George D. Bachand

Motor-protein “roundabouts”: Microtubules moving on kinesin-coated tracks through engineered networks

Nanotechnology promises to enhance the functionality and sensitivity of miniaturized analytical systems. For example, nanoscale transport systems, which are driven by molecular motors, permit the controlled movement of select cargo along predetermined paths. Such shuttle systems may enhance the detection efficiency of an analytical system or facilitate the controlled assembly of sophisticated nanostructures if transport can be coordinated through complex track networks. This study determines the feasibility of complex track networks using kinesin motor proteins to actively transport microtubule shuttles along micropatterned surfaces. In particular, we describe the performance of three basic structural motifs: (1) crossing junctions, (2) directional sorters, and (3) concentrators. We also designed track networks that successfully sort and collect microtubule shuttles, pointing the way towards lab-on-a-chip devices powered by active transport instead of pressure-driven or electroosmotic flow.

Development of a multiplex immunocapture RT-PCR assay for detection and differentiation of tomato and tobacco mosaic tobamoviruses.

Immunocapture (IC) RT-PCR assays were developed for detection of tomato (ToMV) and tobacco mosaic (TMV) tobamoviruses in spruce and pine extracts. When purified viruses were diluted in root or needle extracts of virus-free conifer seedlings, both IC-RT-PCR assays detected their respective target viruses at concentrations of 10-100 fg ml(-1). This compared to ELISA detection sensitivities of 1 ng ml(-1). Primers were designed from regions of high sequence diversity. Specificity of all primer pairs was confirmed by sequencing of PCR products. PCR distinguished more reliably between the two viruses than ELISA. Moreover, a multiplex IC-RT-PCR assay for the simultaneous detection and differentiation of TMV and ToMV was developed. When root extracts were seeded with both viruses simultaneously, the multiplex assay detected each virus at concentrations of 1-10 pg ml(-1). Six TMV and 18 ToMV isolates from various hosts, water samples and a soil sample were amplified and differentiated by multiplex IC-RT-PCR. No amplifications were observed against pepper mild mottle and ribgrass mosaic tobamoviruses and against six viruses belonging to other taxonomic groups.

Detection of tomato mosaic tobamovirus RNA in ancient glacial ice

Abstract Tomato mosaic tobamovirus is a very stable plant virus with a wide host range, which has been detected in plants, soil, water, and clouds. Because of its stability and prevalence in the environment, we hypothesized that it might be preserved in ancient ice. We detected tomato mosaic tobamovirus RNA by reverse-transcription polymerase chain reaction amplification in glacial ice subcores <500 to approximately 140,000 years old from drill sites in Greenland. Subcores that contained multiple tomato mosaic tobamovirus genotypes suggest diverse atmospheric origins of the virus, whereas those containing tomato mosaic tobamovirus sequences nearly identical to contemporary ones suggest that recent tomato mosaic tobamovirus populations have an extended age structure. Detection of tomato mosaic tobamovirus in ice raises the possibilities that stable viruses of humans and other hosts might be preserved there, and that entrapped ancient viable viruses may be continually or intermittently released into the modern environment.

“Smart dust” biosensors powered by biomolecular motors

The concept of a microfabricated biosensor for environmental and biomedical monitoring applications which is composed of environmentally benign components is presented. With a built-in power source (the biological fuel ATP) and driven by biological motors (kinesin), sensing in the microdevice can be remotely activated and the presence of a target molecule or toxin remotely detected. The multifaceted progress towards the realization of such a device is described.

Advanced Optical Imaging Reveals the Dependence of Particle Geometry on Interactions Between CdSe Quantum Dots and Immune Cells

The biocompatibility and possible toxicological consequences of engineered nanomaterials, including quantum dots (QDs) due to their unique suitability for biomedical applications, remain intense areas of interest. We utilized advanced imaging approaches to characterize the interactions of CdSe QDs of various sizes and shapes with live immune cells. Particle diffusion and partitioning within the plasma membrane, cellular uptake kinetics, and sorting of particles into lysosomes were all independantly characterized. Using high-speed total internal reflectance fluorescence (TIRF) microscopy, we show that QDs with an average aspect ratio of 2.0 (i.e., rod-shaped) diffuse nearly an order of magnitude slower in the plasma membrane than more spherical particles with aspect ratios of 1.2 and 1.6, respectively. Moreover, more rod-shaped QDs were shown to be internalized into the cell 2-3 fold more slowly. Hyperspectral confocal fluorescence microscopy demonstrates that QDs tend to partition within the cell membrane into regions containing a single particle type. Furthermore, data examining QD sorting mechanisms indicate that endocytosis and lysosomal sorting increases with particle size. Together, these observations suggest that both size and aspect ratio of a nanoparticle are important characteristics that significantly impact interactions with the plasma membrane, uptake into the cell, and localization within intracellular vesicles. Thus, rather than simply characterizing nanoparticle uptake into cells, we show that utilization of advanced imaging approaches permits a more nuanced and complete examination of the multiple aspects of cell-nanoparticle interactions that can ultimately aid understanding possible mechanisms of toxicity, resulting in safer nanomaterial designs.

Constructing Organic/Inorganic NEMS Devices Powered by Biomolecular Motors

The recognition of many enzymes as nanoscale molecular motors has opened the door for the potential creation of hybrid organic/inorganic nano-electro-mechanical (NEMS) devices. The long-term objective of this research is the integration of F1-ATPase with NEMS to produce useful nanoscale devices. A thermostable F1-ATPase coding sequence has been isolated, cloned, and engineered for high-level protein expression. Precise positioning, orientation, and spacing of individual F1-ATPase molecules were achieved on patterned nickel arrays produced using electron beam lithography. An efficient and accurate assay was developed to evaluate the performance of individual F1-ATPase motors, and confirmed a three-step mechanism of γ subunit rotation during ATP hydrolysis. Further assessment of the biophysical and bioengineering properties of FF1-ATPase currently are being conducted, as well as the construction of a hybrid NEMS device powered by FF1-ATPase. The evolution of this technology will permit the creation of novel classes of nanoscale, hybrid devices.

In vitro Capture, Transport, and Detection of Protein Analytes Using Kinesin‐Based Nanoharvesters

Miniaturization of lab-on-a-chip devices to nanoscale dimensions necessitates a level of systems integration currently found primarily in biological systems. Such devices will require new modes of transportingmacromolecularmaterials at nanometer length scales. In cells, efficient cytoplasmic transport is achieved by energy-consuming, active transport systems in which motor proteins transport cargo along cytoskeletal filaments. For example, the motor protein kinesin-1 carries cell organelles and macromolecules over considerable distances along microtubule filaments. Microtubules are hollow protein polymeric filaments with a diameter of 25 nm and tens of micrometers in length that form a 3D transportation network within the cell. Small groups of kinesin transport cargo at rates up to 12mms , with a catalytic efficiency (i.e., conversion of chemical energy into work) of 50%. Together, this transport system provides a highefficiency means of transporting macromolecular cargo through the highly viscous medium of cytoplasm. The intriguing and powerful properties of kinesin-based transport have spurred its application in hybrid nanoscale systems. Early work focused on applying microfabrication technologies and surface functionalization to guide the kinesinbased transport of molecular shuttles (i.e., stabilized microtubule filaments) and achieve directed transport ofmaterials at the nanoscale. In this mode of application, commonly referred to as the inverted or glidingmotility geometry, kinesin motor proteins are bound on a solid surface such that their catalytic and microtubule-binding domains extend into the solution. In the presence ofATP,microtubule filaments bind to

Biomolecular motors in nanoscale materials, devices, and systems

Biomolecular motors are a unique class of intracellular proteins that are fundamental to a considerable number of physiological functions such as DNA replication, organelle trafficking, and cell division. The efficient transformation of chemical energy into useful work by these proteins provides strong motivation for their utilization as nanoscale actuators in ex vivo, meso- and macro-scale hybrid systems. Biomolecular motors involved in cytoskeletal transport are quite attractive models within this context due to their ability to direct the transport of nano-/micro-scale objects at rates significantly greater than diffusion, and in the absence of bulk fluid flow. As in living organisms, biomolecular motors involved in cytoskeletal transport (i.e., kinesin, dynein, and myosin) function outside of their native environment to dissipatively self-assemble biological, biomimetic, and hybrid nanostructures that exhibit nonequilibrium behaviors such as self-healing. These systems also provide nanofluidic transport function in hybrid nanodevices where target analytes are actively captured, sorted, and transported for autonomous sensing and analytical applications. Moving forward, the implementation of biomolecular motors will continue to enable a wide range of unique functionalities that are presently limited to living systems, and support the development of nanoscale systems for addressing critical engineering challenges.

Controlling kinesin motor proteins in nanoengineered systems through a metal‐binding on/off switch

A significant challenge in utilizing kinesin biomolecular motors in integrated nanoscale systems is the ability to regulate motor function in vitro. Here we report a versatile mechanism for reversibly controlling the function of kinesin biomolecular motors independent of the fuel supply (ATP). Our approach relied on inhibiting conformational changes in the neck‐linker region of kinesin, a process necessary for microtubule transport. We introduced a chemical switch into the neck‐linker of kinesin by genetically engineering three histidine residues to create a Zn2+‐binding site. Gliding motility of microtubules by the mutant kinesin was successfully inhibited by ≥10 µM Zn2+, as well as other divalent metals. Motility was successfully restored by removal of Zn2+ using a number of different chelators. Lastly, we demonstrated the robust and cyclic nature of the switch using sequential Zn2+/chelator additions. Overall, this approach to controlling motor function is highly advantageous as it enables control of individual classes of biomolecular motors while maintaining a consistent level of fuel for all motors in a given system or device. Biotechnol. Bioeng. 2008;101: 478–486.

Interactions between cargo-carrying biomolecular shuttles

Microtubule shuttles propelled by the motor protein kinesin embedded in self-assembled monolayers are being developed for active transport functions in artificial microfluidic systems. As a model system, biotinylated microtubules have been laden with streptavidin-coated particles as cargo. The behaviour of cargo-laden microtubules has been observed using fluorescence microscopy upon activation of kinesin-driven transport processes. Collisions between mobile microtubules and their particulate cargo result in six distinct behaviours: bypass, microtubule bending, particle knock-off, particle transfers between microtubules, co-joining of microtubules to a common particle, and particle-induced severing of microtubules. The distribution of observed events can be described qualitatively on the basis of the mechanics of motor proteins and microtubules, the geometry of the collision events, and the loading rate dependence of the strength of microtubule–particle binding. Implications of the results for the use of motor proteins in active transport and cargo-handling systems for nanomaterials are described.