Determining the Effect of Kinesin-5 Inhibitors on the Nucleation Rates of Microtubules in vitro and Synthesizing, Characterizing, and Purifying Fluorescently Labeled Nucleotides
Open Access
- Author:
- Lingafelt, Colin Mark
- Area of Honors:
- Biomedical Engineering
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Dr. William O Hancock, Thesis Supervisor
Dr. William O Hancock, Thesis Honors Advisor
Dr. Justin Lee Brown, Faculty Reader
Herbert Herling Lipowsky, Faculty Reader - Keywords:
- kinesins
microtubules
fluorescent nucleotides
binding rates
synthesis
Eg5
nucleation
tubulin
critical concentration - Abstract:
- Kinesin proteins are molecular motors in eukaryotic cells that have a variety of intracellular functions including transporting vesicle and protein cargo to different intracellular locations, and aiding in cell division. Motor proteins travel along microtubules to move across the cell using its two heads to "walk" along the filament. In order for a kinesin to "walk", it requires energy derived from ATP hydrolysis. The first half of this thesis is dedicated to designing a protocol for synthesizing, purifying, and characterizing a fluorescently labeled nucleotide by reacting ATP with methylisatoic anhydride. The resulting mATP shows a peak absorbance around 356 nm and a fluorescence around 450 nm that is enhanced by motor binding. The purpose of labeling the nucleotides is to quantitatively study the kinesin chemomechanical cycle by measuring the on- and off- rates for nucleotide binding to the motor. From these data, more information can be discovered about the chemomechanical cycles for different kinesin variants. A cost analysis of producing mATP versus purchasing mATP determined that 85% of the cost can be saved by synthesizing the product in the lab instead of purchasing the same quantity from a commercial producer. The second half of this thesis is dedicated to determining the effects of kinesin-5 motors on microtubule nucleation in vitro. Kinesin-5 (Eg5) is a tetrameric, mitotic kinesin that pushes apart antiparallel microtubules and helps to stabilize the mitotic spindle during mitosis. Because failure to complete mitosis can cause the cell to undergo apoptosis, Eg5 is a target for new cancer drugs that target rapidly dividing cancer cells. Eg5 was recently shown to enhance the microtubule growth rate in vitro, providing a new potential function of this motor as a regulator of microtubule dynamics. Experiments were carried out using fluorescence microscopy and turbidity to measure the influence of Eg5 on microtubule nucleation in vitro. The fluorescence microscopy experiments showed a potential effect of Eg5 on nucleation but more data need to be collected before a definitive conclusion is made. The turbidity experiments did not show evidence that Eg5 affected microtubule nucleation. Therefore, the cumulative results of these experiments were inconclusive in determining whether Eg5 affects microtubule nucleation.