Role of Aurora B-mediated phosphorylation during mitosis and interphase Carmen Taveras Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy under the Executive Committee of the Graduate School of Arts and Science COLUMBIA UNIVERSITY 2017 © 2017 Carmen Taveras All rights reversed ABSTRACT Role of Aurora B-mediated phosphorylation during mitosis and interphase Carmen Taveras Accurate chromosome segregation requires a spindle apparatus composed of microtubules that arise from the spindle to attach to the kinetochore, a protein complex assembled at the centromere of each chromosome. Failure to segregate chromosomes accurately may lead to lethal early developmental defects and tumorigenesis. To achieve proper kinetochore binding to microtubules, mammalian cells have evolved elaborate mechanisms to correct attachment errors and stabilize correct ones. Current models suggest that tension between kinetochore pairs (inter- kinetochore stretch) and tension at the kinetochore (intra-kinetochore stretch) produces a spatial separation of Aurora B kinase from kinetochore-associated and microtubule-binding substrates, subsequently reducing their phosphorylations and increasing their microtubule affinity. However, the tension-based models do not explain how the initial microtubule binding at unattached kinetochores occurs, where there is no tension and kinetochore-associated substrates are highly phosphorylated and, hence unable to bind to microtubules. Therefore, there must be a mechanism that explains how the phosphorylation of kinetochore substrates by Aurora B is reduced in the absence of tension. In the first part of this thesis, I examine the structural features of the coiled-coil domain of the kinetochore-associated kinesin motor protein, CENP-E. Using Single-Molecule High- Resolution Colocalization (SHREC) microscopy analysis of kinetochore-associated CENP-E, I show that CENP-E undergoes structural rearrangements prior to and after tension generation at the kinetochore. Chemical inhibition of the motor motility or genetic perturbations of the coiled-coil domain of CENP-E increases Aurora B-mediated Ndc80 phosphorylation in a tension-independent manner. Importantly, metaphase chromosome misalignment caused by inhibition of CENP-E can be rescued by chemical inhibition of Aurora B kinase. Therefore, CENP-E regulates the initial kinetochore binding to microtubules and the stabilization of kinetochore-microtubule attachments. Formin-dependent actin assembly is known to play a role in multiple processes, including cytokinesis, filopodia formation, cell polarity, and cell adhesion. Thus, formin malfunction is directly linked to various pathologies, including defects in cell migration and tumor suppression. Although the role of formins in actin polymerization has been well described, the mechanistic processes that regulate the actin assembly function of formins remain poorly understood, especially the interplay among the various sub-families of formins and how they are spatiotemporally regulated. In the second part of this thesis, I show that Aurora B-mediated phosphorylation of the formin, mDia3 regulates actin assembly. Previous studies identified two Aurora B phosphorylation sites in the FH2 domain of mDia3. To this end, phosphomimetic and non-phosphorylatable mutants of a constitutively active form of mDia3 were designed to test whether phosphorylation by Aurora B regulates actin assembly. Using an in vitro actin polymerization kinetic assay and expression of fluorescently-tagged constitutively active mDia3 in cells, I show that phosphorylation of mDia3 by Aurora B induces the actin assembly function of mDia3. Furthermore, using a phospho-specific antibody, I show that mDia3 is phosphorylated by Aurora B. Live-cell analysis shows that perturbations of these phosphorylation sites affect cell migration and cell spreading. Therefore, I illustrate a novel regulatory mechanism for the actin assembly function of mDia3 that is dependent on Aurora B kinase activity. TABLE OF CONTENTS List of figures iii Acknowledgements v Chapter 1: Introduction.................................................................................................................1 Part 1: Stabilization of kinetochore-microtubule attachments..............................................2 1. Kinetochore-mediated microtubule capture................................................................2 2. Aurora B corrects kinetochore-microtubule attachment error....................................7 3. Phosphatase recruitment to kinetochores counteract Aurora B kinase activity......... 9 4. The kinetochore phosphorylation/dephosphorylation balance is regulated by tension.......................................................................................................................11 5. CENP-E facilitates the stabilization of kinetochore-microtubule attachments.........16 6. Rationale and objectives for chapter 2......................................................................21 Part 2: Regulation of formin-mediated actin assembly.......................................................23 1. The actin cytoskeleton plays a major role in cell migration.....................................23 2. Formin-mediated actin assembly..............................................................................25 3. Aurora B-mediated phosphorylation of mDia3 regulates mDia3 function...............28 4. Rationale and objectives for chapter 3......................................................................31 Chapter 2: CENP-E regulates Aurora B kinase activity at the kinetochore..........................33 Introduction........................................................................................................................33 Materials and methods.......................................................................................................35 Results................................................................................................................................38 Discussion..........................................................................................................................64 i Chapter 3: Aurora B regulates the actin assembly function of mDia3...................................67 Introduction........................................................................................................................67 Materials and methods.......................................................................................................68 Results................................................................................................................................71 Discussion..........................................................................................................................82 Chapter 4: Discussions and future directions...........................................................................83 Part 1: CENP-E regulates Aurora B kinase activity at the kinetochore.............................83 1. Aurora B-mediated phosphorylation of outer-localized kinetochore substrates are regulated by microtubule attachment not tension.....................................................83 2. The unique structural features of CENP-E facilitate the stabilization of kinetochore- microtubule attachments before and after bi-orientation..........................................89 3. Recruitment of phosphatases to the kinetochore balances the phosphorylation of outer kinetochore-associated components................................................................99 Part 2: Aurora B regulates the actin assembly function of mDia3..................................104 1. Aurora B-mediated phosphorylation activates the actin assembly function of the FH2 domain of mDia3.................................................................104 2. Aurora B-mediated phosphorylation of mDia3 regulates the ‘crosstalk’ between the actin and the microtubule function of mDia3..................................107 3. Aurora B regulates the cytoskeleton in interphase cells......................................108 References...................................................................................................................................111 ii LIST OF FIGURES Chapter 1 Figure 1.1 Initial microtubule capture by kinetochores...................................................................4 Figure 1.2 Illustration of the tension-based models.......................................................................15 Figure 1.3. CENP-E has a unique long and flexible coiled-coil domain.......................................22 Figure 1.4 mDia3 is phosphorylated by Aurora B.........................................................................32 Chapter 2 Figure 2.1 Perturbation of the coiled-coil domain does not affect the kinetochore localization of CENP-E....................................................................................................................................41 Figure 2.2. The long and highly flexible coiled-coil domain of CENP-E is essential for its kinetochore function......................................................................................................................42 Figure 2.3. The length and flexibility of the coiled-coil domain of CENP-E mediate the folding conformation of CENP-E at the kinetochore.................................................................................44 Figure 2.4. Validation of the 3-dimensional SHREC