Fast Vesicle Transport in PC12 Neurites: Velocities and Forces

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Fast Vesicle Transport in PC12 Neurites
Velocities and Forces

• D.B. Hill, M.J. Plaza, K. Bonin, G. Holzwarth
• Department of Physics
• Wake Forest University

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Why Do We Care?

• Much is still unknown about the mechanisms of
  kinesin, especially in-vivo
• A major part of how cells work!
• Kinesin was only theorized but not fully
  discovered until mid-1980s
• Drug delivery to different areas within the cell
• Understanding transport along nerves to better
  understand human neurodegenerative diseases where
  this transport is deficient (Alzheimers disease,
  retinitis pigmentosa)
• http//news-service.stanford.edu/news/2001/july11
  /kinesin-711.html
• Elucidate the mechanical task motor proteins must
  face

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Objectives of this Paper

• To measure the velocities, forces and work
  required to move vesicles in-vivo
• To compare these to the limits established for
  kinesin in solution.
• Specifically, looking at fast transport of
  vesicles by kinesin along microtubules
• To measure the viscoelastic drag forces and
  compare these to buffer, w/ very little drag
  force
• To note patterns in the movement of vesicles that
  may indicate discrete numbers of kinesin proteins
  carrying the vesicle load
• Are the velocities of the vesicles dependent on
  the number of motors carrying them?

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What is Kinesin?

• A single motor protein that moves vesicles,
  organelles, etc. from place to place inside cells
• Converts ATP to mechanical work for each step
• Walks in a foot-over-foot fashion
• Moves along cytoskeletal tracks - microtubules
• From to direction
• (Dynein moves from to )
• Is more delicate in structure than Dynein
• In nerve cells, moves biochemical information,
  food, etc. from cell body to nerve endings
• Is only in eukaryotic cells . Guess why!

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The Ministry of Silly Walks

• How kinesin walks
• http//mc11.mcri.ac.uk/wrongtrousers.html

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http//www-u.life.uiuc.edu/j-roland/artwork.html
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Anterograde vs. Retrograde

• Anterograde Away from the cell body
• Retrograde Towards the cell body
• Axons Take information away from cell body
• Dendrites Bring information to the cell body
• 3 kinds of cellular transport
• Fast anterograde axonal transport
• Slow axoplasmic flow
• Fast retrograde axonal transport.
• In mammal neurons fast 70-300 mm/day

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Key Concerns

• Most kinesin studies were done in-vitro
• Glass slide covered with fixed kinesin proteins,
  moving microtubules along
• Immersed in a watery buffer solution, with little
  drag on the movement
• We want to know how kinesin works in-vivo
• Many more factors to consider here
• Fixed microtubules, kinesin moves
• Viscosity of cytoplasm (differs by a factor of
  10,000-100,000 from buffer!)
• Viscoelastic drag
• Physical obstacles (other vesicles, proteins,
  etc)
• Load on kinesin 3 orders of magnitude greater
  in-vivo

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http//www-u.life.uiuc.edu/j-roland/artwork.html
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The In-Vitro Way

• Microtubule gliding assays
• Kinesin-covered glass coverslip, moving
  microtubules in buffer
• http//www.proweb.org/kinesin/axonemeMTs.html

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The In-Vivo Way (Materials and Methods)

• PC-12 cells cultured, adhered to coverslip
• Placed in a chamber, maintained at 35oC
• Chamber on stage of Nikon E600FN microscope w/
  60x water immersion lens
• DIC (Differential Interference Contrast)
  microscopy
• CCD camera to take movies
• Contrast enhancement and background subtraction
• Series of 128 images saved as one movie
• Vesicle tracking (anterograde and retrograde) and
  determination of vesicle velocity through pattern
  recognition
• Analysis of observed motion
• Viscoelastic properties determined by brownian
  motion of vesicles in cytoplasm near growth cone

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Why PC-12 Cells?

• Rattus Norvegicus adrenal gland cancerous
  immortal cell line
• Very hardy in cell culture
• Can differentiate easily just add nerve growth
  factor!
• Form neurites, which are like axons in neurons in
  that they are long, straight extensions of the
  cell
• Adhere easily to growth plate, easy to see
  transport along long neurites (long stretches in
  focus)

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Background Findings

• In-vitro, with increasing velocity, the retarding
  force on microtubules moved by one kinesin is
  small
• 2 and 3 kinesins moving a microtubule is
  extrapolated to also face diminishing forces with
  increasing velocities
• In vitro, no change in velocity with the number
  of motors expected (forces are very small)
• Using Stokes Law, the drag force experienced by
  a vesicle in vivo goes up rapidly with increasing
  velocity

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Stokes Law

• An expression for the drag force on spherical
  objects moving through a viscous fluid
• F6?RnV
• F is the drag force
• R is the radius of the sphere
• n is the viscosity
• V is the velocity through a continuous fluid.

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Vesicle Movement and Tracking

• Jerky motion saltatory motion
• http//www.wfu.edu/physics/cellmotors/g512.mpg
• Pattern recognition program through
• P pattern
• I subsequent images
• N number of pixels in the pattern

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Vesicle Radius Determination

• Latex beads of known size as a control
• Seen through DIC image
• Then measured diameter through dark/light
  (minimum/maximum) across a vesicle
• Compared magnitude reading on line scan to known
  magnitudes of latex beads using same method
• Vesicle radii around .35 - .40 micrometers

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Results

• Bottom figure with background subtraction and
  contrast enhancement methods
• Movement of vesicles watched in real-time
• Trackable vesicles imaged, recorded, tracked,
  and velocities determined
• Distance vesicle travelled found by sum of
  incremental distances between frames by

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Results Continued

• Total data set
• 57 vesicles
• 9 different PC-12 cells
• 50-128 frames (6-15 seconds of vesicle motion)
• Plotting d/t showed velocities at constant speed
  for 0.5-2 s, and then an abrupt shift to
  different constant speed

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Analysis Model-Free

• Significance of velocity fourier transformed
  with distance (r) and time before and after a
  point
• Same FT velocities, randomized order, plotted
  alongside found vs.
• Below 8 rad/s 0.8 s, vs persist.
• Additionally, velocity data fit to a Lorentzian
  function where ? are the velocities, ?0 is at a
  maximum when ?0 ?, and ? is the full width at
  half the maximum value in the plot of velocities.
• Fit was not good here.
• Best fit occurred when ? 11.3 /s

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Constant Velocity Segments

• Note segments of constant velocity
• Line-segment fit of data, with deviation of data
  from the line as Xr2 (reduced mean-squared error)
• Scanned 2-12 segment fits, lowest Xr2 determined
  best of segments for set
• Inset shows Xr2 values for all 2-12 segments.
  Lowest Xr2 value is the optimal fit.
• Here, 7 segments is best

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Histogram of Scaled Velocities

• Top (experimental velocity/minimum sustained
  speed) for each vesicle
• When single-vesicle data gt randomized data, the
  scaled velocity is significant
• Note discrete significant peaks at 2, 3, and 4 (
  of kinesins)
• Bottom Z is a measure of deviation for each of
  the bars shown in top graph
• Values greater that 2 or less than 2 are
  considered significant v/v0 values.

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Brownian Motion

• Drag force using Stokes Law, needs
  viscoelasticity of cytoplasm
• Viscosity found for vesicles in growth cones near
  neurites
• Saw no brownian motion in neurites
• Inset found viscosities, ?, vs. time

Displacement ?r squared vs. time interval between
observations ?
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Final Results

• 4 anterograde vesicles shown, with their
  respective drag forces calculated with Stokes
  Law
• 3 segments of constant velocity shown for each
  vesicle
• Middle segment (F2) has lowest drag force for all
  4 vesicles
• Proves that in cell (in-vivo) velocity and drag
  force are directly proportional.

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Conclusions

• Vesicles move along microtubules in 1-2 s
  step-like constant-velocity segments
• A constant baseline velocity is usually
  maintained
• Possible explanations
• of motors carrying vesicle changes discretely
  from 1, 2, 3, 4 kinesins at one time
• of microtubules the kinesins travel on change
  and vesicle is being transported (not favored b/c
  vesicles dont change speeds at exactly the same
  place So no microtubule railroad switchyards
  detected)
• Viscosity different in different parts of the
  neurite (not favored for same (not favored b/c
  vesicles dont change speeds at exactly the same
  place)
• Authors favor first argument

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Arguments for Discrete Numbers of Kinesins During
Vesicle Transport

• Anterograde motion in PC12 neurites governed by
  kinesin, as in neural axons, according to
  literature (so weve got the right protein)
• Electron micrographs show transported
  mitochondria with 1-4 kinesins carrying them. (So
  were not terribly off on the of segments)
• Other literature also show step-like velocity
  changes in transport along microtubules
• Found in gliding assay that velocities decrease
  w/ increasing load when low of kinesins, but
  not high (So the number of motors does affect
  velocity)

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My Thoughts (agreements/disagreements)

• Although viscosity may not change markedly at one
  particular place, what about a dynamic flux of
  differing viscosities, that flows randomly
  throughout the cell, affecting velocity?
• What about the possibility that ATP hydrolysis of
  each kinesin step is not at a constant rate
  in-vivo?

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Future Work
(http//faculty.washington.edu/chudler/cells.html)

• Chick embryo neurons
• Same patterns as PC-12?
• Changes over more limited lifetime?

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Acknowledgements

• Dr. Holzwarth
• Dr. Bonin
• Dr. Macosko
• Dr. Carol Milligan
• Dr. Salsbury for the feeling of relief!