Disease related changes in ATAC-seq of iPSC-derived motor neuron lines from ALS patients and controls.

Amyotrophic Lateral Sclerosis (ALS), like many other neurodegenerative diseases, is highly heritable, but with only a small fraction of cases explained by monogenic disease alleles. To better understand sporadic ALS, we report epigenomic profiles, as measured by ATAC-seq, of motor neuron cultures derived from a diverse group of 380 ALS patients and 80 healthy controls. We find that chromatin accessibility is heavily influenced by sex, the iPSC cell type of origin, ancestry, and the inherent variance arising from sequencing. Once these covariates are corrected for, we are able to identify ALS-specific signals in the data. Additionally, we find that the ATAC-seq data is able to predict ALS disease progression rates with similar accuracy to methods based on biomarkers and clinical status. These results suggest that iPSC-derived motor neurons recapitulate important disease-relevant epigenomic changes.

Large-scale differentiation of iPSC-derived motor neurons from ALS and control subjects.

Using induced pluripotent stem cells (iPSCs) to understand the mechanisms of neurological disease holds great promise; however, there is a lack of well-curated lines from a large array of participants. Answer ALS has generated over 1,000 iPSC lines from control and amyotrophic lateral sclerosis (ALS) patients along with clinical and whole-genome sequencing data. The current report summarizes cell marker and gene expression in motor neuron cultures derived from 92 healthy control and 341 ALS participants using a 32-day differentiation protocol. This is the largest set of iPSCs to be differentiated into motor neurons, and characterization suggests that cell composition and sex are significant sources of variability that need to be carefully controlled for in future studies. These data are reported as a resource for the scientific community that will utilize Answer ALS data for disease modeling using a wider array of omics being made available for these samples.

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.

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.

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.

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.

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.

Rise of the Nanorobots: Advances in Control, Molecular Detection, and Nanoscale Actuation Are Bringing Us Closer to a New Era of Technology Enhanced by Nanorobots.

In 1988, a Scientific American article by A.K. Dewdney [1] on the work of nanotechnologist K. Eric Drexler spurred public interest in the nascent field of nanotechnology and its potential for advancing humanity into a new technological age. The article portrayed a world run by nanoscale machines that could operate in any environment (Figure 1), with uses ranging from fighting infections in the human body to building tomorrow's skyscrapers. Nearly 30 years later, these visions of the future are closer than ever. Advances in microscopic imaging, nanobiotechnology, and collective control are ushering in the age of nanoscale robotics.

Proximity does not contribute to activity enhancement in the glucose oxidase-horseradish peroxidase cascade.

A proximity effect has been invoked to explain the enhanced activity of enzyme cascades on DNA scaffolds. Using the cascade reaction carried out by glucose oxidase and horseradish peroxidase as a model system, here we study the kinetics of the cascade reaction when the enzymes are free in solution, when they are conjugated to each other and when a competing enzyme is present. No proximity effect is found, which is in agreement with models predicting that the rapidly diffusing hydrogen peroxide intermediate is well mixed. We suggest that the reason for the activity enhancement of enzymes localized by DNA scaffolds is that the pH near the surface of the negatively charged DNA nanostructures is lower than that in the bulk solution, creating a more optimal pH environment for the anchored enzymes. Our findings challenge the notion of a proximity effect and provide new insights into the role of DNA scaffolds.