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

Yeast Mitotic Spindle Structure Describe questions more (biological) EM indicates most MTs are kinetochore microtubules. Shorter class is the kMT and these are consistent with kinetochore localization. Problem is that we can’t see individual MTs to get information of the dynamics. Therefore we must look at the population using techniques to measure turnover. Point out that you have an unbleached control right next to it. Kubai, 1978

Chromosome Microtubule Attachment Free Tubulin Transition figure for Spindle structure and kt motility to microtubule dynamics. Background of why we need to understand the dynamic properties of MTs within the spindle. The dynamic nature of chromosomes are thought to be dependent upon the dynamics of microtubules. Therefore the identification of molecules involved in this process is essential to understanding how chromosome dynamics occurs. Overview of structure (separated cens, stable - ends at poles, unstable + ends at chromosome, dynamic attachment). Plus end dynamics drive oscillations. We hope to understand the regulation of the plus end dynamics in force generation. Put in Schematic of Chromosome Attachment. 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. Key to point out that many protein interactions have been defined via biochemical and 2 hybrid methods and we would like to determine what the temporal and dynamic nature of these is (unable to be done with the current techniques). The dynamics of proteins can’t be measured however FRAP is an excellent tool for looking at these protein movements. Laser targeted photobleaching and I will describe how we are not disrupting the molecule. Example.

Slide showing fluorescence vs photobleaching Slide showing fluorescence vs photobleaching. (low vs high intensity light).

FRAP Microscope Metamorph Acquisition System Hamamatsu Orca ER CCD Camera Argon Laser Nikon inverted stage stand with a 100X1.4 PlanAPO DIC objective. Motor controlled X, Y and Z stage. Joystick control. Metamorph Image Acquisition for acquisition and analysis. Orca ER CCD camera. 2 W Argon laser greatly attenuated light that is focused on the back aperature of the objective and it shuttered. Creating an laser spot of 488 nm light that has a ½ maximal distribution of ~0.8 um. Stage heater is used for conditional alleles. Nikon E300 Inverted Microscope For more detail: www.bio.unc.edu/faculty/bloom/lab www.bio.unc.edu/faculty/salmon/lab

Orientation to next slides: Time in min Fluorescence Intensity Graph. Spindle

Laser Targeting. 35 msec exposure of 488 nm light Laser Targeting. 35 msec exposure of 488 nm light. Enough light to photobleach the GFP molecule however not enough to damage the tubulin protein. Size of bleach

We are not damaging the tubulin protein We are not damaging the tubulin protein. We are just photobleaching the fluorophore just as we have all seen with normal fluorescence imaging. How do we know that we aren’t hurting the fluorophore: Cells progress normally through mitosis and into the next cell cycle. Rate of anaphase is WT. Make microcolonies on the slab. Bleached fluorophore becomes incorporated into the opposing unbleached ½ spindle indicating that the protein is functional to dissociate and reassociate with the lattice. The rate of loss at the unbleached and gain at the bleached is roughly the same indicating that the is no difference in the dynamics of the bleached vs unbleached ½ spindle.

Timelapse of Fluorescence Recovery After Photobleaching

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) Emphasize the “no destruction”…incorporation of bleached subunits and microcolony… Maddox et al., 2000

May need to simplify…. Remove all of the small text. May want one more

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):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. Kinetochore disruption does not screw up spindle MT dynamics. We have tried multiple alleles and different components of the CBF3 complex and found no change in t1/2.

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. Work on.

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, 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 You Kerry Bloom Ted Salmon Paul Maddox Collaborators Salmon Lab Julie Canman Bonnie Howell Katie Shannon Jennifer Deluca Daniela Cimini Lisa Cameron Jeff Molk Ben Moree Bloom Lab Elaine Yeh Dale Beach Mythreye Karthikeyan Leanna Topper Ted Zarzar Jennifer Stemple David Bouck Goldstein Lab Kerry Bloom Ted Salmon Paul Maddox Collaborators Tim Huffaker Karena Kosco Mention the Tatchell Lab poster using FRAP to look at septin function.