link http://precedings.nature.com/documents/4469/version/1/files/npre20104469-1.pdf Speed effects in gliding motility assays due to surface passivation, water isotope, and osmotic stress. Andy Maloney1, Lawrence J. Herskowitz1 & Steven J. Koch1 Correspondence: (Login to view email address) University of New Mexico, Dept. Physics and Astronomy and CHTM PDF (4 MB) Document Type: Poster Date: Received 18 May 2010 21:04 UTC; Posted 19 May 2010 Subjects: Molecular Cell Biology Tags: kinesin kinesin-1 gliding motility assay casein heavy water heavy-oxygen water Osmotic stress osmotic pressure Abstract: The 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 cargos 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 use of gliding assays to study kinesin and use of 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. Presented at: Thermodynamics and Kinetics of Molecular Motors, 20 May 2010 Discussion Comments: 0 comments Share: (Login to share with a colleague) Additional information License: This document is licensed to the public under the Creative Commons Attribution 3.0 License How to cite this document: Maloney, Andy, Herskowitz, Lawrence, and Koch, Steven. Speed effects in gliding motility assays due to surface passivation, water isotope, and osmotic stress.. Available from Nature Precedings (2010) Version info: Other versions of this document in Nature Precedings v3 Posted 09 March 2011 v2 Posted 01 June 2010 Other versions of this document elsewhere on the web None known.