Till Korten

Towards the application of cytoskeletal motor proteins in molecular detection and diagnostic devices.

Over the past ten years, great advancements have been made towards using biomolecular motors for nanotechnological applications. In particular, devices using cytoskeletal motor proteins for molecular transport are maturing. First efforts towards designing such devices used motor proteins attached to micro-structured substrates for the directed transport of microtubules and actin filaments. Soon thereafter, the specific capture, transport and detection of target analytes like viruses were demonstrated. Recently, spatial guiding of the gliding filaments was added to increase the sensitivity of detection and allow parallelization. Whereas molecular motor powered devices have not yet demonstrated performance beyond the level of existing detection techniques, the potential is great: Replacing microfluidics with transport powered by molecular motors allows integration of the energy source (ATP) into the assay solution. This opens up the opportunity to design highly integrated, miniaturized, autonomous detection devices. Such devices, in turn, may allow fast and cheap on-site diagnosis of diseases and detection of environmental pathogens and toxins.

Dynamic guiding of motor-driven microtubules on electrically heated, smart polymer tracks.

Biomolecular motor systems are attractive for future nanotechnological devices because they can replace nanofluidics by directed transport. However, the lack of methods to externally control motor-driven transport along complex paths limits their range of applications. Based on a thermo-responsive polymer, we developed a novel technique to guide microtubules propelled by kinesin-1 motors on a planar surface. Using electrically heated gold microstructures, the polymers were locally collapsed, creating dynamically switchable tracks that successfully guided microtubule movement.

Parallel computation with molecular-motor-propelled agents in nanofabricated networks

Significance Electronic computers are extremely powerful at performing a high number of operations at very high speeds, sequentially. However, they struggle with combinatorial tasks that can be solved faster if many operations are performed in parallel. Here, we present proof-of-concept of a parallel computer by solving the specific instance {2, 5, 9} of a classical nondeterministic-polynomial-time complete (“NP-complete”) problem, the subset sum problem. The computer consists of a specifically designed, nanostructured network explored by a large number of molecular-motor-driven, protein filaments. This system is highly energy efficient, thus avoiding the heating issues limiting electronic computers. We discuss the technical advances necessary to solve larger combinatorial problems than existing computation devices, potentially leading to a new way to tackle difficult mathematical problems. The combinatorial nature of many important mathematical problems, including nondeterministic-polynomial-time (NP)-complete problems, places a severe limitation on the problem size that can be solved with conventional, sequentially operating electronic computers. There have been significant efforts in conceiving parallel-computation approaches in the past, for example: DNA computation, quantum computation, and microfluidics-based computation. However, these approaches have not proven, so far, to be scalable and practical from a fabrication and operational perspective. Here, we report the foundations of an alternative parallel-computation system in which a given combinatorial problem is encoded into a graphical, modular network that is embedded in a nanofabricated planar device. Exploring the network in a parallel fashion using a large number of independent, molecular-motor-propelled agents then solves the mathematical problem. This approach uses orders of magnitude less energy than conventional computers, thus addressing issues related to power consumption and heat dissipation. We provide a proof-of-concept demonstration of such a device by solving, in a parallel fashion, the small instance {2, 5, 9} of the subset sum problem, which is a benchmark NP-complete problem. Finally, we discuss the technical advances necessary to make our system scalable with presently available technology.

Studying Kinesin Motors by Optical 3D-Nanometry in Gliding Motility Assays

Recent developments in optical microscopy and nanometer tracking have facilitated our understanding of microtubules and their associated proteins. Using fluorescence microscopy, dynamic interactions are now routinely observed in vitro on the level of single molecules, mainly using a geometry in which labeled motors move on surface-immobilized microtubules. Yet, we think that the historically older gliding geometry, in which motor proteins bound to a substrate surface drive the motion microtubules, offers some unique advantages. (1) Motility can be precisely followed by coupling multiple fluorophores and/or single bright labels to the surface of microtubules without disturbing the activity of the motor proteins. (2) The number of motor proteins involved in active transport can be determined by several strategies. (3) Multimotor studies can be performed over a wide range of motor densities. These advantages allow for studying cooperativity of processive as well as nonprocessive motors. Moreover, the gliding geometry has proven to be most promising for nanotechnological applications of motor proteins operating in synthetic environments. In this chapter we review recent methods related to gliding motility assays in conjunction with 3D-nanometry. In particular, we aim to provide practical advice on how to set up gliding assays, how to acquire high-precision data from microtubules and attached quantum dots, and how to analyze data by 3D-nanometer tracking.

Kinesin-1 Motors Can Circumvent Permanent Roadblocks by Side-Shifting to Neighboring Protofilaments

Obstacles on the surface of microtubules can lead to defective cargo transport, proposed to play a role in neurological diseases such as Alzheimers. However, little is known about how motor proteins, which follow individual microtubule protofilaments (such as kinesin-1), deal with obstacles on the molecular level. Here, we used rigor-binding mutants of kinesin-1 as roadblocks to permanently obstruct individual microtubule binding sites and studied the movement of individual kinesin-1 motors by single-molecule fluorescence and dark-field scattering microscopy in vitro. In the presence of roadblocks, kinesin-1 often stopped for ∼ 0.4 s before either detaching or continuing to move, whereby the latter circumvention events occurred in >30% after a stopping event. Consequently, and in agreement with numerical simulations, the mean velocity, mean run length, and mean dwell time of the kinesin-1 motors decreased upon increasing the roadblock density. Tracking individual kinesin-1 motors labeled by 40 nm gold particles with 6 nm spatial and 1 ms temporal precision revealed that ∼ 70% of the circumvention events were associated with significant transverse shifts perpendicular to the axis of the microtubule. These side-shifts, which occurred with equal likelihood to the left and right, were accompanied by a range of longitudinal shifts suggesting that roadblock circumvention involves the unbinding and rebinding of the motors. Thus, processive motors, which commonly follow individual protofilaments in the absence of obstacles, appear to possess intrinsic circumvention mechanisms. These mechanisms were potentially optimized by evolution for the motors specific intracellular tasks and environments.

Sample solution constraints on motor-driven diagnostic nanodevices

The last decade has seen appreciable advancements in efforts towards increased portability of lab-on-a-chip devices by substituting microfluidics with molecular motor-based transportation. As of now, first proof-of-principle devices have analyzed protein mixtures of low complexity, such as target protein molecules in buffer solutions optimized for molecular motor performance. However, in a diagnostic work-up, lab-on-a-chip devices need to be compatible with complex biological samples. While it has been shown that such samples do not interfere with crucial steps in molecular diagnostics (for example antibody-antigen recognition), their effect on molecular motors is unknown. This critical and long overlooked issue is addressed here. In particular, we studied the effects of blood, cell lysates and solutions containing genomic DNA extracts on actomyosin and kinesin-microtubule-based transport, the two biomolecular motor systems that are most promising for lab-on-a-chip applications. We found that motor function is well preserved at defined dilutions of most of the investigated biological samples and demonstrated a molecular motor-driven label-free blood type test. Our results support the feasibility of molecular-motor driven nanodevices for diagnostic point-of-care applications and also demonstrate important constraints imposed by sample composition and device design that apply both to kinesin-microtubule and actomyosin driven applications.

Selective control of gliding microtubule populations.

First lab-on-chip devices based on active transport by biomolecular motors have been demonstrated for basic detection and sorting applications. However, to fully employ the advantages of such hybrid nanotechnology, versatile spatial and temporal control mechanisms are required. Using a thermo-responsive polymer, we demonstrate the selective starting and stopping of modified microtubules gliding on a kinesin-1-coated surface. This approach allows the self-organized separation of multiple microtubule populations and their respective cargoes.

Fluorescence Imaging of Single Kinesin Motors on Immobilized Microtubules

Recent developments in optical microscopy and nanometer tracking have greatly improved our understanding of cytoskeletal motor proteins. Using fluorescence microscopy, dynamic interactions are now routinely observed in vitro on the level of single molecules mainly using a geometry, where fluorescently labeled motors move on surface-immobilized filaments. In this chapter, we review recent methods related to single-molecule kinesin motility assays. In particular, we aim to provide practical advice on: how to set up the assays, how to acquire high-precision data from fluorescently labeled kinesin motors and attached quantum dots, and how to analyze data by nanometer tracking.

Kinesin-1 Expressed in Insect Cells Improves Microtubule in Vitro Gliding Performance, Long-Term Stability and Guiding Efficiency in Nanostructures

The cytoskeletal motor protein kinesin-1 has been successfully used for many nanotechnological applications. Most commonly, these applications use a gliding assay geometry where substrate-attached motor proteins propel microtubules along the surface. So far, this assay has only been shown to run undisturbed for up to 8 h. Longer run times cause problems like microtubule shrinkage, microtubules getting stuck and slowing down. This is particularly problematic in nanofabricated structures where the total number of microtubules is limited and detachment at the structure walls causes additional microtubule loss. We found that many of the observed problems are caused by the bacterial expression system, which has so far been used for nanotechnological applications of kinesin-1. We strive to enable the use of this motor system for more challenging nanotechnological applications where long-term stability and/or reliable guiding in nanostructures is required. Therefore, we established the expression and purification of kinesin-1 in insect cells which results in improved purity and-more importantly-long-term stability 24 h and guiding efficiencies of 90% in lithographically defined nanostructures.

Control and gating of kinesin-microtubule motility on electrically heated thermo-chips.

First lab-on-chip devices based on active transport by biomolecular motors have been demonstrated for basic detection and sorting applications. However, to fully employ the advantages of such hybrid nanotechnology, versatile spatial and temporal control mechanisms are required. Using a thermo-responsive polymer, we demonstrated a temperature controlled gate that either allows or disallows the passing of microtubules through a topographically defined channel. The gate is addressed by a narrow gold wire, which acts as a local heating element. It is shown that the electrical current flowing through a narrow gold channel can control the local temperature and as a result the conformation of the polymer. This is the first demonstration of a spatially addressable gate for microtubule motility which is a key element of nanodevices based on biomolecular motors.