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Molecular shuttles based on motor proteins: Active transport in synthetic environments

Henry Hess and Viola Vogel

Department of Bioengineering, University of Washington, Seattle, WA 98195

Active transport in cells, utilizing molecular motors like kinesin and myosin, provides the

inspiration for the integration of active transport into synthetic devices. Hybrid devices,

employing motor proteins in a synthetic environment, are the first prototypes of

molecular shuttles, until the development of superior synthetic motors succeeds.

Here the basic characteristics of motor proteins are discussed from an engineering point

of view, and the experiments aimed at incorporating motor proteins such as myosins and

kinesins into devices are reviewed. The key problems for the construction of a molecular

shuttle are guiding the direction of the motion, controlling the speed, and loading and

unloading of cargo. Various techniques, relying on surface topography and chemistry as

well as flow fields and electric fields, have been developed to guide the movement of

molecular shuttles on surfaces. The control of ATP concentration, acting as fuel supply,

can serve as a means to control the speed of movement. The loading process requires the

coupling of cargo to the shuttle, ideally by a strong and specific link. Applications of

molecular shuttles can be envisioned e.g. in the field of Nano-Electro-Mechanical-

Systems (NEMS), where scaling laws favor active transport over fluid flow, and in the

bottom-up assembly of novel materials.

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Introduction The construction of nanoscale devices has the potential to radically change technology. Assembling materials from nanoscale building blocks and creating devices, which operate on the single molecules level, promises savings in mass, energy consumption and an increase in functionality. Based on the concept of “self assembly”, utilizing energetic and kinetic control of chemical reactions, a lot of progress has been made in creating new materials. For the nanotechnologist who wants to steer the assembly process through positional control of the parts though, the problem of “big fingers” poses a major hurdle. An AFM or STM moving a nanotube or a small molecule (Eigler-DM & Schweizer-EK, 1990) translates ~1023 atoms in the process, consumes a thousand Watts of power and does not allow the simultaneous control of a second molecule. This illustrates that a large part of the functionality of a current nanomanipulator resides on a macroscopic level, rather than on the scale of the object, which is manipulated. A nanoscale nanomanipulator, termed a “molecular shuttle”, would be a nanometer-sized machine capable of transporting single molecules over small distances under user-control. In order to construct a molecular shuttle an appropriate motor or actuator has to be found. While physicists have suggested mechanisms how to build a motor (Porto, Urbakh & Klafter, 2000), chemists have found phenomena similar to active transport (Schmid, Bartelt & Hwang, 2000),(Brouwer et al., 2001) or designed rotating molecules activated by light (Kelly, De Silva & Silva, 1999),(Koumura et al., 1999), and engineers have shrunk conventional actuators to a few micrometers (Jager, Smela & Inganas, 2000), no man-made load-carrying motor smaller than 1 µm has been demonstrated. In contrast, cells employ more than 100 different designs of nanoscale motors assembled from one to seven protein subunits and operating under close control. Well-known examples are the rotary motor F1-ATPase and the myosin and kinesin families of linear motors. More exotic motor designs include motors pulling on double-stranded DNA to package it into the protein shell of a virus (Davenport, 2001), ribosomes moving along RNA while synthesizing a new protein, or even an electrostrictive membrane protein (Zheng et al., 2000). A straightforward approach to build a molecular shuttle though is to employ the motor proteins, which naturally evolved for the task of nanoscale

transport within the cell (Figure 1). These motor proteins already possess the necessary structural features: They move along the cytoskeleton in a directed manner, they have the ability to bind cargo, and mechanisms regulating their activity exist. Consequently, all studies aimed at building a nanoscale transport system on the basis of motor proteins have chosen as engines motors from the well-known kinesin or myosin family (Vale & Milligan, 2000), rather than the more exotic proteins mentioned above. These motor proteins are fueled by ATP and convert its chemical energy into linear motion with an efficiency exceeding 50%. Kinesin for example moves in discrete steps of 8 nm against an opposing force of 5 pN (Meyhofer & Howard, 1995), (Svoboda & Block, 1994), while hydrolyzing one ATP molecule per step (Howard, 2001), (Schnitzer & Block, 1997).

Figure 1: top – a vesicle-carrying kinesin bound to a microtubule, adapted from (Hirokawa, 1998), bottom - myosin V walking on an actin filament imaged by electron microscopy, adapted from (Walker et al., 2000). The scale bar is 50 nm, a motor is highlighted by false-coloring it in green, a vesicle in yellow, part of the fiber in red. Note the different step size of the motors, given by the distance between the heads, and the structural differences between microtubules (top – red) and actin filaments (bottom – red).

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Choosing motor proteins as engines has far-reaching consequences for the design of molecular shuttles, but is dictated by the lack of alternatives. While motor proteins have impressive characteristics in some respects, their need for a very defined environment, which closely resembles the conditions in cells, limits their applications. In the same way as the transition from horses to cars did not change all transportation concepts, we believe that the eventual replacement of motor proteins by synthetic motors can be done at a later stage and will profit from the experiences in nanoscale transport gained with motor proteins. That motor proteins and inorganic parts can be integrated has been shown e.g. for the rotary motor F1-ATPase. Noji et al. (Noji et al., 1997) assembled a structure resembling a propeller by connecting the motor to an actin filament (Figure 2). Soong et al. successfully replaced the actin filament with a nickel bar and mounted the motor protein on a nanometer-sized support structure (Soong et al., 2000). In our work we place an emphasis on the nanotechnology aspect of motor protein utilization. It has been demonstrated that the combined action of many motor proteins can generate the forces necessary to transport small microchips of 20 µm length (Limberis & Stewart, 2000). There also exists some interest in the development of artificial muscles based on motor proteins. In these applications the focus is diverted from the unique size scale of the motor proteins, towards applications that could be realized using more conventional engineering on a larger scale. In order to structure the problems faced in the development of molecular shuttles the analogy to a railway is helpful.

A railway has trains, a complex network of tracks, stations and ways to control the traffic. The result of the interaction of these elements is that cargo is loaded, moved to a specified point according to a timetable, and unloaded again. The design of a molecular shuttle also has to address the questions of guiding, loading and control of the movement simultaneously. After reviewing what is known about motor proteins from an engineering point of view and a short description of the experimental setup commonly used, we discuss the aspects of guiding the motion, loading the cargo and controlling the speed. Potential applications are sketched then, and an outlook on possible future developments is given.

Figure 2: The rotation of a fluorescently labeled actin filament driven by the motor ATPase can be observed with fluorescence microscopy. His-tags, genetically engineered to the N-terminals of the β-subunits, are used to bind the motor to a coverslip in the right orientation. Adapted from (Noji et al., 1997).

Kinesins and Myosins in vivo In order to evaluate the application of kinesins, myosins and related proteins in nanotechnology as force-generating building blocks, we need a general understanding of the functions, mechanisms and working conditions of these nanoscale machines. Kinesin and myosin generate force by hydrolysing ATP, thus they are ATPases (EC 3.6.4 Hydrolases acting on acid anhydrides; involved in cellular and subcellular movement). The hydrolysis reaction cycle with the general steps of binding ATP, hydrolyzing ATP to ADP and Pi, and releasing ADP and Pi, leads to a “powerstroke” against a load (Howard, 1996). The free energy gained from hydrolysis is the upper limit for the work performed

during the powerstroke. Differing rate-constants for the reaction steps and different morphology of the protein leads to the diversity of speeds and step-sizes found in motor proteins. Common to these enzymes is the high efficiency of energy utilization, which can surpass 50% for kinesin (measured by the ratio between performed work and free energy of ATP) (Kawaguchi & Ishiwata, 2000). With time a large number of kinesins and myosins charged with specialized tasks and different morphologies were discovered, leading to a complex family tree of motor proteins with distinct properties (Hodge & Cope, 2000), (Kim & Endow, 2000).

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Figure 3: Different motor proteins. Myosins move along actin filaments consisting of two strands polymerized from actin subunits twisted around each other. The defined orientation of the actin subunits leads to different properties of the filament ends (commonly referred to as pointed end and barbed end). Myosin II and myosin V move into the direction of the barbed end. Kinesins move along microtubules, which consist of 9 to 18 protofilaments polymerized from tubulin. The protofilaments are strictly parallel for microtubules assembled from 13 protofilaments and twisted around each other for microtubules assembled from more or less than 13 protofilaments. The asymmetry of the tubulin subunits influences the polymerization process. This results in a fast growing (plus) end and a slow growing (minus) end. Conventional kinesin moves towards the plus end. The morphology of the motor influences step sizes and speeds. In both motor protein families we can find processive motors (kinesin, Myosin V), which remain attached at all times with at least one head (“walkers”), and non-processive motors (Myosin II), which detach after the powerstroke (“runners”). Prominent members of these protein families are myosin II, causing muscle contraction, and conventional kinesin, facilitating fast anterograde transport. What kinesins and myosins have in common is an active site in the motor “head” (blue in Figure 3), which undergoes a small conformational change during the hydrolysis cycle. This conformational change is amplified by a “lever arm” subunit (red in figure 3) , which connects the “head” binding to the actin or microtubules to the tail region (green in figure 3). Parallel to the conformational change the affinity of the “head” to the microtubule or actin filament changes during the hydrolysis cycle, which leads to coordinated attachment and detachment of the motor and a displacement in a defined direction. Changes in the length of the lever-arm, in the attachment and detachment rates and the formation

of protein dimers from monomers lead to a variety of motor characteristics. While some motors like myosin II are not permanently in an attached state and usually act collectively (as in striated muscles), others like kinesin or myosin V have two heads (or rather “legs”) and “walk” with at least one head attached at any point in time (Hancock & Howard, 1999). This ability, termed “processivity”, allows a single motor to follow a filament or microtubule in a continuous, uninterrupted motion The most marked difference between the kinesin family and the myosin family is that they move along different “roads”. Kinesins bind to microtubules, while myosins attach to actin filaments. Microtubules as well as actin filaments are polymer filaments assembled from smaller protein subunits (tubulin and actin monomers).

+

thick filament

Barbed end

Barbed end

Pointed end

Pointed end

Myosin II

Myosin V

Kinesin (conventional)

~ 50 nm

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While the 8 nm long tubulin monomer is polymerized into hollow tubes with 25 nm diameter and up to 100 µm length usually composed of 13 parallel strands, the 5.5 nm long actin monomers form double-stranded ropes twisted every 72 nm (Howard, 2001). In the cell a dynamic, regulated equilibrium exists between polymerization and depolymerization, leading to a constant recycling of tubulin and actin into new polymer strands. There, microtubules are unbranched and form a structure only in special situations such as spindle formation during mitosis, whereas actin filaments branch out and form a dense mesh. Nanoscale transport in the cell is mainly associated with processive motors. Kinesin transports freshly synthesized synaptic vesicles with a diameter of 20-50 nm from the soma of a neuron to the synapses at the end of the axons up to a meter away (Figure 4). In different circumstances, kinesin, moving on the “highway” formed by microtubules, hands cargo over to myosin V moving on the actin mesh at the periphery of the cell (Brown, 1999).

2 µm

Figure 4: Electron microscopy images (adapted from (Bahadoran et al., 2001)) show that active transport occurs in very crowded environments. Shown is the transport of melanosomes (arrows) through the dendrites of a melanocyte towards a synapse.

Motor proteins possess a sense of direction. Their morphology specifies into which direction on the asymmetric support (microtubules or actin filaments) they move. Kinesin for instance is moving from the center of the cell to the periphery, the so-called “plus-end” of the microtubules. A complementary class of motors, named Dyneins, accomplishes motion in the reverse direction. In the case of kinesin, a 50 nm long tail with high rotational flexibility (Hunt & Howard, 1993) facilitates the binding of cargo. The selectivity of cargo binding is controlled by specific functional marker molecules (Terada & Hirokawa, 2000) and binding proteins (Goldstein, 2001). There are still many open questions associated with motor proteins. Measuring the forces exerted by single motors and recording the associated step sizes, as well as the ATP consumption per step are recent accomplishments. What regulates the activity of the motors and how they bind to cargo is not always clear, even though some information is available (Goldstein & Philp, 1999). The detailed mechanism of motion is subject of ongoing research (Schief & Howard, 2001). It is instructive, that within cells cargo often consists of larger organelles such as synaptic vesicles or melanosomes (see figure 4) and not single molecules (Goldstein, 2001). For small distances and small particles like nucleotides passive transport due to diffusion is faster and less complicated than active transport by motor proteins (Weisiger, 1998). For larger particles, the cell is gel-like rather than liquid-like due to the extended cytoskeleton and high protein content (Luby-Phelps, 1994). Therefore diffusion constants are smaller than in liquids and decrease sharply as particle size increases. Kinesins have evolved to drag cargo through this gel with a mesh size of roughly 50 nm and not through water. The abundance and diversity of motor proteins testifies to their eminent usefulness in the cellular environment. Cells are a proof of principle for active nanoscale transport based on motor proteins.

Motor proteins in-vitro For a technical application of the motor proteins one has to carefully choose between different species and presumably try to match in-vivo functions and the function in the device. Subsequent adaptation to the intended function can be achieved by genetic engineering (Figure 5). The choices for experiments aimed at utilizing motor proteins in synthetic devices have been either

conventional kinesin or the heavy meromyosin subunit of MyosinII. The advantages of these motors are (1) the close match between the function in-vivo and in the device in the case of kinesin, (2) the breadth of knowledge available (myosin II was first isolated in 1864 - (Huxley, 1957)), and (3) the possibility to produce significant quantities of protein by recombinant methods.

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Figure 5: Genetically engineered kinesins. Deletion of certain regions in the gene produces mutant proteins with different mechanical and chemical properties. Removal of the hinge regions changes the stiffness of the tail. Removal of the tail also influences the ATPase rate (Coy et al., 1999). Tail length and specific tags at the end of the tail influence the binding to surfaces and cargo. The main limitation of devices based on motor proteins is that these proteins have to be kept in an environment similar to the cytosol. This is much more stringent than merely an “aqeous environment”, since pH, salt content and temperature have to stay within a certain range and denaturing substances have to be avoided (Böhm, Stracke & Unger, 2000). The concentration of the “fuel” ATP as well as the concentrations of the “exhaust” ADP and Pi also have a strong influence on the enzyme activity (Wang et al., 2000). Contact between proteins and bare synthetic surfaces frequently leads to the denaturation of the protein, so surface properties have to be controlled (Howard, Hudspeth & Vale, 1989). Last but not least proteins, if not kept frozen or lyophilized, degrade within hours to days due to oxidation and the action of proteases present in trace amounts. The development of in-vitro “motility” assays, which satisfy the environmental requirements of motor proteins, was a major breakthrough (Yanagida et al., 1984). In these assays, the inside of a flow cell was coated with motor proteins and already polymerized microtubules or actin filaments were added together with a reagent hindering the depolymerization reaction (e.g.

Taxol). This removed the complexity associated with the dynamic equilibrium between polymerization and depolymerization present in-vivo and allowed the direct observation of motion caused by motor proteins under the fluorescence microscope. Motility assays employ two different geometries, called the “bead assay” and the “gliding assay” (Figure 6). In the bead assay the microtubules or filaments are fixed to the surface and the respective motors move on them. Often a bead is bound to one or more motors to allow visualization of the movement or the exertion of force in an optical trap. In a gliding assay the tails of the motors are adsorbed to the surface and the heads of the motors move the actin filaments or microtubules across the surface. Both geometries can be used for molecular shuttles, each requiring a different set of concepts with regard to guiding and loading.

Microtubule

KinesinCasein Glass

Microtubule

KinesinKinesin

silanized Glass

Gliding geometry

Bead geometry

Figure 6: In-vitro motility assay in gliding and bead geometry. Casein reduces the denaturation of kinesin upon adsorption. The simple flow cell of the classic motility assay is still at the core of most experimental setups (Howard, Hunt & Baek, 1993). It allows simple imaging of beads and fibers using fluorescence or DIC microscopy, easy exchange of solutions, illumination with light, utilization of flow fields and can be built from a variety of materials. The unit price of less than one US-Dollar and the assembly in less than 5 min also allows using a new cell for each experiment, which avoids contamination problems.

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The experimental procedure for a gliding assay usually includes four basic steps: 1) adsorbing a protein monolayer onto the surface by flushing in a solution containing casein or albumin to reduce denaturation, 2) adsorbing the molecular motors to the pretreated surface by exchanging the solutions, 3) adsorbing the microtubules or the actin filaments to the motors from a third solution, 4) imaging motility with optical microscopy. In a bead assay motors and filaments/microtubules are interchanged in step 2) and 3). In most experiments the resolution limit imposed by optical microscopy does not allow us to directly image the events on the nanoscale. Single steps of the motors cannot be resolved, and e.g. microtubules appear ten times wider than they actually are. Advanced optical microscopy techniques, imaging e.g. just one bead at a time (Visscher, Schnitzer & Block, 1999), can resolve single 8-nm steps of kinesins, which ultimately limit the positional accuracy of a transport system based on motor proteins. However, the advantage of basic optical microscopy over electron microscopy or atomic force microscopy is its ability to image non-destructively multiple transport events in real time.

Objective

slide

spacers

coverslip

Flow in

Flow out

Figure 7: The basic flow cell for the in-vitro motility assay. In-flow is driven by capillary forces for the first filling and by holding a piece of filter paper to the opposite side for the exchange of solutions. The thickness of the spacers is ~100 µm. The flow cell has to be sealed to avoid dehydration (e.g. with immersion oil).

Guiding molecular shuttles along tracks The bulk of the experimental studies published are concerned with the problem of guiding. The general question is: How can we exert a guiding force of sufficient amount and specified direction? As suggested by the railroad analogy, one solution lies in an appropriate modification of the surface resulting in “tracks”. This is not the only solution though, since mechanisms of dynamic steering analogous to a “sailing boat” have been demonstrated, where e.g. magnetic, electric or flow fields control the direction of motion. The big advantages of surface modification are that it does not require additional equipment in the setup and that lithographical or imprinting methods allow the easy reproduction of a particular surface pattern. For the gliding geometry guiding can be achieved by controlling surface topography as well as surface chemistry. In the bead geometry, only the chemical properties of the surface influence the adsorption of microtubules or actin filaments. The guiding methods involving flow fields and electric fields allow more real-time user-control and achieve unidirectionality of movement. This crucial advantage over surface modification vanished though when Hiratsuka et al. introduced an arrow-

shaped surface pattern (Figure 11), which sorts microtubules in a gliding geometry according to their direction of motion (Hiratsuka et al., 2000 & 2001). Böhm et al. mixed dynamic and static guiding by fixing a microtubule configuration aligned by flow fields in a gliding assay, in order to obtain a pattern of tracks for a subsequent experiment in bead geometry (Böhm et al., 2001) combining the advantages of both approaches. Irrespective of the guiding method, we prefer the gliding geometry for transport on the nanoscale. The reasons are that adsorption of microtubules or actin filaments into complex patterns does not seem possible and that in the bead geometry the uninterrupted movement of a single motor is limited to the length of a fiber (either a microtubule or an actin filament). The motor density on the surface in the gliding geometry has to be high enough to ensure that at least three motors can simultaneously bind to a fiber to ensure uninterrupted transport. In practice the motor density is chosen sufficiently high to bind dozens of motors to a single fiber.

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Table 1: Overview of guiding methods.

Can we estimate the amount of force required to change the direction of the gliding fiber? What is the minimal bending radius? How much force is exerted through the various approaches? First of all, even in the absence of external forces will the fiber divert from a straight path on a perfectly flat surface uniformly covered with motors due to the action of Brownian motion. The distance, which the fiber can move on a perfectly plane surface with a uniform density of motor proteins until the initial direction is lost, is on the order of its persistence length, measured to be 5 mm for microtubules and 20 µm for actin filaments (Gittes et al., 1993). The high flexural rigidity of the microtubules makes them much more resistant against the inherent “positional” noise caused by Brownian motion compared to actin filaments. A lower limit for the bending radius is dictated by the stability of the fibers, since actin filaments have been shown to break, when bent too sharply (Arai et al., 1999). The minimum bending radius is found to be 200 nm, well below the radii achieved in guiding structures. The equivalent radius for microtubules is not measured, but certainly smaller than 1 µm since 180O turns within 2 µm channels do not cause breaking. In the following we will discuss the guiding methods one by one. Selective adsorption of motors to create tracks. Positioning the motors in lines leads to guiding since fibers bind preferentially along the lines when first adsorbing and tend to move in the direction of highest motor density (Figure 8). The motors themselves are not oriented, but have the ability to bind to a microtubule in the right orientation due to their flexible tail (Figure 9). Protein binding to hydrophobic surfaces is stronger than binding to hydrophilic regions, so creating a pattern with two different surface chemistries leads to tracks of

motors. Such surface patterns can be created using electron beam writing (Nicolau et al., 1999), microcontact printing (Xia et al., 1999) or lithographic methods (Turner et al., 1995),(Suzuki et al., 1997). The feature size of these patterns is typically 1 to 10 µm, which is much larger than the 25 nm diameter of e.g. a microtubule, and defines the orientation of short fibers poorly. These limitations can be overcome by creating tracks with a width of 10 to 100 nm.

10 µm

Figure 8: Microtubules moving on a PTFE surface with a nanoscale pattern of parallel grooves creating parallel tracks of kinesin motors (Dennis et al., 1999). A low-cost method of building such nanoscale tracks is the adsorption of motor proteins to shear-deposited PTFE (Teflon) surfaces (Wittmann & Smith, 1991). Shear-deposition involves sliding a block of PTFE across a heated glass slide, which leaves a thin film of PTFE behind with parallel grooves a few nanometers deep. PTFE is a material fairly resistant to protein adsorption, so these

Guiding method Geometry Motor Tracks of motors using selective adsorption on patterned Teflon or PMMA

gliding Myosin (Suzuki et al., 1995) Kinesin (Dennis, Howard & Vogel, 1999) Myosin (Nicolau et al., 1999)

Guiding channels, uniform motor density

gliding Myosin (Riveline et al., 1998) Kinesin (Hess et al., 2001)

Electric fields, uniform motor density gliding Myosin (Riveline et al., 1998) Flow field, uniform motor density gliding Kinesin (Stracke et al., 2000) Ordered microtubule arrays (flow field) bead Kinesin (Böhm et al., 2001),(Limberis &

Stewart, 2001) Ordered microtubule arrays, selective adsorption

bead Kinesin (Turner et al., 1995)

Tracks of motors using selective adsorption plus guiding channels

gliding Kinesin (Hiratsuka et al., 2000 & 2001)

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grooves represent defect sites facilitating protein adsorption. The result are parallel, nanometer-wide tracks of motor proteins, which are readily followed by the fibers (Suzuki et al., 1995),(Dennis et al., 1999). The shortcoming of the shear deposition method is that it cannot be adapted to complex patterns.

Figure 9: Selective adsorption of motor proteins into the parallel grooves of a PTFE (Teflon) surface leads to the formation of tracks. Notice that the orientation of the heads is not defined upon adsorption. The rotationally flexible tail allows the heads to assume the right orientation for binding with respect to a microtubule. The general method of creating tracks of motor proteins takes direct advantage of the Brownian motion, since the advancing tip of the fiber has to find the next motor. In a curved track this motor is placed sideways, requiring Brownian motion to bend the tip far enough, so that it can attach to this motor. By equating the energy available from Brownian motion (roughly kT/2) with the energy required to bend the tip of the fiber modeled as a slender rod (EIs/2R2, with EI – flexural rigidity, s - length of rod, R – radius of curvature), we estimate a lower limit for the bending radius depending on the distance between motors (e.g. s = 60 nm in (Dennis et al., 1999)). Therefore a microtubule with a flexural rigidity of 2.2 x 10-23 Nm2 can follow a track with a radius of curvature not smaller than 20 µm. The 300 times more flexible actin filaments can follow tracks with a bending radius down to 1 µm, since the minimum bending radius scales with the root of the flexural rigidity. This shows that the guiding mechanism has to be understood in order to match the motor protein and its fiber with the application, which might require sharp turns on the nanoscale.

10 µm10 µm

Figure 10: Guiding of microtubules in channels. The side walls of channels with a depth of 1 µm exert a guiding force on the microtubules (Hess et al., 2001). The inset shows an AFM image of the surface topography. Using guiding channels (Hess et al., 2001),(Riveline et al., 1998) (Figure 10) has the advantage that the tip of the fiber is pushed against an inflexible sidewall, which is able to exert large guiding forces. In this case, the force available from the motors pushing the tip into the turn limits the bending radius. The force pushing the fiber forward, which is required for a given turning radius, can be found by differentiating the bending energy (EIs/2R2) with respect to the length s. Three kinesins with a combined force of 15 pN can push the stiff microtubule into a turn with 1 µm radius of curvature. This technique is therefore ideally suited to the kinesin/microtubule system. Adsorbing motors only to the bottom of guiding channels combines the approaches and improves the efficiency of guiding, since the fibers cannot slowly climb the sidewalls anymore (Figure 11).

Figure 11: Using lithographic techniques Hiratsuka et al. produced a sophisticated guiding channel (left) acting as a rectifier for the direction of microtubule motion (adapted from Hiratsuka et al., 2000). The arrowheads sort the microtubules according to the rotational sense of direction, which can be observed using fluorescence microscopy (right). Since only the bottom of the channel adsorbs kinesin, the escape of microtubules from the channel is effectively prevented.

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Flow fields (Figure 12) are capable of exerting substantial drag forces depending on flow velocity and viscosity of the solution. The drag force for a flow parallel to the microtubule orientation is given by Fdrag=2πηLvcosh(h/r) with L and r - length and radius of the fiber, h-distance surface to fiber-axis ((Howard, 2001), p106, ex.6.6) and twice that force for a flow field perpendicular to the fiber-axis. At a flow speed of 1 µm/s, which corresponds to the speed of the microtubule movement, the drag force is 0.1 pN, which means it is negligible in relation to the forces exerted by the motors or necessary for guiding. Increasing the flow velocity thousand fold gives a drag force of 100 pN. While a flow velocity of 1 mm/s may not seem high, the parabolic profile of the flow field in the flow cell requires an average flow velocity of 50 mm/s in the cell to obtain a velocity of 1 mm/s at a distance of 100 nm from the surface. In our view, the guiding influence of the flow field stems from its action on the free tip of the advancing microtubule, which is shorter than 0.1 µm (depending on motor density). So only about 1 % of the exerted force is expended on guiding, the rest is balanced by the tight attachment of the microtubule to the surface-bound motors. This small guiding force implies that only wide turns are feasible. Since it is not possible to structure flow fields on a scale of <10 µm anyway (due to the viscosity of the liquid), this limitation can be accepted.

Figure 12: A flow field induces a preferential movement of microtubules in the direction of the flow (from left to right), which leads to alignment. Adapted from (Stracke et al., 2000). Electric fields can also be applied to exert forces on fibers, similar to their utilization in gel electrophoresis (Figure 13). A field strength a of 104 V/m exerts a force per length of 2 pN/µm

(Riveline et al., 1998) on the negatively charged actin filaments (isoelectric point 5.4 (Zechel & Weber, 1978)) and roughly the same force on microtubules (isoelectric point 5.1, David Wilson website). This force is enough to align actin filaments along the electric field lines, but might be insufficient for the guiding of the stiff microtubules. This field strength translates into a voltage of only 0.1 V between two electrodes spaced 10 µm apart! This suggests a merger between motility assays and microfabricated electrodes to obtain dynamic control of guiding in the future. However, two problems can be anticipated: (1) Applying an electric field across the buffer solution leads not only to forces acting on the actin filaments, but on all ions present, causing a flow field as well. (2) Oxygen created at the electrodes might accelerate the degradation of the proteins, and electrolysis might change the buffer conditions.

Figure 13: Traces of short (4 µm), moving actin filaments under the influence of an electric field. Left - on a plane surface the electric field induces a movement towards the cathode. Right – on a surface with 1 µm wide guiding channels oriented parallel to the field lines the filaments move in straight lines but do not turn towards the cathode. The length of the traces is proportional to the speed. Adapted from (Riveline et al., 1998). Magnetic fields exert forces on microtubules, since they have an inherent magnetic dipole moment due to the presence of oriented Pi-electron systems. This dipole moment allows aligning a free-floating microtubule in strong fields (B ~ 10 Tesla) (Bras et al., 1998), but the forces necessary for this are far smaller than 1 pN. An option to obtain forces up to 5 pN is to bind the fibers to superparamagnetic beads (Dynabeads, 2.8 µm diameter) (Uchida et al., 1998). The minimum diameter of the beads is given by ~2 µm though, since the force exerted by the magnetic field scales with the volume of the bead (Grimsehl, 1988). For the development of a nanoscale device with feature sizes below 1 µm this might not be acceptable.

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Tracks of motors, guiding channels, flow fields, electric and magnetic fields and combinations of these approaches are available to guide molecular shuttles. Scalability is an issue here: Some approaches lend themselves to miniaturization more easily than others. Microscopic sources of positional noise entered our discussion and the

stiffness of the “trains” was shown to be an important property. Nature evolved “highways” based on microtubules and “sideroads” based on actin filaments. Will synthetic devices based on motor proteins reflect that distinction? Guiding motion on the nanoscale is still a challenge, but the first tools have been developed.

Loading of Cargo Loading and unloading of cargo received far less attention than the complex of questions associated with guiding. One reason is, that it is seen as a second step, which can be taken once guiding is mastered. The other reason is that in a flow cell in bead geometry one can attach motors to micrometer-sized cargo through nonspecific adsorption, the same mechanism, which binds the motors to a surface in a flow cell in gliding geometry (Block, Goldstein & Schnapp, 1990). Nonspecific adsorption though, as the name implies, offers no control over the binding of cargo to the motors, limiting its use in molecular shuttles. Motors will adsorb everywhere and denature if the surface is not yet covered by protein. This highlights the importance of suppressing unwanted binding when loading cargo. Specificity is desired, since we do not want to bind anything adsorbing protein, but a specific cargo ideally in a specific orientation. The helical structure of actin filaments poses an additional problem for oriented binding, since actin filaments rotate once per micrometer moved (Sase et al., 1997). Bound cargo would presumably follow the filament rotation and potentially get stuck between filament and surface or knock the filament off the surface. Microtubules probably do not rotate if the number of protofilaments equals 13, since then the protofilaments are parallel (Chretien & Wade, 1991). However, polymerization of microtubules in-vitro leads to a distribution of the number of protofilaments (Chretien et al., 1992). This necessitates the selection of the microtubules with the right number of protofilaments. The research on binding of kinesin to cargo in-vivo offers little help, since most questions associated with in-vivo kinesin-cargo interactions are still debated. In vesicle transport in neurons it is assumed that kinesin can either bind directly or through a scaffold protein to vesicle transmembrane proteins. These proteins also act as an “address tag” and control the delivery to specific destinations.(Hurtley, 2001),(Bahadoran et al., 2001). The surface of these vesicles, consisting of hydrophilic headgroups, suppresses non-specific

protein binding. Due to the scarcity of information and the difficulties associated with reconstituting the transmembrane proteins, the integration of the in-vivo system for cargo binding into the molecular shuttles does not seem feasible at the moment. The general concept of a specific tag, placed on the otherwise adsorption-resistant cargo, which is recognized by the appropriate transporter has to be mimicked using different approaches. Ideally we would like to have a strong, specific and reversible connection between transporter and cargo. Biotechnology offers a number of tools to specifically bind proteins to surfaces. Examples are poly-histidine-tags binding to nickel-coated surfaces, and various antigens binding to their antibodies, such as the binding of the vitamin biotin to avidin (egg-white) or its bacterial analogue streptavidin. Based on these tools, techniques to functionalize the shuttles and the cargo each with one of the linkers have to be developed. In this context we showed that the transport of cargo coated with streptavidin by biotinylated microtubules in a motility assay in gliding geometry is feasible (Hess et al., 2001) (Figure 14). This concept is valuable, because it integrates with the current efforts to guide molecular shuttles (based mostly on the gliding geometry). In addition, the strong interaction of biotin and streptavidin is widely used to separate proteins, thus making the key components commercially available as well as subject of on-going research. The complexity of designing links on a molecular level, which withstand applied forces between 1 pN and 1 nN should not be underestimated. At this size scale Brownian motion applies a wide spectrum of forces to the bond. This background of forces leads to a limited lifetime of the bond, which is further decreased by additional forces applied by the cargo. So force-dependent rates of binding and unbinding characterize the link, rather than a specified maximum force (Evans, 2001). For example, the non-covalent interaction between streptavidin and biotin withstands a force of 5 pN for 1 min, while breaking within 1 ms if 200 pN are applied (Merkel et al., 1999).

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Biotin

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Figure 14: Loading of cargo to microtubules can be accomplished using streptavidin and biotin as specific linkers. In a proof-of-principle experiment streptavidin-coated beads were loaded onto biotinylated microtubules (left). Beads in solution were picked up by microtubules and transported across the surface (center). Clumps of beads bound to multiple microtubules and moved randomly across the surface until colliding and fusing with other clumps (right) (Hess et al., 2001). Unloading the cargo requires a mechanism to break the link between cargo and shuttle under user-control. Bond-breaking can be triggered by the release of certain chemicals and enzymes, changes in pH, or in some cases UV-light. The cleavage or creation of bonds based on photochemistry seems to be ideally suited for our purposes since the illumination can be confined to a spot of only 1 µm^2, thus controlling binding or release locally and non-invasively. Exerting forces on a linker connected to a tubulin or actin subunit also tests the integrity of the polymer structure. actin filaments have been shown to withstand high tensile forces of up to 500 pN applied with a microneedle (Kishino & Yanagida, 1988). For microtubules the force required to remove a subunit is not known, but is probably on the same order of magnitude for each

protofilament. The sustained forces are therefore most likely larger than the drag forces, which have to be overcome during transport. However, if ~50 kinesins with a combined force of 250 pN act collectively on a 1 µm long section of a microtubule, its structural integrity may be seriously tested. A difficult question is how fast we can bind cargo to the shuttles. Will we rely on Brownian motion to orient the cargo with respect to the shuttle and initiate binding? Can we design docking stations where preoriented, fixed cargo is picked up? How close does a microtubule come to the surface during its movement and can it pick up molecules from the surface? Future research will address these questions and increase our fundamental understanding of engineering at the nanoscale.

Controlling the motion Regulating the speed of a molecular shuttle is as important as controlling its direction. In a railway two separate systems are used to control the speed: the engine and the brakes. On the nanoscale inertia is negligible, so a braking system is not required to reduce the kinetic energy of the shuttle. A braking system might be used to oppose the force provided by the motors, but as in a railway it seems reasonable to reduce the activity of the motors to slow down instead of applying brakes with the engine running at full speed. Therefore the first question again is: How is motor activity regulated in-vivo? The regulation of myosin II in skeletal muscle has been studied in great detail. In the resting state of

the muscle attachment sites of myosin II on the actin filaments are blocked by tropomyosin. A rise in the Ca2+ concentration above 1 to 5 µM, regulated by the nervous system, leads to binding of Ca2+ by tropomyosin. Ca2+ binding causes a conformational change in tropomyosin exposing the myosin binding sites on the actin filaments and allowing the movement of myosin along the actin filaments (Alberts et al., 1994). For the regulation of kinesin activity a similar mechanism has not been found. The binding of kinesin heads to microtubules increases the activity thousand-fold, while binding of the kinesin tail to cargo increases kinesin activity six-fold (Coy et al., 1999). Microtubule activation and activation by

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cargo make economic sense, since the unnecessary expenditure of ATP by unattached kinesins is avoided. Other parameters like ATP concentration, concentration of divalent cations, or temperature influence kinesin activity but do not closely regulate intracellular transport. The ideal control system in a synthetic device would regulate the motor activity not only for defined times but also for a defined region on the surface. We would also prefer to control the speed over a wide range instead of having only one “ON” and one “OFF” state. For a molecular shuttle based on myosin it seems feasible to emulate the natural control system, even though it has not yet been demonstrated in this context. For a kinesin-based shuttle system, which is favored by others and us for reasons related to guiding and loading, the task is less straightforward. The kinesin activity could be controlled by regulating the concentration of Mg2+ between 0 and 0.5 mM (50% of maximum activity), regulating the concentration of ATP between 0 and 0.05 mM (Km = 50 µM) (Figure 15), or introducing reversible inhibitors.

Figure 15: The dependence of microtubule speed on ATP concentration in a motility assay in gliding geometry measured by Böhm et al. (Böhm et al., 2000). While regulation of the concentration of Mg2+ is technically similar to the control of myosin activity by Ca2+, kinesin activity is 100 times less sensitive to changes in Mg2+ compared to the tropomyosin “switch”. Finding a compound similar to tropomyosin but binding to microtubules would be required to follow this approach, since introducing and removing 0.5 mM of Mg2+ seems very difficult, unless the whole buffer solution is exchanged. The regulation of the concentration of ATP has the advantage that it can be achieved in many different ways. The ATP concentration can be reduced by the action of enzymes, hydrolyzing it

into ADP and Pi, and also increased by enzymes utilizing different sources of energy to form ATP from ADP and Pi. For instance a method to keep ATP concentrations at constant levels is to couple it to a reservoir of phosphoenolpyruvate through an ATP regenerating system (Wettermark, Borglund & Brolin, 1968). Introducing reversible inhibitors is elegant, since the ATP concentration could be kept at saturating levels, while the reversible inhibitors act as “switches”. Local anesthetics such as lidocaine have been shown to inhibit kinesin movement on microtubules as well as myosin movement on actin filaments at millimolar concentrations (Miyamoto et al., 2000). Changing the inhibitor concentration requires exchanging the buffer solution though. While controlling Mg2+ concentration or introducing inhibitors may be advantageous in setups, which utilize flow-fields and exchange the solution rapidly, the control of the ATP concentration seems to us the most intriguing approach. In the first experiment addressing the question of control (Hess et al., 2001), we utilized light-activation of caged ATP to increase the ATP concentration in a flow cell. In order to “cage” ATP an aromatic molecule is connected to the γ-phosphate group of ATP via a bond, which is cleaved after illumination with UV-light. This “caged” ATP does not correctly fit the enzymatic site of the motor protein and cannot be hydrolyzed. Illumination with UV-light is an elegant way to release ATP, since the area of illumination can be defined and the illumination time conveniently controlled. The disadvantage of caged ATP is that only a limited amount can be stored in the flow cell, so the operation time without exchange of the buffer solution is limited. A complication arises from the high fuel efficiency of the motor proteins. Since the motor concentration in the flow cell is only a few nM/L, and more than 99% are not activated by binding to microtubules, the motor activity reduces the ATP concentration by less than 1 nM/L/s. Therefore shuttles slow down only after hours of movement. Decreasing the height of the flow cell, thus increasing motor concentration while leaving the surface density constant, can reduce this time-constant. Another option is to introduce an ATP consuming enzyme, which reduces the ATP concentration on a shorter time-scale. In our experiment hexokinase was introduced, allowing us to tailor the deceleration of the molecular shuttles by changing the hexokinase concentration. The combination of caged ATP and ATP-consuming enzyme provided a system, where the molecular shuttles are stopped in the absence of light and

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move a defined distance after light exposure before returning to rest (Figure 16). This experiment is the proof-of-principle that user control of molecular shuttles is possible. A variety of alternative approaches have been discussed though, and it remains to be seen, which paths are ultimately taken.

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Figure 16: The speed of microtubule movement rises immediately after uncaging caged-ATP with UV-light and falls exponentially as ATP is hydrolyzed by the enzyme hexokinase. The peak in the velocity-time diagram corresponds to a discrete step of a few micrometers, which can be repeated until the reservoir of caged-ATP is exhausted (Hess et al., 2001).

Applications of molecular shuttles In the preceding paragraphs we reviewed the available know-how concerning the assembly of a molecular shuttle. How can we harness the unique characteristics of this transport system? As potential applications of molecular shuttles sensors, self-healing materials, molecular sorters and nanoscale actuators have been mentioned. The usefulness of a functional molecular shuttle is almost considered self-evident, so no thorough discussion has been devoted to the actual benefits of active nanoscale transport. In our opinion, active nanoscale transport has two major advantages: (1) It can bridge passive transport by diffusion and active transport by fluid flow, (2) it can provide nanometer positional control, solving the problem of “big fingers” mentioned in the introduction. Passive transport by diffusion is fast and effective on the nanoscale, and is utilized for many self-assembly processes. Fluid flow is effective on the micrometer to millimeter scale, and is employed in the cardiovascular system or in MEMS devices. Nature employs active transport based on motor proteins on an intermediate scale. Examples are the transport of supramolecular structures like synaptic vesicles with a 20-50 nm diameter through axons with a diameter of ~1 µm, or the accurate positional

control of large cellular structures such as chromosomes during mitosis. Active transport of single proteins over short (<1 µm) distances probably occurs only in the most densely packed regions of the cell, such as focal adhesions. The integration of molecular shuttles based on motor proteins into synthetic devices is therefore advantageous, when device dimensions fall below 10 µm but are larger than 100 nm, so fluid flow is not feasible anymore and diffusion not yet dominant (Figure 17). Since the rate of diffusion depends on particle size as well as on the viscosity of the liquid, this range depends on the application. Molecular shuttles could therefore extend the miniaturization process of analytical equipment, which started with MEMS devices (lab on a chip), to the nanoscale. MEMS-devices are developed e.g. for flow cytometry, transporting blood samples through complex optical and chemical detectors with channel sizes of roughly 100 µm. Here, the minimum channel size is limited to ~10 µm, since for a given pumping pressure flow speed slows dramatically with decreasing channel size. Since molecular shuttles would transport only the analyte and not the solvent, energy requirements would be dramatically reduced.

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Figure 17: Different scaling laws govern diffusion, fluid flow, and active transport by motor proteins. The speed of diffusion (sphere with 1 µm diameter) falls off rapidly over larger distances (<v>= 2 D/<x>, D= kT/6πηr ), while pressure-driven fluid flow (in a 1 meter-long channel, pressure drop of 100 Torr) slows down drastically as channel diameters decrease (v = r2/8η*∆p/∆x). For high viscosity η of the liquid (solid lines), active transport by motor proteins (conventional kinesin in-vitro) is faster if channel diameters are limited and distances sufficiently large. For a 10 times lower viscosity (equal to the viscosity of water, dotted lines) this advantageous region vanishes.

For self-healing materials it is clearly advantageous to fix defects while they are small. Material and energy requirements for “healing” a defect site fall with the size of the defect and the material properties are kept within close tolerances. Molecular shuttles may “survey” a surface and transport the required replacement materials in a directed manner. These applications require fast, “intelligent” transport with low energy consumption but do not take full advantage of molecular shuttles. Unlike diffusion, which is a stochastic process, molecular shuttles are able to create order on the molecular scale. This is a key advantage over other transport processes and could be harnessed in a variety of ways. “Weaving” polymers into a complex fabric could create a revolutionary material in the more distant future. Transporting a piece of nanoscale machinery along various assembly stations in a predetermined order would overcome limitations of self-assembly. The read-out of a molecular computer by a molecular shuttle could solve the problem of interconnects. For these applications nature does not offer a template solution, requiring us to “invent the wheel”. The potential pay-off of nanoscale “fingers” may exceeds the usefulness of a miniaturized MEMS device by far.

Outlook/conclusion The unique characteristics of motor proteins and the existence of a template solution in the form of nanoscale transport in cells makes motor proteins an ideal base for the assembly of molecular shuttles. Inspired by nature, solutions for the key problems of a nanoscale transport system based on motor proteins have been demonstrated. With state of the art techniques, guiding and loading of a molecular shuttle as well as control of its movement is possible. The motor protein kinesin is currently the workhorse, taking 8 nm steps per ATP hydrolyzed and pulling with a force of 5 pN. A variety of mechanisms to guide the shuttles have been studied including patterned surfaces, flow fields and electrical fields. The selective coupling of biotinylated microtubules to streptavidin antibodies attached to cargo is a model for the

loading procedure. Defined exposure of caged ATP to UV-light can control the fuel supplied to motor proteins and is a strategy to control the speed of molecular shuttles. The utilization of motor proteins as engines is not without disadvantages. The strict requirements on environmental conditions and the natural degradation of proteins are serious problems. Potential solutions lie in genetic engineering or the utilization of more stable proteins from extremophile organisms. If devices based on motor proteins will be ubiquitous is an open question. However, the principles of nanoscale transport, explored using motor proteins, will still apply after an eventual transition to artificial motors.

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