CONTROLLED ASSEMBLY OF MICROTUBULES AND MANIPULATION OF KINESIN DRIVEN MICROTUBULE MOTION
Open Access
- Author:
- Uppalapati, Maruti Chandra Mouli Varma
- Graduate Program:
- Bioengineering
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- March 31, 2008
- Committee Members:
- William O Hancock, Committee Chair/Co-Chair
Thomas Nelson Jackson, Committee Chair/Co-Chair
Richard Cyr, Committee Member
Ahmed A Heikal, Committee Member
Ryan S Clement, Committee Member - Keywords:
- protein patterning
microfabrication
AC electrokinetics
microtubule
kinesin
mitosis - Abstract:
- Kinesins are microtubule based motor proteins that play important roles in intracellular transport and cell division in eukaryotic cells. Because this motion can be recapitulated in synthetic environments using purified components, the kinesin-microtubule system is an excellent model for biologically derived nanoscale motion. The first objective of this thesis was to develop microscale transport systems that use kinesin-driven microtubules as carriers to transport and sort cargo in lab-on-a-chip devices. The second objective was to develop in vitro models that mimic the microtubule organization found in cells, to study the role of kinesin motors in cellular processes such as cell division. By optimizing materials, surfaces and geometries, functional kinesins motors and microtubules were successfully integrated into enclosed microchannels. A three-tier hierarchical system of microfluidic channels that links the microscale transport channels to macroscopic fluid connections was used. Shallow microchannels (5 micron wide and 1 micron deep) were etched in a glass substrate and bonded to a cover glass using PMMA as an adhesive. Intermediate channels (~100 micron wide) serve as reservoirs and connect to 250 micron deep microchannels that hold fine gauge tubing for fluid injection. High surface-to-volume ratio in microchannels results in gradients of motor adsorption. To solve the problems of high surface-to-volume ratio in these systems, we developed an approach, using a headless kinesin construct, to eliminate gradients in motor adsorption and microtubule binding in the enclosed channels. This enables precise control of kinesin density in the microchannels and maximizes microtubule movement. The confinement geometry (channel cross-section 5 micron x 1 micron) enables long-distance movement (~5 mm) and directional control, which are prerequisites for developing hybrid lab-on-chip devices using biomotor-driven transport. We further showed that constructing a circular ring using these microchannels generates a high density ensemble of isopolar microtubules driven by kinesin motors. These aligned microtubules can be used for microscale transport applications or as a model in vitro system for studying kinesin-driven microtubule organization in cells. The stability and limited shelf life of these motor proteins and their associated protein filaments is a barrier to implementing of kinesin-driven transport in devices. We demonstrated that freeze-drying or critical point drying kinesins adsorbed to glass surfaces extends their lifetime from days to more than four months. Further, photoresist deposition and removal can be carried out on these motor-adsorbed surfaces without loss of motor function. The methods developed here are an important step towards realizing the integration of biological motors into practical devices, and these approaches can be extended to patterning and preserving other proteins immobilized on surfaces. A number of tools have been developed to manipulate microtubules in solution, including optical tweezers, fluid flow, magnetic fields, and DC electrophoresis, however each approach has its limitations. AC electrokinetics provides a novel tool for manipulating and organizing microtubules in solution, enabling new experimental geometries for investigating and controlling the interactions of microtubules and microtubule motors in vitro. By fabricating microelectrodes on glass substrates and generating AC electric fields across solutions of microtubules in low ionic strength buffers, we were able to collect and align bundles of microtubules and to measure the electrical properties of microtubules in solution. We found that AC electric fields result in electroosmotic flow, electrothermal flow and dielectrophoresis of microtubules, which can be controlled by varying the solution conductivity, AC frequency, and electrode geometry. These experiments demonstrate that AC electrokinetics provides a powerful new tool for kinesin-driven transport applications. Furthermore, by maximizing dielectrophoretic forces and minimizing electroosmotic and electrothermal flows, microtubules could be assembled into opposed asters, reminiscent of the mitotic spindle in dividing cells. However the microtubules in these asters were not sorted for polarity. We developed kinesin motor patterning and neutravidin patterning techniques and used them to organize microtubules in the asters an isopolar manner, similar to microtubule organization in a mitotic spindle. These assembled structures serve as an in vitro model for investigating the role of microtubule motors in development and maintenance of the mitotic spindle.