Fluorescence Recovery After Photobleaching (FRAP) to Study the
Kinetochore Microtubule Interaction.
Chad G. Pearson,
Paul S. Maddox, E.D. Salmon and Kerry Bloom
University of North
Carolina, Biology Department, 623 Fordham Hall CB#3280, Chapel Hill,
NC 27599-3280
To understand the kinetochore microtubule interaction, essential for
proper chromosome segregation during mitosis, it is necessary to
observe the attachment and dynamic movement of these structures within
living cells. Budding yeast contain a single kinetochore microtubule
for each chromosome. However, individual microtubule dynamics cannot
be observed within the mitotic spindle because of the lack of spatial
resolution of the light microscope. We have used fluorescence recovery
after photobleaching (FRAP) to measure the turnover of GFP labeled
tubulin (GFP-Tub1) within the mitotic spindle. Using a focused laser,
GFP-Tub1 in one half of the mitotic spindle is photobleached while
protein function is not disrupted. Microtubule turnover is facilitated
by the growth and shortening of microtubule plus ends to exchange
fluorescently labeled tubulin in the nucleoplasm and opposing half
spindle with photobleached or unlabeled tubulin in the microtubule
lattice. The rate and extent of this turnover provides a quantitative
assay for measuring kinetochore microtubule dynamics. Cells depleted
of the microtubule binding and kinetochore associated protein, Stu2p,
have severely decreased microtubule turnover (Kosco et al,
2001). Chromosome oscillations are dependent upon turnover of spindle
microtubules and are also decreased in cells lacking Stu2p. Stu2p is
therefore a key coupler between microtubule and chromosome
dynamics. However, loss of chromosome attachment, generated by
conditional alleles of the core kinetochore component ndc10-2, does
not alter spindle microtubule fluorescence recovery after
photobleaching. These results indicate that alterations in kinetochore
components differentially regulate microtubule dynamics. The FRAP
analysis is critical in distinguishing the role of essential
kinetochore components in a dynamic process.
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