Experimental overview of DTRA kinesin projectKoch Lab, UNM Dept. Physics and Center for High Technology Materials (CHTM)<b...
KochLab Overview / Acknowledgments<br />Single-molecule manipulation<br />Optical tweezers; magnetic tweezers; MEMS<br />K...
Microtubules are polymers of tubulin heterodimers<br />Fast polymerizing end<br />Plus end<br />8 nanometers<br />25 nanom...
Our goal is to gain atomistic insight through a variety of experiments and simulations…especially focusing on water<br />S...
Gliding motility assay<br />Buffer includes ATP, antifade cocktail<br />Andy is currently leading the GMA project<br />100...
Gliding motility assay is initially our main assay<br />Buffer includes ATP, antifade cocktail<br />Passivated glass surfa...
Heavy water background<br />Naturally abundant 1 / 6600 hydrogen molecules is deuterium<br />	17 mM deuterium in “standard...
Gliding assays in D2OSquiggly Microtubules; MT-MT interactions<br />(Show movie externally)<br />“Motility in 100% D2O”<br />
Gliding assays in D2OSignificantly more stable microtubules (and maybe kinesin)  Activity lasts &gt; 24 hours<br />(Show m...
Microtubule velocity in gliding assay is measured viaLabVIEW image tracking software written by Larry<br />Open source sof...
Gliding motility assay—Deuterium Isotope Effects<br />7. Guydosh, Nicholas R, and Steven M Block. “Direct observation of t...
Implications of DTRA research so far<br />Basic Research<br />	Gaining insights into fundamental mechanochemistry of kines...
Next steps – Study isotope effect; osmotic stress<br />18-oxygen water (does not exchange with protein groups; hydrogen bo...
Acknowledgments<br />Our Lab—Larry Herskowitz, Andy Maloney, Anthony Salvagno,Brian Josey, Emmalee Jones, Linh Le, Brigett...
End<br />
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2010 DTRA Update

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Brief progress update and project overview (experimental side only) for DTRA visitor to UNM.

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2010 DTRA Update

  1. 1. Experimental overview of DTRA kinesin projectKoch Lab, UNM Dept. Physics and Center for High Technology Materials (CHTM)<br />“Kiney”<br />Steve Koch, DTRA Co-PI, Experimental LeadAsst. Prof. Physics and Astronomy<br />Larry HerskowitzPhysics Ph.D. Student<br />Andy Maloney<br />Physics Ph.D. Student<br />Anthony Salvagno, IGERT FellowPhysics Ph.D. Student<br />Brian Josey<br />Physics B.S. Student<br />SJK Note: For most of the embedded movies, try thispublic directory:<br />http://kochlab.org/files/Movies/2010%20Feb%20DTRA%20Presentation<br />Email: sjkoch@unm.edu<br />Emmalee Jones, (rotating)NSMS Ph.D. Student<br />
  2. 2. KochLab Overview / Acknowledgments<br />Single-molecule manipulation<br />Optical tweezers; magnetic tweezers; MEMS<br />Kinesin / mictrotubules<br />Osmotic stress; isotope effects<br />Protein-DNA interactions; transcription<br />Collaborations<br />Susan Atlas—Lead of the DTRA projectUNM Physics / Cancer Center / Director of CARC<br />HaiqingLiu (G. Mantano lab)—Microdeviceapplications of kinesin<br />LANL & Center for Integrated Nanotechnology (CINT)<br />Evan Evans Lab—Single-molecule thermodynamics and kinetics<br />U. New Mexico / U. British Columbia / Boston U.<br />Funding<br />DTRA—Basic Science; CHTM—Startup; ACS—Jan Oliver IRG<br />
  3. 3. Microtubules are polymers of tubulin heterodimers<br />Fast polymerizing end<br />Plus end<br />8 nanometers<br />25 nanometers<br />Slow polymerizing end<br />Minus end<br />Tubulin can be purified from, for example, cow brains<br />Microtubules can be reliably polymerized in vitro<br />Stabilized with anti-cancer drug Taxol<br />
  4. 4. Our goal is to gain atomistic insight through a variety of experiments and simulations…especially focusing on water<br />Susan Atlas (PI) and Steve Valone (LANL)<br />“Charge transfer embedded atom model” (CT-EAM)<br />Atomistic modeling of kinesin catalytic core<br />Molecular dynamics<br />Kochlab: Biophysical experimental studies of kinesin<br />
  5. 5. Gliding motility assay<br />Buffer includes ATP, antifade cocktail<br />Andy is currently leading the GMA project<br />100 microns<br />Passivated glass surface (casein)<br />Parameters we can measure<br />Speedspeed distribution<br />MT morphology (straight; circles; length)<br />Assay longevity (activity; photobleaching)<br />(Show movie externally)<br />“motility in regular water”<br />
  6. 6. Gliding motility assay is initially our main assay<br />Buffer includes ATP, antifade cocktail<br />Passivated glass surface (casein)<br />Operate in the high motor density regime<br />Main experimental result is transport velocity<br />Heavy water <br />Osmotic stress<br />Temperature, metal ions, ATP concentration<br /> Site-directed mutagenesis<br />Fascinating early results!<br />Experimental “knobs” to obtain datathat can be compared with theory in the iterative loop<br />
  7. 7. Heavy water background<br />Naturally abundant 1 / 6600 hydrogen molecules is deuterium<br /> 17 mM deuterium in “standard mean ocean water”<br />11% denser than H2O. Freezes at 3.8C. D-bonds stronger.<br />Toxic to eukaryotes. The toxic effects are similar to chemotherapeutic drugs.<br />D2O has been used to stabilize viral vaccines.<br />D2O stabilizes tubulin and microtubules.<br />D2O stimulates tubulin assembly formation.<br />(Other fascinating factoids…)<br />Effects on kinesin motility has not yet been studied<br />
  8. 8. Gliding assays in D2OSquiggly Microtubules; MT-MT interactions<br />(Show movie externally)<br />“Motility in 100% D2O”<br />
  9. 9. Gliding assays in D2OSignificantly more stable microtubules (and maybe kinesin) Activity lasts &gt; 24 hours<br />(Show movie externally) (“motility after 1 day in D2O”)<br />Also reduces photobleaching and possibly the “opticution” effects.<br />
  10. 10. Microtubule velocity in gliding assay is measured viaLabVIEW image tracking software written by Larry<br />Open source software<br />Preparing publication<br />(Show movie externally)<br />
  11. 11. Gliding motility assay—Deuterium Isotope Effects<br />7. Guydosh, Nicholas R, and Steven M Block. “Direct observation of the binding state of the kinesin head to the microtubule.” Nature 461, no. 7260 (September 3, 2009): 125-128. doi:10.1038/nature08259. Supplemental information.<br />Preparing publication<br />
  12. 12. Implications of DTRA research so far<br />Basic Research<br /> Gaining insights into fundamental mechanochemistry of kinesin/MTs<br /> Properties of water ideal connection with theory group<br /> (didn’t show): Have built stochastic simulation to interpret data<br />Applications<br /> D2O Results point towards strategies for improving device robustness<br /> Fundamental understanding will guide directed engineering of motors<br /> Open source software will help community<br />
  13. 13. Next steps – Study isotope effect; osmotic stress<br />18-oxygen water (does not exchange with protein groups; hydrogen bonding same)<br />Osmolytes (e.g. betaine, sucrose)<br />Is this a real effect?<br />
  14. 14. Acknowledgments<br />Our Lab—Larry Herskowitz, Andy Maloney, Anthony Salvagno,Brian Josey, Emmalee Jones, Linh Le, Brigette Black, Igor Kuznetsov<br />Collaborations<br />Susan Atlas—Lead of the DTRA projectUNM Physics / Cancer Center / Director of CARC<br />Steve Valone—Co-PI (LANL)<br />Haiqing Liu—Microdevice applications of kinesin<br />LANL & Center for Integrated Nanotechnology (CINT)<br />Evan Evans Lab—Single-molecule thermodynamics and kinetics<br />U. New Mexico / U. British Columbia / Boston U.<br />Funding<br />DTRA—Basic Science; CHTM—Startup; ACS—Jan Oliver IRG<br />
  15. 15. End<br />

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