Abstract
AbstractThe osmotic pressure and kinetic properties of water play important roles in biomolecular interactions. As pointed out by Parsegian, Rand, and Rau, these crucial roles are often overlooked[1]. In some fields, osmotic stress and isotope effects have been exploited for probing the role water plays in binding interactions of biomolecules. To our knowledge, there have been no studies of osmotic stress and water isotope effects for kinesin, and only a handful for myosin. We're currently using the gliding motility assay to see whether we can extract new information about kinesin-1 / microtubule interactions by changing osmotic stress and water isotopes. We will describe our open-source, automated analysis platform for extracting microtubule gliding speeds from image series. We will also show our preliminary analyses of the changes seen in gliding assays when done in heavy water (either heavy-hydrogen or heavy-oxygen) or osmolytes (betaine). We will discuss whether osmotic stress and isotopes, particularly heavy-oxygen water, might be an important tool for probing effects of water on binding interactions between kinesin and microtubules. We will also discuss potential applications of deuterium water for stabilizing microtubules and kinesin for lab or device applications.[1] Parsegian, V. A., Rand, R. P., & Rau, D. C. (1995). Macromolecules and water: probing with osmotic stress. Methods in Enzymology, 259.This work was supported by the DTRA CB Basic Research Program under Grant No. HDTRA1-09-1-008 in collaboration with Dr. Susan Atlas lab (UNM).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!2011 March Correction: Using temperature stabilization and much-improved technique, Andy has taken higher-quality data for the heavy-hydrogen and heavy-oxygen experiments. See the added slides, 6-7.
Highlights
Speed differences seen in different caseins almost surely an artifact due to increase in microscope temperature over the course of many hours
Andy plotted the data versus absolute time, and they support the notion of a heating effect http://openwetware.org/wiki/User:Andy_Maloney/N otebook/Lab_Notebook_of_Andy_Maloney/2010/05/ 27/Casein_data_revisited
Andy ran other experiments earlier this week to support the temperature effects, and the notion that it’s the microscope body, not the photons coming from lamp
Summary
See above correction slides. differences and the slow inc with time are almost surely slowly increasing temperatu microscope due to the arc la. Differences and the slow inc with time are almost surely slowly increasing temperatu microscope due to the arc la. 2. Preliminary results isotope and osmotic stress effects in kinesin-1 gliding assays. Assay details Dmk401 ug / ml MTs 29% TRITC cytoskeleton bovine Room temp. Andy can finish one sample in 1 hour from start to finish 30 minutes of data, ~9,000 images Primary measurement: speed other qualitative measurements Advantage: lots of data quickly; robust assay. We’ve developed an open-source, automated microtubule tracking and speed analysis platform
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