KochLab Kinesin Project Intro, Summer 2009

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Information about KochLab Kinesin Project Intro, Summer 2009
Technology

Published on August 18, 2009

Author: skoch3

Source: slideshare.net

Description

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

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

Experimental group expertise Single-molecule manipulation Optical tweezers; magnetic tweezers; MEMS Kinesin / mictrotubules Thermostable kinesin; microdevice applications Protein-DNA interactions; transcription 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

Single-molecule manipulation

Optical tweezers; magnetic tweezers; MEMS

Kinesin / mictrotubules

Thermostable kinesin; microdevice applications

Protein-DNA interactions; transcription

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

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) 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

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

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

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 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. Step 1: ATP binding to leading head initiates neck-linker docking with catalytic core Step 2: Neck-linker docking is completed by leading head, throwing trailing head forward by 16 nm toward next tubulin binding site. 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 . 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. R. D. Vale and R. A. Milligan, Science 288 , 88 (2000).

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.

Step 1: ATP binding to leading head initiates neck-linker docking with catalytic core

Step 2: Neck-linker docking is completed by leading head, throwing trailing head forward by 16 nm toward next tubulin binding site.

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 .

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.

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

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

We will utilize two independent experimental platforms “ Easy” Robust Many experimental “knobs” Limited readout More difficult Many experimental “knobs” Many readout variables

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

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

Bead motility assay Andrian Fehr, Science 2003 Steve Block Lab, Stanford Single-molecule kinesin transport Steve Block Lab, Stanford

Optical tweezers are formed by shining laser light into a high numerical aperture objective Optical Trap “ Laser tweezers” Microsphere Biomolecular “Tether” Coverglass

Piezoelectric stage moves coverglass relative to trap center 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 Newton’s third law Force on bead = force on laser collect exit light onto photodiode to measure force, displacement Dielectic particles (500 nm polystyrene) attracted to laser focus

Piezoelectric stage moves coverglass relative to trap center

Newton’s third law

Force on bead = force on laser collect exit light onto photodiode to measure force, displacement

Using optical tweezers, we can apply and measure forces on single tethered biomolecules Microsphere Biomolecular “ Tether” Coverglass Forces from < 1 pN to 100s pN Length precision ~ 1 nm Thermal energy (k B T) 4 pN – nm = 1/40 eV Kinesin 8 nm step, 6 pN stall RNA Polymerase 0.3 nm step, 25 pN stall DNA Unzipping 15 pN OT feedback control software is crucial component We have a user-friendly LabVIEW application with a variety of feedback modes

Forces from < 1 pN to 100s pN

Length precision ~ 1 nm

Thermal energy (k B T)

4 pN – nm = 1/40 eV

Kinesin 8 nm step, 6 pN stall

RNA Polymerase 0.3 nm step, 25 pN stall

DNA Unzipping 15 pN

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

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

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

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

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

 

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)

Osmotic stress increases myosin-actin affinity Highsmith et al. Biophys. J. 1996 No data exist for kinesin-MT Potentially many high-impact results

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

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

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

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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