Functional states of kinetochores revealed by laser microsurgery and fluorescent speckle microscopy
Movie 2: Control experiment. Time-lapse alternating frame FSM/DIC movie after a spot ablation that drilled a hole in a chromosome arm but did not detach the arm from its kinetochore. (980.8Kb)
Movie 3: Time-lapse alternating frame FSM/DIC movie after generating a K-fragment at anaphase onset. (1.127Mb)
LaFountain, James R.
Cohan, Christopher S.
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The impact of mechanical forces on kinetochore motility was investigated using laser microsurgery to detach kinetochores with associated chromatin (K fragment) from meiotic chromosomes in spermatocytes from the crane fly Nephrotoma suturalis. In spermatocytes, elastic tethers connect telomeres of homologues during anaphase A of meiosis I, thus preventing complete disjunction until mid- to late anaphase A. K fragments liberated from tethered arms moved at twice the normal velocity toward their connected poles. To assess functional states of detached and control kinetochores, we loaded cells with fluorescently labeled tubulin for fluorescent speckle microscopy on kinetochore microtubules. Control kinetochores added fluorescent speckles at the kinetochore during anaphase A, whereas kinetochores of K fragments generally did not. In cases in which speckles reappeared in K-fragment K fibers, speckles and K fragments moved poleward at similar velocities. Thus detached kinetochores convert from their normal polymerization (reverse pac-man) state to a different state, in which polymerization is not evident. We suggest that the converted state is “park,” in which kinetochores are anchored to plus ends of kinetochore microtubules that shorten exclusively at their polar ends.
© The Author(s), 2011. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Molecular Biology of the Cell 22 (2011): 4801-4808, doi:10.1091/mbc.E11-06-0494.
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