Dynamics of Centromeres during Metaphase–Anaphase Transition in Fission Yeast: Dis1 Is Implicated in Force Balance in Metaphase Bipolar Spindle

Abstract

In higher eukaryotic cells, the spindle forms along with chromosome condensation in mitotic prophase. In metaphase, chromosomes are aligned on the spindle with sister kinetochores facing toward the opposite poles. In anaphase A, sister chromatids separate from each other without spindle extension, whereas spindle elongation takes place during anaphase B. We have critically examined whether such mitotic stages also occur in a lower eukaryote, Schizosaccharomyces pombe. Using the green fluorescent protein tagging technique, early mitotic to late anaphase events were observed in living fission yeast cells. S. pombe has three phases in spindle dynamics, spindle formation (phase 1), constant spindle length (phase 2), and spindle extension (phase 3). Sister centromere separation (anaphase A) rapidly occurred at the end of phase 2. The centromere showed dynamic movements throughout phase 2 as it moved back and forth and was transiently split in two before its separation, suggesting that the centromere was positioned in a bioriented manner toward the poles at metaphase. Microtubule-associating Dis1 was required for the occurrence of constant spindle length and centromere movement in phase 2. Normal transition from phase 2 to 3 needed DNA topoisomerase II and Cut1 but not Cut14. The duration of each phase was highly dependent on temperature.

pkl1+andklp2+: Two Kinesins of the Kar3 Subfamily in Fission Yeast Perform Different Functions in Both Mitosis and Meiosis

“…These include the kinetochore proteins Mis12p and Mis6p, because loss-of-function alleles of the corresponding genes show a large increase in spindle length (Goshima et al, 1999). The establishment of a metaphase spindle requires a restraining activity from the MT-associated protein, Dis1p and the related Mtc1p; loss of function of Dis1p leads to a precocious anaphase-like elongation of the spindle (Nabeshima et al, 1998;Nakaseko et al, 2001). In addition, Pkl1p interacts with ␥-tubulin, a protein found at the spindle pole body that is essential for MT nucleation (Paluh et al, 2000).…”

“…We have selected the fission yeast Schizosaccharomyces pombe for our research on chromosome motion because of its suitability for molecular and genetic work (Moreno et al, 1991) and its useful mitotic cytology (Hagan and Hyams, 1988;Ding et al, 1993Ding et al, , 1997Hagan, 1998;Nabeshima et al, 1998). It carries only three comparatively large chromosomes, each of which contains a centromere that includes many tens of kilobases (kb) of DNA (Fishel et al, 1988;Chikashige et al, 1989).…”

Untimely expression of gametogenic genes in vegetative cells causes uniparental disomy

“…Deletion strains for the genes summarized in Supplementary Table 1 were obtained from the Bioneer haploid deletion library version 4.0. The following arrays, gene fusions and mutant alleles were previously described: lacO at pericentromeric regions lys1 and cen2-D107 and lacI-GFP at his7 loci 32 , tetO at cen2 and tetR-Tomato adjacent to zfs1 (Z locus) 27 , Padh1-rec8 + -3HA and Padh1-rec8 + -GFP 25 , mei4∆C<NAT 4 , and rec12 ∆ 33 . Deletion of mmi1 was performed in cells carrying a previously described truncated non-functional mei4 ( mei4∆C<NAT ) allele 4 to alleviate growth defects caused by derepression of mei4 in mitotic cells 15 .…”

Cell cycle control of spindle elongation

“…1B and D). [25][26][27][28] The reliance on anaphase B can be explained by the relative dimensions of the mitotic spindle and chromosomes. Both yeast models sport an extremely short metaphase spindle of less than 2 µm and kMTs of ∼400-800 nm of length.…”