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.

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.

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.

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.

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.

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.

Engineering with Biomolecular Motors.

Biomolecular motors, such as the motor protein kinesin, can be used as off-the-shelf components to power hybrid nanosystems. These hybrid systems combine elements from the biological and synthetic toolbox of the nanoengineer and can be used to explore the applications and design principles of active nanosystems. Efforts to advance nanoscale engineering benefit greatly from biological and biophysical research into the operating principles of motor proteins and their biological roles. In return, the process of creating in vitro systems outside of the context of biology can lead to an improved understanding of the physical constraints creating the fitness landscape explored by evolution. However, our main focus is a holistic understanding of the engineering principles applying to systems integrating molecular motors in general. To advance this goal, we and other researchers have designed biomolecular motor-powered nanodevices, which sense, compute, and actuate. In addition to demonstrating that biological solutions can be mimicked in vitro, these devices often demonstrate new paradigms without parallels in current technology. Long-term trends in technology toward the deployment of ever smaller and more numerous motors and computers give us confidence that our work will become increasingly relevant. Here, our discussion aims to step back and look at the big picture. From our perspective, energy efficiency is a key and underappreciated metric in the design of synthetic motors. On the basis of an analogy to ecological principles, we submit that practical molecular motors have to have energy conversion efficiencies of more than 10%, a threshold only exceeded by motor proteins. We also believe that motor and system lifetime is a critical metric and an important topic of investigation. Related questions are if future molecular motors, by necessity, will resemble biomolecular motors in their softness and fragility and have to conform to the "universal performance characteristics of motors", linking the maximum force and mass of any motor, identified by Marden and Allen. The utilization of molecular motors for computing devices emphasizes the interesting relationship among the conversion of energy, extraction of work, and production of information. Our recent work touches upon these topics and discusses molecular clocks as well as a Landauer limit for robotics. What is on the horizon? Just as photovoltaics took advantage of progress in semiconductor fabrication to become commercially viable over a century, one can envision that engineers working with biomolecular motors leverage progress in biotechnology and drug development to create the engines of the future. However, the future source of energy is going to be electricity rather than fossil or biological fuels, a fact that has to be accounted for in our future efforts. In summary, we are convinced that past, ongoing, and future efforts to engineer with biomolecular motors are providing exciting demonstrations and fundamental insights as well as opportunities to wander freely across the borders of engineering, biology, and chemistry.

Reversibly Bound Kinesin-1 Motor Proteins Propelling Microtubules Demonstrate Dynamic Recruitment of Active Building Blocks.

Biological materials and systems often dynamically self-assemble and disassemble, forming temporary structures as needed and allowing for dynamic responses to stimuli and changing environmental conditions. However, this dynamic interplay of localized component recruitment and release has been difficult to achieve in artificial molecular-scale systems, which are usually designed to have long-lasting, stable bonds. Here, we report the experimental realization of a molecular-scale system that dynamically assembles and disassembles its building blocks while retaining functionality. In our system, filaments (microtubules) recruit biomolecular motors (kinesins) to a surface engineered to allow for the reversible binding of the kinesin-1 motors. These recruited motors work to propel the cytoskeletal filaments along the surface. After the microtubules leave the motors behind, the trail of motors disassembles, releasing the motors back into solution. Engineering such dynamic systems may allow us to create materials that mimic the way in which biological systems achieve self-healing and adaptation.

Aldolase Does Not Show Enhanced Diffusion in Dynamic Light Scattering Experiments.

Recent experimental studies have measured a 30-80% increase of the diffusion coefficient when various enzymes, including aldolase, are catalytically active. This observation has been supported by several theoretical explanations; however, other theoretical studies argue against the possibility of enhanced diffusion, and two of them ascribe the experimental observations to potential artifacts arising in fluorescence correlation spectroscopy (FCS) measurements. Here, we utilized dynamic light scattering (DLS) to measure the diffusion coefficient of aldolase in the absence and presence of its substrate. The DLS measurements have an experimental error of 3% and do not find a statistically significant change of the aldolase diffusion coefficient even at a saturating substrate concentration. This finding lends support to the contention that photophysical artifacts may have affected the FCS measurements and challenges the idea that enzymes can be self-propelled by their catalytic activity.

A mouse model of systemic lupus erythematosus responds better to soluble TACI than to soluble BAFFR, correlating with depletion of plasma cells.

The TNF family cytokines B-cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) support plasma cell survival. It is known that inhibitors of BAFF only (BAFFR-Fc) or BAFF and APRIL (TACI-Fc) administered early enough in an NZB/NZW F1 mouse model of systemic lupus erythematosus (SLE) ameliorate clinical outcomes, pointing to a pathogenic role of BAFF. In the present study, TACI-Fc administrated at a later stage of disease, after onset of autoimmunity, decreased the number of bone marrow plasma cells and slowed down further formation of autoantibodies. TACI-Fc prevented renal damage during a 12-week treatment period regardless of autoantibody levels, while BAFFR-Fc did not despite a similar BAFF-blocking activity in vivo. TACI-Fc also decreased established plasma cells in a T-dependent hapten/carrier immunization system better than single inhibitors of BAFF or APRIL, and sometimes better than combined single inhibitors with at least equivalent BAFF and APRIL inhibitory activities. These results indicate that TACI-Fc can prevent symptoms of renal damage in a mouse model of SLE when BAFFR-Fc cannot, and point to a plasticity of plasma cells for survival factors. Targeting plasma cells with TACI-Fc might be beneficial to prevent autoantibody-mediated damages in SLE.

Antibodies That Block or Activate Mouse B Cell Activating Factor of the Tumor Necrosis Factor (TNF) Family (BAFF), Respectively, Induce B Cell Depletion or B Cell Hyperplasia.

B cell activating factor of the TNF family (BAFF), also known as B lymphocyte stimulator, is a ligand required for the generation and maintenance of B lymphocytes. In this study, the ability of different monoclonal antibodies to recognize, inhibit, or activate mouse BAFF was investigated. One of them, a mouse IgG1 named Sandy-2, prevented the binding of BAFF to all of its receptors, BAFF receptor, transmembrane activator and calcium modulating ligand interactor, and B cell maturation antigen, at a stoichiometric ratio; blocked the activity of mouse BAFF on a variety of cell-based reporter assays; and antagonized the prosurvival action of BAFF on primary mouse B cells in vitro A single administration of Sandy-2 in mice induced B cell depletion within 2 weeks, down to levels close to those observed in BAFF-deficient mice. This depletion could then be maintained with a chronic treatment. Sandy-2 and a previously described rat IgG1 antibody, 5A8, also formed a pair suitable for the sensitive detection of endogenous circulating BAFF by ELISA or using a homogenous assay. Interestingly, 5A8 and Sandy-5 displayed activities opposite to that of Sandy-2 by stimulating recombinant BAFF in vitro and endogenous BAFF in vivo These tools will prove useful for the detection and functional manipulation of endogenous mouse BAFF and provide an alternative to the widely used BAFF receptor-Fc decoy receptor for the specific depletion of BAFF in mice.

CCR6-dependent positioning of memory B cells is essential for their ability to mount a recall response to antigen.

Chemokine-dependent localization of specific B cell subsets within the immune microarchitecture is essential to ensure successful cognate interactions. Although cognate interactions between T cells and memory B cells (B(mem)) are essential for the secondary humoral immune responses, the chemokine response patterns of B(mem) cells are largely unknown. In contrast to naive B cells, this study shows that Ag-specific B(mem) cells have heightened expression of CCR6 and a selective chemotactic response to the CCR6 ligand, CCL20. Although CCR6 appears be nonessential for the initial clonal expansion and maintenance of B(mem), CCR6 is essential for the ability of B(mem) to respond to a recall response to their cognate Ag. This dependency was deemed intrinsic by studies in CCR6-deficient mice and in bone marrow chimeric mice where CCR6 deficiency was limited to the B cell lineage. Finally, the mis-positioning of CCR6-deficient B(mem) was revealed by immunohistological analysis with an altered distribution of CCR6-deficient B(mem) from the marginal and perifollicular to the follicular/germinal center area.

Stoichiometry of Heteromeric BAFF and APRIL Cytokines Dictates Their Receptor Binding and Signaling Properties.

The closely related TNF family ligands B cell activation factor (BAFF) and a proliferation-inducing ligand (APRIL) serve in the generation and maintenance of mature B-lymphocytes. Both BAFF and APRIL assemble as homotrimers that bind and activate several receptors that they partially share. However, heteromers of BAFF and APRIL that occur in patients with autoimmune diseases are incompletely characterized. The N and C termini of adjacent BAFF or APRIL monomers are spatially close and can be linked to create single-chain homo- or hetero-ligands of defined stoichiometry. Similar to APRIL, heteromers consisting of one BAFF and two APRILs (BAA) bind to the receptors B cell maturation antigen (BCMA), transmembrane activator and CAML interactor (TACI) but not to the BAFF receptor (BAFFR). Heteromers consisting of one APRIL and two BAFF (ABB) bind to TACI and BCMA and weakly to BAFFR in accordance with the analysis of the receptor interaction sites in the crystallographic structure of ABB. Receptor binding correlated with activity in reporter cell line assays specific for BAFFR, TACI, or BCMA. Single-chain BAFF (BBB) and to a lesser extent single-chain ABB, but not APRIL or single-chain BAA, rescued BAFFR-dependent B cell maturation in BAFF-deficient mice. In conclusion, BAFF-APRIL heteromers of different stoichiometries have distinct receptor-binding properties and activities. Based on the observation that heteromers are less active than BAFF, we speculate that their physiological role might be to down-regulate BAFF activity.

Microscale Rockets and Picoliter Containers Engineered from Electrospun Polymeric Microtubes.

Chemically functional core/shell microtubes made of biodegradable polymers are fabricated using coaxial electrospinning. The luminal walls are chemically functionalized, allowing for regioselective chemical binding or adsorption inside the microtube. Attaching catalytic nanoparticles or enzymes to the luminal walls converts the microtubes into bubble-propelled microrockets. Upon exposure to ultrasound, the microtubes undergo shape shifting, transforming them into picoliter-scale containers.

Velocity Fluctuations in Kinesin-1 Gliding Motility Assays Originate in Motor Attachment Geometry Variations.

Motor proteins such as myosin and kinesin play a major role in cellular cargo transport, muscle contraction, cell division, and engineered nanodevices. Quantifying the collective behavior of coupled motors is critical to our understanding of these systems. An excellent model system is the gliding motility assay, where hundreds of surface-adhered motors propel one cytoskeletal filament such as an actin filament or a microtubule. The filament motion can be observed using fluorescence microscopy, revealing fluctuations in gliding velocity. These velocity fluctuations have been previously quantified by a motional diffusion coefficient, which Sekimoto and Tawada explained as arising from the addition and removal of motors from the linear array of motors propelling the filament as it advances, assuming that different motors are not equally efficient in their force generation. A computational model of kinesin head diffusion and binding to the microtubule allowed us to quantify the heterogeneity of motor efficiency arising from the combination of anharmonic tail stiffness and varying attachment geometries assuming random motor locations on the surface and an absence of coordination between motors. Knowledge of the heterogeneity allows the calculation of the proportionality constant between the motional diffusion coefficient and the motor density. The calculated value (0.3) is within a standard error of our measurements of the motional diffusion coefficient on surfaces with varying motor densities calibrated by landing rate experiments. This allowed us to quantify the loss in efficiency of coupled molecular motors arising from heterogeneity in the attachment geometry.

Directed transport by surface chemical potential gradients for enhancing analyte collection in nanoscale sensors.

Nanoscale detectors hold great promise for single molecule detection and the analysis of small volumes of dilute samples. However, the probability of an analyte reaching the nanosensor in a dilute solution is extremely low due to the sensor's small size. Here, we examine the use of a chemical potential gradient along a surface to accelerate analyte capture by nanoscale sensors. Utilizing a simple model for transport induced by surface binding energy gradients, we study the effect of the gradient on the efficiency of collecting nanoparticles and single and double stranded DNA. The results indicate that chemical potential gradients along a surface can lead to an acceleration of analyte capture by several orders of magnitude compared to direct collection from the solution. The improvement in collection is limited to a relatively narrow window of gradient slopes, and its extent strongly depends on the size of the gradient patch. Our model allows the optimization of gradient layouts and sheds light on the fundamental characteristics of chemical potential gradient induced transport.

Autonomic molecular transport by polymer films containing programmed chemical potential gradients.

Materials which induce molecular motion without external input offer unique opportunities for spatial manipulation of molecules. Here, we present the use of polyacrylamide hydrogel films containing built-in chemical gradients (enthalpic gradients) to direct molecular transport. Using a cationic tertiary amine gradient, anionic molecules were directionally transported up to several millimeters. A 40-fold concentration of anionic molecules dosed in aerosol form on a substrate to a small region at the center of a radially symmetric cationic gradient was observed. The separation of mixtures of charged dye molecules was demonstrated using a boronic acid-to-cationic gradient where one molecule was attracted to the boronic acid end of the gradient, and the other to the cationic end of the gradient. Theoretical and computational analysis provides a quantitative description of such anisotropic molecular transport, and reveals that the gradient-imposed drift velocity is in the range of hundreds of nanometers per second, comparable to the transport velocities of biomolecular motors. This general concept of enthalpy gradient-directed molecular transport should enable the autonomous processing of a diversity of chemical species.