Microtubule transport, concentration and alignment in enclosed microfluidic channels

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 cargo-functionalized 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 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.