The p97-cofactor p37 regulates spindle orientation by limiting cortical NuMA recruitment via PP1/Repo-Man

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Thesis

Reference

The p97-cofactor p37 regulates spindle orientation by limiting cortical NuMA recruitment via PP1/Repo-Man

LEE, Byung Ho

Abstract

Spindle orientation determines the axis of division and is crucial for cell fate, tissue morphogenesis and for the development of an organism. In animal cells, spindle orientation is regulated by the conserved Gαi/LGN/NuMA complex, which targets the force generator dynein/dynactin to the cortex. Here we show that p37/UBXN2B, a cofactor of the p97 AAA ATPase, regulates spindle orientation in mammalian cells by limiting the levels of cortical NuMA. p37 controls cortical NuMA levels via the phosphatase PP1 and its regulatory subunits Repo-Man, but acts independently of Gαi, the kinase Aurora A, or the phosphatase PP2A.

Our data show that in anaphase, when the spindle elongates, PP1-Repo-Man promotes the accumulation of NuMA at the cortex. In metaphase, p37 negatively regulates this function of PP1 resulting in lower cortical NuMA levels and correct spindle orientation.

LEE, Byung Ho. The p97-cofactor p37 regulates spindle orientation by limiting cortical NuMA recruitment via PP1/Repo-Man. Thèse de doctorat : Univ. Genève, 2018, no. Sc.

5190

URN : urn:nbn:ch:unige-1032001

DOI : 10.13097/archive-ouverte/unige:103200

Available at:

http://archive-ouverte.unige.ch/unige:103200

Disclaimer: layout of this document may differ from the published version.

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UNIVERSITÉ DE GENÈVE

Département de Biologie Moleculaire FACULTÉ DES SCIENCES Professeur Robbie Joseph Loewith Département de Physiologie Cellulaire et Métabolisme FACULTÉ DE MEDECINE

Professeur Monica Gotta Professeur Patrick Meraldi

The p97-cofactor p37 Regulates Spindle Orientation By Limiting Cortical NuMA Recruitment Via PP1/Repo-Man

THÈSE

présentée à la Faculté des Sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie

Par Byung Ho LEE

de

Nuku’alofa (TONGA)

Thèse n° 5190

^Oncology,

Milan, ltaly), autorise

I'impression

de la

^présente

thèse,

sans exprimer d'opinion sur les propositions qui y sont énoncées.

Genève, le 9 mars 2018

Thèse -5190

-

Le Doyen

N.B.

-

La thèse doit porter la déclaration précédente et remplir les conditions énumérées dans les "lnformations relatives aux thèses de doctorat à I'Université de Genève".

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i

Figure List

Figure 1.1. Schematic representation of Mitosis 2

Figure 1.2. Orientation of the spindle in symmetric and asymmetric cell division 5 Figure 1.3. The Gαi/LGN/NuMA and dynein/dynactin complexes regulating spindle positioning 8

Figure 1.4. Domains and phosphorylation sites on NuMA 11

Figure 1.5. Protein Phosphatase 2A complex 15

Figure 1.6. Cortical and Spindle pole localization of NuMA regulated by phosphorylation 17

Figure 1.7. Protein Phosphatase 1 complexes 19

Figure 1.8. The mechanism, multicellular processes, and structure of p97 24 Figure 2.1. p37 regulates spindle orientation by limiting cortical NuMA levels 33 Figure 2.2. High cortical NuMA levels in p37 depleted cells depend on a phosphatase, but are

independent of Gαi or Aurora A. 36

Figure 2.3. p37 regulates cortical NuMA levels via PP1 38

Figure 2.4. PP1/Repo-Man controls cortical NuMA localization in metaphase 41 Figure 2.5. p37 controls the PP1/Repo-Man/NuMA pathway specifically in metaphase 43

Supplementary Figure 2.1 51

Figure 3.1. p37 regulates cortical NuMA independent of actin cytoskeleton stabilization 59

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v Figure 3.2. p37 negatively regulates PIP2 dependent cortical NuMA 61 Figure 3.3: Further characterization of PP1/Repo-Man regulation on NuMA 63

Figure 3.4. Further characterization of p37 in mitosis 67

Figure 4.1. p97/p37 regulates NuMA/PP1/Repo-Man in the cytoplasm 73 Figure 4.2. p37 prevents the PP1/Repo-Man dependent pathway of cortical NuMA in Metaphase 78

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vi

Abstract

Mitosis is part of the cell cycle that gives rise to two daughter cells from one parental cell. Key mitotic processes are carried out by a large microtubule-based structure, the spindle. The orientation and positioning of the spindle determines the axis of division. Therefore, spindle orientation determines the type of division, an asymmetric or symmetric cell division. During development or adult life, the homeostatic balance between asymmetric and symmetric cell division contributes to the formation and maintenance of tissues.

The most well-known and conserved regulators of spindle orientation is the heterotrimeric complex, that is composed of Gαi, LGN and NuMA. The Gαi/LGN/NuMA complex are localized at the cortex, where they recruit and regulate the cortical-pulling forces that is mediated by the motor complex, dynein/dynactin. The localization and functional properties of the complexes are regulated by kinases and phosphatase to ensure correct spindle orientation in mitosis.

p97/VCP/Cdc48 is a hexameric complex that segregates target proteins from complexes or intracellular structures and subjects them for proteasomal degradation or recycling. The biological roles of p97 is diversified by its multiple cofactors, in which they determine the activity, localization, and substrate specificity of p97. Cofactors p37 and p47 have been reported for the role in the reassembly of the Golgi apparatus and Endoplasmic Reticulum after mitosis. However, our lab has discovered that the p37 and p47 are important for a proper spindle orientation in metaphase.

The aim of my PhD thesis project was to understand how p37 is involved in regulating spindle orientation in metaphase. To investigate this, we carried out our experiments on HeLa cells, which are recurrently utilized model system for studying spindle orientation. Combining cell biology and biochemistry techniques, we tested specific pathways and regulators of spindle orientation that were in question.

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vii Our experimental results show that 37 depleted cells display misoriented spindle during metaphase and an increased level of NuMA at the cortex. Reducing NuMA levels in p37 depleted cells, via co-depletion, were sufficient to rescue the spindle misorientation of caused by the lack p37. This finding allowed us to conclude that p37 prevents excessive cortical-pulling forces to ensure proper spindle orientation. In an attempt to understand how NuMA is recruited in p37 depleted cells, we found that the upstream cortical recruiters of NuMa, Gαi and LGN, were not influenced nor involved in the excessive recruitment of NuMA in p37 depleted cells.

Alternative to the Gαi/LGN pathway, we found that p37 regulates NuMA via Protein Phosphatase 1(PP1) and Repo-Man. PP1 is famously known to carry out its major role at the kinetochore, the interface between the microtubules of the spindle and chromosomes, to counter the activity of Aurora B kinase. While Repo- Man is responsible for targeting PP1 to the kinetochore. Our depletion and overexpression experiments of PP1 and Repo-Man indicate that these proteins are regulators spindle orientation by promoting cortical NuMA recruitment. We further show that this is under the control of p37 for their co-depletions with p37 resulted in the restored proper spindle orientation and cortical NuMA levels. We found that p37 limits the interplay between PP1/Repo-Man and NuMA by spatially promoting PP1 towards the kinetochore.

However, p37 depletion does not have any influence in cortical NuMA levels during anaphase, while PP1 and Repo-Man depletions results in a decrease in cortical NuMA levels.

To avoid defects in spindle orientation, we conclude that p37 prevents excessive cortical pulling forces.

p37 carries this out by limiting the cortical recruitment of a population NuMA that is dependent on PP1/Repo-Man, but independent of Gαi/LGN. Furthermore, we introduce PP1/Repo-Man promotes cortical accumulation of NuMA and p37 inhibits this during metaphase to allow proper spindle orientation.

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viii

Résumé

Lors de la division cellulaire, la mitose est le processus permettant à une cellule de se diviser en deux cellules filles. Les étapes clés de la mitose sont effectuées par une structure composée de microtubules appelée le fuseau mitotique. Durant la métaphase, les chromosomes sont alignés sur la plaque équatoriale de fuseau mitotique déterminant l’axe de division. Ainsi l’orientation du fuseau mitotique va déterminer le type de division ; symétrique ou asymétrique. Ce mécanisme est essentiel pendant le développement et la vie adulte afin de générer de la diversité cellulaire et la maintenance des différents tissus de l’organisme.

Plusieurs études ont permis de déterminer les facteurs et mécanismes moléculaires clés régulant l’orientation du fuseau mitotique. Parmi les plus conservés, on retrouve un complexe hétérotrimérique de protéines constitué de Gαi, LGN et NuMA. Le complexe Gαi/LGN/NuMA est localisé au niveau de la membrane plasmique (cortex) où il recrute et régule les forces de traction au cortex par l’intermédiaire des microtubules via des protéines moteurs, Dynein/Dynactin. Des protéines kinases et phosphatases vont réguler la localisation et les fonctions de ce complexe pour permettre la bonne orientation du fuseau mitotique.

p97/VCP/Cdc48 est un hexamère protéique qui permet de ségréguer des protéines d’intérêts à partir d’autres complexes protéiques ou de structure intracellulaire à fin de les dégrader par le protéasome ou les recycler.

p97 possède divers rôles biologiques de par l’association de multiples cofacteurs qui vont déterminer l’activité, la localisation et la spécificité de substrats de p97. Précédemment notre laboratoire a mis en évidence le rôle des cofacteurs p37 et p47 (connu pour leur implication dans la formation de l’appareil de Golgi et du réticulum endoplasmique) sur l’orientation du fuseau mitotique lors de la métaphase.

L'objectif du projet de thèse est de comprendre par quels mécanismes p37 régule l’orientation du fuseau mitotique. Pour nos expériences, nous avons utilisé des cellules humaines HeLa. Ces cellules sont utilisées fréquemment en laboratoire de recherche dans l’étude de l’orientation du fuseau mitotique. En combinant

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ix des techniques de biologie cellulaire et de biochimie, nous avons testé différents régulateurs et les voies spécifiques contrôlant l’orientation du fuseau mitotique.

Nous avons mis en évidence qu’en l’absence de p37, le fuseau mitotique est mal orienté et NuMA est accumulée au cortex. La réduction de NuMA en l’absence de p37 est suffisante pour restaurer la mauvaise orientation du fuseau mitotique observé quand seul p37 est absent. Ce rétablissement d’orientation du fuseau mitotique nous permet de conclure que p37 empêche une partie de la population de NuMA d’être recrutée au cortex. Pour comprendre comment NuMA est recrutée au cortex en l’absence de p37, nous avons étudié les facteurs qui recrutent NuMA, Gαi et LGN. Nos résultats montrent que ces facteurs ne sont pas impliqués dans l’excès de recrutement de NuMA au cortex.

Cependant, nous avons mis en évidence que p37 régule NuMA par l’intermédiaire d’une protéine phosphatase et de sa sous-unité régulatrice, la Protéine Phosphatase 1 (PP1) et Repo-Man. L’inactivation de PP1 et Repo-Man entraine la perte de la localisation de NuMA au cortex. De plus, leur double inactivation avec p37 permet de rétablir l’orientation du fuseau mitotique ainsi que le niveau de NuMA au cortex. La surexpression de GFP-Repo-Man^RVXF (contrôle) entraine un défaut d’orientation du fuseau mitotique et une accumulation de NuMA au cortex alors que la surexpression du mutant GFP-Repo- Man^RAXA(mutant ne pouvant pas s’associer à PP1) ne présente aucun effet. Ces résultats indiquent que PP1 et Repo-Man fonctionnent ensemble pour permettre le recrutement de NuMA au cortex. La perte de p37 entraine aussi une diminution de la localisation de PP1 au niveau des kinetochores, indiquant que p37 régule spatialement PP1.

Par conséquent, nos résultats permettent de conclure que p37 empêche l’excès des forces de traction au cortex en limitant la quantité de la protéine NuMA. De plus, la régulation de NuMA par p37 se fait indépendamment de Gαi/LGN mais est dépendante de PP1/Repo-Man. En conclusion, nous avons montré que p37 empêche la localisation de NuMA au cortex via PP1/Repo-Man en favorisant la localisation de PP1 aux kinetochores.

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1

1. Introduction

1.1. Mitosis

The cell cycle is made up of G1, S, G2, and M phase. The G1, S, G2 phases are known as interphase. The M phase or mitosis is where the cell gives rise to two daughter cells. Mitosis is composed of five phases:

prophase, prometaphase, metaphase, anaphase, and telophase (Figure 1.1A). Each mitotic phase regulates key processes that are involved in the regulation of an error-free segregation of the chromosomes, organelle, and other cytoplasmic content. These processes are regulated by the activity of protein kinases, phosphatases and ubiquitin ligases, which cooperatively control mitotic entry and exit.

1.1.1. Mitotic entry

As the G2 phase comes to an end, cells that have successfully duplicated their DNA with no damage and errors can progress onto mitosis. Meanwhile, the mitosis-promoting kinase Cdk1 (Cyclin dependent kinase 1) associates to its activating subunits Cyclin B1 and B2 (Malumbres and Barbacid, 2005). In G2-M transition, a series of phosphorylation and dephosphorylation feedback events leads to the activation of the Cdk1-Cyclin B complex. Most notably, Aurora A kinase activates via phosphorylation Polo-Like kinase 1 (Plk1) which then activates Cdk1-activating phosphatase Cdc25 and deactivates Cdk1-inhibiting kinases Wee1 and Myt1 (Lindqvist et al., 2009; Malumbres and Barbacid, 2005). Upon mitotic entry, Cdk1-Cyclin B complex and a network of multiple kinases, such as Plk1 and Aurora kinases, phosphorylates key factors to regulate processes that drive mitotic progression (Ma and Poon, 2011; Nigg, 2001). In addition, the cell undergoes an extensive morphological reorganization where it begins to round up due to the changes in cytoskeleton network and disassembly of focal adhesion. Intracellular organelles, such as the Endoplasmic Reticulum and Golgi apparatus, breakdown and undergo structural changes and, more importantly, the nucleus begins reorganizing to prepare for Nuclear Envelope Break Down (NEBD) (Champion et al., 2017).

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2 Figure 1.1. Schematic representation of Mitosis. (A) Phases of mitosis; prophase, prometaphase, metaphase, anaphase, and telophase. Dark blue represents the DNA, light blue represents the nuclear envelope, green lines represent the microtubules, yellow circle represents centrosomes/spindle poles, red circle presents the kinetochore, red-dotted line represent the axis of division (B) Chromosomes in metaphase are bipolarly attached by the K-fibers. (C) Cortical regulators of motor proteins that exert cortical-pulling force onto the spindle via the astral microtubule.

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3 1.1.2. Chromosome congression and bipolar spindle assembly

During prophase the chromatin condenses and become rigid individual fiber-like structures that are captured by microtubule upon NEBD and physically separated in anaphase (Antonin and Neumann, 2016). In addition, the duplicated centrosomes nucleate the microtubules (MTOC: Micro-Tubule Organizing Centers) and starts migrating away from each other to form the bipolar spindle (Petry, 2016). This process is assisted by motor proteins such as Eg5, Kif15, and dynein, which also play a role in maintaining the spindle polarity in successive stages of mitosis. At the end of prophase, NEBD occurs and the chromosomes are now in the cytoplasm. This allows the microtubules, centrosomes, and other cytoplasmic proteins to have access to the chromosomes (Figure 1.1A).

The chromosomes are captured via the kinetochores by microtubules originating from the centrosomes;

these microtubules are known as the kinetochore fibers (K-fibers). The kinetochore is a structure composed of protein complexes localized at the centromeric region of the chromosomes, which acts as the main interface between the chromosomes and the mitotic spindle (Figure 1.1B) (Guttinger et al., 2009). In prometaphase, as all chromosomes are captured via the kinetochores, the chromosomes congress to form the metaphase plate, an equatorial position between the two spindle poles (Figure 1.1A) (Cheeseman and Desai, 2008). For proper chromosome congression, kinetochores that are laterally-attached to the K-fibers can be transported towards the equatorial region of the spindle via plus-end-directed kinesin CENP-E (Kapoor et al., 2006). Microtubules and chromokinesins such as hKid and KIF4A (localized at the chromosome arms) can also push chromosomes to the equator, this mechanism is known as polar ejection forces (Wandke et al., 2012). The mentioned mechanisms, coupled with microtubule dynamics lead to the formation of the metaphase plate and progression onto metaphase in mitosis. In metaphase, the whole spindle moves around the central position of the cell to contribute in determining the axis of division.

Spindle positioning is mainly regulated by motor proteins at the cortex that exert cortical-pulling forces on to the spindle (Figure 1.1A and C).

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4 Before the cell can progress into anaphase, there are mechanisms in place to prevent premature anaphase onset and to ensure an error-free segregation of the sister chromatid. To guarantee that the sister chromatids are biorientated, there is a tension sensing mechanism which arises from microtubules pulling on the sister kinetochores from opposite sides (Figure 1B) (Lampson and Cheeseman, 2011). The second mechanism involves the Spindle Assembly Checkpoint (SAC), which needs to be satisfied to trigger the anaphase onset.

The SAC is composed of the Mitotic Checkpoint Complex (MCC; consisting of Mad2, BubR1, Bub3, and CDC20) that inhibits the Anaphase Promoting Complex/Cyclosome (APC/C). The MCC complex carries this out by sequestering CDC20 (the activating subunit of APC/C), thereby preventing recognition and degradation of APC/C substrates; Cyclin B and Securin (Manic et al., 2017).

1.1.3. Chromosome segregation and mitotic exit

The SAC is satisfied when all the kinetochores are bipolarly attached to the microtubules of the spindle, thus ensuring proper chromosome segregation. Following this, the mitotic cell can progress towards anaphase (Sullivan and Morgan, 2007). As the criteria for SAC are met, the APC/C^CDC20 complex ubiquitinates Cyclin B and subjects it for degradation (Jia et al., 2013). The degradation of Cyclin B leads to the inactivation of Cdk1, allowing the counteracting phosphatases to dephoshorylate mitotic-entry promoting substrates and stimulate mitotic exit (Bollen et al., 2009; Jeong and Yang, 2013; Wurzenberger and Gerlich, 2011). In order for the chromosome to initiate separation, Securin degradation via APC/C^CDC20 is crucial. Prior to SAC satisfaction, Securin inhibits the protease Separase from proteolysizing of the Cohesin Complex, which is responsible for physically connecting the two sister-chromatids (Jellapali et al 2001, Hagting et al 2002).

Now that it is possible to segregate the sister-chromatids due to the activity of APC/C^CDC20, the spindle elongates with the help of collective forces that act upon the spindle (Figure 1.1A). Forces that are generated at the cortex (which positions the spindle in metaphase) intensify during anaphase due to the inactivation of Cdk1 (Collins et al., 2012; Gehmlich et al., 2004; Kotak et al., 2013). In addition, the central spindle is formed by antiparallel microtubules, which are used by motor proteins to push the half spindle

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5 apart(Douglas and Mishima, 2010). When telophase begins, chromatin starts to decondense, the central spindle disassembles to form the microtubule based midbody, and the nuclear envelope and other organelles initiate reformation to give rise to the two daughter cells (Guttinger et al., 2009; Hutterer et al., 2009). Upon exiting mitosis, each daughter cell progresses through to become interphase cells.

1.2 Spindle orientation and positioning

Spindle orientation and positioning is a process that occurs during metaphase. The bipolar spindle is positioned in the center of the cell, where the position of the metaphase plate contributes in determining the axis of the division. This was first observed in 1916, where a displaced spindle led to an asymmetric division in the eggs of Crepidula (Conklin, 1916). Subsequently, the spindle was observed to be not static but rather moving around in the cell and that its final orientation correlated with the fate of the daughter cells (Fujita,

Figure 1.2. Orientation of the spindle in symmetric and asymmetric cell division. (A) Spindle axis oriented perpendicular to the polarity axis resulting in symmetric cell division. (B) Spindle axis oriented parallel to the polarity axis resulting in asymmetric cell division.

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6 1960; Martin, 1967). Briefly, the positioning and orientation of the spindle is mainly regulated by proteins and complexes that are localized at the cortex of the mitotic cell (hereafter called ‘cortical regulators’). The cortical regulators are involved in spindle positioning by directly recruiting motor proteins to the cortex.

These specialized motor proteins generate the cortical-pulling forces onto the spindle via the astral microtubules (Figure 1.1C; further detail of the cortical regulators and spindle orientation will be described in the coming sections).

During development and in various types of cells of tissues, the homeostatic balance between symmetric and asymmetric cell divisions plays a crucial role in forming and maintaining the tissue architecture. In tissue, almost all cells are polarized where intracellular components and shape or structure of the cell are unevenly distributed along a polarity axis. A typical example would be the epithelial cells that have an apical-basal polarity axis. The orientation and positioning of the spindle determines the type of cell division:

symmetric (SCD) or asymmetric cell division (ACD) (Panousopoulou and Green, 2014). An orientation of the spindle that is perpendicular to the apical-basal polarity axis leads to a SCD. This results in two daughter cells with identical cellular content (Figure 1.2A). On the other hand, a spindle orientation that is parallel to the apical-basal polarity axis leads to an ACD. This results in the unequal inheritance of cellular content and difference in sizes between the daughter cells (Figure 1.2B). For instance, in the tissue epithelium, a spindle angle that is parallel to the basement membrane gives rise to two identical daughter cells that are positioned in the same layer of the epithelium. On the contrary, a perpendicular angle of the spindle to the basement membrane would result in one of the daughter cell that is positioned outside this layer. The homeostatic balance between ACD and SCD ensures the correct combination of differentiation and proliferation required for tissue formation and maintenance (Fujita, 1960; Lechler and Fuchs, 2005; Martin, 1967; Poulson and Lechler, 2010).

The understanding of ACD today is highly contributed by studies carried out in invertebrate systems (briefly described below). Spindle orientation and positioning are coupled with polarity, where polarity cues regulate the polarization of the cortical regulator. The Caenorhabditis elegans (C.elegans) one cell embryo,

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7 has an anterior-posterior (AP) polarity due to the polarity proteins (anterior: PAR-3/PAR-6/PKC-3, posterior: PAR-2/PAR-1). These polarity proteins mutually inhibit each other for their asymmetric localization. The AP polarity cues are important for an asymmetric positioning of the spindle (Gonczy et al., 2001). During prophase, the cortical regulators direct the cortical-pulling forces towards the anterior side of the embryo. This leads to the rotation and movement of the pronuclei/centrosomes towards the center of the embryo. In early anaphase, the polarity proteins (PAR-3, PAR-2, and PKC-3) promotes the posterior localization of the cortical regulators (Galli et al., 2011; Gotta et al., 2003). The resulting posterior cortical-pulling forces promote the movement of the elongating spindle towards the posterior half of the embryo (Cheng et al., 1995; Colombo et al., 2003; Grill et al., 2001; Kemphues et al., 1988). This ensures an asymmetric cell division, where the daughter cells are different in size and cytoplasmic content.

During the development in Drosophila melanogaster, the neuroblasts (NB), stem cells of the nervous system, divide asymmetrically due to the link between polarity and cortical regulators. The ACD of the NB gives rise to a new NB and ganglion mother cell (GMC). The polarity proteins (Bazooka/Par6/aPKC) are apically localized while the cell fate determinants (e.g. Prospero and Numb) are localized at the basal end of the NB. The polarity proteins directly promote the apical localization of the cortical regulators to orient the spindle along the polarity axis(Yu et al., 2006). This results in the asymmetric segregation of the cell fate determinants (Kraut et al., 1996). The NB daughter cells retain the polarity proteins for further ACD and the GMC contain cell fate determinants for differentiation.

A SCD in the epithelium results from a spindle axis that is parallel to the basement, as previously mentioned (Figure 1.2B). The pathways and mechanism involved in SCD have been mostly characterized in mammalian cells. Cultured cells have been widely used to study many mitotic parameters in addition to spindle orientation. Madin-Darby Canine Kidney (MDCK) cysts, which are a 3D model of the epithelium, are spheres with a monolayer of cells. MDCK cysts have a well-defined polarity axis with the apical domain facing the central lumen. Mitotic cells within the monolayer orient their spindle against the polarity axis due to the lateral localization of the cortical regulator, thus resulting in a SCD (di Pietro et al., 2016; Zheng

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8 et al., 2010). Moreover, cultured HeLa cells symmetrically position the spindle within the center and orient the spindle axis parallel to the culture surface (di Pietro et al., 2016; Toyoshima and Nishida, 2007).

1.3 Cortical regulators of spindle orientation

The cortical forces that pull on the spindle are generated by the dynein/dynactin motor complex that exerts force onto the astral microtubule that originates from the spindle poles. The Gαi/LGN/NuMA complex is the key cortical regulator of spindle positioning due to its role in recruiting and maintaining the dynein/dynactin motor complex at the cortex (Du and Macara, 2004; Kotak et al., 2012).

1.3.1 Force generating complex: Dynein/Dynactin

Dynein is an AAA (ATPase Associated with diverse cellular Activities) ATPase motor protein that moves along microtubules towards the minus end. Dynein carries out its various cellular functions by converting chemical energy obtained by hydrolyzing ATP into mechanical energy that allows it to walk on microtubules (Paschal et al., 1987; Vallee et al., 1989; Vallee et al., 1988). Dynactin is a protein complex with multiple subunits that directly interacts with dynein. Dynactin tethers dynein onto microtubules and transport cargoes, thus dynactin is required for dynein functions (King et al., 2003; Moore et al., 2008;

Schroer, 2004; Sharp et al., 2000; Skop and White, 1998).

For spindle positioning, the dynein/dynactin complex acts as the cortical force generator that positions and elongates the spindle in metaphase and anaphase, respectively (Figure 1.3A-C) (Collins et al., 2012; di Pietro et al., 2016; Izutsu and Yoshida, 1989; Kiyomitsu and Cheeseman, 2012; Kiyomitsu and Cheeseman, 2013; Kotak et al., 2012). Dynein at the cortex can interact with astral microtubules in two different manners: side-on versus end-on interactions. Astral microtubules that polymerize and bend along the cortex interact with dynein in a side-on manner. This mechanism allows dynein to glide on the side of microtubules to generate cortical-pulling forces (Lee et al., 2005; Paschal et al., 1987; Vallee et al., 1988). It was demonstrated in C.elegans that during anaphase the cortical-pulling force are generated from an end-on interaction between dynein and astral microtubules. These astral microtubules remain at the cortex but

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9 undergo depolymerization that generate the pulling force onto the spindle (Kozlowski et al., 2007; Labbe et al., 2003).

Apart from spindle positioning, the dynein/dynactin complex has been reported to be involved in the transport of SAC proteins (e.g. MAD1 and MAD2 of the MCC complex) from the kinetochore to the centrosomes (Barisic and Maiato, 2015; Famulski et al., 2011; Silva et al., 2014; Zheng et al., 2013). The dynein/dynactin complex is also known for its role in the assembly of the bipolar spindle in prometaphase (Merdes et al., 1996; van Heesbeen et al., 2014). Outside mitosis, the dynein/dynactin complex is involved in vesicle and organelle trafficking and transport along microtubules (Carter et al., 2016; Faulkner et al., 2000; Hirokawa, 1998; Melkov et al., 2016; Xiang et al., 2015).

1.3.2 Dynein/Dynactin recruitment complex: Gαi/LGN/NuMA complex

The Gαi/LGN/NuMA complex is localized at the cortex throughout mitosis and is the main platform for dynein/dynactin to bind at the cortex (Figure 1.3A) (Du and Macara, 2004). Because it recruits and regulates dynein/dynactin at the cortex, their spatiotemporal abundance directly influences the amount of force exerted onto the spindle (Du and Macara, 2004; Kotak et al., 2012).

Gαi/LGN subcomplex

Gαi belongs to the Gα subfamily (other subfamily involves Gβ and Gγ) of subunits of the heterotrimeric Guanine nucleotide binding proteins (G protein). Gαi can be associated with either GDP (inactive) or GTP (active) nucleoside to carry out its role in the G-protein signaling pathways. This depends on its guanine exchange factors (GEF) or GTPase-activating proteins (GAP). It was shown in C.elegans (GOA1/GPA16) and Drosophila (Gαi/o) that Gα is involved in spindle positioning and this role was later found to be conserved in mammals. Ric-8, a conserved GEF for Gαi, is required for the dissociation of Gαi-GDP from Gβ and Gγ, it is hypothesized that this allows a sequential hydrolysis of the GTP to form Gαi-GDP that is

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10 available for LGN interaction. Gαi-GDP form interacts directly with LGN, and acts as an anchor for the Gαi/LGN/NuMA complex via its myristolyated tail (Afshar et al., 2005; Couwenbergs et al., 2007;

Couwenbergs et al., 2004; David et al., 2005; Tall et al., 2003; Woodard et al., 2010) (Figure 1.3A).

Throughout the early stages of mitosis in mammalian cells and C.elegans, Gαi localizes around the entire cortex and does not contribute to the polarization of the downstream components of the complex

Figure 1.3. The Gαi/LGN/NuMA and dynein/dynactin complexes regulating spindle positioning.

(A) Schematic representation of the cortex, showing Gαi inserted into the membrane, LGN in open conformation and interacting with Gαi and NuMA, and NuMA bound dynein/dynactin complex exerting force onto the astral microtubule. (B) Ran-GTP and Plk1 signaling, negatively influences the indicated cortical regulators to spatially distribute cortical forces along the spindle axis. (C) Spatiotemporal dependent positioning of the spindle by cortical regulators (Blue dots).

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11 (LGN/NuMA). On the contrary, in Drosophila, Gαi is apically localized and contributes to the apical recruitment of Pins/Mud (LGN/NuMA) (di Pietro et al., 2016; Kokame et al., 1992; Kulukian and Fuchs, 2013; Linder et al., 1991; Schaefer et al., 2001).

LGN, Leucine–glycine–asparagine, (also known as GPSM2; G protein signaling modulator 2; Pins in Drosophila; GPR1/2 in C.elegans) was first identified as an interacting protein of Gαi (Mochizuki et al., 1996). Prior to mitosis the expression of LGN is low, suggesting that its major function is carried out in mitosis where it acts as a key regulator of spindle positioning (Du and Macara, 2004). During mitosis, cortical LGN interacts with Gαi and NuMA, acting as the link of the complex. LGN has a higher affinity towards the Gαi-GDP form through its GoLoco motif and prevents Gαi from releasing GDP, thereby regulating its activity (Jia et al., 2012). The conformational state of LGN can switch between an ‘open’ and

‘closed’ state. LGN interacts with NuMA via the TPR motif (Tetratrico Peptide Repeats) and this interaction results in the ‘open’ conformational state of LGN that has a higher binding affinity towards Gαi (Figure 1.3A) (Culurgioni et al., 2011; Diz-Munoz et al., 2013; Jia et al., 2012; Zhang et al., 2015). LGN is

Figure 1.4. Domains and phosphorylation sites on NuMA. Schematic representation of domains or regions of binding (with indicated proteins) and phosphorylation sites (with indicated kinases and phosphatases) of NuMA. MT = microtubule, NLS = nuclear localization signal, S = serine, T = threonine, Y = tyrosine.

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12 recruited to the cortex by Gαi, and together they behave as a sub-complex of their own, with the goal of recruiting and maintaining NuMA at the cortex (Figure 1.3A).

NuMA, the platform for cortical force

NuMA, Nucleus Associated Mitotic Apparatus, also known as NMP-22 (nuclear matrix protein 22) was first discovered due to its localization changes from the nucleus (during interphase) to the spindle (during mitosis), hence its name (Lydersen and Pettijohn, 1980). Subsequently, the localization of NuMA was found to be at the spindle poles and the cortex during mitosis. Orthologues of NuMA, LIN-5 (C.elegans) and Mud (Drosophila), do not localize in the nucleus but share similar localizations during mitosis.

NuMA is a large protein with a tripartite structure; the globular N-terminal head group and C-terminal tail domain flank a large coiled-coil domain (Figure 1.4). The nuclear localization of NuMA is dependent on a NLS (Nuclear Localization Signal) at the C-terminal tail. During mitosis, the N-terminal domain is responsible for the interaction of NuMA with the dynein/dynactin complex, while the C-terminal tail is important for the localization of NuMA. In mitosis, the recruitment of NuMA to the cortex is highly dependent on its C-terminal tail. NuMA interacts with LGN via a specific EPE-motif (1896-1898) within the LGN binding domain (LGN-BD: 1886-1958aa) (Culurgioni et al., 2011; Zhu et al., 2011). The LGN- BD of NuMa partially overlaps with one of the microtubule binding domains (MT-BD: 1914-1985aa), and it was initially reported that LGN and microtubules bind to NuMA in a mutually exclusive-manner (Du et al., 2002). Although a recent study discovered a new MT-BD (2002-2115aa) that does not overlap with the LGN-BD, and have demonstrated that C-terminal tail of NuMA can associate with both LGN and microtubules, simultaneously (Gallini et al., 2016). In addition, NuMA carries a binding domain and regions for its interaction with 4.1R/G (an actin associated protein) and phosphoinositide of the plasma membrane (Figure 1.4) (Kiyomitsu and Cheeseman, 2013; Kotak et al., 2014; Zheng et al., 2014). These interactions regulate the cortical and spindle pole localization of NuMA.

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13 NuMA has been long believed to be crucial for bipolar spindle assembly, as microinjection of antibodies disrupting the function of NuMA during prophase-metaphase but not anaphase, results in the disruption of the spindle (Kallajoki et al., 1993). Subsequently, it was confirmed that NuMA, together with dynein/dynactin, physically interacts with microtubules (via MT-BD) to regulate bipolar spindle assembly (Haren et al., 2009; Merdes and Cleveland, 1998; Merdes et al., 1996). Furthermore, NuMA is involved in the focusing/tethering of microtubules to the centrosome (spindle pole focusing) and the dynein/dynactin- dependent poleward transport of chromosomes (Elting et al., 2014; Famulski et al., 2011; Silk et al., 2009).

1.4 Regulation of cortical NuMA in metaphase: Gαi/LGN dependent

In interphase and prophase NuMA is kept sequestered in the nucleus, having no association with Gαi and LGN. This keeps the LGN conformation in a ‘closed’ state and unable to bind to Gαi (Diz-Munoz et al., 2013; Du and Macara, 2004; Gallini et al., 2016; Jia et al., 2012; Zhang et al., 2015). Upon NEBD, NuMA enriches at the spindle poles and, with dynein/dynactin, contributes to bipolar spindle assembly (Haren et al., 2009). In the meantime, the Gαi/LGN/NuMA complex begins to accumulate at the cortex.

Spatio-regulation of Cortical LGN/NuMA

As the chromosomes are properly aligned, LGN/NuMA is cleared from cortical regions that are adjacent to the metaphase plate, while becoming concentrated at the regions proximal to the spindle poles. This is due to the Ran-GTP gradient emanating from the chromosomes which negatively regulates LGN/NuMA at the cortex. This polarization of LGN/NuMA distributes the cortical-pulling forces along the spindle axis, allowing the spindle to rock side-ways (Figure 3B and C) (Bird et al., 2013; Kalab and Heald, 2008;

Kiyomitsu and Cheeseman, 2012). Spindle pole-associated Plk1 phosphorylates the dynein/dynactin complex and thus dissociates it from cortical NuMA when the spindle poles move too close to the cortex (approximately 2µm) (Kiyomitsu and Cheeseman, 2012). This is a sensing mechanism of the cell, to promote a central position of the spindle. Furthermore, kinetochore-associated Plk1 on polar chromosomes displaces cortical LGN and causes spindle misorientation (Tame et al., 2016). The responsive localization

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14 of LGN/NuMA and dynein/dynactin complexes to the constantly changing spindle positions indicates that they provide a fine-tuning mechanism of spindle positioning prior to anaphase.

Phosphorylation of NuMA

As NuMA is found at the cortex and the spindle poles during mitosis, there is a shuttling between the two populations which is regulated by phosphorylation. The first sign of NuMA as a phosphoprotein was when multiple Cdk1 sites were reported, however whether Cdk1 was directly involved was not confirmed(Compton and Luo, 1995). Collective studies reported that Cdk1 phosphorylates NuMA on Threonine 2055 residue at the C-terminal tail and directs it to the spindle poles. Due to the overwhelming activity of Cdk1 prior to anaphase onset, NuMA is highly accumulated at the spindle poles and mildly enriched at the cortex. A non-phosphorylateable mutation of Threonine 2005 to Alanine, results in spindle orientation defects that can be rescued by partially inactivating dynein. Thus, Cdk1 is important in maintaining low levels of NuMA at the cortex to prevent excessive cortical-pulling forces (Kiyomitsu and Cheeseman, 2013; Kotak et al., 2013; Kotak et al., 2014; Seldin et al.; Zheng et al., 2014).

Aurora A is a centrosome-associated kinase, and important for mitotic entry processes such as centrosomes maturation and separation, and bipolar spindle formation (Nikonova et al., 2013). To counterbalance the spindle pole localization of NuMA that is mediated by Cdk1, Aurora A kinase phosphorylates NuMA on Serine 1969 and 2047 (located within the MT-BD) at the spindle poles, independent of Cdk1. It was demonstrated that Aurora A does not regulate the ability of NuMA to be localized at the cortex but rather the mobility of NuMA at the spindle poles. Inactivation of Aurora A results in a decrease of cortical NuMA and a higher immobile fraction of NuMA at the spindle poles, resulting in a spindle orientation defect (Gallini et al., 2016). Therefore, Aurora A regulates spindle orientation by maintaining sufficient levels of cortical NuMA.

Phosphorylation of NuMA for controlling spindle positioning is a conserved mechanism in other model organisms. In the C.elegans embryo, LIN-5 contains multiple phosphorylation sites regulated by multiple

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15 kinases. The anteriorly localized kinase PKC-3 phosphorylates LIN-5 to promote a posterior directed spindle movement to ensure ACD. While other kinases (GSK3, casein kinase 1, and CDK1) phosphorylate LIN-5 to regulate its interaction with GPR1/2(LGN) and cortical dynein to regulate spindle orientation (Galli et al., 2011; Portegijs et al., 2016). In Drosophila, Warts kinase, known for its involvement in the Hippo pathway, phosphorylates Mud (NuMA) on Serine 1862 for cortical recruitment in a Pins-dependent manner to regulate spindle orientation (Dewey et al., 2015).

Dephosphorylation of NuMA by PP2A

Unlike the phosphorylation pathways, little has been reported about the phosphatases acting on NuMA. The serine/threonine Protein Phosphatase 2A (PP2A) has been reported to dephosphorylate NuMA during mitosis (Kotak et al., 2013). PP2A belongs to the PPP superfamily of protein phosphatases that complexes with its own set of regulatory subunits (subunit B o(B55), B´ (B56), B´´ (PR72)) to dephosphorylate specific substrates (Figure 1.5) (Bollen et al., 2009). In mitosis, PP2A is known as the counteracting phosphatase for Cdk1 and Aurora A substrates. However, from mitotic entry to anaphase onset, the activity of PP2A is restricted via Cdk1 pathways (Jeong and Yang, 2013; Mochida and Hunt, 2012; Okumura et al., 2014).

PP2A is involved in regulating spindle positioning by directly dephosphorylating NuMA at the Cdk1 site, favoring a cortical recruitment of NuMA opposed to the spindle poles. This activity is important for cortical NuMA localization during metaphase and anaphase, where phosphomimic mutants of threonine 2055 on NuMA does not localize to the cortex throughout mitosis (Kotak et al., 2013; Kotak et al., 2014).

Collectively, PP2A and Cdk1 directly contributes to the equilibrium of NuMA between the cortex and spindle poles during metaphase.

The balance of cortical and spindle pole of NuMA

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16 The amount of cortical force applied on the astral microtubule needs to be sufficient for correct spindle positioning; for too weak or too strong pulling forces lead to spindle orientation defects during metaphase.

This has been exemplified in conditions where abolishment or overexpression of exogenous cortical Gαi, LGN, or NuMA results in spindle orientation and positioning defects (Du and Macara, 2004; Kotak et al., 2012).

The Gαi/LGN subcomplex is the cortical receptor responsible for accommodating the cortical population of NuMA in metaphase. Together, the Gαi/LGN/NuMA complex spatially guides the positioning of the spindle. Since cortical NuMA directly regulates the dynein/dynactin-dependent force, the cells have devised an additional layer of regulation via phosphorylation to tightly control cortical-pulling forces. The regulation by kinases and phosphatase ensures that the precise localization and amount of NuMA at the spindle poles and cortex during metaphase: low levels at the cortex for correct spindle positioning and high levels at the spindle poles for spindle pole focusing (Figure 1.6).

Figure 1.5. Protein Phosphatase 2A complex. PP2A complex consists of a catalytic subunit (purple), a scaffold subunit (beige), and a regulatory subunit B or B´ or B´´. Edited from (Bollen et al., 2009).

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17

1.5 Regulation of cortical NuMA in anaphase: Gαi/LGN independent

Upon anaphase-onset, Cdk1 becomes inactive due to the APC^CDC20 dependent degradation of Cyclin B. The first observation of the change in localization of NuMA was when it was found to be cleared from the spindle poles when Cyclin B was degraded upon anaphase onset (Gehmlich et al., 2004). Cdk1 inactivation results in the increase of PP2A activity, allowing the phosphatase to counteract the bulk of Cdk1 substrates (Bollen et al., 2009; Grallert et al., 2015). This results in the displacement of NuMa from the spindle poles, which has been proposed to reduce pole-focusing (Ban et al., 2007). Meanwhile, NuMA accumulates at the cortex to intensify cortical-pulling forces to elongate the spindle (Figure 1.6) (Kiyomitsu and Cheeseman, 2013; Kotak et al., 2013; Seldin et al., 2013). Cortical Gαi/LGN is dispensable during anaphase for cortical NuMA recruitment, where individual or combination of knock down(s) Gαi/LGN during anaphase did not perturb cortical NuMA levels. In addition, expression of exogenous mutant of NuMA that are non- phosphorylatable at the Cdk1 site is insensitive to the levels of Gαi/LGN even during metaphase (Collins et al., 2012; Kiyomitsu and Cheeseman, 2013; Kotak et al., 2013). The additional population of cortical NuMA that is predominant during anaphase interacts with other cortical receptors to regulate cortical- pulling forces in anaphase. Instead of Gαi/LGN, NuMA interacts with protein 4.1R/G and phosphoinositide (PIP/PIP2) of the plasma membrane (Figure 1.6).

Phosphoinositide: PIP/PIP2/PIP3

Phosphoinositide have been reported to carry out numerous functions in mitosis, particularly in membrane elongation and cytokinesis after anaphase onset (Cauvin and Echard, 2015). There has been evidence of phosphoinositide being involved in spindle positioning. For instance, the levels of phosphoinositide in the plasma membrane indirectly regulates dynein/dynactin levels at the cortex. NuMA has been demonstrated to have a strong binding affinity towards PIP and PIP2, and weaker for PIP3 (Kotak et al., 2014; Zheng et al., 2014). With PIP and PIP2 being equally expressed throughout mitosis, their role as cortical receptors of NuMA have been proposed to be dependent on Cdk1 activity and specific to anaphase. However, chemically-induced increase of PIP2 levels during metaphase lead to NuMA-dependent spindle orientation

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18 defects, independent of Gαi/LGN and the cortical actin network (Kotak et al., 2014). In addition, perturbations of phosphoinositide modulating kinases and phosphatases (for example PTEN and PI3K) have also been reported to result in spindle positioning defects during metaphase (Cauvin and Echard, 2015;

Kotak et al., 2014; Lancaster et al., 2013; Toyoshima et al., 2007).

Protein 4.1

Protein 4.1 is a cytoskeletal protein that is important for the actin organization. In in vitro 4.1 can form mitotic asters in the Xenopus egg and mammalian mitotic extracts (Krauss et al., 2004). During mitosis, 4.1 is phosphorylated by Cdk1 and acquires an additional role at the centrosomes, where it contributes to the spindle pole stabilization with NuMA and dynein/dynactin (Huang et al., 2005; Mattagajasingh et al., 1999).

As previously mentioned, NuMa carries a specific 4.1-binding domain in the C-terminal, and 4.1 has been reported to recruit NuMA to the cortex, therefore acting as an additional cortical receptor for NuMA during mitosis. It was first reported that 4.1 is required for cortical NuMA specifically during anaphase. However, later reports indicate that 4.1 stabilizes cortical NuMA during metaphase, in combination with LGN (Kiyomitsu and Cheeseman, 2013; Kotak et al., 2014; Seldin et al., 2013).

1.6 Associated consequences of uncontrolled spindle orientation

Collective studies observed the loss of the homeostatic balance between ACD and SCD in pathologies.

Pathogenic mutations in proteins that are responsible for neurological diseases and disorders (such as microcephaly, lissencephaly, and Huntington’s disease) lead to spindle misorientation that favor ACD over SCD (Noatynska et al., 2012). For example, Huntingtin, a protein that is often mutated in patients with Huntington’s disease, interacts with microtubules and dynein (Borrell-Pages et al., 2006). The depletion of Huntingtin in cultured cells leads to a spindle misorientation that is caused by the loss of NuMA and dynein/dynactin at the centrosomes (Godin et al., 2010; Godin and Humbert, 2011). For cancer, studies have shown the loss of tissue organization that results from uncontrolled SCD. Differential expression of

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19 the members of the Gαi/LGN/NuMA complex have been observed in cancer. For instance, LGN and NuMA are overexpressed in luminal breast cancer cells. Furthermore, the inactivation of PTEN (tumor suppressor gene) in cultured cells results in spindle positioning defects by influencing cortical dynein (Toyoshima et al., 2007). Collective evidence in investigations carried out in Drosophila indicates that defects in spindle orientation and positioning are closely associated with pathologies, however, studies carried out in mammals are at best correlative (Seldin and Macara, 2017).

Figure 1.6. Cortical and Spindle pole localization of NuMA regulated by phosphorylation.

Phosphorylation by Cdk1 promotes spindle pole localization of NuMA. Aurora A phosphorylation and PP2A dephosphorylation promotes cortical localization of NuMA. Cortical NuMA recruitment relies on Gαi/LGN in metaphase and 4.1/phosphoinositides in anaphase.

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1.7 Serine/Threonine Protein Phosphatase 1

Protein Phosphatase 1 (PP1), like PP2A, is a ubiquitously expressed serine/threonine phosphatase that belongs to the PPP superfamily of phosphatases. Catalytic sites of PP1 and PP2A show structural similarities which may explain why they are redundant in many of their pathways (Bollen et al., 2009;

Bollen et al., 2010; Guo et al., 2014). PP1 is expressed as three different isoforms (α, β, and γ) which all share similar enzymatic properties. PP1 is highly conserved with 80% sequence identity between human and yeast, and the expression of human PP1 can rescue the lethality caused from the loss of PP1 in yeast (Gibbons et al., 2007).

Like PP2A, PP1 forms complexes with its regulatory subunits which determine their functional properties.

However, PP1 has approximately 200 validated regulatory subunits or PP1-Interacting-Proteins (PIPs)(Bollen et al., 2010). These regulatory subunits are responsible in regulating PP1 activity, localization, and substrate specificity (Figure 1.7) (Heroes et al., 2013). There are multiple reported binding motifs for the PP1 regulatory subunits (Heroes et al., 2013); some of the better characterized are the RVxF, SILK, MyPhoNE, SpiDoC and IDoHA. Approximately 90% of the 200 validated regulatory subunits harbor the RVxF motif while the remaining population shares the other motifs (Bollen et al., 2010; Heroes et al., 2013). The regulatory subunits of PP1 contribute to the diversity of PP1, that is required for multiple cellular processes; for example cell cycle progression, DNA damage response, glycogen metabolism, and transcription(Ceulemans and Bollen, 2004; Heroes et al., 2013).

The role of PP1 in mitosis was first described in S.cerevisiae, where mutations on Glc7 (PP1) showed arrest in M phase and chromosome missegregation. It was later found that Glc7 opposes Ipl1 (Aurora B) kinase to regulate chromosome segregation (Chan and Botstein, 1993; Francisco et al., 1994; Pinsky et al., 2006;

Pinsky et al., 2009; Tu and Carlson, 1994). Aurora B kinase is involved in the regulation of SAC by destabilizing erroneous kinetochore-microtubule attachments. Kinetochore-associated PP1 stabilizes kinetochore-microtubule attachments by counteracting Aurora B to ultimately silence SAC (Rosenberg et al., 2011). PP1 is able to do this by inactivating or removing Aurora B from the kinetochore and

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21 dephosphorylating Aurora B substrates (Lesage et al., 2011; Liu et al., 2010; Murnion et al., 2001;

Vanoosthuyse and Hardwick, 2009). Due to the mentioned role of PP1, the targeting and activation of PP1 at the kinetochore is important for SAC. At the kinetochore, the major binding partner of PP1 is KNL-1, of the KMN network. KNL-1 carries the RVxF motif for its interaction with PP1, and mutations of the RVxF motif results in the hyper-activation of SAC (Rosenberg et al., 2011). Motor proteins, CENP-E and Kif18A (both carry the RVxF motif) target PP1 to the kinetochore during chromosome congression (De Wever et al., 2014; Kim et al., 2010). Furthermore, Mytp1 (Myosin phosphatase 1) carries the MyPhoNE motif for its PP1 interaction that allows it to target PP1 to kinetochore-Plk1(Lesage et al., 2011). Subsequently, Repo- Man has been reported to recruit PP1 to the kinetochores in mitosis but more robustly in anaphase (further details of Repo-Man will be described below) (Trinkle-Mulcahy et al., 2006). Activity regulating subunits Sds22 and Inhibitor-3 are reported to regulate the activity of PP1 at the kinetochore and cytoplasm (Eiteneuer et al., 2014; Posch et al., 2010; Wurzenberger et al., 2012). Altogether, there are multiple ways of regulating PP1 targeting and activity at the kinetochore, and perturbing any of these regulatory processes results in higher activity of Aurora B.

Figure 1.7. Protein Phosphatase 1 complexes. PP1 complex is composed of the catalytic subunit (grey), and its multiple regulatory subunits (beige, brown, purple), which dictate PP1 activity, localization, and substrate specificity (yellow and green). Adapted from (Bollen et al., 2009)

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22 Apart from kinetochore-associated PP1, functions of PP1 at the centrosomes and cortex has been reported.

In C.elegans, GSP-1/2 (PP1) negatively regulates ZYG-1 (Plk4) levels at the centrosomes to limit centriole duplication (Peel et al., 2017). In mammalians cells, Centrosomal Protein 192 (CEP192; also known as PP1 regulatory subunit 192) carries a PP1 binding motif and is responsible for recruiting PP1 to the centrosomes to control the activity of Aurora A and Plk1 (Joukov et al., 2014; Nasa et al., 2017). Moreover, centrosome- associated regulatory subunit, inhibitor-2, stimulates Aurora A activity by inhibiting PP1 activity at the centrosomes (Satinover et al., 2004). It has been illustrated in S.cerevisiae, that Glc7 (PP1) is recruited to the bud cortex by its regulatory subunit Bud14, where Glc7 stabilizes microtubule interactions for dynein functions (Knaus et al., 2005). In Drosophila and mammalian cells, when the kinetochore PP1-Sds22 complex is in close proximity to the cortex (in mid-anaphase) it induces polar membrane relaxation by dephosphorylating moesin (a crosslinker of actin at the membrane) to aid in membrane elongation (Ramkumar and Baum, 2016; Rodrigues et al., 2015).

According to the majority of studies characterizing PP1 and PP2A in mitosis, these two phosphatases are considered to be the counteracting phosphatases for Cdk1, Aurora A, and Aurora B substrates, and even acting upon the kinases themselves (Bollen et al., 2009; Heroes et al., 2013; Jeong and Yang, 2013; Kotak et al., 2013; Lesage et al., 2011). While kinase activity is essential for mitotic entry processes, collective studies indicate that phosphatases are crucial for mitotic exit processes that are required for progression of the daughter cells into interphase: such as chromosome segregation and decondensation, spindle disassembly, and nuclear reformation, (Ceulemans and Bollen, 2004; Wurzenberger and Gerlich, 2011).

DNA targeting subunit, Repo-Man

Repo-Man (REcruits PP1 onto Mitotic chromatin at ANaphase or Cell Division Cycle Associated 2:

CDCA2) was first characterized in mammalian cells as a nuclear protein that targets PP1 to the chromatin throughout the cell cycle (Trinkle-Mulcahy et al., 2006). Repo-Man carries the RVxF motif and is found to physically interact with all PP1 isoforms (Hein et al., 2015; Huttlin et al., 2017; Huttlin et al., 2015; St- Denis et al., 2016). Recent reports have shown that Repo-Man recruits PP1γ to the chromatin, while

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23 overexpression of exogenous Repo-Man results in elevated levels of chromatin PP1α as well (Trinkle- Mulcahy et al., 2006). Prior to mitosis, Repo-Man is responsible for the PP1-dependent attenuation of DNA Damage Response (DDR). By recruiting PP1 to the chromatin, it regulates the phosphorylation state of ATM serine/threonine kinase (Ataxia Telangiectasia Mutated; a crucial activator of the DDR) and thus its activity (Peng et al., 2010). Additional reports specify the involvement of Repo-Man in the chromosome decondensation and nuclear reassembly processes, all via its targeting role of PP1 (de Castro et al., 2017;

Vagnarelli, 2014; Vagnarelli and Earnshaw, 2012; Vagnarelli et al., 2011).

Repo-Man activity and kinetochore-localization is regulated by phosphorylation. At early stages of mitosis, a dynamic but small fraction of Repo-Man localizes at the chromosomes. This is negatively regulated by Cdk1 and Aurora B phosphorylation; where chemical inhibition of both kinases and non-phosphorylated mutants of Repo-Man accumulate rapidly at the chromosomes during prometaphase and metaphase. In addition, Cdk1 phosphorylates Repo-Man with a second role, where it hinders the binding of Repo-Man to PP1 via the RVxF motif. On the other hand, Aurora B phosphorylates Repo-Man on an independent site of the RVxF motif that prevents PP2A association (Qian et al., 2013; Vagnarelli and Earnshaw, 2012;

Vagnarelli et al., 2006; Vagnarelli et al., 2011).

PP1/Repo-Man complex has been demonstrated to counteract Aurora B activity during metaphase and anaphase. The knockdown of Repo-Man results in increased levels of Aurora B activity on the centromeres and thus, Aurora B substrates remain phosphorylated; such as histone H3 (at Threonine 3) and Dsn1 (at Threonine 100) (Wurzenberger et al., 2012). Ultimately, during mitosis, PP1/Repo-Man regulates proper chromosome segregation, for its knockdown results in lagging chromosomes, chromosome bridges, and a defect in the poleward movements of chromosomes (Wurzenberger et al., 2012).

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1.8 p97 and cofactor p37

1.8.1 p97/VCP/CDC48 and its cofactors

p97, also known as Vasolin Containing Protein (VCP) (CDC-48 in C.elegans and Cdc48 in S.cerevisiae), is a highly conserved member of the AAA family that was first discovered in yeast as a regulator of cell cycle progression (Moir et al., 1982). p97 is an essential protein and its knockout in mice have been shown to be embryonically lethal (Muller et al., 2007). p97 is an ubiquitous and highly abundant protein that localizes at the nucleus, cytoplasm, and membranous structures (Erzurumlu et al., 2013; Zeiler et al., 2012).

p97 hydrolyzes ATP as a source of energy to segregate target proteins from complexes or membrane structures, and subjects them to degradation or recycling (Figure 1.8A) (Meyer et al., 2012; Yamanaka et al., 2012).

Structurally, the p97 hexamer is made of six p97 proteins that assemble like a barrel structure with a pore at the center (Figure 1.8B).The p97 protein is composed of an N-terminal domain, D1 and D2 ATPase domain, and a C-terminal tail. The ATPase domains are responsible for hydrolyzing ATP to generate mechanical energy to disassemble or extract proteins from complexes or membrane structures (Meyer et al., 2012). The D1 domain is mainly required for the hexamerization of p97, while the D2 domain primarily mediates the ATP hydrolysis (Song et al., 2003; Wang et al., 2003).

The cofactors of p97 guide the localization and substrate specificity of p97. The p97 N-terminal and C- terminal domains are the binding sites for its cofactors. The cofactors harbor different motifs and domains for their interaction with p97; UBX (ubiquitin regulatory X) domain, UBX-L domain, SHP box (also known as BS1), VBM or VIM (VCP binding/interacting motif), PUB (PNGase/UBA or UBX domain) (Buchberger et al., 2015; Schuberth and Buchberger, 2008). Many p97 cofactors share common binding motifs or domains, this makes p97-cofactor interactions to be a puzzling concept. To date, biochemical and structural studies indicate that these cofactors can have different modes of binding: a competitive mode between cofactors, a bipartite mode of binding, and a hierarchical association with different cofactors specifying different pathways (Hanzelmann et al., 2011; Schuberth and Buchberger, 2008). In addition, it has been

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25 reported that the nucleotide-binding state of p97 can contribute to the affinity towards a preference of cofactors (Ewens et al., 2014).

Figure 1.8. The mechanism, multicellular processes, and structure of p97. A) p97 and its cofactors (UBX) recognizes ubiquitinated substrates (S), extracts substrates upon ATP hydrolysis, and subjects them for recycling or proteasomal degradation. B) Structure of the p97 hexamer and its domains C) Schematic representation of the biological roles of p97 and its associated cofactors.

A

B

C

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26 Ever since its discovery, the role and function of p97 has been expanding, confirming it as more than just a regulator of the cell cycle. Collective data indicate that p97 is important for mulitcellular processes during interphase (Figure 1.8C); such as Endoplasmic Reticulum and Golgi apparatus biogenesis, DNA replication, and autophagy (Cao et al., 2003; Dobrynin et al., 2011; Meyer et al., 2012; Meyer et al., 2010;

Ramadan et al., 2007; Torrecilla et al., 2017).

New roles of p97 in cell division, particularly during mitosis, has been emerging in recent years with multiple studies indicating that p97 regulates processes ranging from G2/M transition to mitotic exit. During meiosis I in C.elegans, CDC-48 is important in securing proper meiotic chromosome segregation by limiting the amount and activity of chromosomal AIR-2 (Aurora B) (Sasagawa et al., 2012). p97 and its cofactors Npl4/Ufd1 promote CDC25A (Cdk1-activating phosphatase) degradation as a response to DNA damage/replication stress in late G2. This ensures that cells with DNA damage do not progress into mitosis (Riemer et al., 2014). Subsequently, our lab reported that p97 cofactors p37 and p47 are involved in mitotic entry processes. This was illustrated in C.elegans and mammalian cells, where cofactors p37/p47 and UBXN2 (in C.elegans) regulate centrosome maturation and dynamics by limiting the recruitment of centrosomal Aurora A (Kress et al., 2013). Cofactors Ufd1-Npl4 limit the levels and therefore the activity of chromosomal Aurora B and Survivin to promote proper chromosome segregation (Cao et al., 2003;

Dobrynin et al., 2011; Vong et al., 2005). While in mitotic exit, it was demonstrated in the early embryos of C.elegans and extracts of Xenopus laevis egg that p97 extracts ubiquitinated Aurora B from chromatin to promote chromatin decondensation and nuclear membrane reformation (Meyer et al., 2010; Ramadan et al., 2007). Collectively, p97 and its cofactors are a much conserved and needed factor throughout mitosis.

1.8.2 Cofactors p37 and p47

Among the cofactors of p97, the UBX domain-containing cofactors are categorized into subfamilies according to their structure and sequence similarities outside the UBX domains: UBXD7, UBXD8, FAF1, SAKS1, and p47-subfamilies (Schuberth and Buchberger, 2008; Stach and Freemont, 2017). The homologue of p47-subfamily in other organisms are Shp1 (Suppressor of High copy of PP1) in S.cerevisiae,

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27 UBXN2 in C.elegans, Ubx3 in Schizosaccharomyces pombe (S.pombe), while in mammals the expression of the subfamily has expanded to p47, p37, UBXD4, and Socius (or UBXD5) (Schuberth and Buchberger, 2008). All the proteins of the p47-subfamily carry two binding motifs for their interaction with p97 (via the UBX domain and SHP box motif), a SEP domain that is important for the trimerization, while only p47 has a UBA domain (for ubiquitin interaction) (Beuron et al., 2006; Schuberth and Buchberger, 2008; Yuan et al., 2004). The most characterized, p37 and p47 share the most homology (63%) outside the UBX domain and both are involved in the maintenance and reformation of the Golgi apparatus and Endoplasmic Reticulum after mitosis (Kaneko et al., 2010; Kano et al., 2005a; Kano et al., 2005b; Schuberth and Buchberger, 2008; Uchiyama and Kondo, 2005; Uchiyama et al., 2006).

In yeast, Shp1 was first discovered in S.cerevisiae to rescue the high lethality caused by the overexpression of Glc7 (PP1) during mitosis (Zhang et al., 1995). Ever since, Shp1 has been shown to positively regulate the nuclear localization of Glc7 through direct interactions during mitosis. This ensures that Glc7 counteracts Ipl1 (Aurora B) to promote error-free chromosome segregation (Bohm and Buchberger, 2013;

Cheng and Chen, 2010; Hu et al., 2012). Although this mechanism has not yet been demonstrated to be conserved in mammals, there has been biochemical and proteomic studies reporting the interaction of p37 and PP1 isoforms (Huttlin et al., 2017; Huttlin et al., 2015; Raman et al., 2015).

Our lab has previously demonstrated that p37 and p47 play an important role during mitotic entry in mammalian cells as well as in C.elegans. During the first cell division of the C.elegans embryo, UBXN2 is involved in centrosome maturation during prophase by limiting the recruitment of AIR-1(Aurora A) to the centrosomes. In addition, the depletion of UBXN2 resulted in a spindle misorientation that was partially rescued by co-depleting AIR-1 (Kress et al., 2013). Along the same line, in HeLa cells, p37 and p47 were shown to limit centrosomal Aurora A levels during prophase to regulate centrosome separation. As in C.elegans, p37/p47 depleted cells displayed spindle orientation defects. These data suggest that p37/p47/UBXN2 act via a conserved mechanism to regulate the centrosomes (Kress et al., 2013).

The p97-cofactor p37 regulates spindle orientation by limiting cortical NuMA recruitment via PP1/Repo-Man

Thesis

Reference

The p97-cofactor p37 regulates spindle orientation by limiting cortical NuMA recruitment via PP1/Repo-Man

The p97-cofactor p37 Regulates Spindle Orientation By Limiting Cortical NuMA Recruitment Via PP1/Repo-Man

UNIVERSITÉ DE GENÈvr

DOCTORAT ES SCIENCES, MENTION BIOLOGIE Thèse de Monsieur Byung Ho LEE

<<The p97-cofactor p37 Regulates Spindle Orientation By Limiting Cortical NuMA Recruitment Via PPl/Repo-Mâhn

directrice de thèse (Faculté de

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R. J.

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de biologie

Monsieur B.

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Monsieur P.

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Thèse -5190

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Contents

Figure List

Abstract

Résumé

1. Introduction

1.1. Mitosis

1.2 Spindle orientation and positioning

1.3 Cortical regulators of spindle orientation

1.4 Regulation of cortical NuMA in metaphase: Gαi/LGN dependent

1.5 Regulation of cortical NuMA in anaphase: Gαi/LGN independent

1.6 Associated consequences of uncontrolled spindle orientation

1.7 Serine/Threonine Protein Phosphatase 1

1.8 p97 and cofactor p37