Function and regulation of the mitotic kinesin Kif18A

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Function and regulation of the mitotic kinesin Kif18A

Dissertation submitted for the degree of Doctor of Natural Science

(Dr.rer.nat.) At the

Faculty of Sciences Department of Biology

Presented by Julia Häfner

Day of oral examination: 20. March 2015 1st referee: Prof. Dr. Thomas U. Mayer 2nd referee: Prof. Dr. Martin Scheffner

Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-286548

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Erklärung der Selbstständigkeit

Ich erkläre hiermit, dass ich die vorliegende Arbeit ohne unzulässige Hilfe Dritter und ohne Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe. Die aus anderen Quellen direkt oder indirekt übernommenen Daten und Konzepte sind unter Angabe der Quelle gekennzeichnet. Weitere Personen, insbesondere Promotionsberater, waren an der inhaltlich materiellen Erstellung dieser Arbeit nicht beteiligt. Die Arbeit wurde bisher weder im In- noch im Ausland in gleicher oder ähnlicher Form einer anderen Prüfungsbehörde vor- gelegt.

Julia Häfner Konstanz, den 11.12.2014

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1 Summary

During congression, vertebrate chromosomes align at the spindle equator and upon alignment these bi-oriented chromosomes continue to oscillate around the metaphase plate.

Before sister chromatids are separated upon anaphase onset, these oscillatory movements are dampened resulting in the tight alignment of the chromosomes and the establishment of a thin metaphase plate. The formation of a thin metaphase plate is the key for accu- rate and faithful chromosome segregation. While it is well established that chromosome movements are coupled to the dynamic behaviour of kinetochore-microtubules and forces acting on the chromosomes, the molecular mechanism regulating dampening of chromosome oscillations and metaphase plate thinning remains largely unknown. RNA interference (RNAi)-mediated depletion of Kif18A, a member of the kinesin-8 family of motor proteins, increases the amplitude of chromosome oscillations, whereas over expression of Kif18A decreases it, suggesting that Kif18A dampens oscillations of bi-oriented chromosomes before anaphase. This function of Kif18A depends on its ability to accumulate at the plus-tips of kinetochore microtubules to act cooperatively in altering dynamics of kinetochore-microtubules.

To dissect the regulation of Kif18A in its function to suppress chromosome oscillations, we analysed the post-translational modifications of Kif18A. By combining in-vitro assays with time-lapse microscopy imaging, we demonstrate that Kif18A is phosphorylated by cyclin-dependent kinase 1 (Cdk1) in complex with cyclin-B1 in cells andin-vitroand identified S674 and S684 as major Cdk1-phosphorylation sites on Kif18A. Cdk1 mediated inhibitory phosphorylation on Kif18A during early metaphase prevents Kif18A from accumulating at the plus-tips of kinetochore-microtubules. Consequently Kif18A is unable to suppress chromosome oscillations. Protein phosphatase 1 (PP1) removes these phosphorylations in metaphase in a reaction that depends on a conserved PP1-binding motif in the C-terminus of Kif18A. This dephosphorylation is required for correct accumulation of Kif18A at plus-tips of kinetochore-microtubules resulting in the suppression of oscillations.

Based on our studies we propose a model in which a Cdk1/PP1 phosphorylation switch modulates Kif18A’s ability to accumulate at the plus-tips of kinetochore-microtubules to suppress chromosome oscillations, when kinetochore-microtubules are stably attached and under tension. This idea is in perfect agreement with the observation that metaphase plate thinning coincides with the global inactivation of Cdk1 and temporal localization of PP1 at kinetochores.

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2 Zusammenfassung

Während der Prometaphase bewegen sich die Chromosomen zur Mitte der bipolaren Spindel und bilden die Metaphasenplatte aus. Anschließend oszillieren diese bi-orientierten Chro- mosomen entlang der Pol-zu-Pol Achse um die Metaphasenplatte. Für die korrekte Trennung von Schwester-Chromatiden in Anaphase ist es wichtig, dass die oszillierenden Bewegun- gen unterdrückt werden. Durch diese Unterdrückung kommt es zu einer engen Anordnung der Chromosomen und die Breite der Metaphasenplatte nimmt kontinulierlich ab. Es ist bekannt, dass die Bewegung der Chromosomen auf dem dynamischen Verhalten der Kine- tochore-Mikrotubuli und den Kräften, die auf die Chromosomen wirken, basiert. Über den molekulare Mechnaismus, der die Unterdrückung der Oszillation und den Aufbau einer dün- nen Metaphasenplatte reguliert, ist hingegen nur sehr wenig bekannt. Dem mitotische kinesin Kif18A wird eine wichtige Rolle in der Unterdrückung der Chromosomen-Oszillation vor Eintritt in die Anaphase zugeschrieben. Während seine Depletion über RNA interferenz (RNAi) die Amplitude der Oszillation erhöht, bewirkt die Überexpression von Kif18A eine Reduzierung dieser Amplitude. Diese Funktion von Kif18A hängt von seiner Akkumulierung an den plus-Enden der Kinetochore-Mikrotubuli ab. Hier agieren mehrere Kif18A Moleküle kooperativ zusammen, um die Dynamik der Kinetochore-Mikrotubuli zu beeinflussen.

Um zu verstehen wie sich die Regulierung von Kif18A auf seine Funktion in der Unter- drückung der Oszillation auswirkt, wurde die posttranslationale Modifikation von Kif18A untersucht. Dafür wurdenin-vitroAssays mit mikroskopischen Lebendzell Aufnahmen kom- biniert. Wir konnten zeigen, dass Kif18A von der Cyclin-abhängigen kinase 1 (Cdk1) in Kom- plex mit Cyclin-B1 sowohl in Zellen als auchin-vitrophosphoryliert wird und dass S674 und S684 die beiden Hauptphosphorylierungsstellen von Cdk1 sind. Während der frühen Meta- phase wirkt die Cdk1-vermittelte Phosphorylierung inhibitorisch auf Kif18A, sodass Kif18A nicht an den plus-Enden der Kinetochore-Mikrotubuli akkumulieren kann und dies führt da- zu, dass die Unterdrückung der Oszillation verhindert wird. In später Metaphase entfernt die Protein Phosphatase 1 (PP1) die hemmenden Phosphorylierungen in einer Reaktion, die von der konservierten PP1-Bindestelle im C-Terminus von Kif18A abhängt. Die Dephospho- rylierung ist für die korrekte Akkumulierung von Kif18A an den plus-Enden der Kinetocho- re-Mikrotubuli wichtig, was zur Folge hat, dass die Oszillation unterdrück wird.

Wir postulieren ein Model demzufolge Cdk1 und PP1 antagonisitsch die Fähigkeit von Kif18A an den Mikrotubuli plus-Enden zu akkumulieren, modulieren. Damit regulieren sie über Kif18A die Unterdrückung der Ozillation. Mit dem Aufbau stabiler Kinetochore-Mikrotubuli Interaktionen wird Cdk1 global inaktiviert und PP1 kann am Kintochore lokalisieren.

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4 2 Zusammenfassung

Die Unterdrückung der Oszillation wird somit an die Anheftung der Mikrotubuli an die Kine- tochore und den Aufbau der Spannung zwischen den Schwester-Kinetochoren gekoppelt.

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3 Introduction

The cell cycle is the process, which ensures the duplication and segregation of chromosomes. It can be divided into four major phases (Figure 3.1). In the first gap phase, G1-phase, the cell prepares for DNA-replication and decides based on extra- and intracellu- lar signals to progress to the next cell-cycle stage (S-phase). When extracellular conditions arrest proliferation the cells can enter a prolonged non-dividing state, called G0-phase.

During S-phase the DNA is replicated and each pair of the homologous chromosomes is duplicated resulting in two sister chromatids per chromosome. Sister chromatids are held together by sister chromatid cohesion, which is mediated by cohesin a complex of four subunits: Smc1 and Smc3 (structural maintenance of chromosomes) as well as Scc1 and Scc3 (sister chromatid cohesion). It forms a ring around the sister chromatids to link them together (Gruberet al., 2003). During S-phase also the centrioles are duplicated and form daughter centrosomes (Morgan, 2007). Each centrosome consists of a centriole pair and pericentriolar material including the microtubule-nucleatingγ-TuRC (γ-tubulin ring complex) (Scholey et al., 2003). In the second gap phase, G2-phase, the cells grow and prepare for the entry into mitosis. In mitosis (M-phase) the duplicated chromosomes are segregated and equally distributed into two daughter nuclei. Interphase describes the period from one M-phase to the beginning of the next one (Morgan, 2007).

DNA-damage checkpoint (DDC) Spindle assembly

checkpoint (SAC) interphase

S-phase

G1-

phase G

2-phase

M-phase Restriction

point (Start)

G0-phase

Figure 3.1. The phases of the eukaryotic cell cycle.

The eukaryotic cell cycle can be divided into four phases: the G1-phase, S-phase, G2-phase and M-phase (mitosis).

During S-phase the chromosomes are duplicated and subsequently segregated during M-phase. The cell cycle is controlled by three checkpoints (shown in orange), which ensure entry into the cell cycle in the presence of favourable environmental conditions (restriction point), entry into mitosis upon correct DNA replication (DNA- damage checkpoint) and initiate chromosome separation once all chromosomes are attached to microtubules from opposite spindle poles (spindle assembly checkpoint). Modified from (Morgan, 2007).

The sequence of the cell cycle is a one-way route, which is controlled by three checkpoints, points-of-no-return, and regulated by reversible phosphorylations and proteasome dependent proteolysis (Morgan, 1999; Pines & Rieder, 2001)(Figure 3.1). The restriction point or Start is the first checkpoint at the G1/S transition and depending on environmental

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6 3 Introduction

conditions the cells progress and enter the cell cycle. At the G2/M transition the DNA- damage checkpoint controls for correctly replicated chromosomes. Problems or errors in DNA-replication inhibit entry into mitosis (Fletcheret al., 2002; Jinet al., 1996). The spindle assembly checkpoint (SAC) at the metaphase-to-anaphase transition monitors the attachment status of microtubules to kinetochores (Kulukian et al., 2009). The SAC generates an anaphase wait signal to inhibit the E3-ubiquitin ligase APC/C (anaphase promoting complex/cyclosome) (Herzog et al., 2009). How unattached kinetochores activate the checkpoint and how the checkpoint is silenced will be discussed in Section 3.4.

3.1 The phases of mitosis

Mitosis is a self-guarding process that ensures the equal distribution of the duplicated chromosomes into two daughter nuclei (karyokinesis) and with the division of the cytoplasm (cytokinesis) two daughter cells are formed (Pines & Rieder, 2001). Mitosis can be divided into five phases: prophase, prometaphase, metaphase, anaphase and telophase. Morpho- logical differences appearing in the behaviour of the mitotic spindle and the chromosomes can be used to define the different mitotic stages (Figure 3.2). The mitotic phases also differ in their activity profile of cell-cycle regulators, such as Cdk1/cyclin-B1 kinase and the anaphase promoting complex/cyclosome (APC/C), which represent an additional level of discrimination (Morgan, 1999; Pines & Rieder, 2001).

In prophase the replicated chromosomes condense within the nucleus and the two chromatids are held together by the centromere. The duplicated sister centrosomes begin to separate and the formation of a mitotic spindle is initiated (Morgan, 2007). The separated centrosomes move to opposite sites of the nucleus and represent as spindle poles the microtubule-organizing centres (MTOCs) of the forming bipolar spindle. The newly nucleated microtubules remain anchored within the centrosome (Bornens, 2002; Heald & Walczak, 2009). During prophase microtubules extend from each spindle pole to form a radial array or aster. The change in dynamic instability of microtubules results in the disassembly of stable, long interphasic microtubules and the assembly of short, highly dynamic astral microtubules (Rieder & Khodjakov, 2003). Prometaphase starts with the breakdown of the nuclear envelope membrane (NEBD) and the chromosomes are now free in the cell (Mor- gan, 2007). Breakdown of the nuclear membrane is facilitated by Cdk1-mediated phosphorylation of lamins and nuclear pore complexes (Peter et al., 1990). Chromosomes become attached by dynamic microtubules and a mitotic spindle is formed (Gadde & Heald, 2004).

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3 Introduction 7

metaphase interphase

prometaphase anaphase

prophase telophase/cytokinesis

kinetochore chromosome

centrosome cleavage furrow midzone microtubules

interpolar microtubule

kintetochore microtubule astral microtubule

nuclear envelope

Figure 3.2. The phases of mitosis.

Mitosis can be divided into five different phases: prophase, prometaphase, metaphase, anaphase and telophase.

During mitosis the duplicated chromosomes are separated and during cytokinesis the cytoplasm is divided resulting in the formation of two daughter cells with identical number of chromosomes. The chromosomes are shown in grey, the kinetochores in red and the microtubules in black. Modified from (Morgan, 2007).

The dynamic properties of microtubules and the function of motor proteins as well as microtubule-associated proteins (MAPs) are required for the formation and function of the mitotic spindle (Scholey et al., 2003). In metaphase all chromosomes are aligned at the spindle equator and form a so called metaphase plate (Gadde & Heald, 2004). Degradation of the anaphase inhibitor securin results in the activation of the protease separase. In anaphase cohesin, which holds the sister chromosomes together, is cleaved in the Scc1 subunit by separase and the two sister chromatids are separated. In anaphase A the separated sister chromatids are segregated and move to opposite spindle poles, as the pulling forces at the sister-kinetochores do not oppose each other (Koshland et al., 1988). In anaphase B the spindle elongates and the poles move further away from each other and thereby pull the chromatids to the attached spindle poles resulting in their final segregation (Gadde &

Heald, 2004; Morgan, 2007). Spindle elongation is mediated by pushing forces on midzone microtubules and pulling forces on astral microtubules (Scholeyet al., 2003). During

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8 3 Introduction

telophase, the mitotic spindle dissembles, chromosomes de-condense again and a new nuclear membrane is formed around each set of chromosomes.

During cytokinesis the two daughter nuclei and cytoplasmic organelles are equally distributed by physical separation. A contractile actomyosin ring and anti-parallel midzone microtubules (central spindle) are formed between the two daughter cells. Upon contrac- tion the actomyosin ring pulls the membrane inwards and a cleavage furrow is generated at which the cytoplasm is divided. The cleavage furrows narrows with compaction of the midzone microtubules and the midbody is formed, which still connects the daughter cells.

Cytokinesis ends with abscission, which results in the physical separation of the two daughter cells (Chenet al., 2012).

3.1.1 The assembly of a bipolar spindle

The assembly of the mitotic spindle is facilitated by the centrosomes, which mature and are now capable of nucleating more microtubules as in interphase. In addition the dynamic instability of microtubules increases. The assembly of the biopolar spindle during mitosis is mediated by changes within the characteristics and properties of the microtubules.

In interphase the dynamic of microtubules is on a basal level. An increase in the catastrophe frequency and a decrease in the rescue frequency results in the degradation of interphasic microtubules (Gadde & Heald, 2004). Catastrophe describes the transition from microtubule growth to shrinkage and rescue defines the opposite process (Morgan, 2007).

With the entry into prophase, the growth-rate of the plus-tips of microtubules increases and stays constantly on a high level during mitosis. In addition the microtubule dynamic instability increases in prophase and is increased even further upon breakdown of the nuclear envelope membrane. The assembly of the mitotic spindle depends on three characteristics of microtubules: intrinsic polarity of microtubules, dynamic instability and that they serve as tracks for motor proteins. The polarity of the microtubules is determined by the structural difference in the microtubules ends. The dynamic instability describes the stochastic changes between polymerization (growth) and de-polymerization (shrinkage) of the microtubule ends (Gadde & Heald, 2004; Wittmannet al., 2001). Kinesins are microtubule-binding motor proteins and members of this family play a role in the organization of the spindle, positioning of the chromosomes and de-polymerization of microtubules. They can regulate the organization and stability of the microtubule-arrangement (Morgan, 2007) or they position mitotic chromosomes by stimulating microtubule dynamics (Jaqamanet al., 2010). In the anti-parallel microtubule-arrangement of the mitotic spindle the minus-ends of microtubules are anchored within the spindle poles and their plus-tips grow in the direction of the

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3 Introduction 9

chromosomes (Wittmannet al., 2001). As microtubule growth is favoured in the proximity of chromosomes the interaction of kinetochores with microtubules is facilitated (Heald &

Walczak, 2009). The mitotic spindle consists of three microtubule populations, which differ in their morphology. Kinetochore-microtubules attach with their plus-tips to the outer region of the kinetochore and connect the chromosomes with the spindle poles (Wittmann et al., 2001)(see Subsection 3.2.1 and Figure 3.4). The interpolar-microtubules from opposite spindle poles overlap anti-parallel and facilitate the formation of a bipolar spindle structure (Scholeyet al., 2003). They are required for the stabilization of the bipolarity of the spindle during prometaphase and metaphase. In anaphase they enable the separation of the spindle poles. Astral microtubules extend from the spindle poles into the cytoplasm, where they attach to the cell cortex and take part in the separation of the spindle poles and in the anchorage and positioning of the mitotic spindle (Gadde & Heald, 2004; Morgan, 2007).

3.1.2 Cyclin-dependent kinase 1 (Cdk1)

Cyclin-dependent kinases (Cdks) are heterodimeric protein kinases, which get activated through binding to their co-activators the cyclins (Meyerson et al., 1991). They represent the core of the cell cycle control system and allow the regulation of the different cell cycle stages depending on their association with different types of cyclins. Mitosis is regulated by Cdk1 in complex with cyclin-A or cyclin-B (Draetta et al., 1989). Cyclin-A2 is the main A-type cyclin in somatic cells (Sweeneyet al., 1996). Cdk1/cyclin-A2 is active from late G2 until prometaphase. Its degradation starts with nuclear envelope breakdown and cyclin-A2 is absent upon chromosome alignment (Figure 3.3). Cyclin-A2 is degraded by APC/C^Cdc20 in a D-box and SAC independent manner (den Elzen & Pines, 2001; Furunoet al., 1999), as cyclin-A2 it able to compete with the MCC (mitotic checkpoint complex) for Cdc20 (cell de- vision cycle 20) binding (Di Fiore & Pines, 2010). Cdk1/cyclin-A2 regulates initiation of chromosome condensation, the breakdown of the nuclear envelope membrane and the nuclear accumulation and activation of cyclin-B1 (Furuno et al., 1999; Gong & Ferrell, 2010; Gong et al., 2007). Recently it was shown that cyclin-A2 degradation in prometaphase is required for the switch from labile kinetochore-microtubule attachments in prometaphase to stable kinetochore-microtubule attachments in metaphase, allowing error correction (Kabeche &

Compton, 2013). These results can explain a previous observation that cyclin-A2 levels correlate with delay in chromosome alignment (den Elzen & Pines, 2001).

Cdk1/cyclin-B1 is the key-regulator of mitosis and also called MPF (maturation promoting factor). Cdk1/cyclin-B1 promotes chromosome condensation, disassembly of the nuclear

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10 3 Introduction

envelope membrane and mitotic spindle assembly (Bentley et al., 2007). Cyclin-B1 activation and cyclin-B1 degradation regulate the entry and exit from mitosis (Clute & Pines, 1999) (Figure 3.3). In early prophase cyclin-B1 localizes in the cytoplasm and accumulates in the nucleus in late prophase upon phosphorylation at its N-terminus (Hagtinget al., 1998;

Pines & Hunter, 1991). Cdk1/cyclin-B1 stays inactive until the phosphatase Cdc25 (cell division cycle 25) also enters the nucleus resulting in the de-phosphorylation and activation of Cdk1/Cyclin-B1, which is a pre-requisite for entry into mitosis (Pines & Rieder, 2001). With the breakdown of the nuclear envelope membrane in prometaphase cyclin-B1 localizes at unattached kinetochores, spindle microtubules and centrosomes (Bentleyet al., 2007; Pines

& Hunter, 1991) (Figure 3.3). Kinetochore localization of cyclin-B1 is attachment-dependent and is lost in metaphase at fully congressed kinetochores (Bentleyet al., 2007), which is mediated by dynein/dynactin-mediated stripping (Chenet al., 2008)(Figure 3.3). Cyclin-B1 starts to disappear with chromosome alignment in metaphase and is absent with anaphase onset. This disappearance correlates with SAC inactivation and reflects its degradation by the 26S proteasome, resulting in the inactivation of Cdk1 (Clute & Pines, 1999).

metaphase anaphase

prometaphase

Cdk1/cyclin-A2

Cdk1/cyclin-B1 cytoplasm

PP1α/γ cytoplasm kinetochores chromosomes

cytoplasm cytoplasm/kinetochores

Figure 3.3. Activity profiles of Cdk1 and PP1 during mitosis.

Cdk1/cyclin-A2 (purple) is active from entry into mitosis until prometaphase, where cyclin-A2 is degraded by the APC/C in a checkpoint independent manner. Cdk1/cyclin-B1 (blue) is active from prophase until its checkpoint dependent degradation by the APC/C at metaphase-to-anaphase transition. Dependent on its recruitment factors PP1α/γ(green) accumulate at the kinetochores in metaphase and re-localizes to the chromosomes in anaphase.

Modified from (Bollenet al., 2009).

3.1.3 Protein phosphatase 1 (PP1)

There are two important phosphatases involved in mitosis called protein phosphatase 2A (PP2A) and protein phosphatase 1 (PP1). Both Ser/Thr phosphatases gain diversity through interaction with regulatory subunits to form dimeric (PP1) or in addition with a scaffold subunit trimeric (PP2A) holoenzymes (Bollenet al., 2009). Recently it was shown that PP2A in complex with B56 localizes to centromeres of uncongressed chromosomes in prometaphase and that this localization is attachment-dependent. Therefore it is proposed that PP2A/B56 is required to establish stable kinetochore-microtubule attachments during prometaphase and to facilitate chromosome alignment (Foleyet al., 2011).

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3 Introduction 11

Mammalian cells express three different catalytic isoforms of PP1: α,βandγ (Barkeret al., 1990, 1993, 1994). During interphase all three isoforms localizes in the nucleus and cytoplasm. PP1γ also localizes in the nucleoli, whereas PP1α and PP1βare excluded from them (Andreassen et al., 1998; Trinkle-Mulcahy et al., 2001). During metaphase PP1α and γ localize at the outer kinetochores and thereby flank the kinase Aurora-B, which localizes to the inner centromere region (Figure 3.3). Upon treatment with nocodazole to de-polymerize the microtubules or with taxol to stabilize the microtubules, PP1γ still localizes to the kinetochores. FRAP-analysis in the presence of nocodazole or taxol confirmed that the recruitment of PP1 to the kinetochores is independent of an active transport mechanism along the microtubules (Trinkle-Mulcahy et al., 2003). With anaphase onset PP1γ re-localizes to the chromosomes and in telophase to the cleavage furrow and the midbody (Figure 3.3).

This is in contrast to PP1α, which is excluded from the chromatin but still localizes to the midbody in telophase (Trinkle-Mulcahyet al., 2003, 2006).

The dynamic localization pattern, substrate specificity and activity of PP1 is regulated by interaction with specific subunits, called PIPs (PP1 interacting proteins) (Bollenet al., 2009).

PP1-targeting to outer kinetochores can be mediated by various interaction partners like KNL1, CENP-E or Sds22, but the complexes exhibit different functions. Upon depletion of KNL1 or a KNL1 version mutated in the PP1-binding motif both PP1αandγ are absent from kinetochores, indicating that KNL1 is required for the bulk recruitment of PP1 to kinetochores in human cells. KNL1 recruits PP1 to counteract Aurora-B activity, which is required to stabilize kinetochore-microtubule attachments (Liu et al., 2010a)(see Section 3.5 and Figure 3.7). The plus-end directed kinesin CENP-E (Centromere protein E) recruits PP1 to allow the end-on attachment of congressed chromosomes (Kimet al., 2010b) (see Subsec- tion 3.3.2). PP1 bound to Sds22 inactivates a small pool of kinetochore localized Aurora-B by antagonizing its auto-phosphorylation (Posch et al., 2010). However, a recent publica- tion came to the opposite conclusion: they suggest that Sds22 inhibits kinetochore-bound pool of PP1 and thereby the de-phosphorylation of Aurora-B. Inhibitor-3 (I-3) binds via the RVXF-motif to PP1 in complex with Sds22 and thereby prevents interaction with KNL1. They postulate that the complex formation is required to stabilize PP1 and would allow activation by binding of metal ions. Upon dissociation of Sds22 and I-3, active PP1 can bind via the RVXF-motif to KNL1 and is now able to de-phosphorylate and inactivate Aurora-B (Eiteneuer et al., 2014). This is in agreement with a previous study that showed the formation of this ternary complex but did not further investigate the functional relevance (Lesage et al., 2007). Repo-Man (recruits PP1 onto mitotic chromatin at anaphase) mediates the afore mentioned re-localization of PP1 to chromosomes at the metaphase-to-anaphase transition

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12 3 Introduction

(Trinkle-Mulcahyet al., 2006).

During mitosis PP1α/γ is kept inactive by Cdk1/cyclin-B1 mediated phosphorylation at T320 (Dohadwalaet al., 1994; Kwon et al., 1997) and binding to Inhibitor-1 (I-1) phosphorylated by PKA (protein kinase A) (Endoet al., 1996). By binding via the RVXF-motif Inhibitor-1 acts as a pseudo substrate. At mitotic exit Cdk1-activity drops and auto de-phosphorylation at T320 results in partial activation of PP1. PP1 then de-phosphorylates Inhibitor-1 (I-1), which results in its dissociation and the full activation of PP1 (Kwonet al., 1997; Wuet al., 2009). Additionally, Inhibitor-2 (I-2) anchors PP1 by binding to the RVXF- and SILK-motifs and inhibits the catalytic activity of PP1 by blocking its active site and displacing metal ions (Hurleyet al., 2007). Upon phosphorylation I-2 exhibits a conformational change in its inhibitory helix and is now unable to block the active site of PP1. This results in the partial activation of the phosphatase and PP1 is now able to de-phosphorylate I-2. I-2 dissociation results in full activation of PP1 regarding other substrates but the exact mechanism is not known. (Hemmingset al., 1982; Hurleyet al., 2007).

3.2 Kinetochore-microtubule interactions

3.2.1 The structure of the kinetochore

Kinetochores are multi-protein complexes that assemble in prophase at the centromeres of chromosomes and thereby physically link centromeric DNA to the plus-tips of spindle microtubules (Cheeseman et al., 2006; DeLuca et al., 2006). Their key functions are the end-on attachment of microtubules to generate or transduce forces that are required for chromosome movement and to generate spindle assembly checkpoint signals to prevent missegregation (DeLuca et al., 2005). Based on transmission electron microscopy studies and immunofluorescence analysis the kinetochore structure can be divided into the inner kinetochore (constitutive centromere associated network, CCAN) and outer kinetochore (KNL1/Mis12/Ndc80-network, KMN network) (Brinkley & Stubblefield, 1966; Cheese- manet al., 2006; DeLucaet al., 2005; Suzukiet al., 2011; Wanet al., 2009). In the absence of microtubule attachment the outer kinetochores are decorated by a fibrous corona con- taining proteins for microtubule attachments as well as components of the spindle assembly checkpoint (Cookeet al., 1997; Rieder & Salmon, 1998; Williamset al., 1996).

Centromeres are chromosomal regions that are specified by the presence of nucleosomes that contain the Histone H3 variant CENP-A (centromeric protein A) (Palmer et al., 1991;

Sullivan et al., 1994). The assembly of the vertebrate kinetochore at the centromeric region is mediated by two parallel pathways (Figure 3.4): CENP-C interacts directly with

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3 Introduction 13

CENP-A nucleosomes (Carroll et al., 2010; Kato et al., 2013) and binds the Nnf1 subunit of the Mis12-complex (Gascoigne et al., 2011; Petrovic et al., 2010; Screpanti et al., 2011). The Nsl1 subunit of Mis12-complex in turn interacts with Spc24/Spc25 heads of the Ndc80-complex and KNL1. Thereby, the Mis12-complex connects the inner and outer kinetochore (Petrovic et al., 2010, 2014) and positions them correctly to stimulate their microtubule-binding activities (Welburnet al., 2010). The second pathway is mediated by CENP-T in complex with CENP-W/-X/-S, which forms a nucleosome-like structure and binds and wraps centromeric DNA (Horiet al., 2008; Nishinoet al., 2012). The N-terminal region of CENP-T interacts with Spc24/Spc25 (Gascoigneet al., 2011; Nishinoet al., 2013) and the interaction of Spc24/Spc25 with the Mis12-complex or CENP-T is mutually exclusive (Nishino et al., 2013).

Whereas CENP-A, CENP-C and CENP-T (CCAN proteins) localize at the centromere during the cell cycle, the Mis12-complex and KNL1 localizes to centromeres during S-phase and the Ndc80-complex is recruited in late G2 (Gascoigne & Cheeseman, 2013). Kinetochore assembly is regulated during the cell cycle by cytoplasmic localization of the Ndc80-complex during interphase. Upon nuclear envelope breakdown the Ndc80-complex can assemble on the centromere (Gascoigne & Cheeseman, 2013) and in addition the Cdk1 mediated phosphorylation of CENP-T enhances its binding affinity to the Ndc80-complex (Gascoigneet al., 2011; Nishinoet al., 2013).

Mis12 complex

Ndc80 complex KMN

C

Dsn1 Mis12 Nsl1 Nnf1

A KNL1 H3

H3 T W X S

H3

checkpoint signalling

microtubule Hec1Nuf2 Spc25

Spc24

Hec1Nuf2 Spc25

Spc24

CCAN

Figure 3.4. Assembly of the outer kinetochore.

The constitutive centromere associated network (CCAN) assembles on nucleosomes composed of CENP-A. CENP- C and CENP-T interact with the Mis12-complex and Ndc80-complex, respectively. For simplicity only the CENP proteins interacting with components of the KMN are depicted. The Mis12-complex binds to KNL1 and the Ndc80- complex, which both mediate microtubule attachment. The microtubule-binding of KNL1 is not shown. In addition KNL1 recruits Bub3/Bub1 and Bub3/BubR1 as well as Zwint-1 for checkpoint signalling. Modified from (Corbett &

Desai, 2014).

The KMN network is the core microtubule-attachment site and forms the bridge between the centromere associated proteins and the plus-tips of microtubules (Figure 3.4). It is composed of KNL1, the Mis12-complex (Nnf1, Mis12, Dsn1 and Nsl1) and the Ndc80-complex (Ndc80/Hec1, Nuf2, Spc24 and Spc25) (Cheesemanet al., 2006; Petrovicet al., 2010).

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14 3 Introduction

The Ndc80-complex is essential for kinetochore-microtubule interactions as it represents the linker that has both microtubule and kinetochore binding sites (Cheesemanet al., 2006;

DeLucaet al., 2006). This complex is composed of four proteins: Ndc80/Hec1, Nuf2, Spc24 and Spc25. Hec1/Nuf2 and Spc24/Spc25 form heterodimers with globular heads at each end and are separated by a large coiled coil region. The C-terminal coiled coils of Hec1/Nuf2 interact with the N-terminal coiled coils of Spc25/Spc24 (Ciferri et al., 2005; Wei et al., 2005). The outer head (Hec1/Nuf2) harbours two calponin homology (CH) domains to bind directly to microtubules (Ciferri et al., 2008; Weiet al., 2005). The affinity to microtubules is increased by an unstructured, highly positively charged N-terminal tail of Hec1. The tail mediates microtubule-binding by electrostatic interactions (Ciferriet al., 2008; Guimaraes et al., 2008; Miller et al., 2008) and forms intermolecular contacts to facilitate oligomerization of Ndc80-complexes to allow cooperative association with the microtubule lattice (Alushin et al., 2010; Ciferri et al., 2008). It is proposed that the Hec1/Nuf2 heads form initial, low affinity contacts with microtubules and that the N-terminal tail promotes stable end-on attachment. Phosphorylation of the N-terminal tail by Aurora-B reduces the microtubule-binding of Hec1in-vitro (Cheeseman et al., 2006; Ciferri et al., 2008) and leads to destabilization of kinetochore-microtubule attachments (DeLuca et al., 2011; Guimaraes et al., 2008; Milleret al., 2008).

Recently it was shown that the N-terminal tail harbours two phosphorylation clusters at the beginning (aa1-20) and the end of the tail (aa41-80), close to the CH domain of Hec1.

Aurora-B mediated phosphorylation in the first cluster alters mainly the tail-mediated oli- ogmerization. Whereas phosphorylation in the second cluster leads to a reduction in microtubule-binding and oligomerization (Alushin et al., 2012). Phosphorylation results in a reduction in microtubule plus-tip stabilization which leads to the disassembly of the bound microtubules and this results finally in their detachment (Umbreit et al., 2012). As previ- ously proposed (Alushinet al., 2010) upon initial microtubule-binding the Hec1/Nuf2 heads are separated from Aurora-B phosphorylation due to increased intra-kinetochore stretching (Maresca & Salmon, 2009). Upon de-phosphorylation of the N-terminus high-affinity Ndc80-complex oligomers are formed into a linear array enabling coupled binding along protofilaments (Alushinet al., 2010, 2012).

Aurora-B phosphorylates both clusters of the N-terminal tail in prometaphase. Upon attachment, phosphorylation sites in the first cluster remain at an intermediate level and phosphorylation in the second cluster is absent (DeLucaet al., 2011). It is proposed that upon attachment the phosphorylation in the second cluster decreases resulting in microtubule binding and cooperative interactions by oligomerization, which prevents the accessibility

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3 Introduction 15

of Aurora-B to these sites. Therefore the phosphorylation-status of Hec1 determines the stability of kinetochore-microtubule interactions. High phosphorylation level in prometaphase promote labile interactions allowing detachment and reattachment. In metaphase phosphorylation is low resulting in the establishment of stable chromosome bi-orientation and silencing of the checkpoint (DeLucaet al., 2011).

Per se the Ndc80-complex has a weak microtubule-binding affinity, which is increased upon interaction with KNL1 and the Mis12-complex (Cheesemanet al., 2006). The Mis12-complex forms a rod-like structure consisting of the Nnf1, Mis12, Dsn1 and Nsl1 subunits (Klineet al., 2006; Petrovicet al., 2010). The Mis12-complex interacts with KNL1 via Nsl1 and Dsn1 (Kiy- omitsuet al., 2007). Dsn1 is regulated by Aurora-B phosphorylation, which influences the microtubule-binding affinity of the KMN network (Welburnet al., 2010). The Mis12-complex links the inner and outer kinetochore (Petrovic et al., 2010). KNL1 (kinetochre null, also called Blinkin, Bub-linking kinetochore protein) (Desaiet al., 2003; Kiyomitsu et al., 2007) contributes to microtubule binding via its N-terminal microtubule-binding region (Cheese- man et al., 2006; Welburn et al., 2010) and is required for checkpoint activation and silencing (Cheesemanet al., 2008; Kiyomitsuet al., 2007). How KNL1 plays a role in checkpoint activation and silencing is described in Section 3.4. Additionally, the N-terminus of KNL1 harbours PP1-binding motifs (Liuet al., 2010a). The C-terminus of KNL1 consists of a globular domain, which mediates direct interaction with the Nsl1 and Dsn1 subunit of the Mis12-complex (Kiyomitsuet al., 2007; Petrovicet al., 2010, 2014).

The Ndc80-complex is only able to bind to the straight microtubule lattice. But upon microtubule de-polymerization tubulin adopts a bent conformation and the microtubule peels backward and shows a curved conformation (Mandelkow et al., 1991). To couple chromosome movement with microtubule de-polymerization additional factors must exist that maintain processive kinetochore-microtubule attachment under these conditions. In verte- brates this is mediated by the Ska (spindle and kinetochore associated)-complex, which is composed of three subunits (Ska1, Ska2 and Ska3), whereas each is present in two copies (Gaitanos et al., 2009; Hanisch et al., 2006). The Ska-complex binds directly to microtubules and is able to form oligomers (Schmidt et al., 2012). This enables the Ska-complex to bind to curved protofilaments (depolymerizing microtubules) and to diffuse along the the microtubule lattice (Schmidtet al., 2012; Welburnet al., 2009). The Ska-complex cooperates with the Ndc80-complex to couple chromosome movement to dynamic microtubule plus-tips (Gaitanos et al., 2009; Schmidt et al., 2012; Welburn et al., 2009) and is required to maintain stable end-on kinetochore-microtubule attachments (Gaitanos et al., 2009; Hanischet al., 2006; Welburnet al., 2009). Aurora-B mediated phosphorylation of the

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16 3 Introduction

Ska-complex prevents its interaction with the subunits of the KMN network. Consequently phosphorylated Ska-complex is not recruited to kinetochores and stable kinetochore-microtubule attachments are not established (Chanet al., 2012; Schmidtet al., 2012).

3.2.2 Initial lateral attachment and conversion to stable end-on attachment Attachment of chromosomes to microtubules is a stochastic process and chromosomes indi- vidually follow transitions from lateral to end-on attachment, bi-orientation and alignment at the spindle equator. Consequently chromosomes exist in different states during prometaphase and metaphase as the attachment and alignment of chromosomes are not a coordinated mechanism (Skibbenset al., 1993) (Figure 3.5).

0 0

500 1000 1500 2000 2500

5 10 15 20 25

Time [sec]

Distance [µm]

(c) (d) (e)

(b) (a)

(a)

(b) (c)

(d) (e)

Figure 3.5. Chromosome movement during mitosis.

The left side shows the kinetochore-microtubule attachment status and the distance versus time plot on the right side illustrates the movement of the indicated chromosome. The coloured arrows indicate the direction of the movement of the depicted chromosome. Lateral attached kinetochores glide poleward (a, purple) and become end-on attached. These mono-oriented chromosomes oscillate around the spindle pole (b, green). During chromosome congression kinetochores are bi-oriented and move towards the spindle equator (c, yellow). Bi- oriented chromosomes oscillate around the spindle equator (d, orange) and move poleward upon separation at anaphase onset (e, blue). Modified from (Khodjakovet al., 1999; Skibbenset al., 1993).

After nuclear envelope breakdown, astral MTs from one pole, selected by chance, associate laterally with kinetochores (Figure 3.5(a)). In this lateral attachment the outer plate of the kinetochore binds to the microtubule lattice, which provides a much larger contact surface compared with microtubule plus-tips and is therefore easier to capture at more positions, resulting in an efficient first encounter (Hayden et al., 1990; Magidsonet al., 2011; Rieder

& Alexander, 1990). The minus-end directed dynein transports them along the microtubule lattice towards the attached spindle pole in a process called lateral gliding or sliding (Yang et al., 2007). It is proposed that the high microtubule density at the spindle poles facilitates conversion from lateral attachment into stable, end-on attachment a process called end-on conversion. Recent studies showed that the end-on conversion is a gradual process that depends on the kinesin-13 member MCAK (mitotic centromere-associated kinesin) and CENP-E. CENP-E tethers the lateral kinetochore to the microtubule wall, while the kineto-

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3 Introduction 17

chore is brought in close proximity to the microtubule plus-tip. Whether this is achieved by CENP-E mediated gliding of the lateral attached kinetochore is not clear. After partial end-on attachment is formed, MCAK is required to remove existing lateral attachments and allow full end-on attachment mediated by Hec1 (Shrestha & Draviam, 2013).

Because kinetochores stabilize their associated microtubules, additional MT-attachments and the formation of a kinetochore-fibre (k-fibre), bundles of 25-30 microtubules, is promoted (Hayden et al., 1990; McEwen et al., 1997). The formation of kinetochore-fibres can be promoted during lateral and end-on attachment. End-on attachment is more stable and thus allows maintaining the association with kinetochores and the coupling of chromosome movement to the changes in the dynamic behaviour of microtubules. End-on attachments can also be formed directly during initial capture of kinetochores by microtubules or during formation of a k-fibre. From prometaphase to metaphase the number of kinetochore-microtubules increases and this increase is mediated by tension. The increase in kinetochore-microtubule interactions results in the stabilization of the attachment (King

& Nicklas, 2000).

3.3 Chromosome movement during mitosis

The movement of chromosomes is based on the dynamic behaviour of microtubules (MTs), structural kinetochore components as the Ndc80-complex and several molecular motors (Heald & Walczak, 2009). The dynamic behaviour of microtubules is mediated by the dynamic instability of kinetochore-microtubule plus-tips (Koshland et al., 1988; Mitchison et al., 1984) and to a lesser extend by poleward microtubule flux (Mitchison, 1989; Zhai et al., 1995). Dynamic instability describes the property of microtubules to switch between growth and shrinkage (Mitchison et al., 1984) and upon attachment to kinetochores, this correlates with conformational changes from straight (growth) to curved/peeling (shrinkage) protofilaments (Mandelkowet al., 1991). The de-polymerization of kinetochore-microtubule plus-tips generates a poleward force that moves chromosomes during oscillations in metaphase and separation of chromosomes in anaphase (Grishchuket al., 2005; Koshland et al., 1988). As kinetochores stay attached to microtubule plus-tips while they shrink or grow chromosomes can move back and forth along the microtubules (Grishchuk et al., 2005; Koshlandet al., 1988). Large conformational changes (curling protofilaments) due to microtubule de-polymerization at the plus-tips of microtubules lead to a pulling forces that drive kinetochore movement (Grishchuk et al., 2005; Koshland et al., 1988). The curling can be regulated by the affinity of kinetochore components to it, the length of the curls or the overall shape of the plus-tips (Asbury et al., 2011). Microtubule de-polymerization

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18 3 Introduction

drives poleward movement by two complementary mechanisms (Koshland et al., 1988).

The Ndc80-complex attaches to the microtubule lattice and upon de-polymerization at the plus-tips the attached complex diffuses along the lattice towards the spindle poles (Pow- ers et al., 2009). The Ska-complex binds to curling protofilaments and further peeling of the protofilaments pushes the kinetochore to the spindle-pole (Joglekar & Hunt, 2002; Man- delkowet al., 1991). This allows coupling of de-polymerization at kinetochore-microtubule plus-tips to poleward chromosome movement (Schmidtet al., 2012; Welburnet al., 2009).

During poleward microtubule flux tubulin is incorporated at the plus-tips of microtubules and moves through the microtubules and is released at their minus-end. This results in pulling of the microtubules with the attached chromosomes to the spindle poles (Mitchison, 1989). It was recently shown that microtubule flux is not required for chromosome movement in human cells, which is mainly mediated by the kinetochores (Ganemet al., 2005).

Kinetochores are located back-to-back at opposite sites of the centromere to facilitate their bipolar attachment and equal segregation of the chromatids (Hauf & Watanabe, 2004). Co- hesin physically connects sister centromeres and thereby facilitates bi-orientation and the establishment of tension (Vagnarelliet al., 2004). By interacting with microtubules, kinetochores define the direction of the chromosome movement and the generation of the force to move the chromosomes (DeLucaet al., 2005).

3.3.1 Chromosome movement of mono-oriented chromosomes

End-on attached, mono-oriented chromosomes oscillate around their attached spindle pole due to pulling (poleward) and pushing (anti-poleward) forces (Figure 3.5(b)). Pulling forces are generated at the kinetochore due to microtubule de-polymerization. Pushing forces are mediated by polar ejection forces (PEFs). If the opposing forces are balanced the mono-oriented chromosomes become stably positioned (Ault et al., 1991; Levesque & Compton, 2001; Rieder et al., 1986). Oscillations appear as triangular wave in a distance versus time plot and are mediated when the kinetochore switches between both distinct states (Figure 3.5(b)). In the de-polymerization state the kinetochore is moved poleward to the attached spindle pole. Depletion of MCAK affects chromosome congression and it is postulated that it uses its depolymerase activity to regulate kinetochore-microtubule dynamics to facilitate chromosome movement (Zhu et al., 2005). This results in shortening of the k-fibre and pulling chromosomes towards the attached spindle pole (Wordemanet al., 2007). The rate at which microtubules within a k-fibre de-polymerize is the limiting factor: a kinetochore can move poleward only as fast as its microtubules shorten (Rieder &

Alexander, 1990). Because k-fibre microtubules are mostly anchored with their minus-ends

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3 Introduction 19

in the spindle-pole, the chromosomes can easily move from side to side (Heald & Walczak, 2009). In the polymerization state the kinetochore is pushed away from the pole by PEFs.

PEFs can be generated by two different mechanisms. The polymerization of microtubules can mechanically push the chromosomes away from the pole toward the spindle center.

Or chromosome-arm associated motor proteins, called chromokinesins, slide the chromosomes along spindle-MTs towards their plus-ends at the spindle equator (Ault et al., 1991;

Ke et al., 2009). As PEFs depend on microtubule density and size of chromosome arms (Keet al., 2009; Rieder & Salmon, 1994), they are high at the spindle equator and flatten towards the spindle pole.

Because k-fibres are microtubule-bundles consisting of 25-30 microtubules, the state of the microtubule end must be partially synchronized in order to allow switching between both states. While individual microtubules within a k-fibre can differ in their dynamic behaviour (VandenBeldtet al., 2006) the mechanism how they are coordinated to allow force production and chromosome movement is not known.

3.3.2 Modes of chromosome congression

Congression describes the process by which chromosomes attach to spindle-MTs and move or “congress“ to the equator of the cell. It depends on microtubule dynamics and kinesin- motor activities that cooperate to align the chromosomes at the spindle equator. Once all chromosomes have congressed, the cell is in metaphase and the chromosomes lie in the metaphase plate (Heald & Walczak, 2009). Chromosome congression is mediated by de-polymerization of kinetochore-microtubules and polar ejection forces, that are low at the spindle pole and high at the spindle equator. These opposing forces act to align bi-oriented chromosomes at the spindle equator (Ke et al., 2009; Rieder & Salmon, 1994). If chromosomes are attached to microtubules from the two opposite poles they become bi-orientated (Figure 3.5(c)). It is not clear whether bi-orientation is necessary for congression or if chromosome congression promotes bi-orientation. There are several models which explains how mono-oriented chromosomes (monotelic attached) are converted into bi-oriented chromosomes (amphitelic attached). Mono-orientated chromosomes can either be attached in end-on manner to microtubules emanating from a single spindle pole or be laterally attached to a microtubule. In an amphitelic attachment the kinetochores are attached to microtubules emanating from both spindle poles.

In the classical “search and capture“ model chromosomes congress after bi-orientation.

This model describes bi-orientation as a result of MT-nucleation from the centrosomes (Hayden et al., 1990; Rieder & Alexander, 1990). As microtubules alter between phases

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20 3 Introduction

of growth and shrinkage they search for the kinetochores (Mitchison et al., 1984). Spin- dle microtubules are captured and stabilized by kinetochores to create k-fibres (Hayden et al., 1990; McEwenet al., 1997). The chromosomes move towards the spindle equator by microtubule de-polymerization at the so called leading kinetochore, which results in poleward pulling (McEwen et al., 1997). This model is limited in time, because this process of mono-orientated chromosomes to become bi-oriented is very slow. Consequently the congression of the 96 human chromosomes would require several hours. But chromosomes become fully matured and congressed in 15 to 20 minutes, which means that other pathways contribute to this process (Wollmanet al., 2005).

The second model is based on the kinetochore-mediated k-fibre formation. Here MTs are nucleated from the kinetochores (Witt et al., 1980). The minus-ends of these k-fibre MTs encounter other spindle-MTs and are transported poleward by minus-end directed dynein.

These motor proteins slide the k-fibre along pre-existing spindle MTs and finally their minus ends are incorporated into spindle microtubules. Similar to the search and capture model, attachment of both kinetochores to k-fibres is required for congression (Khodjakov et al., 2003). This model seems to be more universal because it explains how k-fibres can be generated in cells lacking centrosomes.

In 2006 Kapoor et al. (Kapoor et al., 2006) showed that mono-orientated chromosomes can also congress. The plus-end directed motor CENP-E walks along already formed k-fibres, which are attached to bi-oriented chromosomes. Whereas the trailing kinetochore is end-on attached to kinetochore-microtubules the leading kinetochore is laterally attached to pre-existing kinetochore-fibre. CENP-E mediates the gliding of these mono-orientated chromosomes towards the center of the spindle. Here, interaction with spindle microtubules from opposite poles and thus bipolar attachments are more likely to occur. It is proposed that the probability to position mono-oriented chromosomes at the spindle equator increases with the establishment of kinetochore-fibres which act as tracks (Kapoor et al., 2006). Recently it was shown that CENP-E mediated congression of mono-oriented chromosomes is regulated by Aurora-A/B and PP1. Aurora A/B phosphorylation reduces microtubule-binding affinity of CENP-E and thereby its processivity to ensure that CENP-E binds and walks selectively to/along kinetochore-microtubules, which ensures congression of mono-oriented, polar chromosomes (Kimet al., 2010b). In addition, as shown for KNL1 (Liu et al., 2010a), phosphorylation of CENP-E by Aurora-A/B prevents binding to PP1. It is hypothesized that CENP-E itself recruits the counteracting phosphatase PP1 to allow the establishment of stable end-on attachment of those chromosomes congressed by CENP-E and the formation of bi-orientation (Kimet al., 2010b). It is hypothesized that the flexible

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3 Introduction 21

and long coiled coil region of CENP-E facilitates initial capturing and might also result in the formation of erroneous kinetochore-microtubule attachments. In this last model congression can occur without bi-orientation and may serve to facilitate bipolar attachments.

Consequently this model describes chromosome congression as a cooperative process to ensure that they are all aligned before segregation. Therefore mono-oriented chromosomes exhibit a direct movement towards the spindle equator (Kapoor et al., 2006). It was also shown that CENP-E does not contribute to kinetochore-sliding on microtubule plus-tips after end-on attachment and alignment to the metaphase plate (Jaqamanet al., 2010).

3.3.3 Sister-kinetochore oscillations

Sister-kinetochore oscillations are mediated by switching between poleward and anti-poleward chromosome movements. Based on their position during chromosome oscillations the kinetochore that is moving towards its attached spindle pole is called leading kinetochore and in an active state. Its sister-kinetochore is called trailing or lagging kinetochore, pulled by its sister and in a passive state (Khodjakov & Rieder, 1996; Skibbenset al., 1993; Waters et al., 1996). Skibbens, et al. (Skibbens et al., 1993) postulated that during congression, chromosomes do not oscillate as their anti-poleward movement is of longer duration resulting in the net chromosome movement towards the spindle equator. However, Khodjakov, et al. (Khodjakov & Rieder, 1996) and Waters, et al. (Waters et al., 1996) showed by laser-surgery experiments and video microscopy studies that in vertebrate cells congress- ing chromosomes exhibit short oscillatory movements towards the proximal pole before they align at the metaphase plate (Khodjakov & Rieder, 1996; Waters et al., 1996)(Fig- ure 3.5(c)). Upon cutting with a leaser beam at the centromere between a bi-oriented, congressed sister-kinetochore the leading kinetochore is moving towards the attached spindle pole, while the lagging kinetochore stops movement (Khodjakov & Rieder, 1996).

Chromosomes continue to oscillate around the metaphase plate after they reached the spindle equator (Jaqaman et al., 2010; Skibbens et al., 1993), this requires end-on kinetochore- microtubule attachments and is mediated by microtubule dynamics, which are modulated by tension (Jaqaman et al., 2010; Wan et al., 2012)(Figure 3.5(d)). It is also observed that bi-oriented chromosomes exhibit regular oscillations around the metaphase plate with different amplitudes and consequently some appear to move whereas others seem motionless (Khodjakovet al., 1997).

Oscillations are defined as directional and abrupt switches of chromosomal motion, also termed directional instability (Skibbens et al., 1993). Sister-kinetochore oscillations are thought to occur when kinetochores switch between polymerizing (passive, no force pro-

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22 3 Introduction

duction) and de-polymerizing (active, force production) states. These antagonistic pulling forces create a tug-of-war, which appear as triangular wave in the distance versus time plots (Figure 3.5(c,d)). Therefore, the switching of sister-kinetochores between both states must be coordinated so that both kinetochores are not in the active state at the same time.

When the two kinetochores exchange roles, the chromosomes reverse direction and a new oscillation begins. Consequently the coordination of the switching influences the position of a chromosome within the spindle.

Directional switching during chromosome oscillations of the kinetochores between leading and lagging state is mediated by polar ejection forces (PEFs) and centromere stiffness (link- age between sister chromatids). They control the position of the chromosome and induce a directional switch to reverse movement if a chromosome comes too close to one pole (Jaqaman et al., 2010; Ke et al., 2009; Stumpff et al., 2012). PEFs modulate tension at the leading kinetochore to influence motor activity (Ke et al., 2009; Khodjakov & Rieder, 1996). PEFs are generated by the plus-end directed chromokinesin Kid (kinesin like protein Kif22), which pushes chromosomes away from the spindle poles along non-kinetochore microtubules (Levesque & Compton, 2001; Stumpffet al., 2012; Wandkeet al., 2012). Cutting of chromosome arms by laser-surgery or depletion of Kid reduce polar-ejection forces and chromosome oscillations are increased and irregular. Therefore it is concluded that PEFs control the switching from poleward to anti-poleward movement to limit the oscillation amplitude (Ke et al., 2009; Stumpff et al., 2012; Wandke et al., 2012). As depletion of Kid has only a minor influence on oscillations of congressed chromosomes it is suggested that PEFs facilitate chromosome alignment at the spindle equator (Wandke et al., 2012). This limitation of chromosome oscillations is further achieved by a spatial gradient of PEFs. In metaphase PEFs increase most rapidly near the cell equator and flatten towards the poles as the magnitude of PEFs is proportional to microtubule density and size of the chromosome arms. This ensures that bi-oriented chromosomes oscillate around the metaphase plate. If a chromosomes moves poleward, the PEFs act at the leading kinetochore and induce a directional switch (Keet al., 2009). These forces are inactive in anaphase upon degradation of Kid (Levesque & Compton, 2001) and thereby separated chromosomes can be moved poleward in the absence of opposing forces (Figure 3.5(e)).

It is proposed that the microtubule flux rate determines whether kinetochores oscillate or not. In vertebrate cells kinetochores oscillate and the flux rate is low (Mitchison, 1989) and in the absence of microtubule flux cells show chromosome oscillations in metaphase and chromosome segregation in anaphase implying that microtubule flux plays only a minor role in chromosome movement (Ganem et al., 2005). This leads to the hypothesis that

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3 Introduction 23

microtubule de-polymerization at kinetochores (fast kinetochore movement) drives chromosome segregation in early anaphase and microtubule flux (slow kinetochore movement) is responsible for the chromosome movement in late anaphase (Zhai et al., 1995) (Fig- ure 3.5(e)). The microtubule flux rate is high in drosophila embryos and xenopus egg ex- tract spindles and consequently their kinetochores show no oscillatory movements (Maddox et al., 2003). It is proposed that a high flux rate prevents the kinetochore form switching directions. This suggests that the presence of high microtubule flux rates correlates with the lack of kinetochore oscillations in metaphase and determines chromosome movement in anaphase (Maddoxet al., 2003). This hypothesis comes from the observations that poleward microtubule flux and chromosome movement in Anapahase A occurs with the same rate in Xenopus and Drosophila spindles (Desaiet al., 1998; Maddox et al., 2002). The absence of chromosome oscillations in plant cells correlates with the absence of polar ejection forces (Khodjakovet al., 1996).

The function of sister-kinetochore oscillations is not clear. It is proposed that kinetochore oscillations prevent chromosomes from becoming entangled or damaged during alignment at the spindle equator (Keet al., 2009). Sister-kinetochore oscillations could also be a mechanism to check for proper kinetochore-microtubule-attachments, which result in a balance of forces.

3.4 The Spindle assembly checkpoint (SAC)

The spindle assembly checkpoint inhibits anaphase onset, until all kinetochores are correctly attached. In particular the SAC monitors the microtubule occupancy of a kinetochore:

it senses the microtubule-bindings sites of a kinetochore, which are not or transiently occu- pied by microtubules under low tension (Kulukianet al., 2009). Under this condition spindle checkpoint components namely Mad (mitotic arrest deficient) and Bub (budding uninhibited by benomyl) proteins bind to the kinetochore to generate a wait anaphase signal. The tem- plate model describes the conformational change of open-(O)-Mad2 to closed-(C)-Mad2, which is the active form and able to bind to Cdc20. Mad1-C-Mad2 complexes localize at unattached kinetochores. Upon dimerization with Mad1/C-Mad2 cytosolic O-Mad2 is converted to C-Mad2, which is now able to interact with Cdc20 to form together with BubR1 and Bub3 the mitotic checkpoint complex (MCC)(De Antoni et al., 2005; Kulukian et al., 2009; Mapelliet al., 2007). The E3 ubiquitin ligase APC/C (anaphase promoting complex or cyclosome) is kept inactive by sequestering its co-activator Cdc20 (cell division cycle 20) into the MCC and by direct binding of the MCC to the APC/C to inhibit its ability to bind substrates such as cyclin-B1 and securin (Herzog et al., 2009). Recently, it was shown

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24 3 Introduction

that the spindle-assembly checkpoint has a gradual response rather than an all-or-nothing response, which depends on the amount of kinetochore-recruited Mad2 and formed MCC (Collinet al., 2013; Dick & Gerlich, 2013).

The MELT-motifs in the N-terminal and central region of KNL1 are required for the recruitment of Bub3/Bub1 to kinetochores, which in turn recruits Bub3/BubR1 (Primorac et al., 2013; Yamagishi et al., 2012; Zhang et al., 2014). This interaction is mediated by Mps1 (monopolar spindle 1) phosphorylation of the MELT-motifs (Yamagishiet al., 2012) and this interaction is further enhanced by the two helical KI-motifs in the N-terminus of KNL1 that interact with the TPR-domains of Bub1 and BubR1 (Bolanos-Garciaet al., 2011; Kiyomitsu et al., 2007, 2011). In addition KNL1 interacts with Zwint-1 via its C-terminal coiled coil region (Petrovicet al., 2010). Zwint-1 in turn recruits the Rod-ZW10-Zwilch (RZZ)-complex, which is required for the localization of dynein to kinetochores as well as Mad1 (Kopset al., 2005; Starret al., 2000). Aurora-B mediated phosphorylation of Zwint-1 is required for the recruitment of the RZZ-complex and dynein to kinetochores (Kasuboskiet al., 2011).

The spindle assembly checkpoint is satisfied with full microtubule attachment, which results in stretching within the kinetochore and establishment of intra-kinetochore tension (Uchidaet al., 2009). Recently it was shown that the kinetochore-stretching within the kinetochore (intra-kinetochore distance) is insensitive to changes in the distance between the kinetochores (inter-kinetochore distance) (Suzuki et al., 2014). The silencing of the spindle assembly checkpoint is mediated by removal of the checkpoint proteins Mad1/Mad2, action of counteracting phosphatases and disassembly of the MCC. Mad1/Mad2 as well as the RZZ-complex are directly transported or stripped away by the minus-end directed dynein (Famulski et al., 2011; Howell et al., 2001). The stripping of these complexes prevents formation of new MCCs. Counteracting de-phosphorylations are mediated by PP1.

PP1 de-phosphorylates cytoplasmic dynein, which is then able to remove the checkpoints proteins from the kinetochore (Whyteet al., 2008). In addition PP1 removes the phosphorylation by Mps1 on KNL1, which results in the removal of Bub1/Bub3 and BubR1/Bub3 from the kinetochores (Zhanget al., 2014). This was confirmed in recent publications and it was postulated that PP1 localization is required to ensure efficient checkpoint silencing upon establishment of kinetochore-microtubules (Nijenhuiset al., 2014). The disassembly of free MCC is mediated by p31^comet which binds to Mad2 (Westhorpeet al., 2011) and the APC/C subunit APC15 is required for the turnover of APC/C-bound MCC (Mansfeldet al., 2011).

Upon silencing of the checkpoint the APC/C is activated (Shah & Cleveland, 2000) and now able to induce the degradation of securin (responsible to maintain sister chromatid cohesion) and cyclin-B1 (responsible to keep the mitotic stage) by the 26S proteasome (Morgan,

Function and regulation of the mitotic kinesin Kif18A