It was proposed by Jaqaman et al. that the suppression of chromosome oscillations is cou-pled to the establishment of tension between sister-kinetochores, which in turn affects the activity of kinases that might influence the function of microtubule depolymerases. There-fore, we initially tested whether the activity of the major mitotic kinase Cdk1/cyclin-B1 cor-relates with the suppression of chromosome oscillations (Figure 5.1a-c). For these analysis we induced the expression of cyclin-B1 with tetracycline. As we wanted to use cyclin-B1 as indicator for Cdk1-activity we tried to use an expression level that would not or only slightly influence the timing of mitosis as exogenous cyclin-B1 can act as competitive inhibitor for ubiquitination (Clute & Pines, 1999).
Our attempts to monitor the movement of single chromosomes from prometaphase until anaphase were unsuccessful as HeLa-cells contain an unusual high chromosome number (Bottomleyet al., 1969) and are not flat but round up and move in z-direction during mitosis.
To circumvent this technical issue we monitored the movement of all chromosomes instead of a single chromosome pair. Therefore, we measured the width of the CENP-A-mCherry signal. During prometaphase the microtubules search for chromosomes and are widely dis-tributed in the cell. With chromosome alignment at the spindle equator a broad metaphase plate is formed, indicating high chromosome oscillations around the metaphase plate. The width is reduced when sister-kinetochore oscillations are dampened as consequence of re-duced oscillation amplitude by altering the speed and/or switch rate. In agreement with previous data (Jaqamanet al., 2010), we observed that chromosome oscillations are high during prometaphase and decrease in metaphase (Figure 5.1b).
6.1.1 Kif18A is phosphorylated by Cdk1/cyclin-B1
Based on our observation that the decrease in cyclin-B1 levels correlates with establishment of a thin metaphase plate and that Kif18A is required to dampen chromosome oscillations we tested whether Kif18A is a substrate of Cdk1. By western-blot analysis we could show that Kif18A is specifically phosphorylated in metaphase. Additionally, inhibition of Cdk1 by roscovitine showed that Cdk1 is able to phosphorylate Kif18A in cells (Figure 5.2a). These data are in agreement with previous observations that marrow stromal Kif18A (MS-Kif18A) is modified by phosphorylation. In control treated cells MS-Kif18A migrates in two forms and only in the faster migrating form upon CIP treatment (Zusev & Benayahu, 2008).
As Cdk1 can be activated in mitosis by cyclin-A2 or cyclin-B1, we first tested which co-acti-vator is required for Kif18A phosphorylation. To test, whether Cdk1 phosphorylates Kif18A
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in complex with cyclin-B1 or cyclin-A2, we depleted cyclin-A2 and judged the phosphory-lation level of Kif18A by its migration pattern. As cyclin-A2 depleted cells show a delay in nuclear envelope breakdown we collected the cells after mitotic shake off to ensure a homogeneous, cyclin-A2 depleted cell population (Gong et al., 2007). Upon cyclin-A2 depletion Kif18A still migrates in two forms, indicating that Kif18A is still phosphorylated (Figure 5.2b). These results were confirmed byin-vitrokinase assays using cell extract con-taining high or low cyclin-A2 levels (Figure 5.4d). In summary, both experiments showed that the presence or absence of cyclin-A2 had no major influence on the phosphorylation level of Kif18A. However, we cannot rule out the possibility that Cdk1/cyclin-A2 initiates the phosphorylation on Kif18A in prometaphase, which is then maintained by Cdk1/cyclin-B1 until metaphase-to-anaphase transition.
Previous publications showed that the degradation of cyclin-A2 in prometaphase is required to align the chromosomes at the spindle equator by regulating the dynamics of kineto-chore-microtubules (den Elzen & Pines, 2001; Kabeche & Compton, 2013). Cyclin-B1 degra-dation starts in metaphase (Clute & Pines, 1999) and high expression of non-degradable cyclin-B1 causes a metaphase-arrest phenotype, in which the chromosomes continue to oscillate back and forth out of the metaphase plate (Wolf et al., 2006). Based on these observations and together with our data we suggest that Kif18A is mainly phosphorylated by Cdk1/cyclin-B1 to control its activity in dampening chromosome oscillations.
6.1.2 Cdk1 phosphorylation affects Kif18A’s ability to suppress chromosome os-cillations
To analyse whether the Cdk1-mediated phosphorylation has an impact on the function of Kif18A, we created phospho-mutants and generated stable cell lines expressing CEN-P-A-mCherry and inducible Kif18A-variants. Cells expressing GFP-Kif18AWTcould rescue the Kif18A depletion phenotype and the cells showed comparable timings to establish a thin metaphase plate and to enter anaphase as untreated, control cells (Figure 5.5c). However, cells expressing non-phosphorylatable Kif18A (S674A/S684A→2A) are able to establish a thin metaphase plate prematurely but are then delayed in anaphase onset. 2A-express-ing cells are able to establish a thin metaphase plate with the same efficiency than WT-expressing cells (Figure 5.5c-d). Previous data have shown that the over expression of GFP-Kif18A reduces chromosome oscillations (Stumpffet al., 2008), which results in the for-mation of a thin metaphase plate. We observed that cells expressing GFP-Kif18AWT in the WT- or Kif18A-depleted background showed the same timing to establish a thin metaphase plate (Figure 5.5c). In addition, the expression levels of wildtype and non-phosphorylatable
72 6 Discussion
Kif18A were comparable to each other and to endogenous Kif18A (Figure 5.5b).
During live-cell imaging of our cell lines expressing CENP-A-mCherry we observed that in some cells only the kinetochores were marked and in other cells the kinetochores and chro-mosomes are marked. These observations lead to the conclusion that CENP-A-mCherry is expressed to different extent in single cells. However, independently from the CENP-A expression levels, cells expressing the different Kif18A mutants showed reproducibly their described phenotypes. This is in line with previous data, which showed that over expres-sion of CENP-A results in its incorporation throughout the chromosomes but does not induce ectopic kinetochore formation (Gascoigne et al., 2011) and does not affect chromosome segregation (Sullivan et al., 1994). These data lead to the conclusion that the observed phenotype in cells expressing non-phosphorylatable Kif18A is independent on the expres-sion level of GFP-Kif18A and CENP-A-mCherry and solely depends on the mis-regulation of GFP-Kif18A2A.
Cells expressing phosphomimic Kif18A (S674D/S684D→2D) showed no defect in mitotic timing or the efficiency to establish a thin metaphase plate (Figure 5.5c-d). This indicates that the two single negative charges introduced by mutation of both serines to aspartate were not able to functionally mimic the doubly charged serine upon phosphorylation (Pearl-man et al., 2011). The absence of the full negative charges might affect electrostatic in-teractions, which could be required to induce conformational changes of Kif18A. Therefore, we did not further pursue this approach.
Previous data have proposed that Kif18A functions at bi-oriented chromosomes to suppress sister-kinetochore oscillations (Jaqamanet al., 2010; Stumpff et al., 2008). To further test whether the expression of GFP-Kif18A2A affects the timing of chromosome alignment or the suppression of chromosome oscillations we performed in depth analysis of the mitotic events. These data showed that cells expressing WT and non-phosphorylatable Kif18A have a comparable timing to align their chromosomes. But cells expressing non-phosphorylat-able Kif18A suppress chromosome oscillations prematurely resulting in the establishment of a thin metaphase plate too soon (Figure 5.6a-c).
By co-expressing mCherry-cyclin-B1 we were able to show that cells expressing non-phos-phorylatable Kif18A establish a thin metaphase plate in the presence of high cyclin-B1 lev-els (Figure 5.7a-b). This indicates that the Cdk1-mediated phosphorylation has a negative effect on the activity of Kif18A. Additionally, our data showed that the degradation kinetics of cyclin-B1 are comparable in 2A- or WT-expressing cells. This is in full agreement with a recent observation that the spindle assembly checkpoint is a gradual response and that the strength of the checkpoint and the activation of the APC/C correlate with degradation
6 Discussion 73
kinetics of cyclin-B1 (Collin et al., 2013). It was previously shown that phosphorylation of separase by Cdk1/cyclin-B1 inhibits its proteolytic activity (Gorr et al., 2005; Stemmann et al., 2001). Based on these observations and taken our data, one could imagine that cells expressing non-phosphorylatable Kif18A are unable to resolve cohesion between sis-ter chromatids and to separate their chromosomes even if the chromosomes are tightly aligned. To test this prediction one could either over express a constitutive active separase or check for the appearance of a self-cleavage product of separase upon its activation in 2A-expressing cells (Holland & Taylor, 2006; Stemmannet al., 2001).
We performed initial experiments to investigate how Cdk1 phosphorylation affects the in-trinsic properties of Kif18A usingin-vitroTIRF-microscopy.In-vitrostepping assays revealed that phosphorylated His-Kif18AWT-GFP showed no difference in velocity or processivity in comparison to His-Kif18A2A-GFP (Figure 5.19a-c). Unfortunately, based on the assay con-ditions we were unable to analyse Kif18A’s pausing time at the plus-tips of microtubules.
Therefore, we speculate that Cdk1-phosphorylation affects the pausing-time of Kif18A and thereby its ability to accumulate a the plus-tips of kinetochore-microtubules. As the phos-phomimetic mutant did not functionally mimic phosphorylation we were unable to test this mutant in our in-vitroTIRF-microscopy assays. Additionally, the conditions of our TIRF-mi-croscopy assays were not compatible with ourin-vitrokinase assays and we were unable to analyse directly whether and how pre-phosphorylation of Kif18A affects its intrinsic proper-ties.
6.1.3 Kif18A is de-phosphorylated by PP1
Previous data showed that PP1 is recruited to kinetochores to stabilize kinetochore-mi-crotubule attachments by de-phosphorylating Aurora-B substrates. Thereby it promotes its own accumulation at fully attached kinetochores in metaphase (Kim et al., 2010a; Liu et al., 2010a; Posch et al., 2010; Wurzenberger et al., 2012). PP2A, another important phosphatase, localizes to kinetochores in prometaphase and is absent in metaphase (Fo-ley et al., 2011). In addition, it was recently shown that the yeast homologue of Kif18A Klp5/Klp6 binds directly to PP1 via a C-terminal PP1-binding motif, which is conserved in humans (Meadows et al., 2011). Based on these data, together with the observation that only inhibition of PP1 by Tautomycin or high doses of Ocadaic Acid (Figure 5.10b) induces persistent phosphorylation of Kif18A, we focused on PP1 as counteracting phosphatase.
By co-immunoprecipitation experiments we showed an interaction between endogenous Kif18A and over expressed GFP-PP1α andγ (Figure 5.11a) in cells. Unfortunately, based on the high cytoplasmic pool of PP1 (Trinkle-Mulcahy et al., 2003) we were unable to show a
74 6 Discussion
phase-specific interaction between GFP-PP1 and endogenous Kif18A. Normally, PP1 is kept inactive by binding to different inhibitors (I-1, I-2, I-3), which use the same binding site as Kif18A (Eiteneueret al., 2014; Hurleyet al., 2007; Kwon et al., 1997; Lesageet al., 2007;
Wang et al., 2008; Wu et al., 2009). However, one could imagine that over expression of PP1 results in the appearance of inhibitor-free PP1, which is now able to interact with Kif18A.
We confirmed in-vitro that Kif18A binding to PP1α and γ is direct and depends on its PP1-docking motif in the C-terminus (Figure 5.11c-d). This is in agreement with previous data that showed that mutating the valin or phenylalanin in the PP1-binding motif abolished interaction with PP1 (Egloff et al., 1997; Meadows et al., 2011). The interaction between PP1 and Kif18A via the conserved RVXF-motif was recently confirmedin-vitro and in cells by an independent study (De Weveret al., 2014). Additionally, we showed that PP1 binding is necessary for the de-phosphorylation of the two Cdk1-sites S674/S684 (Figure 5.12a-c).
Besides PP1 itself (Dohadwalaet al., 1994; Kwonet al., 1997; Wuet al., 2009), Kif18A is the first known Cdk1 substrate that is de-phosphorylated by PP1 during mitosis.
Based on the biochemical data, we predict that cells expressing the Kif18A mutant that is unable to bind to PP1, should exhibit a defect in metaphase plate thinning, as Kif18A is con-stantly phosphorylated. Indeed, we observed that upon expression of GFP-Kif18APP1∆ the cells show increased oscillation amplitudes resulting in the formation of a broad metaphase plate (Figure 5.14 and Figure 5.13). Previous data showed that upon addition of MG-132 to prometaphase cells, chromosomes continue to move back and forth around the meta-phase plate (den Elzen & Pines, 2001). Together with our observation in cells expressing persistent phosphorylated Kif18A (GFP-Kif18APP1∆) this leads to the conclusion that MG-132 prevents cyclin-B1 degradation, which results in persistent phosphorylation of Kif18A and continuous chromosome oscillations. This is further supported by the observation that in the presence of non-degradable cyclin-B1 chromosomes continue to oscillate around the metaphase plate (Wolfet al., 2006). The increased sister-kinetochore oscillation phenotype in cells expressing GFP-Kif18APP1∆ depends on the inability of PP1 to de-phosphorylate GFP-Kif18APP1∆ as a double defective mutant (PP1∆+2A), behaves like a non-phosphory-latable mutant alone (Figure 5.13 and Figure 5.14).
6.1.4 Kif18A is not required to recruit PP1 to kinetochores
Previous data have shown that the fission yeast Klp5/Klp6 interacts with PP1 to recruit it to the kinetochores to establish chromosome bi-orientation and to facilitate checkpoint si-lencing. It is important to note that the checkpoint silencing function is independent of the motor activity of Klp5/Klp6. In addition, the checkpoint silencing assays were performed in
6 Discussion 75
the absence of microtubules (Meadowset al., 2011).
Based on these observations the question arises whether Kif18A functions also as a trans-port molecule to deliver PP1 to the kinetochores or whether Kif18A is a sole substrate of kinetochore-bound PP1. From the following data we favour the latter: cells expressing GFP-Kif18APP1∆showed no difference in the phosphorylation levels of Hec1 (Figure 5.21a-b), a kinetochore substrate of PP1 required for microtubule attachment (DeLuca et al., 2011;
Welburn et al., 2010). As Hec1 is the most outer kinetochore protein (Wan et al., 2009) and based on the spatial separation model it should be the most sensitive substrate to Aurora-B phosphorylation upon establishment of stable end-on attachments and tension (Welburn et al., 2010). We confirmed this in KNL1-depleted cells, which showed increased phosphorylation of Hec1 compared to control depleted cells (Figure 5.21a-b).
However, one could also argue that Kif18A bound PP1 is required for de-phosphorylation of an other substrate. In agreement with previous data (Trinkle-Mulcahy et al., 2001), we were unable to analyse the endogenous PP1 localization and levels directly based on un-reproducible stainings using antibodies (data not shown). Therefore, it would be beneficial to generate double stable cell lines expressing GFP-Kif18A variants and mCherry-PP1γ to test the dynamic PP1 levels directly and independently of a specific substrate as readout.
As previously shown, over expression of a targeting subunit can alter the dynamic local-ization pattern of PP1 (Trinkle-Mulcahy et al., 2001). One could argue that expression of GFP-Kif18AWTshould induce a premature kinetochore localization of mCherry-PP1γ if acting as kinetochore PP1-interacting protein (PIP). In addition it was shown that expression of a PIP mutated in the PP1-binding motif can act dominant negative and prevent the recruit-ment of PP1 (Trinkle-Mulcahy et al., 2006). Accordingly, cells expressing GFP-Kif18APP1∆
should show reduced PP1 levels at kinetochores in contrast to WT-expressing cells.
However, previous data lead to the conclusion that PP1 localizes to kinetochores indepen-dent of an active transport mechanism along the microtubules. In FRAP-experiments PP1 was still able to localize to the kinetochores in the presence of nocodazole, a condition that prevents Kif18A from accumulating at kinetochores (Stumpff et al., 2011; Trinkle-Mulcahy et al., 2003). This is a strong indication that in human cells PP1 recruitment to kineto-chores is independent of Kif18A. Whereas Klp5/Klp6 mutated in the PP1-binding motif is still able to localize at the plus-tips of kinetochore-microtubules in metaphase (Meadows et al., 2011), GFP-Kif18APP1∆was unable to efficiently accumulate at microtubule plus-tips (Figure 5.17a-c). Additionally, our data show that artificial targeting of PP1γ to kinetochores by fusion to Mis12 allows cells expressing GFP-Kif18AWTto establish a thin metaphase plate prematurely. In contrast to cells expressing GFP-Kif18A2A, which showed no further
accel-76 6 Discussion
eration in metaphase plate thinning upon expression of Mis12-GFP-PP1γ and they establish a thin metaphase plate with the same timing as 2A-expressing cells alone (Figure 5.22b-c).
Finally, cells expressing catalytic inactive versions of Mis12-mCherry-PP1γ exhibit normal timing to establish a thin metaphase plate (Figure 5.22d) further supporting the depen-dency on the catalytic activity of PP1. From these experiments we draw the conclusion that the premature metaphase plate thinning phenotype upon kinetochore targeting of PP1 depends on the premature de-phosphorylation of GFP-Kif18A.
Additionally, it was recently published that Kif18A is a low-force motor, which makes it un-able to transport cargos along the microtubules (Jannasch et al., 2013). Based on these data we favour the idea that Kif18A is only a substrate of kinetochore-bound PP1 and the timely de-phosphorylation of Kif18A regulates metaphase plate thinning in pre-anaphase cells. From these data it seems unlikely that Kif18A interacts constitutively with PP1 and thereby transports its own phosphatase, as proposed for the function of kinesin-8 in fission yeast (Meadowset al., 2011).
Whether PP1 stays bound to KNL1, while de-phosphorylating Kif18A, or jumps from KNL1 onto Kif18A to mediate the de-phosphorylation is not clear. It is proposed that PP1 is able to bind multiple PIPs as long as they use different PP1-binding motifs (Heroeset al., 2012).
The latter possibility seems unlikely as both KNL1 and Kif18A require the RVXF-motif to be able to bind PP1 (Liu et al., 2010a). But KNL1 harbours a second PP1-binding motif, the SILK-motif and it is an intriguing idea that PP1 binds to KNL1 via the SILK-motif to be able to interact with Kif18A via the RVXF-motif. One could additionally imagine that the affinity of PP1 to bind phosphorylated substrates is higher, which would result in an increased binding to phosphorylated Kif18A, but this remains to be tested.
6.1.5 Kif18A is not involved in checkpoint silencing
It was shown in yeast that cells expressing a Klp5/Klp6 mutant unable to interact with PP1 are delayed in anaphase onset. This is in agreement with our data in human cells express-ing GFP-Kif18APP1∆and might implicate that the checkpoint function is conserved in Kif18A.
However, cells expressing the double defective Kif18A mutant (PP1∆+2A) restore the ana-phase delay to a comparable timing as in cells expressing only GFP-Kif18A2A. This indicates that the observed anaphase delay in cells expressing the Kif18A PP1-binding mutant is mainly mediated by de-regulated Kif18A itself, which is unable to suppress chromosome oscillations. In addition, it was recently investigated, whether kinetochore-recruitment of PP1 plays a role in checkpoint silencing by preventing that PP1 interacts with KNL1, the main kinetochore-recruitment protein for PP1 (Liuet al., 2010a; Zhanget al., 2014). Cells
6 Discussion 77
expressing a PP1-binding deficient KNL1 mutant show a 10 minute delay from metaphase to anaphase and increased levels of Bub1 and BubR1. However no major segregation de-fects were observed indicating that kinetochore targeting of PP1 plays only a minor role in checkpoint silencing (Zhang et al., 2014). This is in contrast to yeast cells, where binding of PP1 to kinetochores is required for checkpoint silencing (Meadowset al., 2011).
6.1.6 Plus-tip accumulation of Kif18A is regulated by Cdk1 and PP1
How Cdk1-meditated phosphorylation prevents Kif18A accumulation and PP1-mediated de-phosphorylation promotes it has to be further investigated. A good candidate would be the second, ATP-independent microtubule-binding region in the very C-terminus of Kif18A.
Via electrostatic interactions it increases the microtubule-binding affinity of Kif18A (Stumpff et al., 2011). These interactions provide a good possibility to be regulated directly or
Via electrostatic interactions it increases the microtubule-binding affinity of Kif18A (Stumpff et al., 2011). These interactions provide a good possibility to be regulated directly or