Highly Efficient Guiding of Microtubule Transport with Imprinted CYTOP Nanotracks

Cheng, Li-Jing; Kao, Ming Tse; Meyhöfer, Edgar; Guo, L. Jay

doi:10.1002/smll.200400109

Cited by 69 publications

(61 citation statements)

References 20 publications

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“…As with the directional rectifier, motors were adsorbed and observed to be functional on all interior surfaces. Blocking motor activity on the side walls, a prerequisite for open channels (Hiratsuka et al, 2001;Moorjani et al, 2003;Cheng et al, 2005), is not required for enclosed channels. The design consists of a circular ring with entry and exit channels that connect to reservoirs (intermediate channels) 100 µm wide where microtubules are introduced.…”

Section: Controlling Motor Density In Enclosed Channelsmentioning

confidence: 99%

Microtubule transport, concentration and alignment in enclosed microfluidic channels

Huang

Uppalapati

Hancock

et al. 2006

Biomed Microdevices

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The kinesin-microtubule system has emerged as a versatile model system for biologically-derived microscale transport. While kinesin motors in cells transport cargo along static microtubule tracks, for in vitro transport applications it is preferable to invert the system and transport cargofunctionalized microtubules along immobilized kinesin motors. However, for efficient cargo transport and to enable this novel transport system to be interfaced with traditional microfluidics, it is important to fabricate enclosed microchannels that are compatible with kinesin motors and microtubules, that enable fluorescence imaging of microtubule movement, and that provide fluidic connections for sample introduction. Here we construct a three-tier hierarchical system of microfluidic channels that links microscale transport channels to macroscopic fluid connections. Shallow microchannels (5 µm wide and 1 µm deep) are etched in a glass substrate and bonded to a cover glass using PMMA as an adhesive, while intermediate channels ( ∼ 100 µm wide) serve as reservoirs and connect to 250 µm deep microchannels that hold fine gauge tubing for fluid injection. To demonstrate Ying-Ming Huang and Maruti Uppalapati contributed equally to this work. the utility of this device, we first show the performance of a directional rectifier that redirects 96% of moving microtubules and, because any microtubules that detach rapidly rebind to the motor-coated surface, suffers no microtubule loss over time. Second, we develop an approach, using a headless kinesin construct, to eliminate gradients in motor adsorption and microtubule binding in the enclosed channels, which enables precise control of kinesin density in the microchannels. Finally, we show that a 60 µm diameter circular ring functionalized with motors concentrates and aligns bundles of ∼ 3000 uniformly oriented microtubules, while suffering negligible ATP depletion. These aligned isopolar microtubules are an important tool for microscale transport applications and can be employed as a model in vitro system for studying kinesin-driven microtubule organization in cells. Electronic Supplementary Material

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Section: Controlling Motor Density In Enclosed Channelsmentioning

confidence: 99%

Microtubule transport, concentration and alignment in enclosed microfluidic channels

Huang

Uppalapati

Hancock

et al. 2006

Biomed Microdevices

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“…[19,20] Howard et al demonstrated in 1989 that observing the attachment of microtubules from solution to surface-adhered kinesin motors enables the determination of motor densities as low as 2 proteins per lm 2 by measuring the rate of microtubule attachment. [21,22] Attachment rate measurements have subsequently been adapted to the determination of relative kinesin motor activity on different surfaces [9] and to the evaluation of guiding structures for microtubule transport [10,23] Since kinesins long tail domain evolved to efficiently connect to cargo, we hypothesize that it can serve as a particularly efficient probe for attachment points on the surface. [24,25] .…”

mentioning

confidence: 99%

Quantifying the Performance of Protein‐Resisting Surfaces at Ultra‐Low Protein Coverages using Kinesin Motor Proteins as Probes

Katira¹,

Agarwal²,

Fischer³

et al. 2007

Advanced Materials

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Surfaces resistant to protein adsorption are very desirable for a variety of applications in biomedical engineering and bionanotechnology, since protein adsorption is often the first step in a cascade of events leading to systems failure. Initial efforts to create adsorption-resistant surfaces succeeded in reducing the adsorption by 80% compared to untreated surfaces to 100 ng cm -2 by employing poly(ethylene glycol) coatings.[1]Recently, optimization of brush density and morphology has reduced adsorption to 1 ng cm -2 or less.[2] These coverages, equal to about one tenth of a percent of a monolayer, represent the detection limit for several characterization techniques, including SPR [2,3] and radiolabeling.[4] However, biological effects can be observed for protein coverages below this detection limit. For example, adsorption of blood proteins can initiate the intrinsic coagulation cascade at low coverage.[5] Therefore more sensitive techniques for the measurement of protein adsorption are needed. Moreover, if protein adsorption is exceedingly slow, higher sensitivity would permit an accelerated quantification of the performance of the surface (e.g. within hours instead of days). Non-fouling surfaces are also a critical part of hybrid nanodevices, which utilize precisely positioned biomolecules in an artificial environment.[6] For example, controlled adsorption of kinesin, [7][8][9][10][11][12][13][14] myosin, [15][16][17] and F1-ATPase motors [18] has been utilized for the design of molecular shuttles and nanopropellers. Adsorption of motor proteins outside the intended regions at densities down to one motor per square micrometer (0.04 ng cm -2) can lead to loss of device function, since individual motors can already bind and transport the associated filaments outside their intended tracks.The binding of associated filaments, such as microtubules or actin filaments, is readily observed by fluorescence microscopy, since these filaments are composed of thousands of protein subunits and carry typically at least a thousand covalently linked fluorophores. [19,20] Howard et al. demonstrated in 1989 that observing the attachment of microtubules from solution to surface-adhered kinesin motors enables the determination of motor densities as low as 2 proteins per lm 2 by measuring the rate of microtubule attachment. [21,22] Attachment rate measurements have subsequently been adapted to the determination of relative kinesin motor activity on different surfaces [9] and to the evaluation of guiding structures for microtubule transport [10,23] Since kinesins long tail domain evolved to efficiently connect to cargo, we hypothesize that it can serve as a particularly efficient probe for attachment points on the surface. [24,25] . Landing rate measurements enable the measurement of absolute coverages of functional kinesins in the range of 0.004 -1 ng cm -2 , thus enabling to differentiate the performance of even the best non-fouling surfaces. While landing rate measurements in effect count individual proteins, their complexity ...

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“…Namely, in order to apply gliding microtubules driven by molecular motors to a nanotransport device for sorting, separation, or assembly of materials, it is essential to guide microtubules along predetermined pathways (van den Heuvel and Dekker, 2007). Various approaches to design guiding tracks have been investigated, including topographically patterned tracks (Hess et al, 2001;Hiratsuka et al, 2001), chemical patterning of the kinesin attachment sites (Clemmens et al, 2003), a combination of physical and chemical approaches (Cheng et al, 2005), and enclosed microfluidic channels as means for directional confinement (van den Heuvel et al, 2006;Yokokawa et al, 2006).…”

mentioning

confidence: 99%

Microtubule shuttles on kinesin-coated glass micro-wire tracks

Kim

Liao

Sikora

et al. 2014

Biomed Microdevices

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Gliding of microtubule filaments on surfaces coated with the motor protein kinesin has potential applications for nano-scale devices. The ability to guide the gliding direction in three dimensions allows the fabrication of tracks of arbitrary geometry in space. Here, we achieve this by using kinesin-coated glass wires of micrometer diameter range. Unlike previous methods in which the guiding tracks are fixed on flat two-dimensional surfaces, the flexibility of glass wires in shape and size facilitates building in-vitro devices that have deformable tracks.

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Highly Efficient Guiding of Microtubule Transport with Imprinted CYTOP Nanotracks

Cited by 69 publications

References 20 publications

Microtubule transport, concentration and alignment in enclosed microfluidic channels

Microtubule transport, concentration and alignment in enclosed microfluidic channels

Quantifying the Performance of Protein‐Resisting Surfaces at Ultra‐Low Protein Coverages using Kinesin Motor Proteins as Probes

Microtubule shuttles on kinesin-coated glass micro-wire tracks

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