelles, cells, and whole animals. In engineered systems, these motors can potentially be used to power actuators and engines, shuttle cargo to sensors, and enable new computing paradigms. Here, we review the progress in the past decade in the integration of biomolecular motors into hybrid nanosystems. After briefly introducing the motor proteins kinesin and myosin and their associated cytoskeletal filaments, we review recent work aiming for the integration of these biomolecular motors into actuators, sensors, and computing devices. In some systems, the creation of mechanical work and the processing of information become intertwined at the molecular scale, creating a fascinating type of "active matter". We discuss efforts to optimize biomolecular motor performance, construct new motors combining artificial and biological components, and contrast biomolecular motors with current artificial molecular motors. A recurrent theme in the work of the past decade was the induction and utilization of collective behavior between motile systems powered by biomolecular motors, and we discuss these advances. The exertion of external control over the motile structures powered by biomolecular motors has remained a topic of many studies describing exciting progress. Finally, we review the current limitations and challenges for the construction of hybrid systems powered by biomolecular motors and try to ascertain if there are theoretical performance limits. Engineering with biomolecular motors has the potential to yield commercially viable devices, but it also sharpens our understanding of the design problems solved by evolution in nature. This increased understanding is valuable for synthetic biology and potentially also for medicine.

Rahman M.A., Reuther C., Lindberg F.W., Mengoni M., Salhotra A., Heldt G., Linke H., Diez S., Månsson A.

The guided gliding of cytoskeletal filaments, driven by biomolecular motors on nano/microstructured chips, enables novel applications in biosensing and biocomputation. However, expensive and time-consuming chip production hampers the developments. It is therefore important to establish protocols to regenerate the chips, preferably without the need to dismantle the assembled microfluidic devices which contain the structured chips. We here describe a novel method toward this end. Specifically, we use the small, nonselective proteolytic enzyme, proteinase K to cleave all surface-adsorbed proteins, including myosin and kinesin motors. Subsequently, we apply a detergent (5% SDS or 0.05% Triton X100) to remove the protein remnants. After this procedure, fresh motor proteins and filaments can be added for new experiments. Both, silanized glass surfaces for actin-myosin motility and pure glass surfaces for microtubule-kinesin motility were repeatedly regenerated using this approach. Moreover, we demonstrate the applicability of the method for the regeneration of nano/microstructured silicon-based chips with selectively functionalized areas for supporting or suppressing gliding motility for both motor systems. The results substantiate the versatility and a promising broad use of the method for regenerating a wide range of protein-based nano/microdevices.

Matusovsky O.S., Mansson A., Persson M., Cheng Y., Rassier D.E.

Significance The advent of high-speed atomic force microscopy (HS-AFM) changed the field of biology considerably. HS-AFM is the only method where in situ dynamics of biological samples and imaging can be coupled with a spatial resolution of 1 to 5 nm in the horizontal direction. Unlike electron or cryo-electron microscopy, HS-AFM does not require fixation or freezing of the samples, and has the ability to derive kinetic parameters by recording the live movements of single-molecule dynamics. In this paper, we used HS-AFM to investigate directly the mechanisms of cardiac muscle activation. We visualized the muscle regulatory tropomyosin–troponin complex movements during activation by calcium or myosin (motor that drives contraction), and the structural transitions that happen during these events.

Garcin C., Straube A.

Abstract Directed cell migration is critical for embryogenesis and organ development, wound healing and the immune response. Microtubules are dynamic polymers that control directional migration through a number of coordinated processes: microtubules are the tracks for long-distance intracellular transport, crucial for delivery of new membrane components and signalling molecules to the leading edge of a migrating cell and the recycling of adhesion receptors. Microtubules act as force generators and compressive elements to support sustained cell protrusions. The assembly and disassembly of microtubules is coupled to Rho GTPase signalling, thereby controlling actin polymerisation, myosin-driven contractility and the turnover of cellular adhesions locally. Cross-talk of actin and microtubule dynamics is mediated through a number of common binding proteins and regulators. Furthermore, cortical microtubule capture sites are physically linked to focal adhesions, facilitating the delivery of secretory vesicles and efficient cross-talk. Here we summarise the diverse functions of microtubules during cell migration, aiming to show how they contribute to the spatially and temporally coordinated sequence of events that permit efficient, directional and persistent migration.

Takatori S.C., Quah T., Rawlings J.B.

Feedback control is essential to the performance of dynamical systems, helping to drive nonequilibrium systems from one state to another. In this review, we discuss feedback control applied to living and synthetic active matter—systems that are constantly dynamical and out of equilibrium. We review the experimental and theoretical work in controlling the trajectory and distribution of active matter, from single particles to collective populations. Modern advances in microscopy and numerical computation have enabled data-rich studies of active systems, aided by data-driven approaches to model, forecast, and control the complex and chaotic behaviors of active matter. We describe the basic mathematical structure of active Brownian particles, with a focus on observability and time delay embedding to control particle motion using density data alone. Finally, we comment on the future outlook of controlling complex systems with multibody interparticle and hydrodynamic interactions.

Melbacke A., Salhotra A., Ušaj M., Månsson A.

AbstractIn the in vitro motility assay (IVMA), actin filaments are observed while propelled by surface-adsorbed myosin motor fragments such as heavy meromyosin (HMM). In addition to fundamental studies, the IVMA is the basis for a range of lab-on-a-chip applications, e.g. transport of cargoes in nanofabricated channels in nanoseparation/biosensing or the solution of combinatorial mathematical problems in network-based biocomputation. In these applications, prolonged myosin function is critical as is the potential to repeatedly exchange experimental solutions without functional deterioration. We here elucidate key factors of importance in these regards. Our findings support a hypothesis that early deterioration in the IVMA is primarily due to oxygen entrance into in vitro motility assay flow cells. In the presence of a typically used oxygen scavenger mixture (glucose oxidase, glucose, and catalase), this leads to pH reduction by a glucose oxidase-catalyzed reaction between glucose and oxygen but also contributes to functional deterioration by other mechanisms. Our studies further demonstrate challenges associated with evaporation and loss of actin filaments with time. However, over 8 h at 21–26 °C, there is no significant surface desorption or denaturation of HMM if solutions are exchanged manually every 30 min. We arrive at an optimized protocol with repeated exchange of carefully degassed assay solution of 45 mM ionic strength, at 30 min intervals. This is sufficient to maintain the high-quality function in an IVMA over 8 h at 21–26 °C, provided that fresh actin filaments are re-supplied in connection with each assay solution exchange. Finally, we demonstrate adaptation to a microfluidic platform and identify challenges that remain to be solved for real lab-on-a-chip applications.

Tu X., Yin S., Zang J., Zhang T., Lv C., Zhao G.

Barakat J.M., Modica K.J., Lu L., Anujarerat S., Choi K.H., Takatori S.C.

Melbacke A., Salhotra A., Usaj M., Månsson A.

Abstract In the in vitro motility assay (IVMA), actin filaments are observed while propelled by surface-adsorbed myosin motor fragments such as heavy meromyosin (HMM). In addition to fundamental studies, the IVMA is the basis for a range of lab-on-a-chip applications, e.g. transport of cargoes in nanofabricated channels in nanoseparation/biosensing or the solution of combinatorial mathematical problems in network-based biocomputation. In these applications, prolonged myosin function is critical as is the potential to repeatedly exchange experimental solutions without functional deterioration. We here elucidate key factors of importance in these regards. Our findings support a hypothesis that early deterioration in the IVMA is primarily due to oxygen entrance into in vitro motility assay flow cells. In the presence of a typically used oxygen scavenger mixture (glucose oxidase, glucose, and catalase), this leads to pH reduction by a glucose oxidase-catalyzed reaction between glucose and oxygen but also contributes to functional deterioration by other mechanisms. Our studies further demonstrate challenges associated with evaporation and loss of actin filaments with time. However, over 8 h at 21-26 oC, there is no significant surface desorption or denaturation of HMM if solutions are exchanged manually every 30 min. We arrive at an optimized protocol with repeated exchange of carefully degassed assay solution of 45 mM ionic strength, at 30 min intervals. This is sufficient to maintain the high-quality function in an IVMA over 8 h at 21-26 oC, provided that fresh actin filaments are re-supplied in connection with each assay solution exchange. Finally, we demonstrate adaptation to a microfluidic platform and identify challenges that remain to be solved for real lab-on-a-chip applications.

Salhotra A., Rahman M.A., Ruijgrok P.V., Meinecke C.R., Ušaj M., Zemsky S., Lindberg F.W., Surendiran P., Lyttleton R.W., Linke H., Korten T., Bryant Z., Månsson A.

Ramm B., Khmelinskaia A., Franquelim H.G., Schwille P.

AbstractSpatial organization on the atomic scale is one of the key objectives of nanotechnology. The development of DNA nanotechnology is a hallmark of material programmability in 2D and 3D, in which the large variety of available DNA modifications allows it to be interfaced with a number of inorganic and organic materials. Nature’s solution to spatiotemporal control has been the evolution of self-organizing protein systems capable of pattern formation through energy dissipation. Here, we show that combining DNA origami with a minimal micron-scale pattern-forming system vastly expands the applicability of DNA nanotechnology, whether for the development of biocompatible materials or as an essential step toward building synthetic cells from the bottom up. We first describe the interaction of DNA origami nanostructures with model lipid membranes and introduce the self-organizing MinDE protein system from Escherichia coli. We then outline how we used DNA origami to elucidate diffusiophoresis on membranes through MinDE protein pattern formation. We describe how this novel biological transport mechanism can, in turn, be harnessed to pattern DNA origami nanostructures on the micron scale on lipid membranes. Finally, we discuss how our approach could be used to create the next generation of hybrid materials, through cargo delivery and multiscale molecular patterning capabilities.

Kalra A.P., Biswas S., Mulrain I., Wang M., Tuszynski J.A., Scholes G.D.

Månsson A.

ABSTRACTSince the late 1990s, efforts have been made to utilize cytoskeletal filaments, propelled by molecular motors, for nanobiotechnological applications, for example, in biosensing and parallel computation. This work has led to in-depth insights into the advantages and challenges of such motor-based systems, and has yielded small-scale, proof-of-principle applications but, to date, no commercially viable devices. Additionally, these studies have also elucidated fundamental motor and filament properties, as well as providing other insights obtained from biophysical assays in which molecular motors and other proteins are immobilized on artificial surfaces. In this Perspective, I discuss the progress towards practically viable applications achieved so far using the myosin II–actin motor–filament system. I also highlight several fundamental pieces of insights derived from the studies. Finally, I consider what may be required to achieve real devices in the future or at least to allow future studies with a satisfactory cost–benefit ratio.

Karan C., Chaudhuri D.

We consider a bio-filament under the collective drive of motor proteins to obtain an active Langevin description of its dynamics. The competition between motor proteins generates self-load captured by an effective temperature.

Kalra A.P., Benny A., Travis S.M., Zizzi E.A., Morales-Sanchez A., Oblinsky D.G., Craddock T.J., Hameroff S.R., MacIver M.B., Tuszyński J.A., Petry S., Penrose R., Scholes G.D.

The repeating arrangement of tubulin dimers confers great mechanical strength to microtubules, which are used as scaffolds for intracellular macromolecular transport in cells and exploited in biohybrid devices. The crystalline order in a microtubule, with lattice constants short enough to allow energy transfer between amino acid chromophores, is similar to synthetic structures designed for light harvesting. After photoexcitation, can these amino acid chromophores transfer excitation energy along the microtubule like a natural or artificial light-harvesting system? Here, we use tryptophan autofluorescence lifetimes to probe energy hopping between aromatic residues in tubulin and microtubules. By studying how the quencher concentration alters tryptophan autofluorescence lifetimes, we demonstrate that electronic energy can diffuse over 6.6 nm in microtubules. We discover that while diffusion lengths are influenced by tubulin polymerization state (free tubulin versus tubulin in the microtubule lattice), they are not significantly altered by the average number of protofilaments (13 versus 14). We also demonstrate that the presence of the anesthetics etomidate and isoflurane reduce exciton diffusion. Energy transport as explained by conventional Förster theory (accommodating for interactions between tryptophan and tyrosine residues) does not sufficiently explain our observations. Our studies indicate that microtubules are, unexpectedly, effective light harvesters.

Kučera O., Gaillard J., Guérin C., Utzschneider C., Théry M., Blanchoin L.

Motility assays use surface-immobilized molecular motors to propel cytoskeletal filaments. They have been widely used to characterize motor properties and their impact on cytoskeletal self-organization. Moreover, the motility assays are a promising class of bioinspired active tools for nanotechnological applications. While these assays involve controlling the filament direction and speed, either as a sensory readout or a functional feature, designing a subtle control embedded in the assay is an ongoing challenge. Here, we investigate the interaction between gliding microtubules and networks of actin filaments. We demonstrate that the microtubule's behavior depends on the actin architecture. Both unbranched and branched actin decelerate microtubule gliding; however, an unbranched actin network provides additional guidance and effectively steers the microtubules. This effect, which resembles the recognition of cortical actin by microtubules, is a conceptually new means of controlling the filament gliding with potential application in the design of active materials and cytoskeletal nanodevices.

van Delft F.C., Månsson A., Kugler H., Korten T., Reuther C., Zhu J., Lyttleton R., Blaudeck T., Meinecke C.R., Reuter D., Diez S., Linke H.

Abstract Network-based biocomputation (NBC) is an alternative, parallel computation approach that can potentially solve technologically important, combinatorial problems with much lower energy consumption than electronic processors. In NBC, a combinatorial problem is encoded into a physical, nanofabricated network. The problem is solved by biological agents (such as cytoskeletal filaments driven by molecular motors) that explore all possible pathways through the network in a massively parallel and highly energy-efficient manner. Whereas there is currently a rapid development in the size and types of problems that can be solved by NBC in proof-of-principle experiments, significant challenges still need to be overcome before NBC can be scaled up to fill a technological niche and reach an industrial level of manufacturing. Here, we provide a roadmap that identifies key scientific and technological needs. Specifically, we identify technology benchmarks that need to be reached or overcome, as well as possible solutions for how to achieve this. These include methods for large-scale production of nanoscale physical networks, for dynamically changing pathways in these networks, for encoding information onto biological agents, for single-molecule readout technology, as well as the integration of each of these approaches in large-scale production. We also introduce figures of merit that help analyze the scalability of various types of NBC networks and we use these to evaluate scenarios for major technological impact of NBC. A major milestone for NBC will be to increase parallelization to a point where the technology is able to outperform the current run time of electronic processors. If this can be achieved, NBC would offer a drastic advantage in terms of orders of magnitude lower energy consumption. In addition, the fundamentally different architecture of NBC compared to conventional electronic computers may make it more advantageous to use NBC to solve certain types of problems and instances that are easy to parallelize. To achieve these objectives, the purpose of this roadmap is to identify pre-competitive research domains, enabling cooperation between industry, institutes, and universities for sharing research and development efforts and reducing development cost and time.

Kučera O., Gaillard J., Guérin C., Théry M., Blanchoin L.

AbstractCytoskeletal motility assays use surface-immobilised molecular motors to propel cytoskeletal filaments. These assays have been widely employed to characterise the motor properties and interactions of cytoskeletal elements with themselves or with external factors. Moreover, the motility assays are a promising class of bio-inspired active tools for nanotechnological applications. While effective utilisation of these assays involves controlling the filament direction and speed, either as a sensory readout or a functional feature, designing a subtle control embedded in the assay is an ongoing challenge. Here we investigate the interaction between motor-propelled microtubules and networks of actin filaments. We demonstrate that the microtubules respond to a network of actin filaments and that this response depends on the network’s architecture. Both linear actin filaments and a network of actin branched by the Arp2/3 complex decelerate microtubule gliding; however, an unbranched actin network provides additional guidance and effectively steers the microtubules. This effect, which resembles the recognition of cortical actin architecture by microtubules, is a conceptually new means of controlling the filament gliding in the motility assay with potential application in the design of active materials and cytoskeletal nano-devices.

Zhang W., Cho W.C., Bloukh S.H., Edis Z., Du W., He Y., Hu H.Y., Hagen T.L., Falahati M.

Targeting microtubules (MTs), dynamic and stable proteins in cells, by different ligands have been reported to be a potential strategy to combat cancer cells. Inorganic nanoparticles (NPs) have been widely used as anticancer, antibacterial and free radical scavenging agents, where they come in contact with biological macromolecules. The interaction between the NPs and biological macromolecules like MTs frequently occurs through different mechanisms. A prerequisite for a detailed exploration of MT structures and functions for biomedical applications like cancer therapy is to investigate profoundly the mechanisms involved in MT-NP interactions, for which the full explanation and characterization of the parameters that are responsible for the formation of a NP-protein complex are crucial. Therefore, in view of the fact that the goal of the rational NP-based future drug design and new therapies is to rely on the information of the structural details and protein-NPs binding mechanisms to manipulate the process of developing new potential drugs, a comprehensive investigation of the essence of the molecular recognition/interaction is also of considerable importance. In the present review, first, the microtubule (MT) structure and its binding sites upon interaction with MT stabilizing agents (MSAs) and MT destabilizing agents (MDAs) are introduced and rationalized. Next, MT targeting in cancer therapy and interaction of NPs with MTs are discussed. Furthermore, interaction of NPs with proteins and the manipulation of protein corona (PC), experimental techniques and direct interaction of NPs with MTs, are discussed, and finally the challenges and future perspective of the field are introduced. We envision this review can provide useful information on the manipulation of the MT lattice for the progress of cancer nanomedicine.