Fluorescent Speckle Microscopy and Cell Division
Paul Maddox Chad Pearson Julie Canman Kerry Bloom E. D. Salmon 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 C. M. Waterman-Storer and E. D. Salmon. (1998)
C. M. Waterman-Storer and E. D. Salmon. (1998).How microtubules get
fluorescent speckles.Biophys Journal 75, ~5% ~0.5% Sites of
Microtubule Assembly/Disassembly Microtubule Translocation How Do
Microtubules get Fluorescent Speckles? 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))0.5=2.15 Note:Microscope
Magnification = (3*Pixel-width)/resolution = (3*6.7 mm) / 0.27 =
74.4 How Do Microtubules get Fluorescent Speckles? FSM Posters and
Talks ASCB 99
Gaudenz Danuser:Sat., 2:20 pm, rm 38, Analysis of Cytoskeletal
(actin) Dynamics Bill Bement and Clare Waterman-Storer:Sun., 5:25
pm, rm 31, Actin and Microtubule Interactions Arshad Desai:Mon.,
4:45 pm, rm 20, Microtubule Motility in Xenopus and Drosophila
Spindles 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
Examples of FSM of Microtubule Poleward Flux in Spindles with
Self-Organized Poles
Xenopus Extract Spindles Ptk1 cells ~ 5 % labeled Tubulin ~ 0.05 %
labeled Tubulin
Waterman-Storer, C. M., A. Desai, J. C. Bulinski and E. D. Salmon
Fluorescent speckle microscopy: Visualizing the movement, assembly
and turnover of macromolecular assemblies in living cells. Current
Biology. 8: ~ 0.3 % labeled Tubulin 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 The
polarity and dynamics of microtubule assembly in the budding yeast
S. cerevisiae.Nature Cell Biology, 2:36-41 Astral and Nuclear
Microtubule Organization During the Yeast Cell Cycle 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? 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,
Imaging System(s) Where do assembly dynamics of astral microtubules
occur? Astral microtubules are dynamic at their plus ends and not
their minus ends. What are the dynamic properties of microtubules
in the Metaphase spindle? 66 % of metaphase spindle microtubules
turnover with a half-life of 53 sec.33% are much more stable. Winey
et al. (1995) Journal of Cell Biology. 129(6):1601-1615.
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):
Therefore we conclude that the kinetochore microtubules are dynamic
while the interpolar microtubules are stable. What are the dynamic
properties of the microtubules in the Anaphase spindle? Anaphase
microtubules are stable and grow from their plus ends. How does the
mitotic spindle breakdown in telophase? Spindle disassembly occurs
by plus end depolymerization. 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. 8: ) In yeast:Astral and Anaphase spindle microtubules
extend from the Spindle Pole Body and have stable minus ends
Acknowledgements: Tim Mitchison Ted Salmon Arshad Desai Shinya
Inoue
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
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