Evidence that kinetochore microtubules in crane-fly spermatocytes disassemble during anaphase primarily at the poleward end.

Abstract

Anaphase chromosome motion involves the disassembly of kinetochore microtubules. We wished to determine the site of kinetochore microtubule disassembly during anaphase in crane-fly spermatocytes. In crane-fly spermatocyte spindles, monoclonal antibody 6-11B-1 to acetylated alpha-tubulin labels kinetochore microtubules almost exclusively, with an area immediately adjacent to the kinetochore being weakly or not labelled. This 'gap' in acetylation at the kinetochore serves as a natural marker of kinetochore microtubules in the kinetochore fibre. We measured the length of the gap on kinetochore fibres in metaphase and anaphase in order to deduce the fate of the gap during anaphase; we used this information to determine where kinetochore microtubules disassemble in anaphase. Gap lengths were measured from confocal microscope images of fixed spermatocytes dual labelled with 6-11B-1 to acetylated alpha-tubulin and YL1/2 to tyrosinated alpha-tubulin, the latter being used to determine the positions of kinetochores. In metaphase the average gap length was 1.7 microns. In anaphase, the gap appeared to decrease in length abruptly by about 0.4 microns, after which it decreased in length by about 0.2 microns for every 1 microns that the chromosome moved poleward. PacMan models of chromosome movement predict that this 'gap' in staining should disappear in anaphase at a rate equal to that of chromosome movement. Thus, our results do not support theories of chromosome motion that require disassembly solely at the kinetochore; rather, in crane-fly spermatocytes kinetochore microtubule disassembly in anaphase seems to take place primarily at the poles.