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Using FRAP to Study the Kinetochore-Microtubule
Interaction
C.G. Pearson, P.S. Maddox, E.D. Salmon and K. Bloom
Yeast Mitotic Spindle Structure
Kubai, 1978
Chromosome Microtubule Attachment
Free Tubulin
Microtubule
Fluorescence Recovery After Photobleaching (FRAP)
• Fluorescence based assay to determine protein dynamics (localized and/or diffusive).
• Photobleaching of GFP tagged proteins without destruction of protein function.
• Determine tubulin turnover within the microtubule by measuring rate and extent of fluorescence recovery.
FRAP Microscope
Hamamatsu Orca ER CCD Camera
Nikon E300 Inverted Microscope
MetamorphAcquisition
SystemArgon Laser
For more detail:www.bio.unc.edu/faculty/bloom/labwww.bio.unc.edu/faculty/salmon/lab
Timelapse of Fluorescence Recovery After Photobleaching
Using FRAP to measure spindle microtubule dynamics.
% Recovery ofBleached ½ Spindle =F(final) – F(t=0)
Rate of turnover = Half-time to recovery (t1/2)
Maddox et al., 2000
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?
Microtubule turnover in kinetochore protein mutants.
CTF13 and STU2 - Essential, mutants delay in metaphase by the spindle checkpoint, chromosome loss mutant, localize to CEN.
CTF13 (ctf13-30) - Core kinetochore component
STU2 (stu2td) –Microtubule binding protein.
Normal microtubule dynamics in ctf13 mutants.
stu2 mutants have decreased microtubule turnover.
Also see Kosco et al, 2001
Microtubule turnover in kinetochore protein mutants.
• FRAP allowed us to discern differences in mutants that show similar morphological phenotypes.
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?
Assembly dynamics of astral microtubules occur at the plus-end
Assay for Dynamic Attachment
Assay for Dynamic Attachment
Assay for Dynamic Attachment
Assay for Dynamic Attachment
Microtubules grow and shorten while attached to the shmoo tip
Which microtubule end, plus or minus contributes to the dynamics
and motility?
Shmoo tip microtubules
add and subtract
subunits from their plus ends and not their minus ends.
Analysis of Protein Dynamics Using FRAP.
• Dynamics of localized and diffuse
proteins in live cells.• Spindle MT FRAP to study
microtubule dynamics and their regulation by chromosomes.
Thank YouThank YouKerry Bloom
Ted Salmon
Paul Maddox
Bloom LabElaine YehDale BeachMythreye KarthikeyanLeanna TopperTed ZarzarJennifer StempleDavid Bouck
Goldstein Lab
Salmon Lab
Julie Canman
Bonnie Howell
Katie Shannon
Jennifer Deluca
Daniela Cimini
Lisa Cameron
Jeff Molk
Ben Moree
Collaborators
Tim Huffaker
Karena Kosco