Monitoring Real-Time Microtubule Spool Formation in a PDMS Microfluidic Device

Abstract

The motor-driven self-assembly of microtubule rings and spools has been reported by several research groups and attributed to one of two potential mechanisms: pinning by inactive motors or mechanical strain from oligomer rotation. Here we used a polydimethylsiloxane (PDMS) microfluidic device that decreases photodamage via deoxygenation to study the initial stages of ring formation in absence of photodamage-related effects to kinesin motors. Specifically, valves on the devices were used to control the addition of biotinylated microtubules and streptavidin-coated quantum dots to the system, while effectively removing oxygen from the sample. This approaches enabled us to use relatively high excitation intensities and high frame rates to characterize the early events of microtubule ring formation. We show that both proposed mechanisms occur, and we characterize the conditions under which each mechanism predominates. In the pinning mechanism, it was observed that a single microtubule may be pinned at the head, leading the tail to loop and contact itself, forming a single ring. Once the initial ring is formed, additional bundling occurred as other microtubules encountered the ring and became attached. For the mechanical strain mechanism, microtubule oligomers were observed to undergo ring formation in the absence of detectable pinning events. Here, the presence of non-13mer microtubules induces oligomers to rotate axially, resulting in twisted nano-domains that introduce mechanical strain, coercing the bundle to move in a curved path and form a ring. Together, these data provide a critical insight as to the mechanism underlying the self-assembly of dynamic material structures based on active transport systems.Sandia National Laboratories is a multi-program laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the U.S. Department of Energy's National Nuclear Security Administration under contract DE-AC04-94AL85000.