Microtubule transport, concentration and alignment in enclosed microfluidic channels

Abstract

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

Transportation of Nanoscale Cargoes by Myosin Propelled Actin Filaments

“…This has inspired development of molecular motor driven lab-on-a-chip devices [3], [4], [5], [6], [7] with cargo pick-up and transportation [8], [9], [10], [11], [12], [13], [14], [15], [16], [17]. Possible applications include periodic chemistry [18], [19], assembly of molecular components [20], [21], [22], sorting, positioning and/or concentration [20], [23], [24], [25], [26], [27] of molecules. Even proof-of-principle diagnostics devices [28], [29], [30], [31], [32] have been described, with significant future potential [3], [7].…”

Molecular communication: Harnessing biochemical materials to engineer biomimetic communication systems

“…In this arrangement, cargoes such as nano-or microspheres [7,14,41,40], virus particles [8], proteins [81,55,23], DNAs [16,91,80] and lipid vesicles [46] have been successfully transported. Considering that the gliding direction of MTs can be controlled by utilizing preconfigured microlithographic tracks [39,47] or by applying electric [95] and magnetic fields [48], the main challenge in this arrangement is to load/unload cargoes onto/from gliding MTs at a sender/receiver in a reversible manner. Tight bindings, such as avidin-biotin or antigen-antibody bindings, that are suitable for permanent bindings may not be suitable for reversible cargo loading/unloading.…”

Comparing Guiding Track Requirements for Myosin- and Kinesin-Powered Molecular Shuttles

“…31 The increase in the maximum channel width from actin filaments to microtubules, however, has a dramatic impact on fabrication. Structures for the control of microtubule guiding can be routinely fabricated by photolithography, [32][33][34][35] while control of actin filament transport is best accomplished by structures fabricated by e-beam lithography. 15,36 Our detailed analysis supports the general statement that flexible filaments and fast motors are desirable.…”