Lab on a Chip

Nano-biopower supplies for biomolecular motors: the use of metabolic pathway-based fuel generating systems in microfluidic devices

Joshua R. Wasylycia^ab, Svetlana Sapelnikova^ab, Hyuk Jeong^c, Jelena Dragoljic^a, Sandra L. Marcus^a and D. Jed Harrison*^ab
aDeptartment of Chemistry, University of Alberta, Edmonton, AB T6G 2G2, Canada
^bNational Institute for Nanotechnology, National Research Council of Canada, Edmonton, Alberta, Canada
^cDeptartment of Chemistry, Sookmyung Women's University, Seoul, 140-742, Korea

We report fuel generation systems for molecular motors based on pyruvate kinase, or for the first time, mitochondria, implemented within microfluidic devices. Intact organelles acted as bio-nanopower supplies for molecular motors, using isolated mitochondria to convert chemical energy from succinate to ATP, harnessing nature's enzymatic transformation cascades directly. Motors were activated essentially equally by ATP produced by pyruvate kinase, mitochondria, or direct addition of ATP.

Molecular motors are active in the presence of adenosine triphosphate (ATP), which provides the fuel for inducing motion as the high energy phosphate bond is hydrolyzed. In the long term, some form of nanopower supply, utilizing a high energy density fuel such as glucose (36–38 ATP are generated per glucose molecule¹) would be appropriate for fueling molecular motor operation in vitro. In living cells, mitochondria are the primary fuel generators, and these can be harnessed to power biomolecular motors.

Biomolecular motors serve various important functions inside the cell, exhibiting many modes of operation. These natural bio-nanomechanical systems are of great interest for the design of nanoscale systems.^2–4 Cytoskeletal motor proteins (kinesin, myosin, dynein)^5,6 produce sliding movement along filamentous structures of complementary tracks, acting as one of the major active transport mechanisms in cells and providing a model linear motor system for nanotechnology. The kinesin–microtubule system has been implemented successfully in synthetic environments for directional movement,^7–14 cargo transport^7,8,15–17 nanostructuring,^18,19 surface imaging²⁰ and sorting of protein assemblies.^11,21

A limited number of steps have been taken to date to address fuel supply and control for molecular motors. Photolytic cleavage of caged ATP²² generates an optical on/off switch, but the reagents remain low in energy density, are relatively costly, and the efficiency was low. Recently, polymer particles loaded with pyruvate kinase (PK) have been attached to microtubules as cargo,²³ supplying a localized source of ATP from phosphoenolpyruvate (PEP). This scheme uses only the last step in initial glycolysis.

The challenge of using higher energy density fuels is addressed through the use of mitochondria, employing nature's intact machinery to generate far more ATP from fuel sources such as PEP, pyruvate, or succinate than the pyruvate kinase reaction delivers. This study shows, for the first time, the ability to use isolated mitochondria to synthesize ATP for powering molecular motors within microfluidic devices. Furthermore, this study examines the use of microfluidic devices to control the on/off cycle of molecular motors, utilizing the PK enzyme system.

Microtubules were polymerized at 37 °C for 30 min from a mixture of 25% rhodamine labeled and 75% unlabeled tubulin (Cytoskeleton, Denver, CO, USA) at a concentration of 1.4 mg mL⁻¹ in BRB80 buffer. Full length drosophila kinesin heavy chain encoded from plasmid pPK113 (gift from J. Howard) was prepared at a concentration of 10 μg mL⁻¹ in BRB80 buffer containing 0.2 mg mL⁻¹ casein.²⁴ Motility assays²⁵ were performed as described below. All experiments were conducted at room temperature (21 °C).

A channel network was fabricated²⁶ in glass to the dimensions specified in Fig. 1a. A suspension of 20 μm enzyme-coated polystyrene beads was introduced to the 60 μm deep channel. Beads were physically constrained at the end of the bed, by the depth of the channel network (<12 μm). An ADP rich solution was introduced to reservoir 1 and incubated for 5–10 min to produce ATP, then mobilized to the detection window, where motility was observed by fluorescence microscopy.

	Fig. 1 (a) Schematic diagram of flow channels and beds in a microfluidic chip. The cartoon illustrates the orientation of the kinesin/microtubule motor system. A fluorescent microscope was used to take images in a small section of the detection window. Channels are 100 μm wide and 10 μm deep. ATP producing bed is 2 cm long, 220 μm wide and 60 μm deep. ATP was transferred from the ATP producing bed to the microtubule/kinesin zone by applying vacuum at the outlet. (b) Diagram of a 15 μL microfluidic chamber device, utilizing filter paper to prevent mixing of the solution components of the microtubule chamber with those of the ATP generating chamber.

Two-chamber devices (15 μL) were assembled using double-sided tape, positioned between a glass microscope slide and a coverslip (Fig. 1b). Filter paper (Grade 2, Whatman International Ltd., Maidstone, England) was positioned in the chamber to divide it into two sections, blocking bead transport, but allowing fluid transport. A motility assay was performed on one side of the filter paper and beads or mitochondria suspension were perfused into the opposite side. Fluid was transferred by simultaneously absorbing the fluid with filter paper on one side of the chamber while injecting a new fluid into the opposite side of the chamber.

Rabbit muscle pyruvate kinase (Sigma–Aldrich) was physisorbed on a 0.5 mL aliquot of 20 μm diameter polystyrene beads (Polysciences, Warrington, PA, USA). A luminometric assay was used to determine the ability of pyruvate kinase to produce ATP in vitro. ATP concentrations were determined with a luminometric method based on firefly luciferase,^27–29 using an ATP determination kit (A-22066, Molecular Probes, Eugene, OR, USA) and BioOrbit 1253 (BioOrbit Oy, Turku, Finland) luminometer.

Liver mitochondria were prepared from male Sprague-Dawley rats weighing 150–200 g according to a standard procedure.³⁰ Mitochondria were used in suspension (final concentration 2.4 mg mL⁻¹ protein) as well as immobilized on 20 μm polystyrene beads. Buffer conditions were adjusted to meet the motility assay demands.

For the luminometric assay of ATP production, mitochondria were immobilized on silica-based C₁₈ beads by physisorption with the use of Tris (pH 7.4) buffer, without taxol and antifade. Immobilization was performed as described for the polystyrene beads. ATP concentrations were determined using the luminometric based assay described above.

We first evaluated the ability of both mitochondria, and the enzyme pyruvate kinase (PK) to generate ATP from appropriate substrates in a cuvette. The conversion efficiency of ADP into ATP was studied using a luciferase luminometric method, to provide an estimate of the efficacy of the planned conversion reactions. Pyruvate kinase was immobilized on polystyrene beads. The luciferase assay showed the enzyme was able to catalyse ATP production with a conversion efficiency of 80%, from 1 to 5 mM initial ADP concentration, with an incubation time of 10 min, using an equal concentration of PEP as the fuel source. Mitochondria are most easily trapped at specific locations within a microfluidic chip if they are first immobilized. We adsorped the mitochondria on either polystyrene or C₁₈ beads, and tested the activity of the preparation using the luminometric method. For a reaction time of 10–30 min, conversion of ADP to ATP of 80% was observed for both 5 mM pyruvate/5 mM malate or 10 mM succinate/2 μg mL⁻¹ rotenone as the fuel, as determined with the luciferase assay. The latter system will only generate ATP from the oxidative phosphorylation process in mitochondria, due to the inhibitory action of rotenone on complex I in the mitochondrial membrane and on the citric acid cycle.^28,31 Further, addition of 10 mM sodium azide, which inhibits the electron transfer by cytochrome oxidase,³² reduced ATP production to less than 10% conversion (oxidative phosphorylation is responsible for ATP production). For both systems the ATP concentrations produced are sufficient to drive in vitro molecular motor reactions (vide infra), and we concluded that ATPase generation of ATP by mitochondria was invoked.

A simple fluidic device consisting of a two-section chamber, separated by a membrane filter to reduce the speed of mixing, was used to evaluate the motility of microtubules on a kinesin coated surface with various ATP sources. The velocity of the microtubules was used as the figure of merit.

Tests in the two-chamber device with immobilized PK, PEP and ADP (1.5 mM each) showed the same microtubule velocities as observed when directly adding standard 1 mM ATP solution. Tracking vectors for microtubules gliding on a kinesin treated surface powered by ATP produced in situ by pyruvate kinase are shown in Fig. 2. In the provided image, microtubule velocity was found to be 170 nm s⁻¹ ± 10%. Reported gliding velocities vary widely, and are known to depend on several factors, including ATP/ADP and Mg²⁺ concentration and temperature.³³ Our observations are in agreement with gliding velocities reported by others (from 100–2000 nm s⁻¹).³⁴

	Fig. 2 Molecular motor motility tracks are shown from a sequence of fluorescent images taken 10 s apart. Each dot and line represents the velocity vector between images. The microtubules in this image were mobilized by ATP produced in situ by mitochondria. Track indicates initial position of microtubules. Electronic supplemental information includes a time lapse movie showing 90 s of motility of microtubules.

PK enzyme-coated polystyrene beads were packed uniformly from suspension into the bed of a microfluidic network fabricated in glass. The device allowed for the selective control of fluid delivery to the microtubule/kinesin zone. We were able to start and stop the movement of microtubules at a given location several times during a single assay, as illustrated by the cycles shown in Fig. 3. Solutions were transferred in less than 1 min and the response of the motors was faster than the time required to view the microtubules after solutions had been transferred (a few seconds). The elapsed time between states was on the order of a few minutes. Initial addition of ATP from the enzyme reactor bed initiated microtubule motion, while flushing the ATP solution back into the packed enzyme reactor bed from the motility bed stopped all measurable motion. Subsequent re-delivery of ATP from the enzyme bed again induced motility. This fluidic switch approach potentially establishes a method to recycle ADP byproduct back to ATP, reducing the ability of ADP to inhibit motility. Luciferase assays showed pyruvate kinase loaded beads retained approximately 70% conversion of ADP through at least four reaction cycles.

	Fig. 3 The average velocity of microtubules within the field of view of the detector is shown under conditions where ATP was delivered from the ATP producing bed (on), and was then flushed away (off). The x-axis represents stages when ATP rich fluid was present (on) and when ATP rich fluid was absent (off). Standard deviations are for 10 microtubules in a single experiment.

The ability to induce motility with mitochondria as the fuel supply system was evaluated in the two-chamber device, using 5 mM succinate as the fuel. Considerable effort went into matching buffer conditions suitable for ATP production by mitochondria with the buffer conditions required for the motility assay. The largest challenge arose because mitochondria require oxygen, while the fluorescent dye bleached in its presence, necessitating the use of oxygen scavenging agents in the molecular motor buffer, with an oxygenated buffer for the mitochondria. Mitochondria were delivered into one of the chambers as a suspension, or immobilized on polystyrene beads. The observed microtubule velocities using ATP generated by mitochondria from succinate, both in suspension as well as immobilized on polystyrene beads, were in the same range as with standard 1 mM ATP. Observed velocities of microtubules were 160 nm s⁻¹ ± 26% (N = 30 microtubules). Luciferase assays showed significant loss in activity of mitochondria after one hour, so remaining activity after the experiment is minimal.

Mitochondria and pyruvate kinase were utilized as the fuel generation systems to power the motility of kinesin powered microtubules in a microfluidic device. Activation of the molecular motors was found to be reversible by pumping solution away from the motors. This study lays the ground work for using high energy density fuel sources (ie. glucose) to produce many molecules of ATP that can be directly used by the motors. Furthermore, the study demonstrates a method to recycle ADP back to ATP by flowing solutions between chambers in the microfluidic device.

Footnotes

The HTML version of this article has been enhanced with colour images.

Electronic supplementary information (ESI) available: Movie showing motility of microtubules. See DOI: 10.1039/b801033a