Molecular Motor or Molecular Clock: A Question of Load.

The output of a motor is work, while the output of a clock is information. Here it is discussed how a molecular motor can produce both, work and information, depending on the load. If the ratio of the backward and forward stepping rates of a molecular motor increases exponentially with load, the change in free energy per step can be used to produce only work (at stall force) or only timing information (at zero force), or anything in between.

The rate of microtubule breaking increases exponentially with curvature.

Microtubules, cylindrical assemblies of tubulin proteins with a 25 nm diameter and micrometer lengths, are a central part of the cytoskeleton and also serve as building blocks for nanobiodevices. Microtubule breaking can result from the activity of severing enzymes and mechanical stress. Breaking can lead to a loss of structural integrity, or an increase in the numbers of microtubules. We observed breaking of taxol-stabilized microtubules in a gliding motility assay where microtubules are propelled by surface-adhered kinesin-1 motor proteins. We find that over 95% of all breaking events are associated with the strong bending following pinning events (where the leading tip of the microtubule becomes stuck). Furthermore, the breaking rate increased exponentially with increasing curvature. These observations are explained by a model accounting for the complex mechanochemistry of a microtubule. The presence of severing enzymes is not required to observe breaking at rates comparable to those measured previously in cells.

Microscale Colocalization of Cascade Enzymes Yields Activity Enhancement.

Colocalization of cascade enzymes is broadly discussed as a phenomenon that can boost the cascade reaction throughput, although a direct experimental verification is often challenging. This is mainly due to difficulties in establishing proper size regimes and in the analytical quantification of colocalization effect with adequate experimental systems and simulations. In this study, by taking advantage of reversible DNA-directed colocalization of enzymes on microspheres, we established a cascade system that can be used to directly evaluate the colocalization effect with exactly the same experimental settings except for the state of enzyme dispersion. In the regime of highly dilute microspheres of particular sizes, the colocalized cascade shows enhanced activity compared with the freely diffusing cascade, as evidenced by a shortened lag phase in the time-course production. Reaction-diffusion modeling reveals that the enhancement can be ascribed to the initial accumulation of intermediate substrate around the colocalized enzymes and is found to be carrier-size-dependent. This work demonstrates the dependence of the colocalization effect of enzyme cascades on an interplay of nano- and microscales, lending theoretical support to the rational design of highly efficient multienzyme catalysts.

Ligand-independent oligomerization of TACI is controlled by the transmembrane domain and regulates proliferation of activated B cells.

In mature B cells, TACI controls class-switch recombination and differentiation into plasma cells during T cell-independent antibody responses. TACI binds the ligands BAFF and APRIL. Approximately 10% of patients with common variable immunodeficiency (CVID) carry TACI mutations, of which A181E and C172Y are in the transmembrane domain. Residues A181 and C172 are located on distinct sides of the transmembrane helix, which is predicted by molecular modeling to spontaneously assemble into trimers and dimers. In human B cells, these mutations impair ligand-dependent (C172Y) and -independent (A181E) TACI multimerization and signaling, as well as TACI-enhanced proliferation and/or IgA production. Genetic inactivation of TACI in primary human B cells impaired survival of CpG-activated cells in the absence of ligand. These results identify the transmembrane region of TACI as an active interface for TACI multimerization in signal transduction, in particular for ligand-independent signals. These functions are perturbed by CVID-associated mutations.

Optical Control of Mitosis with a Photoswitchable Eg5 Inhibitor.

Eg5 is a kinesin motor protein that is responsible for bipolar spindle formation and plays a crucial role during mitosis. Loss of Eg5 function leads to the formation of monopolar spindles, followed by mitotic arrest, and subsequent cell death. Several cell-permeable small molecules have been reported to inhibit Eg5 and some have been evaluated as anticancer agents. We now describe the design, synthesis, and biological evaluation of photoswitchable variants with five different pharmacophores. Our lead compound Azo-EMD is a cell permeable azobenzene that inhibits Eg5 more potently in its light-induced cis form. This activity decreased the velocity of Eg5 in single-molecule assays, promoted formation of monopolar spindles, and led to mitotic arrest in a light dependent way.

Robotic end-to-end fusion of microtubules powered by kinesin.

The active assembly of molecules by nanorobots has advanced greatly since "molecular manufacturing"­that is, the use of nanoscale tools to build molecular structures­was proposed. In contrast to a catalyst, which accelerates a reaction by smoothing the potential energy surface along the reaction coordinate, molecular machines expend energy to accelerate a reaction relative to the baseline provided by thermal motion and forces. Here, we design a nanorobotics system to accelerate end-to-end microtubule assembly by using kinesin motors and a circular confining chamber. We show that the mechanical interaction of kinesin-propelled microtubules gliding on a surface with the walls of the confining chamber results in a nonequilibrium distribution of microtubules, which increases the number of end-to-end microtubule fusion events 20-fold compared with microtubules gliding on a plane. In contrast to earlier nanorobots, where a nonequilibrium distribution was built into the initial state and drove the process, our nanorobotic system creates and actively maintains the building blocks in the concentrated state responsible for accelerated assembly through the adenosine triphosphate­fueled generation of force by kinesin-1 motor proteins. This approach can be used in the future to develop biohybrid or bioinspired nanorobots that use molecular machines to access nonequilibrium states and accelerate nanoscale assembly.

Deformation of microtubules regulates translocation dynamics of kinesin.

Microtubules, the most rigid components of the cytoskeleton, can be key transduction elements between external forces and the cellular environment. Mechanical forces induce microtubule deformation, which is presumed to be critical for the mechanoregulation of cellular events. However, concrete evidence is lacking. In this work, with high-speed atomic force microscopy, we unravel how microtubule deformation regulates the translocation of the microtubule-associated motor protein kinesin-1, responsible for intracellular transport. Our results show that the microtubule deformation by bending impedes the translocation dynamics of kinesins along them. Molecular dynamics simulation shows that the hindered translocation of kinesins can be attributed to an enhanced affinity of kinesins to the microtubule structural units in microtubules deformed by bending. This study advances our understanding of the role of cytoskeletal components in mechanotransduction.

APRIL limits atherosclerosis by binding to heparan sulfate proteoglycans.

Atherosclerotic cardiovascular disease causes heart attacks and strokes, which are the leading causes of mortality worldwide1. The formation of atherosclerotic plaques is initiated when low-density lipoproteins bind to heparan-sulfate proteoglycans (HSPGs)2 and become trapped in the subendothelial space of large and medium size arteries, which leads to chronic inflammation and remodelling of the artery wall2. A proliferation-inducing ligand (APRIL) is a cytokine that binds to HSPGs3, but the physiology of this interaction is largely unknown. Here we show that genetic ablation or antibody-mediated depletion of APRIL aggravates atherosclerosis in mice. Mechanistically, we demonstrate that APRIL confers atheroprotection by binding to heparan sulfate chains of heparan-sulfate proteoglycan 2 (HSPG2), which limits the retention of low-density lipoproteins, accumulation of macrophages and formation of necrotic cores. Indeed, antibody-mediated depletion of APRIL in mice expressing heparan sulfate-deficient HSPG2 had no effect on the development of atherosclerosis. Treatment with a specific anti-APRIL antibody that promotes the binding of APRIL to HSPGs reduced experimental atherosclerosis. Furthermore, the serum levels of a form of human APRIL protein that binds to HSPGs, which we termed non-canonical APRIL (nc-APRIL), are associated independently of traditional risk factors with long-term cardiovascular mortality in patients with atherosclerosis. Our data reveal properties of APRIL that have broad pathophysiological implications for vascular homeostasis.

Chemically-powered swimming and diffusion in the microscopic world.

The past decade has seen intriguing reports and heated debates concerning the chemically-driven enhanced motion of objects ranging from small molecules to millimetre-size synthetic robots. These objects, in solutions in which chemical reactions were occurring, were observed to diffuse (spread non-directionally) or swim (move directionally) at rates exceeding those expected from Brownian motion alone. The debates have focused on whether observed enhancement is an experimental artefact or a real phenomenon. If the latter were true, then we would also need to explain how the chemical energy is converted into mechanical work. In this Perspective, we summarize and discuss recent observations and theories of active diffusion and swimming. Notably, the chemomechanical coupling and magnitude of diffusion enhancement are strongly size-dependent and should vanish as the size of the swimmers approaches the molecular scale. We evaluate the reliability of common techniques to measure diffusion coefficients and finish by considering the potential applications and chemical to mechanical energy conversion efficiencies of typical nanoswimmers and microswimmers.

Power Law Behavior in Protein Desorption Kinetics Originating from Sequential Binding and Unbinding.

The study of protein adsorption at the single molecule level has recently revealed that the adsorption is reversible, but with a long-tailed residence time distribution which can be approximated with a sum of exponential functions putatively related to distinct adsorption sites. Here it is proposed that the shape of the residence time distribution results from an adsorption process with sequential and reversible steps that contribute to overall binding strength resembling "zippering". In this model, the survival function of the residence time distribution of single proteins varies from an exponential distribution for a single adsorption step to a power law distribution with exponent -1/2 for a large number of adsorption steps. The adsorption of fluorescently labeled fibrinogen to glass surfaces is experimentally studied with single molecule imaging. The experimental residence time distribution can be readily fit by the proposed model. This demonstrates that the observed long residence times can arise from stepwise adsorption rather than rare but strong binding sites and provides guidance for the control of protein adsorption to biomaterials.

Kinesin-Recruiting Microtubules Exhibit Collective Gliding Motion while Forming Motor Trails.

Microtubules gliding on surfaces coated with kinesin motors are minimalist experimental systems for studying collective behavior. Collective behavior in these systems arises from interactions between filaments, for example, from steric interactions, depletion forces, or cross-links. To maximize the utilization of system components and the production of work, it is desirable to achieve mutualistic interactions leading to the congregations of both types of agents, that is, cytoskeletal filaments and molecular motors. To this end, we used a microtubule-kinesin system, where motors reversibly bind to the surface via an interaction between a hexahistidine (His6) tag on the motor and a Ni(II)-nitrilotriacetic acid (Ni-NTA) moiety on the surface. The surface density of binding sites for kinesin motors was increased relative to our earlier work, driving the motors from the solution to the surface. Characterization of the motor-surface interactions in the absence of microtubules yielded kinetic parameters consistent with previous data and revealed the capacity of the surface to support two-dimensional motor diffusion. The motor density gradually fell over 2 h, presumably due to the stripping of Ni(II) from the NTA moieties on the surface. Microtubules gliding on these reversibly bound motors were unable to cross each other and at high enough densities began to align and form long, dense bundles. The kinesin motors accumulated in trails surrounding the microtubule bundles and participated in microtubule transport.

Microtubule Detachment in Gliding Motility Assays Limits the Performance of Kinesin-Driven Molecular Shuttles.

The creation of complex active nanosystems integrating cytoskeletal filaments propelled by surface-adhered motor proteins often relies on the filaments' ability to glide over up to meter-long distances. While theoretical considerations support this ability, we show that microtubule detachment (either spontaneous or triggered by a microtubule crossing event) is a non-negligible phenomenon that has been overlooked until now. The average gliding distance before spontaneous detachment was measured to be 30 ± 10 mm for a functional kinesin-1 density of 500 µm-2 and 9 ± 4 mm for a functional kinesin-1 density of 100 µm-2 at 1 mM ATP. Even microtubules longer than 3 µm detached, suggesting that spontaneous detachment is not caused by the stochastic absence of motors or their stochastic release due to a limited run length.

Synthetic Systems Powered by Biological Molecular Motors.

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.

Inhibitors in Commercially Available 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonate) Affect Enzymatic Assays.

ABTS, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate), is a common chromogenic substrate for peroxidase enzymes, which are widely used in biochemical research and diagnostic tests. We discovered that impurities in the commercially available ABTS significantly affect the results of peroxidase activity assays. We show that the impurities inhibit the activity of the peroxidases and the influence varies for different batches of ABTS from the same source. The inhibition of horseradish peroxidase (HRP) is uncompetitive for the substrate H2O2 while it is competitive for the substrate ABTS. By using high-resolution mass spectrometry, potential inhibitors were identified to be precursors or analogs of ABTS. The inhibitors are also capable of inhibiting the GOx-catalyzed reduction of the ABTS radical cation by glucose in anaerobic conditions. As the inhibition is found to be pH-dependent, diagnostic applications, such as ELISA tests based on the peroxidase-H2O2-ABTS system, should be carried out at pH 4.4 to minimize the inhibitory effect of potentially present impurities.

Adaptation of Patterns of Motile Filaments under Dynamic Boundary Conditions.

Boundary conditions are important for pattern formation in active matter. However, it is still not well-understood how alterations in the boundary conditions (dynamic boundary conditions) impact pattern formation. To elucidate the effect of dynamic boundary conditions on the pattern formation by active matter, we investigate an in vitro gliding assay of microtubules on a deformable soft substrate. The dynamic boundary conditions were realized by applying mechanical stress through stretching and compression of the substrate during the gliding assay. A single cycle of stretch-and-compression (relaxation) of the substrate induces perpendicular alignment of microtubules relative to the stretch axis, whereas repeated cycles resulted in zigzag patterns of microtubules. Our model shows that the orientation angles of microtubules correspond to the direction to attain smooth movement without buckling, which is further amplified by the collective migration of the microtubules. Our results provide an insight into understanding the rich dynamics in self-organization arising in active matter subjected to time-dependent boundary conditions.

Enhanced Diffusion of Catalytically Active Enzymes.

The past decade has seen an increasing number of investigations into enhanced diffusion of catalytically active enzymes. These studies suggested that enzymes are actively propelled as they catalyze reactions or bind with ligands (e.g., substrates or inhibitors). In this Outlook, we chronologically summarize and discuss the experimental observations and theoretical interpretations and emphasize the potential contradictions in these efforts. We point out that the existing multimeric forms of enzymes or isozymes may cause artifacts in measurements and that the conformational changes upon substrate binding are usually not sufficient to give rise to a diffusion enhancement greater than 30%. Therefore, more rigorous experiments and a more comprehensive theory are urgently needed to quantitatively validate and describe the enhanced enzyme diffusion.

Assembling Molecular Shuttles Powered by Reversibly Attached Kinesins.

This protocol describes how to create kinesin-powered molecular shuttles with a weak and reversible attachment of the kinesins to the surface. In contrast to previous protocols, in this system, microtubules recruit kinesin motor proteins from solution and place them on a surface. The kinesins will, in turn, facilitate the gliding of the microtubules along the surface before desorbing back into the bulk solution, thus being available to be recruited again. This continuous assembly and disassembly leads to striking dynamic behavior in the system, such as the formation of temporary kinesin trails by gliding microtubules. Several experimental methods will be described throughout this experiment: UV-Vis spectrophotometry will be used to determine the concentration of stock solutions of reagents, coverslips will first be ozone and ultraviolet (UV) treated and then silanized before being mounted into flow cells, and total internal reflection fluorescence (TIRF) microscopy will be used to simultaneously image kinesin motors and microtubule filaments.