During metaphase, sister kinetochores are attached to opposite spindle poles via dynamic microtubule linkages. A balance of forces (microtubule dynamics pulling the sister chromatids apart and cohesive tension holding the arms of the sisters together) positions sister kinetochores on either side of the spindle equator.15,16
Moreover, yeast kinetochores form two clusters, each containing one sister kinetochore from each pair of replicated chromatids.15
Here, we find that Kip3 plays an important role in kinetochore clustering. Deletion of KIP3
slightly decreases the length of kinetochore microtubules while slightly increasing the average distance between kinetochores (as expected, since spindle length is not altered). However, the largest effects of KIP3
deletion are not in these average distances, but in distance variability. In the absence of Kip3, the distance between sister kinetochores is more variable and the variability in kinetochore microtubule length is also increased. Given that spindle length is not increased in the mutant, the changes we detect in interkinetochore and polekinetochore distances are not an indirect consequence of spindle elongation. Instead, as kinetochores are attached to the ends of dynamic microtubules, we conclude that Kip3 is required for tight control of kinetochore microtubule lengths. This is consistent with a published results that Kip3 and Kif18A, Kip3's homolog in mammalian cells, control microtubule length by increasing microtubule rescue and catastrophe frequencies.6,30
Tytell and Sorger8
showed that kip3Δ
mutant cells have prolonged DNA hyperstretching between kinetochores and more instances of lagging chromosomes, compared with wild-type cells. The authors concluded that Kip3 must synchronize anaphase chromosome movements and/or coordinate the dissolution of sister chromatid cohesion. Our results suggest a different interpretation. We observe that kinetochore positions are more varied at metaphase in the absence of Kip3. When these cells enter anaphase, the kinetochores start from different places along the spindle and reach the pole at different times, thus some will appear to lag. Moreover, sister kinetochores with longer interkinetochore distances will have more hyperstretched DNA.
None of the six yeast kinesins are individually essential, and cells can survive with only two kinesin motors: Cin8 plus Kip3 or Cin8 plus Kar3 (kinesin-14).31
Cin8 plays a critical role in spindle assembly, a role not shared with Kip3. Loss of Cin8 alone (even in the presence of the five other kinesin motors), blocks bipolar spindle formation in 60% of cells.27,32
Similarly, deletion or knock down of homologous, but essential, kinesin-5 motors in higher eukaryotes results in the formation of monopolar spindles.5,27,33,34
Although only a subset of cells is amenable to an analysis of Cin8's role in kinetochore clustering, consistent with previous results,19
we found that kinetochores are often declustered in the bipolar spindles that manage to form in cin8Δ
mutants. Although both Cin8 and Kip3 are required to position the kinetochores, their roles are not identical, as the deletion of CIN8
moves kinetochores closer to the spindle equator, on average, whereas deletion of KIP3
moves kinetochores closer to the poles.
The fission yeast kinesin-8 homolog positions kinetochores by acting as a coupler between the kinetochore and depolymerizing kinetochore microtubules.10,20,35
There is no evidence to support a similar role for Kip3 in budding yeast. Kip3 is not essential and although cells grow with a slight metaphase delay, chromosome loss rates in the absence of Kip3 are normal.13
In addition, chromosome detachment is not increased by deletion of KIP3
In vitro, Kip3 is a length-dependent depolymerase.6,36
The simplest model predicts kinetochore microtubule length would be shorter in the presence of Kip3 than in its absence. This is not the case. Instead, we find that the average metaphase kinetochore microtubule is slightly longer in the presence of Kip3 (i.e., deletion of KIP3
decreases kinetochore microtubule length). Consistent with the idea that Kip3 increases microtubule length in vivo, the shortening rate of cytoplasmic microtubules is slower when Kip3 is present.6
In addition, a mathematical model that quantitatively reproduces the in vitro results of Varga and collegues36
shows that lowering the concentration of a processively depolymerizing microtubule motor would increase the standard deviation in microtubule lengths. How the depolymerase activity observed in vitro is regulated to yield the functions observed in vivo will be interesting to discover.
In conclusion, we find that Kip3 does not regulate spindle length and is not required to attach chromosomes to microtubules. Instead, Kip3 clusters kinetochores on the metaphase spindle by tightly regulating kinetochore microtubule length.