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Paul Maddox^1,2, Aaron Straight^1,3, Peg Coughlin^1,3, Timothy J. Mitchison^1,3 and Edward D. Salmon^1,2

¹ Cell Division Group, Marine Biological Laboratory, Woods Hole, MA 02543
² Department of Biology, University of North Carolina, Chapel Hill, Chapel Hill, NC 27599
³ Department of Cell Biology, Harvard Medical School, Boston, MA 02115

Address correspondence to E.D. Salmon, Dept. of Biology, CB3280, University of North Carolina, Chapel Hill, Chapel Hill, NC 27599. Tel.: (919) 962-2354. Fax: (919) 962-1625. email: tsalmon{at}email.unc.edu

Microtubule plus ends dynamically attach to kinetochores on mitotic chromosomes. We directly imaged this dynamic interface using high resolution fluorescent speckle microscopy and direct labeling of kinetochores in Xenopus extract spindles. During metaphase, kinetochores were stationary and under tension while plus end polymerization and poleward microtubule flux (flux) occurred at velocities varying from 1.5–2.5 µm/min. Because kinetochore microtubules polymerize at metaphase kinetochores, the primary source of kinetochore tension must be the spindle forces that produce flux and not a kinetochore-based mechanism. We infer that the kinetochore resists translocation of kinetochore microtubules through their attachment sites, and that the polymerization state of the kinetochore acts a "slip-clutch" mechanism that prevents detachment at high tension. At anaphase onset, kinetochores switched to depolymerization of microtubule plus ends, resulting in chromosome-to-pole rates transiently greater than flux. Kinetochores switched from persistent depolymerization to persistent polymerization and back again during anaphase, bistability exhibited by kinetochores in vertebrate tissue cells. These results provide the most complete description of spindle microtubule poleward flux to date, with important implications for the microtubule–kinetochore interface and for how flux regulates kinetochore function.

Key Words: kinetochore; fluorescent speckle microscopy; mitosis; centromere; anaphase

Abbreviation used in this paper: FSM, fluorescent speckle microscopy.

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