““Myosin V Walks Hand-Myosin V Walks Hand-Over-Hand: Single Over-Hand: Single Fluorophore Imaging with Fluorophore Imaging with 1.5-nm Localization”1.5-nm Localization”

Ahmet Yildiz, Joseph N. Forkey, Sean A. McKinney, Taekjip Ahmet Yildiz, Joseph N. Forkey, Sean A. McKinney, Taekjip Ha, Yale E. Goldman, Paul R. SelvinHa, Yale E. Goldman, Paul R. Selvin

Science Vol 300, 27 June 2003Science Vol 300, 27 June 2003

Presented by: Hanna SeitzPresented by: Hanna Seitz

www.news.uiuc.edu/scitips

OutlineOutline

I.I. background: myosin V structurebackground: myosin V structure

II.II. experimental overviewexperimental overview

III.III. methods methods

IV.IV. resultsresults

V.V. summarysummary

VI.VI. outlookoutlook

I. background: myosin V structureI. background: myosin V structure

myosin Vmyosin V:: dimeric molecular motor that “travels” dimeric molecular motor that “travels”

along actin filamentsalong actin filaments

heads contain catalytic domain that heads contain catalytic domain that bind actin and hydrolyze ATPbind actin and hydrolyze ATP

light chain domain connects to coiled-light chain domain connects to coiled-coil stalk, which is connected to cargo coil stalk, which is connected to cargo binding domainbinding domain

defects lead to neurological and defects lead to neurological and immunological diseasesimmunological diseases

www.rpi.edu/.../mb2/part1/images/

II. experimental overviewII. experimental overview

2 models for myosin V motion2 models for myosin V motion::

1.1. inchworminchworm: step size of head is : step size of head is equal to step size of stalk (~ 37 equal to step size of stalk (~ 37 nm)nm)

2.2. hand-over-handhand-over-hand: :

leading head leading head doesn’tdoesn’t move; move; trailing head moves twice as far as trailing head moves twice as far as stalk (stalk moves ~ 37 nm)stalk (stalk moves ~ 37 nm)

alternating steps of 37-2x nm, alternating steps of 37-2x nm, 37+2x nm for a 37+2x nm for a fluorophorefluorophore at the at the light chain domainlight chain domain

Yildiz et al, Science Vol 300 27 June 2003

II. experimental overview II. experimental overview continuedcontinued

To test the hand-over-hand model, To test the hand-over-hand model, single molecule fluorescence single molecule fluorescence imaging techniquesimaging techniques were used: were used:

localization of molecule in 2D to within 1.5 nm & a 0.5 second localization of molecule in 2D to within 1.5 nm & a 0.5 second resolutionresolution

enhanced photostability of fluorophore (dye) allows for minutes enhanced photostability of fluorophore (dye) allows for minutes of observation (Oof observation (O22-depletion via glucose oxidase and catalase)-depletion via glucose oxidase and catalase)

TIRF (total internal reflection fluorescence microscopy): used to TIRF (total internal reflection fluorescence microscopy): used to excite and image individual fluorophores onto charge coupled excite and image individual fluorophores onto charge coupled device, allowing processing of sequential imagesdevice, allowing processing of sequential images

► ► efficient dye localization via FIONA efficient dye localization via FIONA

III. methods: TIRFIII. methods: TIRF

for imaging of individual fluorophores; for imaging of individual fluorophores; background fluorescence is eliminatedbackground fluorescence is eliminated

use of evanescent waves to excite use of evanescent waves to excite fluorophores (~100 nm deep penetration)fluorophores (~100 nm deep penetration)

evanescent waves (electric field) are created evanescent waves (electric field) are created when the incident light is totally reflected at when the incident light is totally reflected at the boundarythe boundary

total internal reflection when angle of incident total internal reflection when angle of incident light to normal is equal to or greater than light to normal is equal to or greater than critical anglecritical angle

steeper incident angle leads to deeper light steeper incident angle leads to deeper light penetration (bigger field)penetration (bigger field)

1 = specimen in aqueous buffer

2 = evanescent wave range

3 = cover slip

4 = oil

5 = objective

6 = emission light

7 = excitation light

http://en.wikipedia.org/wiki

III. methods: FIONAIII. methods: FIONA

FIONAFIONA = fluorescence imaging with one- = fluorescence imaging with one-nanometer accuracynanometer accuracy center of image able to be precisely located center of image able to be precisely located by collecting large number of photonsby collecting large number of photons

curve-fitting the image (point spread function, curve-fitting the image (point spread function, PSFPSF) to a Gaussian function allowed for center ) to a Gaussian function allowed for center determination of the imagedetermination of the image

the goal via the Gaussian function is to the goal via the Gaussian function is to determine the center of distribution determine the center of distribution μμ and the and the standard error of the mean standard error of the mean σσ

a Gaussian curve-fit to a PSF

http://en.wikipedia.org/wiki

III. methods: FIONAIII. methods: FIONA continuedcontinued

σσ ‘s relation to the number of collected photons (N), pixel size ‘s relation to the number of collected photons (N), pixel size of imaging detector (a), deviation of the background (b) and the of imaging detector (a), deviation of the background (b) and the width of distribution (swidth of distribution (s ii) is given by:) is given by:

with i = index of x and y directionwith i = index of x and y direction

the first term is the the first term is the photon noise (photon noise ( illumination) illumination) (dominant (dominant contributor to contributor to σσ), the second term is the ), the second term is the effect of finite pixel size (effect of finite pixel size ( detection) detection) of the detector, and the third term is the of the detector, and the third term is the effect of effect of background (background ( sample) sample)

IV. results: control experiment for IV. results: control experiment for localization of dyelocalization of dye

Cy3 dye attached to coverslip via a DNA-Cy3 dye attached to coverslip via a DNA-biotin-streptavidin linkagebiotin-streptavidin linkage

Gaussian analysis of the circled PSF led to Gaussian analysis of the circled PSF led to good fit (rgood fit (r22 = 0.994); SNR (signal-to-noise = 0.994); SNR (signal-to-noise ratio) of PSF is 32ratio) of PSF is 32

under oxygen scavenging conditions, the under oxygen scavenging conditions, the highlighted PSF lasted 100 images (50 sec) highlighted PSF lasted 100 images (50 sec)

before photobleaching.before photobleaching.

Yildiz et al, Science Vol 300 27 June 2003

IV. results: control experiment for IV. results: control experiment for

localization of dyelocalization of dye continuedcontinued

horizontal movement of Cy3-DNA coverslip via nanometric-stage horizontal movement of Cy3-DNA coverslip via nanometric-stage tests the ability to measure step sizestests the ability to measure step sizes

~ 30-nm steps observed after moving the coverslip with a nanometric stage and plotting PSF center against time. Red lines give positions between each step.

precision, σ, is ~1 nm

accuracy, μ, (difference between measured and expected step size) also ~1 nm

~ 7-nm steps

Yildiz et al, Science Vol 300 27 June 2003

IV. results: step sizes of myosin VIV. results: step sizes of myosin V

specific myosin V light chain domains targeted and labeled with single specific myosin V light chain domains targeted and labeled with single bifunctional rhodamine (BR) or monofunctional Cy3 (18.5 nm, 7 nm and bifunctional rhodamine (BR) or monofunctional Cy3 (18.5 nm, 7 nm and 2.5 nm from midpoint of axis) 2.5 nm from midpoint of axis)

labeled myosin V added to F-actin filaments immobilized on a coverslip labeled myosin V added to F-actin filaments immobilized on a coverslip and observation with TIRFand observation with TIRF

when no ATP present, no movement of dye spots observed; 300 nM of when no ATP present, no movement of dye spots observed; 300 nM of ATP allowed for visible step movementATP allowed for visible step movement

5,000 – 10,000 photons per spot allow center location to 1.5 nm5,000 – 10,000 photons per spot allow center location to 1.5 nm

3 different3 different myosin V step combinations observed: myosin V step combinations observed: 74 nm steps (with in between 0 nm step)74 nm steps (with in between 0 nm step) alternating 52- & 23-nm stepsalternating 52- & 23-nm steps alternating 42- & 33-nm stepsalternating 42- & 33-nm steps

IV. results: 74-nm stepsIV. results: 74-nm steps

for 32 molecules a total of 231 for 32 molecules a total of 231 steps were observedsteps were observed

histogram determined 73.8 histogram determined 73.8 ± ± 5.3 nm steps with good fit to 5.3 nm steps with good fit to Gaussian (rGaussian (r22 = 0.994) = 0.994)

in hand-over-hand model in hand-over-hand model (37 ± 2x) 74 nm-step is result (37 ± 2x) 74 nm-step is result of dye near catalytic domainof dye near catalytic domain stalk moves 37 nm, dye stalk moves 37 nm, dye (x =) 18.5 nm from midpoint (x =) 18.5 nm from midpoint of motionof motion steps of 3 different myosin V molecules; histogram

with the 32 molecules taking 231 steps

Yildiz et al, Science Vol 300 27 June 2003

IV. results: 52-23 nm stepsIV. results: 52-23 nm steps

for 6 molecules a total for 6 molecules a total of 92 52-23 nm steps of 92 52-23 nm steps were observedwere observed

histogram determined histogram determined averages of 51.7 averages of 51.7 ± 4.2 ± 4.2 nm 23.1 ± 3.4 nm, and nm 23.1 ± 3.4 nm, and 73.6 ± 5.3 nm steps73.6 ± 5.3 nm steps

in hand-over-hand in hand-over-hand model, 52-23 nm steps model, 52-23 nm steps are result of dye on 5are result of dye on 5thth light chainlight chain (x =) ~7 nm from (x =) ~7 nm from midpoint of motionmidpoint of motion

Steps of two myosin V molecules; histogram of the 6 molecules taking 92 steps. Peak at 74 nm due to some missed steps (52 + 23)

Yildiz et al, Science Vol 300 27 June 2003

IV. results: 42-33 nm stepsIV. results: 42-33 nm steps

for 6 molecules 69 for 6 molecules 69 alternating 42-33 nm steps alternating 42-33 nm steps were observedwere observed

histogram determined histogram determined averages of 42.4 averages of 42.4 ± 2.9 nm, ± 2.9 nm, 32.8 ± 2.1 nm, and 74.1 ± 32.8 ± 2.1 nm, and 74.1 ± 2.2 nm steps2.2 nm steps

In hand-over-hand model, In hand-over-hand model, 42-33 nm steps are result of 42-33 nm steps are result of dye on 6dye on 6thth light chain light chain (x =) ~2.5 nm from (x =) ~2.5 nm from midpointmidpoint

Steps of three myosin V molecules; histogram of the 6 molecules taking 69 steps. Peak at 74 nm due to some missed steps (42 + 33)

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IV. results: the 0-nm stepIV. results: the 0-nm step hand-over-hand model would predict a 0-nm step for every 74-hand-over-hand model would predict a 0-nm step for every 74-

nm step (37-2x, x = 18.5)nm step (37-2x, x = 18.5)

0-nm step can’t be seen but 2 kinetic analyses imply it0-nm step can’t be seen but 2 kinetic analyses imply it1.) 74-nm step has half the step rate (0.17 s1.) 74-nm step has half the step rate (0.17 s-1-1) compared to 42-) compared to 42-33 nm and 52-23 nm steps (0.35 s33 nm and 52-23 nm steps (0.35 s-1-1))2.) indirect detection via kinetic rate constant (k) and dwell time 2.) indirect detection via kinetic rate constant (k) and dwell time ((tt); dwell time = no movement due to dissociation, ATP wait, etc.); dwell time = no movement due to dissociation, ATP wait, etc.

probability of dwell times (when kprobability of dwell times (when k11 = k = k22; A ; A B B : k: k11 and B and B A’: k A’: k22))

P(P(tt) = k) = k11ee-k-k11

tt (for 42-33 nm and 52-23 nm steps)(for 42-33 nm and 52-23 nm steps)

P(P(tt) = ) = ttkk22ee-k-ktt (for 74-0 nm steps)(for 74-0 nm steps)

IV. results: the 0-nm stepIV. results: the 0-nm step continuedcontinued

from the equations, an initial increase in dwell time and then a from the equations, an initial increase in dwell time and then a decrease is expected for the 74-0 nm data (decrease is expected for the 74-0 nm data ( single step single step mechanism)mechanism)

for the 42-33 nm and 52-23 nm data a monotonous decay is for the 42-33 nm and 52-23 nm data a monotonous decay is expectedexpected

dwell time histograms are shown

k52-23,42-33 = 0.28 s-1 (r2 = 0.984)

k74-0 = 0.33 s-1 (r2 =0.986)

single rate constant is valid because rate limiting step is initial ATP binding; thus myosin V speed is proportional to concentration of ATP

Yildiz et al, Science Vol 300 27 June 2003

V. summaryV. summary

myosin V takes different step sizes; 74-0 nm, 52-23 nm, 42-33 nm myosin V takes different step sizes; 74-0 nm, 52-23 nm, 42-33 nm due to dye on different positions on the light chain (x distance from due to dye on different positions on the light chain (x distance from the midpoint of motion)the midpoint of motion)

these steps are in line with 37 these steps are in line with 37 ± 2x nm prediction of hand-over-± 2x nm prediction of hand-over-hand model; no 37 nm steps seen (thus not inchworm model)hand model; no 37 nm steps seen (thus not inchworm model)

the specific and sensitive single molecule fluorescence techniques the specific and sensitive single molecule fluorescence techniques with an oxygen scavenging system allowed for with an oxygen scavenging system allowed for extended extended observationobservation, a high photon number for , a high photon number for 1.5-nm localization1.5-nm localization, and a , and a low-noise detector for low-noise detector for high SNRhigh SNR

made step measurement visualization supporting the made step measurement visualization supporting the hand-over-hand model possible!!!hand-over-hand model possible!!!

VI. outlookVI. outlook

other molecular motors, such as kinesin, other molecular motors, such as kinesin, could have similar mode of movementcould have similar mode of movement

2 types of hand-over-hand models:2 types of hand-over-hand models:1.) asymmetric: heads are not 1.) asymmetric: heads are not equivalent and equivalent and nono twisting of stalk twisting of stalk2.) symmetric: heads are functionally 2.) symmetric: heads are functionally the same and stalk twists ~ 180the same and stalk twists ~ 180° on ° on each stepeach step

asymmetric model favored asymmetric model favored because no large cargo twisting; no because no large cargo twisting; no torque required that twists motortorque required that twists motor

movement of kinesin on microtubuli

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Thank You for the keen Thank You for the keen attention!!attention!!

literature and referencesliterature and references

Yildiz et al. Myosin V Walks Hand-Over-Hand: Single Fluorophore Imaging with 1.5 nm-Yildiz et al. Myosin V Walks Hand-Over-Hand: Single Fluorophore Imaging with 1.5 nm-Localization. Localization. ScienceScience Vol Vol 300300. 27 June 2003.. 27 June 2003.

Berg, Jeremy M., Tymoczko, John L., Stryer, Lubert. Berg, Jeremy M., Tymoczko, John L., Stryer, Lubert. BiochemistryBiochemistry. 6. 6thth edition. 2007: W.H. edition. 2007: W.H. Freeman and Company, 41 Madison Avenue, New York, NY 10010.Freeman and Company, 41 Madison Avenue, New York, NY 10010.

Lodish, Harvey, A. Berk, P. Matsudaira, C. Kaiser, M. Krieger, M. Scott, S. Zipursky, J. Lodish, Harvey, A. Berk, P. Matsudaira, C. Kaiser, M. Krieger, M. Scott, S. Zipursky, J. Darnell. Darnell. Molecular Cell BiologyMolecular Cell Biology. 5. 5thth edition. 2004: W.H. Freeman and Company, 41 Madison edition. 2004: W.H. Freeman and Company, 41 Madison Avenue, New York, NY 10010.Avenue, New York, NY 10010.

http://www.olympusmicro.com/primer/techniques/fluorescence/tirf/tirfhome.htmlhttp://www.olympusmicro.com/primer/techniques/fluorescence/tirf/tirfhome.html

http://valelab.ucsf.edu/http://valelab.ucsf.edu/