Experimental Overview  Koch Lab, UNM Dept. Physics and  Center for High Technology Materials (CHTM) Steve Koch, Co-PI, Exp...
Experimental group expertise <ul><li>Single-molecule manipulation </li></ul><ul><ul><li>Optical tweezers; magnetic tweezer...
Kinesin binds to microtubules and uses  ATP hydolysis to walk along tubulin protofilaments An overview of the  two basic c...
Microtubules are polymers of tubulin heterodimers 25 nm 4 nm 8 nm
Microtubules can be reliably polymerized in vitro  In living cells, predominant form of MTs have 13 protofilaments (PFs) I...
Kinesin binds to microtubules and uses  ATP hydolysis to walk along tubulin protofilaments An overview of the  two basic c...
Kinesin is a eukaryotic molecular motor protein with a number of intracellular functions Mitosis Intracellular transport V...
Conventional kinesin-1 “walks” along protofiliments  in hand-over-hand mechanism Sablin and Fletterick, 2004 JBC
Processivity Thorn, Ubersax, Vale JCB 2000
A possible mechanism for kinesin procession <ul><li>Gray :  coiled-coil;  blue:  catalytic core; white/ green :  α ,  β  s...
Truncated, tagged conventional kinesin constructs Coy, Hancock, Wagenbock, Howard (1999) Full length conventional kinesin ...
Striving for atomistic insights into catalytic mechanism Sablin and Fletterick, 2004 JBC Much has been learned about kines...
We will utilize two independent experimental platforms “ Easy” Robust Many experimental “knobs” Limited readout More diffi...
Gliding motility assay Kinesin Microtubule Glass Surface (passivated with casein) + Buffer, ATP Gliding motility assay, Ko...
Gliding motility assay Kinesin Microtubule Glass Surface (passivated with casein) + Buffer, ATP Operate in the high motor ...
Bead motility assay Andrian Fehr, Science 2003 Steve Block Lab, Stanford Single-molecule kinesin transport Steve Block Lab...
Optical tweezers are formed by shining laser light into a high numerical aperture objective Optical Trap “ Laser tweezers”...
<ul><li>Piezoelectric stage moves coverglass relative to trap center </li></ul>Using optical tweezers, we can apply and me...
Using optical tweezers, we can apply and measure forces on single tethered biomolecules Microsphere Biomolecular  “ Tether...
Bead motility assay High kinesin concentration Measure velocity of collective molecular motors (similar to gliding assay) ...
Kinesin-microtubule unbinding forces Kawaguchi, Uemura, Ishiwata 2003 “ Dynamic Strength of Molecular Adhesion Bonds” Evan...
Bead motility assay High kinesin concentration Measure velocity of collective molecular motors (similar to gliding assay) ...
Our initial experiments will pursue effects of osmotic stress and light / heavy water in two experimental assays Water con...
Why is water so important? Each time the kinesin head binds to tubulin, dozens of “hydrating” water molecules must be excl...
 
The osmotic stress method relies on changing water activity by adding high concentration of solutes Parsegian, Rand, Rau, ...
Osmotic stress increases myosin-actin affinity Highsmith et al. Biophys. J. 1996 No data exist for kinesin-MT Potentially ...
Osmotic stress studies of kinesin    untapped Utility proven in protein-DNA studies Protein DNA Non-specific, K nonsp Spe...
Osmotic stress dramatically increases lifetime of bound molecular complexes Osmotic pressure helpful For increasing lifeti...
Our preliminary data showed that osmotic stress effects protein-DNA unbinding forces X-intercept of these curves reveals o...
Next up, Susan will describe novel theoretical methods and look at the impact of these experimental “knobs” Properties of ...
 
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KochLab Kinesin Project Intro, Summer 2009

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Introduction to KochLab's kinesin project, beginning summer 2009.

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  • As one would perhaps guess, molecular motors and microfilaments are key components of these systems.
  • As one would perhaps guess, molecular motors and microfilaments are key components of these systems.
  • Talk generally about: 1. Force detection capabilities. (Conservation of momentum) Also mention the geometry and calibration 2. The biochemical methods for attaching the DNA to the beads.
  • Talk generally about: 1. Force detection capabilities. (Conservation of momentum) Also mention the geometry and calibration 2. The biochemical methods for attaching the DNA to the beads.
  • Talk generally about: 1. Force detection capabilities. (Conservation of momentum) Also mention the geometry and calibration 2. The biochemical methods for attaching the DNA to the beads.
  • KochLab Kinesin Project Intro, Summer 2009

    1. 1. Experimental Overview Koch Lab, UNM Dept. Physics and Center for High Technology Materials (CHTM) Steve Koch, Co-PI, Experimental Lead Asst. Prof. Physics and Astronomy Larry Herskowitz, IGERT Fellow Physics Ph.D. Student Anthony Salvagno, IGERT Fellow Physics Ph.D. Student Brigette Black Physics Ph.D. Student Andy Maloney, NSF IGERT Fellow Physics Ph.D. Student Igor Kuznetsov Postdoc Linh Le Physics B.S. Student “ Kiney” SJK: This is a talk I gave for group meeting summer 2009…please let me know if images or other need attribution
    2. 2. Experimental group expertise <ul><li>Single-molecule manipulation </li></ul><ul><ul><li>Optical tweezers; magnetic tweezers; MEMS </li></ul></ul><ul><li>Kinesin / mictrotubules </li></ul><ul><ul><li>Thermostable kinesin; microdevice applications </li></ul></ul><ul><li>Protein-DNA interactions; transcription </li></ul>Collaborations: Haiqing Liu —Microdevice applications of kinesin LANL & Center for Integrated Nanotechnology (CINT) Evan Evans Lab —Single-molecule thermodynamics and kinetics U. New Mexico / U. British Columbia
    3. 3. Kinesin binds to microtubules and uses ATP hydolysis to walk along tubulin protofilaments An overview of the two basic components of this system: Microtubules Kinesin Microtubules are a key component of the system: kinesin does not move or catalyze ATP hydrolysis in absence of MTs Goldstein Lab
    4. 4. Microtubules are polymers of tubulin heterodimers 25 nm 4 nm 8 nm
    5. 5. Microtubules can be reliably polymerized in vitro In living cells, predominant form of MTs have 13 protofilaments (PFs) In vitro “reassembly” of microtubules was possible by the early 1970s (Borisy, Brinkley, …) Typically performed with purified bovine or porcine brain tubulin Produces an assortment of MTs with varying numbers of PFs (usually not 13) Recombinant tubulin is not readily available MTs are stabilized by taxol … chemical cross-linking is another strategy Easily visualized by fluorescence microscopy - +  tubulin dimer   Protofilament 25 nm
    6. 6. Kinesin binds to microtubules and uses ATP hydolysis to walk along tubulin protofilaments An overview of the two basic components of this system: Microtubules Kinesin Goldstein Lab
    7. 7. Kinesin is a eukaryotic molecular motor protein with a number of intracellular functions Mitosis Intracellular transport Vale, Reese, Sheetz, 1985, Cell 42 39-50. “Identification of a Novel Force-Generating Protein, Kinesin, Involved in Microtubule-Based Motility.” At least 14 families of kinesin across all eukaryotes Dimeric “conventional” kinesin-1 : vesicle transport Kinesin-1, -2, -3, etc… E.g., Kinesin-5 is tetrameric kinesin: spindle formation HHMI Winter Bulletin 2005 Kinesin-5 tetramers
    8. 8. Conventional kinesin-1 “walks” along protofiliments in hand-over-hand mechanism Sablin and Fletterick, 2004 JBC
    9. 9. Processivity Thorn, Ubersax, Vale JCB 2000
    10. 10. A possible mechanism for kinesin procession <ul><li>Gray : coiled-coil; blue: catalytic core; white/ green : α , β subunits of microtubulin heterodimer; red / orange / yellow : neck linker in successively more tightly-docked states on catalytic core; cargo not shown. </li></ul><ul><li>Step 1: ATP binding to leading head initiates neck-linker docking with catalytic core </li></ul><ul><li>Step 2: Neck-linker docking is completed by leading head, throwing trailing head forward by 16 nm toward next tubulin binding site. </li></ul><ul><li>Step 3: After a random diffusional search, new leading head docks tightly onto the binding site, completing 8 nm motion of attached cargo. Polymer binding accelerates ADP release; trailing head hydrolyzes ATP to ADP-P i . </li></ul><ul><li>Step 4: ATP binds to leading head following ADP release, and neck-linker (orange) begins to zipper onto catalytic core. The trailing head, which has released its Pi and detached its neck linker (red) from its core, is in the process of being thrown forward. </li></ul>R. D. Vale and R. A. Milligan, Science 288 , 88 (2000).
    11. 11. Truncated, tagged conventional kinesin constructs Coy, Hancock, Wagenbock, Howard (1999) Full length conventional kinesin self-inhibits by tail binding to motor domain Asbury, Fehr, Block (2003) Recombinant kinesin expressed in E. coli, purified by his-tag methods Limited commercial availability
    12. 12. Striving for atomistic insights into catalytic mechanism Sablin and Fletterick, 2004 JBC Much has been learned about kinesin at the stochastic (mechanical) level But atomistic understanding of mechanochemistry is lacking Our goal is to gain atomistic insight via a variety of experiments and simulations
    13. 13. We will utilize two independent experimental platforms “ Easy” Robust Many experimental “knobs” Limited readout More difficult Many experimental “knobs” Many readout variables
    14. 14. Gliding motility assay Kinesin Microtubule Glass Surface (passivated with casein) + Buffer, ATP Gliding motility assay, Koch @ Sandia Thermophilic fungal kinesin (field of view approx 150 microns) Microtubule velocity is measured either manually or by automatically tracking MT ends Larry Herskowitz (grad) is currently adapting existing tracking software for this purpose Image: George Bachand
    15. 15. Gliding motility assay Kinesin Microtubule Glass Surface (passivated with casein) + Buffer, ATP Operate in the high motor density regime Main experimental result is transport velocity Osmotic stress Light / heavy water Temperature, metal ions, ATP concentration Site-directed mutagenesis Gliding motility assay, Koch @ Sandia Thermophilic fungal kinesin (field of view approx 150 microns) Experimental “knobs” to obtain data that can be compared with theory in the iterative loop Image: George Bachand
    16. 16. Bead motility assay Andrian Fehr, Science 2003 Steve Block Lab, Stanford Single-molecule kinesin transport Steve Block Lab, Stanford
    17. 17. Optical tweezers are formed by shining laser light into a high numerical aperture objective Optical Trap “ Laser tweezers” Microsphere Biomolecular “Tether” Coverglass
    18. 18. <ul><li>Piezoelectric stage moves coverglass relative to trap center </li></ul>Using optical tweezers, we can apply and measure forces on single biomolecules Infrared laser focused through microscope objective piezoelectric stage Quadrant photodiode to measure force Optical Trap Microsphere Biomolecular “Tether” Coverglass <ul><li>Newton’s third law </li></ul><ul><ul><li>Force on bead = force on laser collect exit light onto photodiode to measure force, displacement </li></ul></ul>Dielectic particles (500 nm polystyrene) attracted to laser focus
    19. 19. Using optical tweezers, we can apply and measure forces on single tethered biomolecules Microsphere Biomolecular “ Tether” Coverglass <ul><ul><li>Forces from < 1 pN to 100s pN </li></ul></ul><ul><ul><li>Length precision ~ 1 nm </li></ul></ul><ul><ul><li>Thermal energy (k B T) </li></ul></ul><ul><ul><li>4 pN – nm = 1/40 eV </li></ul></ul><ul><ul><li>Kinesin 8 nm step, 6 pN stall </li></ul></ul><ul><ul><li>RNA Polymerase 0.3 nm step, 25 pN stall </li></ul></ul><ul><ul><li>DNA Unzipping 15 pN </li></ul></ul>OT feedback control software is crucial component We have a user-friendly LabVIEW application with a variety of feedback modes
    20. 20. Bead motility assay High kinesin concentration Measure velocity of collective molecular motors (similar to gliding assay) Low kinesin concentration Single-molecule studies of kinesin: processivity force-velocity pull-off force Block et al. (2003) PNAS
    21. 21. Kinesin-microtubule unbinding forces Kawaguchi, Uemura, Ishiwata 2003 “ Dynamic Strength of Molecular Adhesion Bonds” Evan Evans and Ken Ritchie, 1997 Biophys. J. Brower-Toland et al., 2002
    22. 22. Bead motility assay High kinesin concentration Measure velocity of collective molecular motors (similar to gliding assay) Low kinesin concentration Single-molecule studies of kinesin: processivity force-velocity pull-off force Experimental knobs for iterative theory/experiment loop: Osmotic stress Light / heavy water Temperature, metal ions, ATP concentration Site-directed mutagenesis
    23. 23. Our initial experiments will pursue effects of osmotic stress and light / heavy water in two experimental assays Water connects our experiment / theory iterative loop In addition to coupling experiment / theory these are an exciting, untapped line of experiments Only a couple papers exist for myosin / none for kinesin
    24. 24. Why is water so important? Each time the kinesin head binds to tubulin, dozens of “hydrating” water molecules must be excluded. Each time the kinesin unbinds, water must “rehydrate” Thus, “water activity” strongly impacts binding kinetics (and whole kinetic cycle) Okada, Higuchi, Hirokawa Water excluded Water hydrating
    25. 26. The osmotic stress method relies on changing water activity by adding high concentration of solutes Parsegian, Rand, Rau, Methods in Enzymology 259 (1995) “ Osmolyte” (sucrose, betaine, PEG, …) Reduces the chemical potential of water Molecule of interest has a shell of hydrating water molecules (higher chemical potential)
    26. 27. Osmotic stress increases myosin-actin affinity Highsmith et al. Biophys. J. 1996 No data exist for kinesin-MT Potentially many high-impact results
    27. 28. Osmotic stress studies of kinesin  untapped Utility proven in protein-DNA studies Protein DNA Non-specific, K nonsp Specific complex, K sp Sidorova and Rau, PNAS 1996
    28. 29. Osmotic stress dramatically increases lifetime of bound molecular complexes Osmotic pressure helpful For increasing lifetime too ln(Fraction bound) Sidorova and Rau Kinesin binding / unbinding
    29. 30. Our preliminary data showed that osmotic stress effects protein-DNA unbinding forces X-intercept of these curves reveals off-rate Evans & Ritchie 1997 theory Protein-DNA interactions probed by DNA unzipping is another Koch Lab project We anticipate similar effects of osmotic stress on kinesin-MT forced disruption Specific Non-specific
    30. 31. Next up, Susan will describe novel theoretical methods and look at the impact of these experimental “knobs” Properties of water will provide initial strong ties between theory and experiment Provide a very interesting line of high-impact experiments Also provide a connection to technological applications of kinesin / MT system Long-term stability of kinesin and microtubules Up-modulation of kinesin processivity? velocity? strength?

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