Kinesin-14 and kinesin-5 antagonistically regulate microtubule nucleation by γ-TuRC in yeast and human cells
Bipolar spindle assembly is a critical control point for initiation of mitosis through nucleation and organization of spindle microtubules and is regulated by kinesin-like proteins. In fission yeast, the kinesin-14 Pkl1 binds the g-tubulin ring complex (g-TuRC) microtubule-organizing centre at spindle poles and can alter its structure and function. Here we show that kinesin-14 blocks microtubule nucleation in yeast and reveal that this inhibition is countered by the kinesin-5 protein, Cut7. Furthermore, we demonstrate that Cut7 binding to g-TuRC and the Cut7 BimC domain are both required for inhibition of Pkl1. We also demonstrate that a yeast kinesin-14 peptide blocks microtubule nucleation in two human breast cancer cell lines, suggesting that this mechanism is evolutionarily conserved. In conclusion, using genetic, biochemical and cell biology approaches we uncover antagonistic control of microtubule nucleation at g-TuRC by two kinesin-like proteins, which may represent an attractive anti-mitotic target for cancer therapies.
Results
Figure 2
Co-IPs
[x] cut7:
[x] Co-IP with alp4
[x] cut7HS:
[x] Co-IP with alp4
[x] cut7ST:
[x] Co-IP with alp4
Figure 3
[x] cut7 in gtb1-k5a background:
[x] Reduced in the high molecular weight fraction with alp4
[x] increased localization to spindle MTs (not quantified)
[x] Lost localization to the SPBs (immunofluo)
[x] cut7HS in gtb1-k5a background:
[x] Absent from the high molecular weight fraction with alp4
[x] cut7ST in gtb1-k5a background:
[x] Reduced in the high molecular weight fraction with alp4
[x] It is weird that the cut7 is reduced, but this truncation isn't.
[x] NLS-Cut7ST localizes to SPBs only, not to spindle microtubules. This is quite important although already mentioned in Hagan paper
[x] NLS-Cut7T localizes to SPBs only, not to spindle microtubules.
[x] NLS-Cut7T22 all localization lost.
[x] NLS-Cut7ST22 all localization lost.
Figure 3
[x] cut7D pkl1Dstronger signal of spindle tubulin (not quantified)
[x] Mixed culture?
[x] pkl1D thick spindle
Next figures
[x] Many studies have used the double mutant pkl1D cut7D but they never reported chromosome segreagation errors.
[x] Also cut7D pkl1D has consistently been found to have short metaphase length in multiple backgrounds and delayed anaphase onset.
FYPO
GO
Existing annotations.
[x] Revise refs 17, 22, 23 -> Interaction of pkl1 with gamma-tub
[x] Ref. 23 -> pkl1 replaces HSET in human.
[x] all human g-TuSC protein components are also compatible.
[x] Hagan paper nucleolus -> nucleus in the localization phenotype.
[x] Annotations from Yukawa paper to FYPO:0006339 are a bit confusing. The mislocalized nucleus annotation should maybe replaced by only by diploidisation, and the ones that don't do this by cut phenotype (if curated at all since these are downstream effects of failure to form a spindle). What happens is that the nucleus does not divide and it's pushed to one of the daughter cells. Also some annotations for the Hagan paper are here.
[x] Revise delayed onset of anaphase B, and ana/meta transition. There might be an issue on this already.
[x] Cut7 allele annotations from Hagan's paper, they are the same here:
Name
Aminoacids kept
cut7-ST
443-1085
cut7-H
1-443
cut7-T
888-1085
cut7-HS
1-888
cut7-22
Pro1021->Ser
cut7ST-22
Pro1021->Ser
cut7T-22
Pro1021->Ser
[x] Is localization of fragments somethign that should be curated
[x] Put only the mislocalized ones.
[ ]
Ask the authors:
Discuss with Val
[x] Is the replacement of human function curatable / of interest?
[x] They report normal spindles (no single traces shown). Toda and multiple publications from our lab show cut7D pkl1D has short spindles.
[x] It would be really nice to have all the alleles that are constructs shown in a list, I think. Perhaps we could use something that people mentioned during the conference.
PMID:25348260
Kinesin-14 and kinesin-5 antagonistically regulate microtubule nucleation by γ-TuRC in yeast and human cells
Bipolar spindle assembly is a critical control point for initiation of mitosis through nucleation and organization of spindle microtubules and is regulated by kinesin-like proteins. In fission yeast, the kinesin-14 Pkl1 binds the g-tubulin ring complex (g-TuRC) microtubule-organizing centre at spindle poles and can alter its structure and function. Here we show that kinesin-14 blocks microtubule nucleation in yeast and reveal that this inhibition is countered by the kinesin-5 protein, Cut7. Furthermore, we demonstrate that Cut7 binding to g-TuRC and the Cut7 BimC domain are both required for inhibition of Pkl1. We also demonstrate that a yeast kinesin-14 peptide blocks microtubule nucleation in two human breast cancer cell lines, suggesting that this mechanism is evolutionarily conserved. In conclusion, using genetic, biochemical and cell biology approaches we uncover antagonistic control of microtubule nucleation at g-TuRC by two kinesin-like proteins, which may represent an attractive anti-mitotic target for cancer therapies.
Results
Figure 2
Co-IPs
Figure 3
[x] cut7 in gtb1-k5a background:
increased localization to spindle MTs(not quantified)[x]
cut7HS in gtb1-k5a background:Absent from the high molecular weight fraction with alp4[x]
cut7ST in gtb1-k5a background:Reduced in the high molecular weight fraction with alp4It is weird that the cut7 is reduced, but this truncation isn't.[x] NLS-Cut7ST localizes to SPBs only, not to spindle microtubules. This is quite important although already mentioned in Hagan paper
[x] NLS-Cut7T localizes to SPBs only, not to spindle microtubules.
[x] NLS-Cut7T22 all localization lost.
[x] NLS-Cut7ST22 all localization lost.
Figure 3
cut7D pkl1Dstronger signal of spindle tubulin(not quantified)Mixed culture?Next figures
FYPO
GO
Existing annotations.
[x] Revise refs 17, 22, 23 -> Interaction of pkl1 with gamma-tub
[x] Ref. 23 -> pkl1 replaces HSET in human.
[x] all human g-TuSC protein components are also compatible.
[x] Hagan paper nucleolus -> nucleus in the localization phenotype.
[x] Annotations from Yukawa paper to FYPO:0006339 are a bit confusing. The mislocalized nucleus annotation should maybe replaced by only by diploidisation, and the ones that don't do this by cut phenotype (if curated at all since these are downstream effects of failure to form a spindle). What happens is that the nucleus does not divide and it's pushed to one of the daughter cells. Also some annotations for the Hagan paper are here.
[x] Revise delayed onset of anaphase B, and ana/meta transition. There might be an issue on this already.
[x] Cut7 allele annotations from Hagan's paper, they are the same here:
Ask the authors:
Discuss with Val