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