Nanoscale patterning of kinesin motor proteins and its role in guiding microtubule motility
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- Verma, V., Hancock, W.O. & Catchmark, J.M. Biomed Microdevices (2009) 11: 313. doi:10.1007/s10544-008-9237-9
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Biomolecular motor proteins have the potential to be used as ‘nano-engines’ for controlled bioseparations and powering nano- and microelectromechanical systems. In order to engineer such systems, biocompatible nanofabrication processes are needed. In this work, we demonstrate an electron beam nanolithography process for patterning kinesin motor proteins. This process was then used to fabricate discontinuous kinesin tracks to study the directionality of microtubule movement under the exclusive influence of surface bound patterned kinesin. Microtubules moved much farther than predicted from a model assuming infinite microtubule stiffness on tracks with discontinuities of 3 μm or less, consistent with a free-end searching mechanism. As the track discontinuities exceeded 3 μm, the measured and predicted propagation distances converged. Observations of partially fixed microtubules suggest that this behavior results from the interaction of the microtubules with the surface and is not governed predominately by the microtubule flexural rigidity.
KeywordsElectron beam lithographyKinesin patterningMicrotubule flexural rigidity
In eukaryotic cells, conventional kinesin (Howard 1996; Vale and Fletterick 1997; Warner and McIntosh 1999) motors transport intracellular cargo along microtubules, polymers of tubulin protein that measure 25 nm in diameter and several micrometers in length. Microtubules have a structural polarity, and kinesin motors move unidirectionally toward the plus- or fast-growing end of microtubules. Other biomotors such as dynein move towards the minus end of the microtubules, providing a two way transport system. Conventional kinesin has two heads, each measuring 4 nm × 4 nm × 7 nm, which use the energy from adenosine triphosphate (ATP) hydrolysis to walk in 8 nm steps along microtubules (Howard 1996; Kull et al. 1996; Coy et al. 1999). Individual kinesin motors can exert a maximal force of 6 pN (Svoboda et al. 1993) and work at efficiencies of ∼50%. Kinesin motors can be immobilized on surfaces through their tail domain at densities of ∼103 motors/μm2 (Hancock and Howard 1998) and theoretically deliver cumulative forces in the range of nN/μm2.
Developments in nanofabrication processes have enabled the synthesis of devices with nanometer scale dimensions (Yong Chen 2001; Craighead 2000; Christie and Donald 2003). The biomotor–microtubule system may be an ideal technology for powering and controlling the operation of such devices for drug delivery, biological assays or molecular sorting. However, the creation of such hybrid devices requires nanoscale fabrication processes that are compatible with motor proteins, microtubules, and other associated support proteins such as casein. To this end, several fabrication approaches have been examined to position biomotors, align microtubules or direct the motion of microtubules propelled by immobilized biomotors. For example, Limberis et al. (2001) used fluid flow to align microtubules on a surface, and Brown et al. (2002) immobilized and extended microtubule seeds to produce a linear array of microtubules. To control the direction of microtubule transport, Hiratsuka et al. (2001) and Moorjani et al. (2003) used microscale lithography to create microchannels in photoresist on glass substrates. Cheng et al. (2005) used nanoimprinting of CYTOP on glass to achieve the same ends. These fabricated surfaces were then exposed to kinesin motors, and the resist channels were found to control the direction of microtubule transport. In all of these cases, the ability of the microchannels to guide the direction of microtubule transport relied upon the channel bottoms and sidewalls having different surface chemistries, such that functional motors were present only on the channel bottoms.
To date, approaches for selectively patterning kinesin motors have separated the microfabrication and surface engineering steps from the protein adsorption steps—generally heterogeneous surfaces are created and motors are exposed to the entire surface. One of the major reasons for not using lithography directly in protein patterning has been the perception that fabrication chemicals are lethal to proteins (Ilic and Craighead 2000). To address the incompatibility of proteins with lithography chemicals, we previously developed techniques for removing PMMA photoresist using chemicals that do not affect the functionality casein or kinesin motors (Verma et al. 2005).
2 Materials and methods
Electron beam lithography
Electron beam lithography of poly methylmethacrylate (PMMA, Microchem) was used to pattern circular regions on glass cover slips (Corning, number 1 1/2, 22 mm2). The process consisted of the following steps. Two percent PMMA 495 K was spin coated on several glass cover slips at 4,000 RPM for 40 s and then baked at 180°C for 2 min. A 200-Å thin film of gold was thermally evaporated onto the PMMA layer to conduct away the electrons during electron beam exposure. The gold film was etched away in Gold etch-type TFN (Transene Co. Inc., Danvers) after exposing the resist. The patterns were electron beam written at a dose of 825 μC/cm2 using a Leica EBPG-5HR electron beam lithography system. Patterns were developed in 1:3 MIBK: IPA for 90 seconds and then rinsed in IPA for 1 min.
Microtubule motility assays
Casein was prepared by dissolving casein powder in BRB80 buffer (80 mM PIPES, 1 mM MgCl2, 1 mM EGTA, pH 6.9), centrifuging and filtering to remove insoluble components. A portion of the casein was labeled with Cy-5 dye following standard procedures (Amersham Biosciences, Buckinghamshire). Full-length Drosophila conventional kinesin was bacterially expressed and purified following standard procedures (Hancock and Howard 1998). For fluorescence studies, a mixture of 0.5 mg/ml unlabelled and 0.1 mg/ml Cy-5 labeled casein in BRB80 buffer was incubated in a flow cell containing the patterned PMMA cover slip for 5 min at room temperature. A motor solution consisting of 8.5 μg/ml kinesin motors, 0.2 mg/ml casein and 100 μM Mg-ATP in BRB80 buffer was then incubated in the flow cell for 10 min. To pattern the motors, the flow cell was disassembled by removing the tape, and the patterned cover slip was submerged in pure acetone bath for 30 min. To facilitate effective PMMA lift-off, a custom fixture was used to hold the glass cover slips during PMMA lift-off step, the acetone bath was rocked at 0.75 Hz, and the acetone was changed every 10 min. After PMMA removal step, another flow cell was made from the processed cover slip and the surface was imaged by either epifluorescence or total internal reflection microscopy (TIRF) using a Nikon TE2000 inverted microscope (60×, 1.45 NA, CFI Plan Apo TIRF oil objective).
Microtubules were polymerized bovine brain tubulin purified and rhodamine labeled according to standard procedures (Hyman 1991; Williams and Lee 1982). To polymerize microtubules, 4 mg/ml tubulin, 1 mM GTP, 4 mM MgCl2 and 5% DMSO were combined in BRB80 buffer, and the temperature was raised to 37°C for 20 min. Polymerized microtubules were then diluted 40-fold in BRB80 containing 10 μM paclitaxel. After motors were patterned by PMMA removal, another flow cell was made from the acetone treated glass cover slip and motility solution (BRB80 with 20 mM d-glucose, 20 μg/ml glucose oxidase, 8 μg/ml catalase and 0.5% β-mercaptoethanol) containing 80 μg/ml rhodamine-labeled microtubules was introduced. Fluorescent microtubules were observed under epifluorescence microscopy (Nikon E600, 60×, 1.2 N. A. water immersion Plan Fluor objective).
In the present work we demonstrate direct patterning of the motor protein kinesin and the associated support protein casein using electron beam lithography. Pattering is performed using a lift-off process employing the electron beam sensitive resist poly methyl methacrylate (PMMA). By patterning PMMA on glass, adsorbing casein and kinesin to the surface, and then removing the PMMA, circular regions of motor proteins measuring as small as 250 nm in diameter can be directly patterned. This process exhibits several advantages including direct compatibility with standard fabrication techniques used to manufacture NEMS/MEMS devices, potential scalability to sub-20 nm dimensions, and the inherent ability to create a final planar surface in the unpatterned regions absent of chemical functionalization. These unpatterned regions can subsequently be functionalized with other biomotors or other relevant proteins to create new functional surfaces. This capability can be used, for example, to pattern two types of biomotors with opposite directionalities, presenting the possibility of bidirectionally controlled microtubule transport.
The effectiveness of kinesin patterning was first assessed using microtubule binding studies. Microtubules were sheared using a 30 gauge needle so that their lengths were comparable to the diameter of the patterned kinesin regions. Microtubules were then introduced into the flow cell for 20 min in the presence of 1 mM AMP-PNP (adenylylimidodiphosphate), a non-hydrolysable ATP analog that results in microtubules binding tightly to functional kinesin motors without moving. Cy-5 labeled casein and rhodamine labeled microtubules were imaged on the same processed sample using two different fluorescent filter sets. Figure 2(b) depicts fluorescence images showing that microtubules bound selectively to the regions where Cy-5 labeled casein and kinesin were patterned. This result indicates that there was no non-specific microtubule binding, and that the PMMA lift-off process produces highly specific patterning of kinesin and microtubules.
An important application of biomotor patterning is to create tracks of proteins that guide the motion of microtubules. Controlling the direction of microtubule propagation could enable several applications including molecular sorting and the development of new lab-on-a-chip devices where microtubules carry cargo against fluid flow or concentration gradients. Reuther et al. (2006) recently showed that microtubules can be effectively used as templates to pattern linear tracks of motors on surfaces, but the pattern lengths are limited by the lengths of the microtubule templates used, and because the microtubules land randomly on the surface it is difficult to control the location of the motor tracks. Because electron beam lithography can be used to precisely generate virtually any desired pattern, this approach was implemented to fabricate discontinuous tracks of kinesin motors. The hypothesis is that by creating specific patterns of motors on surfaces, the direction of microtubule transport can be controlled to maximize the distance of transport in selected directions. Microtubule propagation on such small features has not been examined to date.
Due to their relatively high stiffness, microtubules generally move across kinesin-functionalized surfaces in relatively straight lines. However, in regions sparsely decorated with motors, the leading end of a moving microtubule undergoes thermal fluctuations that increase the area over which it “searches” for new motors. Previous studies on the impact of surface-patterned biomotors on microtubule directionality have predicted a minimum microtubule radius of curvature r = πEI / 4kBT where E is the modulus and I is the geometric moment, kB is Boltzmann’s constant and T is temperature (Gittes et al. 1993; Hess et al. 2002; Hunt and Howard et al. 1993). Using a number of different experimental procedures, measured values of EI for microtubules range from 2 × 10−24 Nm2 (Kikumoto et al. 2006) to 2.2 × 10−23 Nm2 (Gittes et al. 1993). These values translate into radii of curvature, r, ranging from 382 to 4200 μm. This approach implies that microtubules traveling along patterned tracks of motors would need to remain almost perfectly parallel to the track or else they would run off of the track (Hess et al. 2002).
Lateral fluctuations in the microtubule leading end is only one determinant of the interaction between microtubules and surface bound motors. Another issue is the probability that the free end of the microtubule will be sufficiently close to motor for binding. For neighboring motors to redirect the motion of a microtubule, the microtubule must come into close proximity to the surface (∼80 nm or less depending upon the length of the kinesin tail and the location of the kinesin head relative to the surface) for an interval of time that allows the motors to bind to the filament. It is expected that for a microtubule being guided along patterned motor regions with small center-to-center spacing, the short length of the microtubule free end will ensure that it remains close to the surface. However, with increased spacing distances, the microtubule free ends are expected to undergo larger magnitude fluctuations, and hence may spend a significant fraction of the time away from the surface. To study this phenomenon, we used the same microtubules shown in Fig. 8(a–b), to qualitatively assess the position of the microtubule with respect to the surface as a function of time. Microtubules were imaged with a Nikon E600 (60× objective, 1.2 N.A.) which exhibits a depth of focus of ∼500 nm. It was recently shown that in a geometry similar to ours that kinesin motors elevate microtubules less than 20 nm above the glass surface (Kerssemakers et al. 2006), so if the objective is focused on the glass surface, then microtubule segments ∼250 nm from the surface or less will appear crisp, while segments farther away from the surface will appear out of focus. Over 100 still images of the microtubules were examined. It was found that ∼70% of the time the free microtubule end is positioned away from the surface, including a 20-s continuous interval. If this microtubule were not immobilized, it would have propagated ∼20 μm over this interval before returning to the surface. Furthermore, this is also an overestimate of the time spent within <80 nm of the surface (the contour length, and hence the expected maximal reach of conventional kinesin; Howard 1996), since our qualitative assessment of the position of the microtubule while near the surface is only accurate to within ∼250 nm. Hence, although the microtubule flexural rigidity allows the free end to search over a relatively wide area, thermal fluctuations of the filament tip normal to the surface decrease the probability that the microtubule will bind to motors immobilized on the surface. In addition, it can’t be ruled out microtubules also have small degrees of curvature that cause the leading end to bend away from the surface. This microtubule curvature has been noted by others (Gittes et al. 1993; Amos and Cross 1997).
In conclusion, the present study demonstrates that functional kinesin motor proteins can be patterned through electron beam lithography and a PMMA lift-off process that is compatible with standard MEMS/NEMS fabrication processes. Protein-containing regions as small 250 nm were patterned on glass substrates with no measurable non-specific binding. Surprisingly, kinesin motors retain their microtubule transport function, demonstrating the biocompatibility of this process with this type of biomotor protein. The use of such conventional nanoscale patterning approaches, which directly integrate biological molecular motors and associated proteins, provides a new capability for engineering new hybrid synthetic and biological devices and systems.
This novel biomotor patterning process was used to investigate the directionality of microtubule propagation on discontinuous kinesin tracks. The percentage of microtubules propagating long distances along the patterned tracks was compared to that predicted by a statistical model based on the track geometry and assuming infinite elastic modulus of the microtubule. Interestingly, on tracks exhibiting no discontinuity or a discontinuity of 3 μm, microtubules traveled much farther than predicted from the statistical model. From observing the microtubule behavior, it is clear that thermal fluctuations in the leading end enables the filaments to interact with subsequent patterned regions, resulting in a change in the propagation direction and continued motion along the patterned track. In contrast, for discontinuities of 6 or 9 μm and for distances exceeding 60 μm, the propagation distances begin to converge with the model predictions. Qualitative studies of the leading microtubule end indicated that the leading segment remains within a few hundred nanometers of the surface approximately one third of the time. It is hypothesized that the relatively fast microtubule transport velocity in conjunction with the small percentage of time the free microtubule end spends in close proximity to the surface is responsible for the inability of patterned motors to guide microtubules indefinitely. These results put an upper limit on the optimal motor spacing that best enables long distance transport on kinesin-patterned surfaces.
This work was supported by The Pennsylvania State University Center for Nanoscale Science, a NSF Materials Research Science and Engineering Center (DMR0213623). It was also supported by the National Nanotechnology Infrastructure Network (NSF Cooperative Agreement No. 0335765 with Cornell University) and The Pennsylvania State University Materials Research Institute. V.V wishes to thank the Haythornthwaite Foundation for their Founder’s Prize and grant for year 2005–06.
We thank Maruti Uppalapati for purifying kinesin and Gayatri Muthukrishnan for purifying and labeling tubulin. We also thank Dr. Edward Basgall and Dr. Khalid Eid for assistance with electron beam lithography.