Recent data (Meadowset al., 2011) postulated that the yeast homologue Klp5/Klp6 is re-quired to recruit PP1 to the kinetochores to silence the spindle assembly checkpoint. Based on these data we analysed whether the observed phenotypes (Figure 5.13, Figure 5.14 and Figure 5.17) are due to the effect of PP1 on Kif18A’s function or whether Kif18A plays a role in the recruitment of PP1 to the kinetochores. We investigated the phosphorylation level of Hec1 at S55, which is sensitive to Aurora-B and PP1 activity (DeLucaet al., 2011; Welburn et al., 2010).
We speculated that a Kif18A mutant that is deficient in PP1 transport should show increased levels of Hec1 phosphorylation since less PP1 would be available at the kinetochore to de-phosphorylate its substrate Hec1. We measured the Hec1 and phospho-Hec1 levels and correlated them to the CREST signal. CREST serum (derived from patients with Calcinosis, Raynaud’s syndrome, Esophageal dysmotility, Sclerodaytyly and Telangiectasia syndrome) can be used to stain for centromeric proteins CENP-A, CENP-B and CENP-C (Earnshaw &
Rothfield, 1985; Moroiet al., 1980).
64 5 Results
a CREST Hec1 pHec1 Hec1/CREST pHec1/CREST
control-siRNAKNL1-siRNA
Figure 5.20. Increase in phospho-Hec1 levels upon depletion of KNL1.
(a)Immunofluorescence images of control or KNL1 depleted cells arrested in mitosis by MG-132 treatment. Cells were fixed and immunostained for Hec1, phospho-Hec1 (S55, pHec1) and CREST. Images are maximum intensity projections of deconvolved z-stacks. Scale bar, 4µm.
(b) Hec1 and pHec1 intensity was set relative to total CREST levels and normalized to Hec1/CREST and pHec1/CREST ratio of control cells. Bar graphs show the mean±SD from two independent experiments with 87 cells (control-siRNA) or 88 cells (KNL1-siRNA).
To test this setup, we depleted KNL1, the main kinetochore-recruitment factor of PP1 (Liu et al., 2010a). The depletion of KNL1 results in the loss of kinetochore-microtubule interac-tions and the absence of chromosome alignment at the metaphase plate (Cheesemanet al., 2008; Kiyomitsuet al., 2007). Analysis of KNL1 depleted cells revealed the described phe-notypes that these cells show reduced kinetochore-microtubule interactions and no chro-mosome alignment (Figure 5.20a). Upon depletion of KNL1 the ratio of Hec1 to CREST stays constant in comparison to control cells (0.97±0.2 (KNL1-siRNA) to 1.0 (control-siRNA), Fig-ure 5.20b), whereas the ratio of pHec1/CREST increases by two-fold (1.0 (control-siRNA) to 1.83±0.05 (KNL1-siRNA), Figure 5.20b). These results suggest that under the tested conditions the phospho-Hec1 antibody is sensitive to changes in PP1 localization at the kinetochores reflected by the detection of an increase in Hec1 phosphorylation.
Next we tested the phosphorylation level of Hec1 in cells expressing the different GF-P-Kif18A variants. These experiments were performed under rescue conditions, where we depleted endogenous Kif18A by siRNA, induced the expression of the different GFP-Kif18A variants and blocked the cells at the metaphase-to-anaphase transition by MG-132 treat-ment (Figure 5.21a).
5 Results 65
Figure 5.21. Kif18A is not required for the KT-recruitment of PP1.
(a)Immunofluorescence images of Kif18A-RNAi cells expressing the indicated GFP-Kif18A variants and arrested in mitosis by MG-132 treatment. Cells were fixed and immunostained for Hec1, phospho-Hec1 (S55, pHec1) and CREST. Images are maximum intensity projections of deconvolved z-stacks. Scale bar, 4µm.
(b) Hec1 and pHec1 intensity was set relative to total CREST levels and normalized to Hec1/CREST and pHec1/CREST ratio of wild-type (WT) GFP-Kif18A. Bar graphs show the mean±SD from three independent ex-periments with more than 40 cells per condition.
The quantifications show that there is no difference in the levels of phospho-Hec1 at the kinetochores in cells expressing persistent phosphorylated GFP-Kif18APP1∆ (Figure 5.21b).
Based on these results we suggest that binding of PP1 to Kif18A is required for the control of Kif18A’s function in regulating metaphase plate thinning but not for the kinetochore re-cruitment of PP1.
The recruitment of PP1 to the outer kinetochores is mainly mediated by binding to KNL1, which is regulated by tension: under low centromere tension KNL1 is phosphorylated by Au-rora-B and PP1 is unable to bind. With chromosome bi-orientation tension is increased and PP1 can bind to KNL1 and de-phosphorylate further Aurora-B substrates (Liuet al., 2010a).
Based on our data that cells expressing non-phosphorylatable Kif18A initiate metaphase plate thinning prematurely we speculated that uncoupling the tension dependent localiza-tion of PP1 to KNL1 should cause a similar phenotype to 2A-expressing cells. To test this idea we constitutively targeted PP1 to the outer kinetochores by fusion to Mis12. GFP-PP1γ localizes only in metaphase to the kinetochores (Trinkle-Mulcahyet al., 2003).
66 5 Results
Time from NEBD to thinnest MP [min]
WT 2A GFP-Kif18A
Time from NEBD to thinnest MP [min]
d
Figure 5.22. Cells expressing Mis12-GFP-PP1γ mimic the 2A-phenotype in establishing a thin meta-phase plate prematurely.
(a)Time-lapse images of cells expressing GFP-PP1γ fused to Mis12 (Mis12-GFP-PP1γ) to show the constitutive kinetochore targeting of GFP-PP1γ. Time stamp at nuclear envelope breakdown (NEBD) was set to 0:00 minutes.
Scale bar, 5.6µm.
(b)Still images of movies showing HeLa-cells stably expressing CENP-A-mCherry and inducibly expressing WT or 2A GFP-Kif18A transiently transfected with Mis12-GFP-PP1γ. Time stamp at nuclear envelope breakdown (NEBD) was set to 0:00 minutes. Scale bar, 5.6µm.
(c)Quantification of time from nuclear envelope breakdown (NEBD) to establishment of a thin metaphase plate (tMP). Bars show mean±SD from four independent experiments with n = 27 and 50 cells (WT±Mis12-GFP-PP1γ) or n = 51 and 43 cells (2A±Mis12-mCherry-PP1γ).
(d)Quantification of timing to establish a thin metaphase plate (tMP) in cells inducibly expressing wild-type or catalytic inactive Mis12-mCherry-PP1γvariants. Bars show mean±SD from two independent experiments with 56 (WT), 50 (D64N), 44 (R96E) and 28 (H125A) cells.
The fusion to Mis12, allows Mis12-GFP-PP1γ to localize to the kinetochores from entry into mitosis until anaphase onset (Figure 5.22a). To analyse how co-expression of Mis12-GFP-PP1γ influences the timing in WT- or 2A-expressing cells we transiently transfected Mis12-GFP-PP1γ
5 Results 67
into the stable cell lines expressing CENP-A-mCherry and inducible GFP-Kif18A variants. We analysed the timing of metaphase plate thinning by live-cell microscopy (Figure 5.22b,c).
Upon co-expression of Mis12-GFP-PP1γ, GFP-Kif18AWT-expressing cells are able to establish a thin metaphase plate with the same timing as 2A-expressing cells (22.6±1.1, (WT+Mis12-GFP-PP1γ) compared to 22.1±2.3, (2A) Figure 5.22c). Cells expressing both GFP-Kif18A2A and Mis12-GFP-PP1γdo not further accelerate timing of metaphase plate thinning (22.3±2.0 Figure 5.22c).
Our data suggest that PP1 controls chromosome oscillations by de-phosphorylating Kif18A at S674/S684 and that Kif18A does not serve as kinetochore-recruiter for PP1. In order to confirm that the observed phenotypes are due to the phosphatase activity of PP1 we cre-ated three different catalytic inactive PP1 mutants. We introduced different mutations in or-der to abrogate or reduce the catalytic activity by directly mutating the active site (H125A), by introducing a mutation in a metal binding pocket (D64N) or by alternating the cataly-sis by affecting the hydrogen bond to phosphorus oxygen (R96A) (Helpset al., 2000; Kim et al., 2010a; Sheppecket al., 1997; Trinkle-Mulcahyet al., 2001; Zhanget al., 1996). Cells expressing Mis12-mCherry-PP1γWT establish a thin metaphase plate in 21.5±0.2 min upon NEBD, whereas cells expressing the different catalytic inactive mutants need 31.5±3.8 min (D64N), 30.4±2.0 min (R96E) and 30.2±3.4 min (H125A) to establish a thin metaphase plate, which is comparable to cells expressing GFP-Kif18AWT (Figure 5.22c). These results support our idea that PP1-mediated de-phosphorylation of Kif18A supports metaphase plate thinning.
6 Discussion
After chromosome congression, bi-orientated kinetochores typically oscillate around the spindle equator (Skibbens et al., 1993). These oscillatory movements facilitate chromo-some alignment and protect them from becoming damaged or entangled (Keet al., 2009).
It is further proposed that chromosome oscillations around the spindle equator are a control process to ensure that both sister-kinetochores are properly attached (Liuet al., 2009). As cells progress towards anaphase these movements are suppressed resulting in the tight alignment of the chromosomes (Jaqaman et al., 2010; Liu et al., 2008; Magidson et al., 2011).
During metaphase, Kif18A shows a gradual accumulation at the plus-tips of kinetochore-mi-crotubules (Mayret al., 2007; Stumpffet al., 2008). Its intrinsic activities, namely high pro-cessivity and dwell time at the microtubule plus-tips, enable Kif18A to reach and stay at the plus-tips to a certain threshold concentration (Stumpffet al., 2011; Vargaet al., 2009). Only when enough motor proteins are localized to the plus-ends, the threshold concentration is reached, microtubule dynamics are altered and oscillations are suppressed.
In a recent study it was shown that dampening of sister-kinetochore oscillations results in the establishment of a thin metaphase plate (Jaqaman et al., 2010). They proposed a model in which gradual changes in the centromere stiffness between prometaphase and metaphase could affect breathing dynamics of sister-kinetochores, which in turn affect the phoshorylation status of microtubule regulators. But the exact mechanism was not clear (Jaqamanet al., 2010).
Our data presented in this work show that Kif18A is such a microtubule regulator, whose localization and activity is regulated by (de)phosphorylation in an tension-dependent man-ner. In agreement with the described model by Jaqaman et al. we showed that Cdk1 phosphorylation prevents Kif18A accumulation. Binding of PP1 to Kif18A via a C-terminal binding motif results in the de-phosphorylation of the Cdk1-sites and allows Kif18A to lo-calize to the plus-tips of kinetochore-microtubules. As the activity of Cdk1 and kinetochore localization of PP1 is altered with increasing centromere stiffness, changes in tension be-tween sister-kinetochores is coupled to Kif18A localization. This results in a reduction of the oscillation speed to establish a thin metaphase plate. In the following chapter I will describe our data in relation to published ideas and present the regulation of Kif18A’s function in a working model. I will additionally discuss open questions and propose future directions how to address them.
70 6 Discussion