Fluorescent Speckle Microscopy and Cell Division

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Fluorescent Speckle Microscopy and Cell Division

• Paul Maddox
• Chad Pearson
• Julie Canman
• Kerry Bloom
• E. D. Salmon

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What is Fluorescent Speckle Microscopy (FSM)?

• Fluorescent discontinuities, speckles in
  biological polymers (e.g. microtubules, actin
  filaments)
• Caused by stochastic incorporation of
  fluorescently tagged subunits into the polymer
• Allows visualization of assembly dynamics and
  motility of the polymer

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C. M. Waterman-Storer and E. D. Salmon. (1998).
How microtubules get fluorescent speckles.
Biophys Journal 75, 2059-2069.
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5
0.5
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Sites of Microtubule Assembly/Disassembly
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Microtubule Translocation
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How Do Microtubules get Fluorescent Speckles?
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How Do Microtubules get Fluorescent Speckles?

• For resolution of 1.4 NA obj, r 0.27 mm (N
  440 dimers)
• Fraction of labeled tubulin, f 1
• Mean of fluorophores M fN 4.4
• Standard deviation, SD (fN(1-f))0.52.15
• Note Microscope Magnification
  (3Pixel-width)/resolution
  (36.7 mm) / 0.27 74.4

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How Do Microtubules get Fluorescent Speckles?
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FSM Posters and Talks ASCB 99

• Gaudenz Danuser Sat., 220 pm, rm 38, Analysis
  of Cytoskeletal (actin) Dynamics
• Bill Bement and Clare Waterman-Storer Sun.,
  525 pm, rm 31, Actin and Microtubule
  Interactions
• Arshad Desai Mon., 445 pm, rm 20, Microtubule
  Motility in Xenopus and Drosophila Spindles

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FSM Posters and Talks ASCB 99

• Tarun Kapoor et al. Mon. Session 2.
  Investigating bipolar spindle formation and a
  kinesin inhibitor. Poster B-30.
• Chloe Bulinski et al. Wed. Session 4. Sparkling
  Speckles of GFP-Ensconsin on Microtubules Poster
  B-107
• Paul Maddox et al. Wed. Poster B-108

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Examples of FSM of Microtubule Poleward Flux in
Spindles with Self-Organized Poles

• Xenopus Extract Spindles
• Ptk1 cells

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5 labeled Tubulin
0.05 labeled Tubulin
Waterman-Storer, C. M., A. Desai, J. C. Bulinski
and E. D. Salmon. 1998. Fluorescent speckle
microscopy Visualizing the movement, assembly
and turnover of macromolecular assemblies in
living cells. Current Biology. 81227-1230.
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0.3 labeled Tubulin
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Analysis of the polarity of assembly dynamics of
astral and nuclear microtubules in the vegetative
cell cycle
P. Maddox, K. Bloom, and E.D. Salmon. 2000. The
polarity and dynamics of microtubule assembly in
the budding yeast S. cerevisiae. Nature Cell
Biology, 236-41
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Astral and Nuclear Microtubule Organization
During the Yeast Cell Cycle
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Questions

• Where do assembly dynamics of astral microtubules
  occur?
• What are the dynamic properties of microtubules
  in the Metaphase spindle? The Anaphase spindle?
• How does the central spindle break down in
  telophase?

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Why worry about minus end assembly dynamics?
Minus ends are at the SPB. Several microtubule
motor proteins (Kar3p, Kip3p, Kip2p, Dhc1p) are
localized to the SPB and have been proposed to
function there to control microtubule length and
produce force.
e.g. Huyett et al. (1998). J. Cell Science 111,
295-301
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Imaging System(s)
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Where do assembly dynamics of astral microtubules
occur?
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Astral microtubules are dynamic at their plus
ends and not their minus ends.
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What are the dynamic properties of microtubules
in the Metaphase spindle?
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66 of metaphase spindle microtubules turnover
with a half-life of 53 sec. 33 are much more
stable.
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There are 24 microtubules per half spindle. 16
(66 ) are kinetochore microtubules. While 8
(33 ) are overlapping interpolar microtubules.
Winey et al. (1995) Journal of Cell Biology.
129(6)1601-1615.
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Therefore we conclude that the kinetochore
microtubules are dynamic while the interpolar
microtubules are stable.
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What are the dynamic properties of the
microtubules in the Anaphase spindle?
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Anaphase microtubules are stable and grow from
their plus ends.
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How does the mitotic spindle breakdown in
telophase?
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Spindle disassembly occurs by plus end
depolymerization.
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Summary

• FSM is a powerful tool for studying polymer
  dynamics in living cells
• FSM allows visualization of microtubule assembly
  dynamics and motility within the mitotic spindle
• In vertebrates Poleward flux and minus end
  disassembly occurs for non-centrosomal spindle
  microtubules Centrosomal microtubule minus ends
  are stable (Waterman-Storer et al., 1998 Cur.
  Biol. 81227-1230.)
• In yeast Astral and Anaphase spindle
  microtubules extend from the Spindle Pole Body
  and have stable minus ends

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Acknowledgements

• Tim Mitchison
• Aneil Mallavarapu
• Aaron Straight
• Arshad Desai
• Shinya Inoue
• MBL
• Nikon
• Hamamatsu Photonics
• Universal Imaging
• Yokagawa

• Ted Salmon
• Clare Waterman-Storer
• Julie Canman
• Bonnie Howell
• Kerry Bloom
• Sid Shaw
• Elaine Yeh
• Dale Beach
• Chad Pearson
• Doug Thrower