Abstract
AbstractThe molecular motor kinesin-1, an ATPase, and the substrate it walks along, microtubules, are vital components of eukaryotic cells. Kinesin converts chemical energy to linear motion as its two motor domains step along microtubules in a process similar to how we walk. Cells create systems of microtubules that direct the motion of kinesin. This directed motion allows kinesin to transport various cargoes inside cells.During the stepping process, the kinesin motor domains bind and unbind from their binding sites on the microtubules. Binding and unbinding rates of biomolecules are highly dependent on hydration and exclusion of water from the binding interface. Osmotic stress will likely strongly affect the binding and unbinding rates for kinesin and thus offers a tool to specifically probe those steps. We will report the effects of different osmolytes on microtubule speed and other observables in the gliding motility assay.Kinesin’s kinetic core cycle hydrolyzes ATP with the help of a water molecule. Any modification to the water molecules the kinesin is in will change how ATP hydrolyzes and will ultimately affect how kinesin moves along microtubules. We will report preliminary results showing how kinesin is affected when the solvent it is in is changed from light water to heavy water.When used in a surface assay or in devices, the kinesin and microtubule system is also dependent on substrate passivation. Kinesin motor domains do not transport microtubules in the gliding motility assay if kinesin is added to a glass microscope slide that has not been functionalized. Functionalization of the glass slides and slips is typically performed with bovine milk proteins called caseins. Bovine casein is a globular protein that can be broken up into four constituents: αs1, αs2, β, and κ. Each casein constituent affects how kinesin adheres to the glass and ultimately the speed at which microtubules are observed to glide at. Building on the work of Verma et.al., we have found that each constituent individually produces different outcomes in gliding assays. We will present these findings and discuss implications they have for the use of gliding assays to study kinesin and the use of the kinesin-microtubule system in microdevices.[1] Chaen, S, N Yamamoto, I Shirakawa, and H Sugi. 2001. Effect of deuterium oxide on actomyosin motility in vitro. Biochimica et biophysica acta 1506, no. 3: 218-23.[2] Vivek Verma, William O Hancock, Jeffrey M Catchmark, "The role of casein in supporting the operation of surface bound kinesin," J. Biol. Eng. 2008; 2: 14. Acknowledgements: This work was supported by the DTRA CB Basic Research Program under Grant No. HDTRA1-09-1-008.CORRECTIONS: At the workshop, Erik Schaffer pointed out to us that some of our speed differences (casein data) were surely due to microscope increasing in temperature. I've edited the poster to correct for this. Thanks, Erik!
Highlights
For kinesin to function properly in a gliding motility assay, the substrate to with which kinesin adheres must first be passivated[1]
In order to determine if osmotic stress is the key descriptor as to why gliding speeds are changed, viscosity measurements must be still done on the assay solutions
Except for the wiggles which we believe is due to the osmotic stress affect
Summary
For kinesin to function properly in a gliding motility assay, the substrate to with which kinesin adheres must first be passivated[1]. Each casein constituent affects how kinesin adheres to the glass (or possibly how kinesin adheres to casein) and they affect the speed at which microtubules are observed to glide. Building on the work of Verma et al.[1], we have found that each constituent of bovine casein produces different outcomes in gliding assays. An interesting feature of the data is that the microtubules speed up over time in all assays.
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