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). How
microtubules get fluorescent speckles. Biophys Journal 75,
2059-2069.
~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. 1998.
Fluorescent speckle microscopy: Visualizing the movement, assembly and
turnover of macromolecular assemblies in living cells. Current Biology.
8:1227-1230.
~ 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. 2000. 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, 295-301
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.
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.
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 (WatermanStorer et al., 1998 Cur. Biol. 8:1227-1230.)
• In yeast: Astral and Anaphase spindle microtubules extend
from the Spindle Pole Body and have stable minus ends
Acknowledgements:
• Ted Salmon
– Clare Waterman-Storer
– Julie Canman
– Bonnie Howell
• Kerry Bloom
–
–
–
–
–
Sid Shaw
Elaine Yeh
Dale Beach
Chad Pearson
Doug Thrower
• Tim Mitchison
– Aneil Mallavarapu
– Aaron Straight
•
•
•
•
•
•
•
Arshad Desai
Shinya Inoue
MBL
Nikon
Hamamatsu Photonics
Universal Imaging
Yokagawa