Advanced Review

Biomolecular motors in nanoscale materials, devices, and systems George D. Bachand,∗ Nathan F. Bouxsein, Virginia VanDelinder and Marlene Bachand 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. © 2013 Wiley Periodicals, Inc.

How to cite this article:

WIREs Nanomed Nanobiotechnol 2014, 6:163–177. doi: 10.1002/wnan.1252

INTRODUCTION

B

iomolecular motors are broadly defined as a class of proteins that consume energy to perform useful work inside a cell. Many of the emergent and distinguishing phenomena found in living organisms are enabled through the action of this exquisite group of proteins. For example, a cell uses the cooperative actions of the DNA polymerase, helicase, and topoisomerase biomolecular motors in order to replicate its genomic DNA prior to cell division.1 Similarly, the coordinated transport of pigmentcontaining organelles by cytoskeletal motors and filaments at the cellular level enables the ability of organisms, such as certain species of fish, to change ∗ Correspondence

to: [email protected]

Center for Integrated Nanotechnologies, Sandia National Laboratories, Albuquerque, NM, USA Conflict of interest: The authors have declared no conflicts of interest for this article.

Volume 6, March/April 2014

color at the mesoscopic level.1 Fundamental to both of these examples is the ability of biomolecular motors to push the system away from equilibrium dynamics through the dissipation of energy, which in turn enables an array of emergent phenomena (e.g., self-healing) that are unique to living organisms. The biochemistry, biophysics, and physiological functions of biomolecular motors have been widely studied over the past 30 years. This research has established a considerable scientific understanding of the structure–function relationships that underpin the incredible properties of these proteins, which are reviewed elsewhere.2,3 A generalized property shared by most, if not all, biomolecular motors is their ability to undergo complex chemomechanical conformational changes that, in turn, convert chemical energy into mechanical work with an enviable efficiency (e.g., 50–90%). Knowledge concerning the exact mechanisms underpinning this level of efficiency is still lacking and remains a hot

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topic for enzymologists. Moreover, understanding the collective behaviors of biomolecular motors in physiological processes that span from the molecular and nanometer-level to the organismal and mesoscale also remains an active area of research. During the rapid rise of nanotechnology in the late 1990s and early 2000s, researchers in this area quickly recognized the potential of bimolecular motors to serve as molecular actuators in integrated or hybrid nanosystems. Moreover, integration of these motors in nanosystems has largely been driven by their intrinsic biological functions, which include: (1) membrane transporters, (2) rotary motors, (3) nucleic acid motors, and (4) cytoskeletal motors. The earliest examples of integrated, biomolecular powered nanosystems include a rotary device powered by ATP synthase4 and kinesin-powered molecular shuttles.5 Advances in the ability to interface biomolecular components with engineered materials and devices further enabled the development of more advanced systems such as polymerase-powered nanodevices that move along DNA tracks,6 optoelectronic devices based on bacteriorhodopsin ion transporters,7 ATP synthase-powered DNA sensors,8 and swimming lipid vesicles powered by a rotary flagellar motor from bacteria.9 While all classes of bimolecular motors perform critical cellular functions, this review will focus on one class of biomolecular motors, the cytoskeletal motor proteins, and summarize their use in dissipative nanomaterials assembly and nanoscale sensing applications.

dynamics of these two intracellular transportation systems are briefly discussed.

Actin-Based Transport Actin filaments (AF) play a critical role in biological functions including muscle contraction, vesicle transport, cell locomotion, and cytokinesis. Polymerization of globular-actin (G-actin) monomers leads to mature, filamentous structures that consist of a two-stranded, right-handed helix with a halfpitch of 36 nm, diameter of approximately 10 nm, and persistence length of 15–20 μm.10 Additional structural morphology is imparted by actin-related proteins (e.g., ARP2/3 complex) and actin-binding proteins (e.g., fascins and formins) that induce branching and bundling of AFs, respectively. AFs have structural polarity in that one end (i.e., the minus end) of the filament will possess a G-actin monomer which has the nucleotide-binding site exposed. Upon monomer addition, this binding site possesses an adenosine triphosphate (ATP) molecule that is hydrolysed to ADP quickly after the monomer is incorporated into the filament. This hydrolytic activity is related to the dynamic nature of AFs, which is characterized by alternating cycles of assembly and disassembly. Assembly is initiated by the aggregation of G-actin into unstable oligomers (i.e., nucleation), followed by a rapid elongation phase, and finally a steady-state phase characterized by monomer exchanged but without a net change in filament length.10 While assembly and disassembly of AFs is tightly controlled by a number of actinbinding proteins, AF dynamics can be regulated ex vivo by molecules such as cytochalasins and phalloidin. AFs interact with a superfamily of proteins, myosin, which consists of more than 35 classes and includes single- and double-headed motors, as well as plus- and minus-end directed motors (i.e., motors that move toward the plus- and minus-end of the filament, respectively).3 The myosin II class (Figure 1(a)), which is responsible for muscle actuation, has been a primary focus for nanotechnological applications.11

CYTOSKELETAL TRANSPORT SYSTEMS The cytoskeleton of eukaryotic cells is composed of three types of proteinaceous polymer filaments: actin, microtubule (MT), and intermediate filaments. In addition to their roles in mechanical stabilization, two of these filaments, actin and MTs, also serve as ‘tracks’ for biomolecular motors (Table 1) to transport macromolecular cargo and organelles. In the following sections, the structure, properties, and

TABLE 1 Properties of Various Biomolecular Motors Responsible for Cytoskeletal Transport Within Eukaryotic Cells Motor

Filament

ATP/motor

Step size (nm)

Force (pN)

Work (pN·nm)

Efficiency (%)

AF

2

6

6

36

Approximately 50

Myosin V

AF

2

36

2

72

Approximately 90

Kinesin-1

MT

2

8

5

40

Approximately 50

Cytoplasmic dynein

MT

Up to 8

8–24

1–7

N/A

N/A

Myosin II

N/A, not available.

164

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(a)

Biomolecular motors in nanoscale materials, devices, and systems

Myosin II Motor domain (N-terminus) Coiled-coil tail

Neck region ~150 nm

(b)

Kinesin-1 Motor domain (N-terminus) Neck-linker region

Coiled-coil tail

Light chains ~ 60 nm

FIGURE 1 | Artistic representation of the structures of (a) myosin II and (b) kinesin-1 biomolecular motors.

The motor domains of myosin II possess actin binding and ATP catalytic sites, and are connected by an α-helical neck region and a long coiled-coil tail. As ATP is hydrolysed, the motor moves non-processively (i.e., it rapidly detaches from the AF) with a step size of approximately 6 nm corresponding to the distance between G-actin monomers. Approximately 36 pN·nm of work is performed with each step, corresponding to a thermodynamic efficiency of approximately 50% based on the free energy available from ATP hydrolysis (i.e., approximately 80 pN·nm).12 A second motor, myosin V, has also been of potential interest based on its role in intracellular trafficking of vesicles, as well as its ability to generate approximately 72 pN·nm of work per step with a thermodynamic efficiency of 90%.13 In contrast to myosin II, myosin V motors move processively along AFs with a step size of 36 nm, which corresponds to the half-pitch of the filament.13 Thus, whereas myosin II follows the helical pitch of AFs, myosin V can move linearly along AFs without translation about its axis, an important advantage for transport in nanoscale systems.

MT-Based Transport MTs are cytoskeletal polymeric filaments that serve two primary roles: (1) structural support for the cell, and (2) intracellular network for motor protein transport. The fundamental building blocks of MTs are α- and β-tubulin, which bind together tightly to form a heterodimer approximately 8 nm in length. Assembly of these heterodimers in the presence of guanosine triphosphate (GTP) forms protofilaments Volume 6, March/April 2014

with an intrinsic polarity where one end is terminated by an α-tubulin (i.e., minus-end) and the other end is terminated by a β-tubulin (i.e., plus-end). Lateral association of protofilaments, with similar polarity, leads to the formation of extended sheets and eventually the mature MT filament. If a sheet contains fewer than or more than 13 protofilaments, the MT experiences lattice rotation where the protofilaments follow a helical path around the surface of the MT as opposed to running parallel to the MT axis.14 MTs, unlike AFs, do not have accessory proteins that enable the formation of branched architectures. The hollow, tubular nature of MTs imparts a high flexural rigidity to these filaments, resulting in characteristic persistence lengths ranging from 1 to 10 mm.15 While MTs possess great mechanical stability, they are highly nonequilibrium structures that undergo cyclic polymerization and depolymerization, a phenomenon known as dynamic instability. The polymerization and depolymerization of MTs can produce forces up to 40 pN and −15 pN, respectively.10 Catastrophic depolymerization of MTs has been attributed to loss of GTP from the growing end, or ‘cap’ of the MT. Depolymerization of MTs is inhibited by MT-associated proteins (e.g., MAP2, ® and Tau) and paclitaxel (i.e., Taxol ); the latter, in particular, has been widely used to stabilize MTs for use in integrated nanomaterials, devices, and systems. Two classes of motor proteins, kinesin (Figure 1(b)) and dynein, enable the bidirectional transport of macromolecules and organelles along MT networks in the cells. The kinesin superfamily is divided into fourteen separate families based on phylogenetic analyses. While each family has unique structural aspects and cellular functions, they all share a highly conserved motor domain that contains an ATP-catalytic site and a MT-binding site. Kinesin motors also possess a ‘tail’ of variable length that is used for cargo binding and can serve as an inhibitory regulator.10 The majority of kinesin are plus-end directed motors with the notable exception of the kinesin-14 family (c-terminal motors) that are minusend directed. For conventional kinesin (i.e., kinesin-1), a number of studies have confirmed that the motor domains move processively in an asymmetric ‘handover-hand’ manner, taking an 8-nm step, consistent with the spacing of αβ tubulin dimers, for every molecule of adenosine triphosphate (ATP) that is hydrolysed.16,17 The force associated with the kinesin stepping has been measured at approximately 5 pN per step, which translates to approximately 40 pN·nm of energy expended in this process and thermodynamic efficiency of approximately 50%.

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Virus cargo Nanoparticle cargo

Microtubule shuttle

Kinesin motor

Casein

Substrate

FIGURE 2 | Schematic representation of the inverted (gliding) motility assays in which kinesin motors are adsorbed to a substrate, and support the transport of MT filaments across a surface. MTs, in turn, may be functionalized with receptors to enable the attachment and transport of both biotic and abiotic cargoes.

The second class of MT-based biomolecular motors, dynein, is commonly divided into two subclasses: (1) cytoplasmic dynein, which is responsible for minus-end directed transport of organelles, and (2) axonemal dynein, which is found in the axoneme of cilia and flagella. Structurally, cytoplasmic dynein is a homodimer, similar to many myosin and kinesin, and possesses five functional elements: MT-binding domain, stalk, linker, motor domain, and tail.18 While it is known that dynein motors are exclusively minusend directed motors, studies have not provided a consensus with regard to step size and force production. Step sizes from 8 to 24 nm and forces of 1–7 pN per step have been reported in the literature.19 In addition, cytoplasmic dynein can bind up to eight ATP molecules, but it is believed that only one ATP is hydrolysed per step. Thus, due to these uncertainties, the work performed by and efficiency of cytoplasmic dynein cannot be accurately determined.

limitations, however, concern the restricted size of the tracks (i.e., tens of microns) and inability to easily and logically organize filaments into the complex architectures necessary to realize useful applications. The vast majority of nanotechnological applications of cytoskeletal transport have adapted the inverted or gliding motility geometry in which the biomolecular motors are adsorbed to a solid surface, and transport the filament in a manner analogous to crowd surfing at a rock concert (Figure 2). Advantages of the inverted motility system include (1) long, extended run lengths that are dependent primarily on the availability of ATP and (2) relatively simple approaches to functionalize cytoskeletal filaments with a variety of biological and synthetic cargoes. Based on its ubiquitous application, this review will primarily focus on the nanotechnological advancements using the inverted motility of kinesin-driven MT filaments, but also will highlight key examples using the natural geometry, as well as examples involving actin-based transport.

Ex Vivo Cytoskeletal Transport In natural systems, AFs and MTs serve as the molecular railroad tracks on which myosin and kinesin/ dynein motors, respectively, transport a range of macromolecular and organelle cargoes. Minimalistic reconstruction of these systems has been achieved by surface adhesion of AFs and MTs to a solid surface (e.g., glass), followed by the addition of purified motor proteins capable of moving along these static filaments. This geometry, which mimics the intracellular architecture, has also been used for specific nanotechnological applications. The primary 166

MATERIALS ASSEMBLY AND MANIPULATION Cytoskeletal active transport has been proposed as a means of dynamically self-assembling nanoscale structures through the dissipation of energy by the biomolecular motors. Self-assembly has been widely explored as a means of fabricating nanoscale architectures from the bottom-up in a faster and more simplistic way than common top-down fabrication (e.g., electron beam lithography).20 However, because

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Biomolecular motors in nanoscale materials, devices, and systems

Dynamic Assembly of Biomimetic Structures The mitotic spindle that forms during cell division represents an elegant biological example of a dissipatively self-assembled system that relies heavily on the dynamic interactions among a number of biomolecular motors. Specifically, motor proteins of both the kinesin and dynein family are critical to the assembly and maintenance of the spindle and the MT-based structures that power chromosome segregation during mitosis and meiosis. A wide array of self-organized structures resembling the asters of the mitotic spindle has been shown to self-assemble based on the in vitro reconstitution of a simple system consisting of only MTs and motor proteins (Figure 3).24 The morphology of these structures could be altered by adjusting the concentration and combination of motors in the system. Such experiments demonstrate the ability to dynamically self-assemble biomimetic structures outside the complex environment of a living cell through basic reconstitution of kinesin motors and MTs. Bundled structures of AFs and MTs may be self-assembled though simple electrostatics25 or applied osmotic forces,26 or by the addition of a crosslinking agent, resulting in compact arrays of parallel filaments and/or networks that exhibit unique properties and behaviors. For example, the Volume 6, March/April 2014

(b)

(c)

Kinesin

(a)

0.8

(d)

1.0

(e)

1.6

(f)

Ncd

of the reliance on short range interactions, the building blocks of traditional self-assembly require thermally-activated diffusion to bring subunits together and facilitate equilibrium interactions, or to disassemble subunits that are nonspecifically bound. Recently, dissipative (dynamic) self-assembly has been discussed as an alternative approach to nanomaterials assembly, whereby nonequilibrium structures are maintained through the dissipation of energy by entropy producing processes.21 Biological examples of dissipative self-assembly are ubiquitous, and commonly involve biomolecular-active transport to remove the limitations of diffusional and thermodynamic equilibrium.22,23 Building on this concept, cytoskeletal transport has been explored as a means of assembling and manipulating biological, synthetic, and composite nanomaterials outside the confines of a living cell. Here, the activity of the motor proteins allows for a dynamic and adaptive self-assembly pathway relying on the availability of an energy source but otherwise operating without outside manipulation. Additionally, the collective force generation by the motors enables rapid and efficient transport of building blocks, as well as the ability to separate nonspecifically bound components.

100 µm 3.0

5.3

6.5

Motor concentration (µM)

FIGURE 3 | Self-assembly of complex MT structures based on the interactions between MTs and multimeric complexes of two different kinesin motor proteins. (Reprinted with permission from Ref 24. Copyright 2001 AAAS)

mechanical properties of AFs crosslinked with filamin A into networks structures can be modulated with the addition of myosin motors.27 These activated networks show a strong stiffening response similar to a shear-hardening material, indicating that the motion of the molecular motors can impart internal stress mechanics to control global properties of the composite. Additionally, recent work combined osmotically assembled MTs with oligomeric kinesin (i.e., two or more kinesin motors bound together) to generate structures that resemble the core structure of eukaryotic cilia and flagella (Figure 4).29 Remarkably, when the motors are activated though the addition of ATP, the bundle assembly transitions into a nonequilibrium phase that mimics the self-sustained wave-like beating patterns of isolated axonemes.28 Such structures highlight the ability of these systems to self-assemble into functional, biomimetic devices with coordinated molecular motion. Further, the coordination of motors in these ex vivo systems can be used to generate force actuations that far exceed single motor forces, as discussed in the next section.30

Dissipative Self-Assembly of Nanocomposites As noted above, the inverted motility assay (Figure 2) is an ideal platform for the construction of nanobiotechnological structures from the bottom-up. Commonly, the gliding MTs in these assays adopt tightly bent or buckled configurations31 with radii of curvature that far exceed the natural curvature of freely fluctuating MTs.32 The induced strain imparted into the MTs by the surface-adsorbed motors allows for non-equilibrium, nanoscale structures to be

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(a) +

Biotin-labeled kinesin:



+

Depletion force

Streptavidin: + Microtubules: − Polyethelene glycol:



+

− Boundary Motor force

(b)

0s

42 s

84 s

126 s

168 s

210 s

252 s

294 s

FIGURE 4 | (a) Schematic representation of motor-driven, self-assembled MT bundles. (b) Fluorescence photomicrographs of MTs bundles exhibiting wave-like beating patterns that are similar to that observed for isolated axonemes. (Reprinted with permission from Ref 28. Copyright 2011 AAAS)

actively self-assembled. For example, kinesin-driven transport of biotinylated MTs results in the selfassembly of nanospools and nanorings in the presence of streptavidin or streptavidin-coated nanoparticles (Figure 5).33–42 Here, the collective work performed by the kinesin motors overcomes the bending energy of the stiff MT filaments, and ensures polar alignment of overlapping MTs by breaking initially opposed contacts between mis-oriented MTs. Detailed examination of motor-assembled nanocomposites of MTs and streptavidin-coated nanoparticles has revealed two remarkable materials properties:38 (1) the stored elastic energy of the nanospools can exceed 33,000 kb T; and (2) ring formation is mechanistically driven by MTs that have a twisted protofilament assembly (i.e., any MT that has more or less than 13 protofilaments). During selfassembly of tubulin dimers into MTs, the number of protofilaments can vary based on the assembly conditions,43 where 12- and 14-protofilament MTs display small amounts of right- and left-handed supertwist, respectively. Because kinesin motors follow along the protofilament’s axis, transport of 12and 14-protofilament MTs by surface-bound motors results in axial rotation in accord with the direction of the supertwist. It has been suggested that, during activated assembly of the nanospools, adjacent MT filaments form strained coiled-coil domains following the oligomerization of MTs containing at least one 12- or 14-protofilament MT unit (Figure 5). Supporting this hypothesis, the rotational directions of the nanospools 168

can be biased, either clockwise or counter-clockwise, by using populations of MTs that have predominately 12- or 14- protofilaments, respectively.35,38,44 Moreover, the assembly of the structures can be modulated by controlling the thermodynamic contributions and rate of energy-dissipation, as well as through microscale confinement.34,36,38 The dissipative self-assembly of nanorings and millimeter-scale wires45 represents excellent examples of biomanufactured nanomaterials built primarily through the work of the motor proteins. Moreover, implementation of this assembly strategy has been proposed to power nanofluidic pumps by attaching kinesin motors containing large beads to the ring structures.46

Motor-Driven Assembly and Organization of Lipid Networks A second example of how kinesin transport can be used to dissipatively self-assemble complex structures involves the formation of lipid nanotube networks, similar to those commonly produced by mechanical micropipetting techniques.47 Extrusion of lipid nanotubes from lipid vesicles has previously been demonstrated using the force actuation of kinesin motors in the natural motility geometry (i.e., motors transport of cargo along MTs; Figure 6(a)). Giant unilamellar vesicles were functionalized with kinesin motors though a biotin–streptavidin linkage and deposited on a surface containing immobilized MTs.48 When the motors attach to a MT, their

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14-protofilament (left-handed, CCW)

Translations and axial rotations of microtubules

13-protofilament

Nucleation

12-protofilament (right-handed, CW)

i. Thermodynamic 'gluing' and strain built

(a)

(b)

5 μm

50 nm

ii. Coiled coil and linked domain formation

(c)

(d)

100 nm

100 nm

(e)

(f)

5 μm

100 nm

(g)

(i)

iii. 'Glued' structure bending

iv. Circle winding

NANOFLUIDICS TRANSPORT AND SENSING

5 μm

Growth

(h)

v. Circle formation

5 μm

500 nm

(j)

(k)

5 μm

500 nm

(l)

(m)

500 nm

50 nm

FIGURE 5 | Proposed mechanism for the biomolecular motor-driven self-assembly of nanocomposite rings consisting of biotinylated MT filaments and streptavidin-coated nanoparticles. (Reprinted with permission from Ref 38. Copyright 2008 John Wiley & Sons, Inc.)

motion extracts lipid nanotubes from the vesicle, where multiple kinesin motors act in coordination to generate the forces required to overcoming vesicle surface tension and allow nanotube formation.49,50 The morphology of these lipid nanotubes is similar to the recently reported tunneling nanotubes found between cells,51 proposed to play an important role in cell-to-cell signaling and sharing of cellular proteins. Complex, large-scale networks of lipid nanotubes have recently been fabricated using the kinesin-driven, inverted motility of MTs (Figure 6(b)). In this system, large multilamellar lipid vesicles, which provide a near infinite source of lipid material, Volume 6, March/April 2014

were functionalized with biotin and attached to motile biotinylated MTs though a streptavidin bridge. Because multiple surface-bound motors act on individual MTs, one MT is able to generate enough force to not only extract lipid nanotubes from the source vesicle, but also create network structures by generating new nanotubes from the existing nanotubes.52 The resulting structures are therefore highly bifurcated with cumulative branch lengths exceeding 10 mm. These networks morphologically resemble neuronal cells where the vesicle represents the soma, and the lipid nanotubes are the axons and dendrites. Moreover, the continual transport of MTs in this system enable networks to dynamically assemble, disassemble, and reorganize over time in a manner analogous to the continuous remodelling of the endoplasmic reticulum of eukaryotic cells.

Reducing the size of bioanalytical devices and sensors from the micro- to nanoscale has been widely pursued as a means of reducing reagent usage, increasing detection efficiency, and decreasing processing time. Nanofluidic devices, however, are practically much more difficult to operate as they require very high pressures or voltages to drive flow, and are exceedingly susceptible to clogging. The use of cytoskeletal transport in these applications has been explored based on the fundamental role of biomolecular motors in intracellular materials trafficking. In particular, biomolecular motors offer a novel means for mitigating many engineered issues, as well as obviating the need for external forces to drive flow altogether. Realization of practical sensing applications based on cytoskeletal transport, however, requires key enabling elements including imparting selectivity and directing transport for sorting, separation, and detection of target analytes, as well as controlling motor speed and function to facilitate efficient cargo loading and unloading. The sections below summarize recent progress with regard to developing these elements and realizing prototype sensing applications.

Directing Cytoskeletal Transport The natural motility assay mimics the transport of organelles and other cargo (e.g., viruses) throughout the cell, and serves as a model system for ex vivo transport. Whereas MTs are commonly organized around a centrosome in the cell, realization of sensing platforms based on cytoskeletal transport requires

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(a)

(b)

FIGURE 6 | (a) Fluorescence photomicrograph of a lipid network extracted from kinesin motor-functionalized vesicles as they move along

surface-adhered MTs (not visible). Scale bar = 10 μm. (Reprinted with permission from Ref 49. Copyright 2003 National Academy of Sciences, USA) (b) Fluorescence photomicrograph showing a highly bifurcate lipid nanotube network (red) formed by the transport of MTs by surface-adsorbed kinesin motors. Scale bar = 25 μm.

the ability to logically pattern and/organize polaroriented bundles of MTs on solid surfaces. To this end, several methods have been developed to create mimics of oriented MT arrays in devices, including the use of flow to align pinned MTs, and controlled polymerization from oriented ‘seed’ MTs.53 Of these methods, flow alignment has shown the greatest promise for generating polar-aligned arrays of MTs. For example, lithographic patterns along with electrical or fluid flow were used to pin and orient AFs on a surface, which in turn supported the directed transport of myosin coated beads in the natural geometry.54 Recently, analytical models were developed to optimize the formation of floworiented MT arrays. Here, the degree of MT alignment was shown to be limited by thermal forces, whereas the kinetics of the process was regulated by factors including the flow strength, MT stiffness, MT velocity, and tip length.55 Approaches have also been developed for creating 3D centrosome-like structures;56,57 implementation of these structures for sensing applications, however, has not been achieved to date. A large number of strategies have been developed to direct MT and AF transport in the inverted motility geometry. For example, chemical patterning and anti-fouling coatings have been used to regulate adsorption of kinesin to specific regions, while physical patterns consisting of walls, overhangs, and enclosed microchannels have enabled directed transport of MTs.58–62 Mechanical rectifiers and concentrators have been constructed based on refinement of these approaches,63 which exploit the collective forces exerted by multiple kinesin motors to bend MTs into radii of curvature well below their intrinsic persistence length. Recently, optimal guiding of MT transport has been characterized 170

using analytic models of the stochastic motion,64 dispersion in MT velocity,65,66 and optimal track geometries.67 Passive steering approaches have been applied to direct the transport of AFs in the inverted motility geometry. Here, the lower persistence length and higher gliding speeds of AF apply different constraints on the chemical and physical barriers required for efficient guiding and rectification.68 Recently, circumvention of persistence length issues has been achieved by bundling AFs with the protein fascin, thereby increasing the stiffness of the filaments and enhancing guided transport.69 Overall, these approaches to steer cytoskeletal transport show great promise with respect to ‘autonomous’ analytical and sensing systems (e.g., smart dust) as they do not require outside operator control. Active steering mechanisms have also been developed based on the intrinsic, as well as engineering properties of the filaments, to develop more sophisticated and complex systems. For example, electric fields have been used to manipulate the velocity and direction of motion,65,66,68 based on their net negative charge. Similarly, control over MT transport was demonstrated by attaching magnetic nanoparticles to the leading end of MTs and applying an external magnetic field to steer MT transport.70 Recently, active guiding of MT transport was achieved using electrically heated gold microstructures to control the phase transition of the thermo-responsive polymer poly(N-isopropylacrylamide) (PNIPAM), as shown in Figure 7.71 In this example, the local heating of PNIPAM on the surfaces enabled kinesin motors to become accessible to MTs and thus direct their transport through junctions. In these examples, active steering enables more refined control over transport, but restricts highly parallel processing based on the

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(a)

e

Microtubule Kinesin PNIPAM

Vo lta g

(b)

T < LCST

Biomolecular motors in nanoscale materials, devices, and systems

(c)

Collapsed Extended

T < LCST

T < LCST

15 μm

Heating line

FIGURE 7 | (a, b) Active steering of kinesin-transported MTs may be achieved using heat from gold electrodes to control the phase transition of the thermo-responsive polymer PNIPAM, which in turn regulates access to kinesin motors in the different paths. (c) Trajectory traces of MTs moving through a junction based on the conformation of PNIPAM at this intersection. (Reprinted with permission from Ref 71. Copyright 2013 American Chemical Society)

inability to observe and simultaneously manipulate large numbers of filaments.

Controlling Transport Velocity Application of cytoskeletal transport in hybrid systems requires the ability to control the transport function (i.e., stop/start motor function), as well as regulate velocity of the biomolecular motors. With regard to the former, genetic engineering approaches have been used to mutate kinesin with chemical ‘on/off switches’, responding to divalent ions including Ca2+ , Zn2+ , and Mg2+ .72–74 Here, the addition of metal ions allosterically inhibits processing by the motors, which can be reversed through chelation, re-activating transport. The ability to turn motor function on and off has also been realized using the thermal phase transition of PNIPAM attached to gliding MTs,75 as well as use of photolysis on caged inhibitory peptides.76 Recently, a graphenepolymer hybrid device was used to control the release of ATP, which in turn started and stopped the transport of AF in this system.77 Because biomolecular motors follow typical Michaelis-Menten enzymes kinetics, regulating transport velocity may be achieved by relatively simple means, such as altering the concentration of fuel (i.e., ATP) present in the system. This approach has been demonstrated using an ATP generation system and the photo-activated release of caged-ATP.78,79 More advanced methods involving the interfacial properties of the support surface have also been explored as a means of controlling velocity. For example, the velocity of kinesin transport was shown to be regulated by switching the charge state of a conductive polymer.80 More recently, MT velocity was regulated through Volume 6, March/April 2014

the photosiomeriation of an azobenzene monolayer, which resulted in the reversible presentation of a cationic amine group, changing the charge state of the monolayer.81,82

Selective Capture and Transport Fundamental to sensing applications is the ability to selectively capture and detect a target analyte. Within this context, immunological, double-antibody sandwich approaches have been widely applied for active transport-based sensing platforms due to the high specificity and ability to optically detect captured analytes (Figure 8).55,61 Application of this approach first requires the ability to impart highly selective interactions between the transport elements (i.e., motors or filaments) and the molecules to be detected. Relatively few methods have been developed to functionalize biomolecular motors for selective capture, as chemical modifications often result in the inactivity of motor function. The approaches applied to date have mainly involved selective modification of the tail region, commonly via genetic engineering, to include His-tags or biotinylation regions with which Ni-NTA or streptavidin-functionalized molecules can interact, respectively.84,85 In contrast, a larger number of strategies for functionalizing AFs and MTs have been developed, and have generally involved the reactivity of lysine residues on the surface of the G-actin monomers or tubulin dimers. These approaches have allowed filaments to be functionalized with constituents including biotin, fluorescent dyes, DNA, and antibodies.86–91 Functionalization of the filaments can, however, adversely impact transport and lead to decreased velocities, depending on the size and density of the linker and/or cargo.42,92

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Capture

Transport

Tag

Transport

Analyte

Detect

Overhang

Antibody Tag - antibody Kinesin Microtubule 20 μm

Excitation / Emission light

800 μm

FIGURE 8 | Example of a sandwich-based approach for analyte capture, labeling, and detection using kinesin-directed transport of MTs in a micro-patterned device. (Reprinted with permission from Ref 83. Copyright 2009 Nature Publishing)

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FIGURE 9 | Multiplexed capture and bidirectional transport of two different analytes using kinesin and dynein motors moving along arrays of MT tracks. (Reprinted with permission from Ref 95. Copyright 2013 American Chemical Society)

Examples of Sensing Applications Several proof-of-principle demonstration systems based on the natural geometry of cytoskeletal transport have been reported. In the first example, the combination of microfluidic flow and cytoskeletal transport was used to process and detect analytes, where kinesin-functionalized beads captured target analytes as they moved along MT tracks in the main channel of the device.93 Microfluidic flow in two sets of side channels crossing a main channel was then used to separate the captured analytes into the different side channels.93 Using a similar 172

approach, kinesin-functionalized quantum dots were used to selectively capture a target protein, TNF-α, which in turn was visualized by a second selectively functionalized quantum dot.85 Most recently, the use of kinesin and dynein motors was used to achieve simultaneous anterograde (i.e., toward the plus end) and retrograde transport (i.e., toward the minus end) of different analytes and cargoes.94 In this elegant example, multiplexed capture, separation, and detection were achieved by selectively functionalizing the different motors to bind different cargoes (Figure 9).95 An advantage of this approach is the

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ability to separate cargoes along the same MT track, as opposed to relying on multiple sets of oriented MT tracks to accomplish this task. In addition, dynein, despite its slower velocity and lower proccessivity, is better able to navigate roadblocks,53 which may enhance the overall system efficiency. In summary, while useful, the scope of sensing applications based on the natural motility geometry is rather narrow based on challenges including limited filament lengths, decreased velocity based on cargo size,96 and traffic jams associated with molecular crowding.97 The inverted motility geometry of AFs and MTs has also been used to develop prototype sensing and detection systems. Both the myosin- and kinesinbased transport systems possess advantages and disadvantages with respect to sensing applications. For example, while myosin has the advantage of a 10fold faster rate of transport, axial rotation of the AFs during transport has raised questions regarding its ability to effectively capture and transport cargo.68 This issue (i.e., axial rotation of the filaments) also applies to kinesin-transport of MTs when the number of protofilaments is greater or less than thirteen. Similarly, the effectiveness of the different guiding methods is dependent on the vastly different flexural rigidities of AFs and MTs. Despite these differences, both kinesin and myosin motility has been used to selectively capture, process, and transport a range of analytes including protein, viruses, and bacteria in proof-of-principle systems.88,90,91,98,99 More sophisticated systems have been developed in which principles of guiding transport were integrated with selective analyte capture. For example, different geometry channels were used to sort, transport, concentrate, and detect fluorescently labeled proteins captured by gliding biotinylated MTs.100,101 Further, a ‘smart dust’ biosensor (Figure 8) was developed in which all device functions (e.g., capture, sorting) were dictated by the device geometry and performed without the need for user input.83 The potential use of such devices for autonomous sensing applications has been proposed,102 and likely will require an increasingly level of engineering complexity to realize specific practical applications.

CONCLUSION The ex vivo application of cytoskeletal transport systems has broadly impacted two areas of nanobiotechnology: (1) dissipative self-assembly of nanomaterials, and (2) autonomous, nanofluidic sensing and diagnostic devices. With respect to the former, a central challenge moving forward Volume 6, March/April 2014

involves the translation of nanoscale phenomena into mesoscopic behaviors. A key enabling step will be developing systems in which active transport enables dissipative assembly of composite nanomaterials in three dimensions. A second key aspect involves coordinated interactions across multiple length scales and time scales to realize macroscopic phenomena. Considering the example of how organisms can change color, the dynamic reorganization of pigmentcontaining organelles by cytoskeletal motors enables a cell to alter its color. Translation of this cellular phenomenon to the organismal level, however, requires many cells to simultaneously undergo this change in a highly coordinated and collective manner. Complex signaling pathways are responsible for such coordination and not easily translated into synthetic and hybrid systems. Thus, to realize more relevant materials systems, it will be critical to design novel, bioinspired means in which information may be shared across multiple length scales to amplify phenotypic behaviors to macroscopic levels. By doing so, new classes of materials may be realized, including those capable of self-healing, adaptive morphology, and self-directed re-organization. While great strides have been achieved with respect to engineering analytical and sensing devices using biomolecular transport systems, the realization of practical nanomedical devices will require more sophisticated component integration. A primary limitation of the current systems is the reliance on microscopic interrogation and read-outs. Preferably, nanoscale sensors will provide a visible and/or quantitative read-out, such as is common in the formation of a visible band in lateral flow immunoassays. In particular, the development of novel device designs will be required to limit signal from unbound tags, as they significantly reduce the signal-to-noise ratio. In addition, more advanced onchip detection methods such as surface-acoustic wave or electrochemical-based sensors may be integrated into the devices to achieve a more sensitive and user-friendly read-out. Here, active transport may be used to move beyond lateral flow geometries by replacing microfluidic flow and achieving novel means for analyte capture, mixing, and separation, while detection is attained with on-chip sensors. While nature provides a vast library of biomolecular motors, relatively few of these have been applied as molecular actuators in hybrid and/or integrated nanoscale systems. Key examples, however, demonstrate the viability of this approach to power nanoscale systems while addressing unique engineering challenges at the nanoscale. Thus,

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biomolecular motors will continue to provide a means of achieving biomimetic functionalities in nanoscale systems, many of which will be well-positioned to

address critical technological problems such as solar energy harvesting, water desalination, self-repairing materials, and electrochemical energy storage.

ACKNOWLEDGMENTS Preparation of this manuscript was supported by the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Materials Sciences and Engineering, Project KC0203010. Sandia National Laboratories is a multi-program laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the U.S. Department of Energy’s National Nuclear Security Administration under contract DE-AC04-94AL85000.

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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 ...
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