In Vitro Assembly of Artificial Mitotic Spindles Using Dielectrophoresis

Open Access
Author:
Aravamuthan, Vidhya
Graduate Program:
Bioengineering
Degree:
Master of Science
Document Type:
Master Thesis
Date of Defense:
None
Committee Members:
  • William O Hancock, Thesis Advisor
  • Richard Cyr, Thesis Advisor
Keywords:
  • Artificial spindles
  • dielectrophoresis
Abstract:
The separation of duplicated chromosomes during mitosis is achieved by the mitotic spindle which consists of microtubules and associated motor proteins. Many motor proteins are known to interact with microtubules and affect their dynamics and organization. Knockout strategies have been used to probe the spindle in cells, but due to the inherent redundancy built into this complex process, deleting individual motor proteins often has only subtle effects. Single molecule studies have been used to study the properties of isolated motor proteins. However, they do not provide any information on the interaction of motors with complex microtubule assemblies like the spindle. The goal of this project is to develop novel engineering approaches for the study of mitosis that bridges single molecule studies and in vivo studies in cells. The approach is to assemble artificial spindles in vitro to study and characterize interactions of single motors with the microtubules that make up the spindle. To achieve this goal, we have used microelectrodes fabricated on a quartz substrate. AC electric fields were then applied across the electrodes, which results in electroosmotic flows and dielectrophoretic forces. Microtubules assemble at the electrode tip due to the resultant electrokinetic forces and are then immobilized on the neutravidin patterned electrodes. These assembled microtubules were then extended with rhodamine tubulin to obtain overlapping opposed microtubules. The ability to extend microtubules in the flow cell facilitates the study of interesting phenomena like plus tip tracking of motor proteins and the influence of motors on polymerization kinetics. This assembly of microtubules then provides a platform to study the function of single motors and populations of motors and their effect on the spindle. The scope of this research can be further expanded by using beads coated with a single motor type in conjunction with optical traps to impose external forces. This novel experimental tool should provide important clues towards understanding force generation and microtubule rearrangements by molecular motors in the mitotic spindle.