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Using FRAP to Study the Kinetochore-Microtubule Interaction

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).

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Using FRAP to Study the Kinetochore-Microtubule Interaction

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  1. Using FRAP to Study the Kinetochore-Microtubule Interaction C.G. Pearson, P.S. Maddox, E.D. Salmon and K. Bloom

  2. Yeast Mitotic Spindle Structure Kubai, 1978

  3. Chromosome Microtubule Attachment Free Tubulin Microtubule

  4. 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.

  5. FRAP Microscope Metamorph Acquisition System Hamamatsu Orca ER CCD Camera Argon Laser Nikon E300 Inverted Microscope For more detail: www.bio.unc.edu/faculty/bloom/lab www.bio.unc.edu/faculty/salmon/lab

  6. Timelapse of Fluorescence Recovery After Photobleaching

  7. Using FRAP to measure spindle microtubule dynamics. % Recovery of Bleached ½ Spindle = F(final) – F(t=0) Rate of turnover = Half-time to recovery (t1/2) Maddox et al., 2000

  8. What are the dynamic properties of microtubules in the Metaphase spindle?

  9. 66 % of metaphase spindle microtubules turnover with a half-life of 53 sec. 33% are much more stable.

  10. 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.

  11. Therefore we conclude that the kinetochore microtubules are dynamic while the interpolar microtubules are stable.

  12. What are the dynamic properties of the microtubules in the Anaphase spindle?

  13. 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.

  14. Normal microtubule dynamics in ctf13 mutants.

  15. stu2 mutants have decreased microtubule turnover. Also see Kosco et al, 2001

  16. Microtubule turnover in kinetochore protein mutants. • FRAP allowed us to discern differences in mutants that show similar morphological phenotypes.

  17. 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

  18. C. M. Waterman-Storer and E. D. Salmon. (1998). How microtubules get fluorescent speckles. Biophys Journal 75, 2059-2069.

  19. ~5% ~0.5%

  20. Sites of Microtubule Assembly/Disassembly

  21. Microtubule Translocation

  22. How Do Microtubules get Fluorescent Speckles?

  23. Assembly dynamics of astral microtubules occur at the plus-end

  24. Assay for Dynamic Attachment

  25. Assay for Dynamic Attachment

  26. Assay for Dynamic Attachment

  27. Assay for Dynamic Attachment

  28. Microtubules grow and shorten while attached to the shmoo tip

  29. Which microtubule end, plus or minus contributes to the dynamics and motility?

  30. Shmoo tip microtubules add and subtract subunits from their plus ends and not their minus ends.

  31. 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.

  32. Kerry Bloom Ted Salmon Paul Maddox Bloom Lab Elaine Yeh Dale Beach Mythreye Karthikeyan Leanna Topper Ted Zarzar Jennifer Stemple David Bouck Goldstein Lab Salmon Lab Julie Canman Bonnie Howell Katie Shannon Jennifer Deluca Daniela Cimini Lisa Cameron Jeff Molk Ben Moree Thank You Collaborators Tim Huffaker Karena Kosco

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