Live analysis of lagging chromosomes during anaphase and their effect an spindle elongation rate in fission yeast

The fission yeast Schizosaccharomyces pombe is widely used as a model syste m for studies of the cell cycle and chromosome biology To enhance these stu dies we have fused GFP to the chromodomain protein Swi6p, thus allowing nuc lear and chromosome behaviour to be followed in living cells using time-lap se fluorescence microscopy. Like endogenous Swi6p, GFP-Swi6p localises to t he nucleus and is concentrated at the heterochromatic centromeres and telom eres, The nucleus is highly dynamic during interphase: the clustered centro meres, in particular, are highly mobile, By expressing GFP-alpha2-tubulin a nd GFP-Swi6p in the same cells we observe that the clustered centromeres mo ve in concert with the cytoplasmic microtubules, which is likely to reflect their association with the spindle pole body, Drug treatment indicates tha t this movement is dependent on intact cytoplasmic microtubules, We have al so used GFP-Swi6p to investigate the properties of lagging chromosomes obse rved in mutants with defects in chromosome segregation. Lagging chromosomes display a variety of behaviours on anaphase spindles, most surprisingly, c hromosomes appear to initiate microtubule interactions and move to the pole s late in anaphase B, Interestingly, in cells displaying lagging chromosome s, the rate of spindle elongation is slowed by a factor of two, This sugges ts that cells are able to sense the presence of a lagging chromosome and sl ow anaphase B in order to allow it extra time to reach the pole, However, t his mechanism is not dependent on the spindle checkpoint proteins Bub1p or Dma1p, raising the possibility that a novel checkpoint mechanism operates t o retard spindle elongation if lagging chromosomes are detected, An alterna tive model is also discussed in which single defective kinetochores on lagg ing chromatids are able to interact simultaneously with microtubules emanat ing from both poles and affect spindle dynamics by counteracting the spindl e elongation force.