title: Microtubule assembly and kinetochore directional instability in vertebrate monopolar spindles: implications for the mechanism of chromosome congression.
abstract: We have proposed previously a kinetochore motor-polar ejection model for chromosome congression to the metaphase plate where forces generated at the kinetochore are antagonized by away-from-the pole forces generated within each half-spindle on the chromosome arms. This model was based in large part on observations of the behavior of chromosomes on monopolar spindles. In these cells chromosomes typically become attached to the pole by only one kinetochore fiber. These mono-oriented chromosomes move to positions away from the pole even though they are pulled poleward at their kinetochores. Their arms are also ejected away from the pole when severed from the centromere. Here we have characterized further the properties of monopolar spindles in newt lung epithelial cells to determine the similarities between monopolar and bipolar spindles. We found no significant differences between monopolar and bipolar spindles over the parameters examined, which included: microtubule dynamics as measured by fluorescence redistribution after photobleaching; the ability of polar microtubule arrays to push chromosome arms away from the pole; the dependence of chromosome position relative to the pole on microtubule assembly; the number of kinetochore microtubules per kinetochore; and the directional instability of kinetochore motion during chromosome oscillations poleward and away-from-the-pole. As in bipolar spindles, kinetochore directional instability is characterized by abrupt switching between constant velocity phases of poleward and away-from-the-pole motion. From these data we conclude that the mechanism(s) responsible for chromosome positioning in monopolar spindles are fundamentally the same as those in bipolar spindles; only the geometry of the two spindle forms and the interplay between sister kinetochore directional instabilities are different. We also found no correlation in the kinetochore-to-pole distance with kinetochore microtubule number in monopolar spindles, but a strong qualitative correlation with microtubule density. This finding indicates that oscillations of mono-oriented chromosomes in both monopolar and bipolar spindles occur because chromosomes persist in poleward motion until they reach a density of polar microtubules sufficiently high to promote switching to away-from-the-pole motion. As the kinetochore and chromosome arms move away-from-the-pole, microtubule density decreases and the kinetochore switches to poleward motion, pulling the chromosome arms back into regions of higher microtubule density. The mechanism regulating kinetochore switching between poleward and away-from-the-pole motion is poorly understood, but may depend on tension at the kinetochore generated by pushing forces on the chromosome arms produced by the polar microtubule arrays.
authors.affiliation: Department of Molecular Biology, Lehigh University, Bethlehem, PA 18015.
authors.forename: L
authors.initials: L
authors.lastname: Cassimeris
authors.forename: C L
authors.initials: CL
authors.lastname: Rieder
authors.forename: E D
authors.initials: ED
authors.lastname: Salmon
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D002875:Chromosomes / Q000648:ultrastructure*; D004847:Epithelial Cells; D004848:Epithelium / Q000648:ultrastructure; D007700:Kinetics; D008168:Lung / Q000166:cytology* / Q000648:ultrastructure*; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000648:ultrastructure*; D012455:Salamandridae; D008941:Spindle Apparatus / Q000648:ultrastructure*; D013997:Time Factors; D014714:Vertebrates