Controlling the Direction of Kinesin-Driven Microtubule Movements along Microlithographic Tracks

Hiratsuka, Yuichi; Tada, Tomofumi; Oiwa, Kazuhiro; Kanayama, Toshihiko; Uyeda, Taro Q.P.

doi:10.1016/s0006-3495(01)75809-2

Cited by 333 publications

(323 citation statements)

References 23 publications

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“…While there has been extensive work using patterned surfaces to redirect kinesin-driven microtubule transport (Hiratsuka et al, 2001;Hess et al, 2002;Moorjani et al, 2003), three dimensional confinement is a prerequisite for harnessing this biological system for microscale transport applications and for combining motor driven transport with pressuredriven microfluidics. Although kinesins immobilized on engineered surfaces can faithfully direct microtubules and their associated cargo, diffusion and convection in the bulk solution above the surface can easily cancel out any motordriven transport.…”

Section: Resultsmentioning

confidence: 99%

“…A number of groups have shown that three dimensional patterns on motor-coated surfaces will bend or buckle microtubules and reorient their direction (Hiratsuka et al, 2001;Hess et al, 2002;Clemmens et al, 2003;Moorjani et al, 2003;van den Heuvel et al, 2005). However, because the channels are open on their top, detachment from the surface prevents complete redirection and requires an influx of new microtubules to compensate for microtubules lost from the surface.…”

Section: Enclosed Microchannels Redirect Moving Microtubulesmentioning

confidence: 99%

“…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%

“…A number of groups have shown that microfabricated surface features such as walls and channels can be used to redirect and control the trajectory of kinesin-driven microtubules (Hiratsuka et al, 2001;Hess et al, 2002;Clemmens et al, 2003;Moorjani et al, 2003). However, in most of these demonstrations the top of the channels are open to the bulk solution and because filaments can detach from the kinesin coated surface and diffuse away, microtubules are lost from the surface over time (half of the observed population in some designs (Clemmens et al, 2004;van den Heuvel et al, 2005)).…”

Section: Introductionmentioning

confidence: 99%

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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: Resultsmentioning

confidence: 99%

Section: Enclosed Microchannels Redirect Moving Microtubulesmentioning

confidence: 99%

Section: Controlling Motor Density In Enclosed Channelsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Microtubule transport, concentration and alignment in enclosed microfluidic channels

Huang

Uppalapati

Hancock

et al. 2006

Biomed Microdevices

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show abstract

“…Given the potential applications of this system, it is thus necessary to develop a way to restrict the movement of filaments to one dimension along linear tracks for extended periods of time. Restricting kinesin-driven movement of microtubules along linear tracks was achieved by using micrometer-scaled grooves lithographically fabricated on glass surfaces [48][49][50]. In the presence of detergent, kinesin selectively adsorbed onto a glass surface from which the photoresist polymer has been removed, not on the photoresist polymer itself [50].…”

Section: Controlling the Direction Of Protein Filament Movement Usingmentioning

confidence: 99%

How Linear Motor Proteins Work

Oiwa

Manstein

Controlled Nanoscale Motion

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Most animals perform sophisticated forms of movement such as walking, running, flying and swimming using their skeletal muscles. Although directed movement is not generally associated with plants, cytoplasmic streaming in plant cells can reach velocities greater than 50 µm/s and thus constitutes one of the fastest forms of directed movement. Unicellular eukaryotic organisms and prokaryotes display diverse mechanisms by which they are able to actively move towards a food source, light or other sensory stimuli. On the cellular level active transport of vesicles and organelles is required, since the cytoplasm resembles a gel with a mesh size of approximately 50 nm, which makes the passive transport of organelle-sized particles impossible. For elongated cells such as neurons, even proteins and small metabolites have to be actively transported.Linear motor proteins, moving on cytoskeletal filaments such as actin filaments and microtubules, are predominantly responsible for the motile activity in eukaryotic cells. They are chemo-mechanical enzymes that use the chemical energy from adenosinetriphosphate (ATP) hydrolysis to generate force and to move cargoes along their filament tracks. Under physiological conditions, the energy input per molecule of ATP corresponds to the chemical free energy liberated by its hydrolysis to adenosinediphosphate (ADP) and inorganic phosphate (P i ), ca. 10 −19 J (100 pNnm). The thermodynamic efficiency of motor proteins varies between 30 and 60%. As machines, motor proteins are unique since they convert chemical energy to mechanical work directly, rather than through an intermediate such as heat or electrical energy. Structural Features of Cytoskeletal Motor ProteinsThree families of linear, cytoskeletal motor proteins have been described: kinesin, dynein, and myosin ( Fig. 3.1). Kinesin and dynein family members K. Oiwa and D

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