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Investigation of the functions of the NDC80 kinetochore complex in budding yeast Ma, Lina 2010

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 INVESTIGATION OF THE FUNCTIONS OF THE NDC80 KINETOCHORE COMPLEX IN BUDDING YEAST   by LINA MA   A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES  (Genetics)   THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  October 2010 © Lina Ma, 2010 ii  Abstract  Kinetochores are multi protein complexes comprised of inner kinetochore proteins that assemble on centromeric DNA sequences and outer kinetochore proteins that bind to microtubules. Kinetochores are responsible for accurate transmission of genetic information during each cell division. In the budding yeast Saccharomyces cerevisiae, the highly conserved Ndc80 kinetochore complex has been well characterized in terms of its roles in chromosome-microtubule attachment and spindle checkpoint.  The work presented in this thesis suggests that the Ndc80 complex has additional cellular roles. The kinetochore is required to prevent spindle expansion  during S phase in budding yeast, but the mechanism of how the  kinetochore maintains integrity of the bipolar spindle in S phase is not well understood. In Chapter 2, I demonstrate that a  mutation in Spc24, a component of the Ndc80 complex, causes lethality when cells are exposed to the DNA  replication inhibitor hydroxyurea (HU) due to premature spindle  expansion and segregation of incompletely replicated DNA. Overexpression  of Stu1, a CLASP-related MT-associated protein or a truncated  form of the XMAP215 orthologue Stu2 rescues spc24-9 HU lethality  and prevents spindle expansion. Stu1 and Stu2  localize to the kinetochore early in the cell cycle and Stu2  kinetochore localization depends on Spc24. I propose that mislocalization  of Stu2 results in premature spindle expansion in S phase stalled  spc24-9 mutants. In Chapter 3 and Chapter 4, I present data suggesting that the Ndc80 complex has a role iii  in the cAMP/PKA glucose signaling pathway and actin regulation. Firstly, I identified genetic interactions between Spc24 and the components of cAMP/PKA pathway and demonstrated that spc24 mutants have defects in PKA signaling in response to glucose depletion. Interestingly, most of the temperature sensitive mutant alleles of the Ndc80 complex are rescued by non-glucose carbon sources.  Finally, I present actin defects in spc24 mutants and genetic interactions between spc24 mutants and mutants of proteins involved in actin turnover, nucleation or polarity establishment. Further investigation on the mechanism and rationale behind the kinetochore-cytoskeleton interactions will be an intriguing avenue for future research.    iv  Preface Chapter 2 is modified from a published paper ―Ma L., McQueen J., Cuschieri L., Vogel J., Measday V, Spc24 and Stu2 Promote Spindle Integrity When DNA Replication is Stalled, Molecular Biology of the Cell, 18(8): 2805-2816 (2007)‖. I did the experiments described in this chapter with the following exceptions: The spot assay in Figure 2.1(A) and images in Figure 2.1(B) and 2.14 were performed by Vivien Measday. Jennifer McQueen performed all the experiments shown in Figure 2.4. Timelapse imaging in Figure 2.9, 2.10 and 2.11 were performed by Jennifer McQueen (Measday lab), Lara Cuschieri (Vogel lab) and Jackie Vogel at McGill University.  The paper was written by Vivien Measday. Copyright of the paper belongs to American Society for Cell Biology (ASCB) and the link to the publication on-line is: http://www.molbiolcell.org/cgi/content/full/18/8/2805?maxtoshow=&hits=10&RESULTFORMA T=&author1=Lina+Ma&andorexacttitle=and&andorexacttitleabs=and&andorexactfulltext=and& searchid=1&FIRSTINDEX=0&sortspec=relevance&resourcetype=HWCIT. v  Table of contents Abstract ..................................................................................................................................... ii Preface...................................................................................................................................... iv Table of contents ........................................................................................................................v List of tables ............................................................................................................................. ix List of symbols and abbreviations ......................................................................................... xiii Nomenclature ...........................................................................................................................xv Acknowledgements ................................................................................................................ xvi Chapter 1  General Introduction ..............................................................................................1 1.1 Cell division .............................................................................................................1 1.1.1 Life cycle in the budding yeast ..................................................................1 1.1.2 Mitotic cell cycle ........................................................................................2 1.2  Kinetochore ..............................................................................................................4 1.2.1  Centromere .................................................................................................4 1.2.2  Kinetochore assembly ................................................................................5 1.2.3  Regulation of kinetochore-microtubule attachment ...................................9 1.2.4  The Ndc80 complex .................................................................................10 1.3  Summary ................................................................................................................13 Chapter 2  Spc24 and Stu2 Promote Spindle Integrity when DNA Replication is Stalled ....24 2.1 Introduction ............................................................................................................24 2.2 Materials and methods ...........................................................................................27 2.2.1 Strain construction....................................................................................27 2.2.2 HCS screen ...............................................................................................28 vi  2.2.3 Plasmid construction - subcloning of HCS genes ....................................28 2.2.4 Microscopic analyses ...............................................................................29 2.2.5 Live cell analysis ......................................................................................30 2.2.6 Spindle length measurements ...................................................................31 2.2.7 Stu2-VFP fluorescence measurement ......................................................31 2.2.8 ChIP assays (Chromatin immunoprecipitation) .......................................32 2.3 Results ....................................................................................................................33 2.3.1 Spc24 is required for viability and preventing spindle expansion during HU arrest ..............................................................................................................33 2.3.2 Characterization of the kinetochore in spc24-9 mutants ..........................34 2.3.3 Bipolar attachment is not a requirement for maintaining a short spindle in HU treated cells ................................................................................................34 2.3.4 Identification of HCS genes that rescue spc24-9 HU lethality ................36 2.3.5 HCS rescue occurs through restraining spindle expansion ......................37 2.3.6 Stu1 localizes to kinetochores prior to anaphase .....................................40 2.3.7 Stu2 localizes to kinetochores early in the cell cycle ...............................41 2.3.8 Stu2 is mislocalized in HU treated spc24-9 cells .....................................42 2.4 Discussion ..............................................................................................................45 Chapter 3  Determination of a Novel Role for the Ndc80 Kinetochore Complex in the Ras2/cAMP/PKA Pathway ......................................................................................................68 3.1 Introduction ............................................................................................................68 3.2 Materials and methods ...........................................................................................70 3.2.1 Strains, plasmids and media .....................................................................70 vii  3.2.2 Trehalose determination ...........................................................................70 3.2.3 Msn2 Western blot ...................................................................................71 3.3 Results ....................................................................................................................71 3.3.1 The temperature sensitivity of spc24-9 is rescued by reducing cAMP/PKA activities ...........................................................................................71 3.3.2 spc24-9 cells have less trehalose compared to wild type .........................73 3.3.3 Msn2 dephosphorylation is compromised in spc24 upon glucose depletion ...............................................................................................................74 3.3.4 Non-glucose carbon sources rescue the ts of the mutants of Ndc80 complex components ............................................................................................75 3.4 Discussion ..............................................................................................................77 Chapter 4  Identification of Actin Defects in Kinetochore Mutants in the Budding Yeast S. cerevisiae .................................................................................................................................89 4.1 Introduction ............................................................................................................89 4.2 Materials and methods ...........................................................................................93 4.2.1 Strains, plasmids and microbial techniques .............................................93 4.2.2 Timecourse and western blot ....................................................................93 4.2.3 Fluorescence microscopy .........................................................................94 4.3 Results ....................................................................................................................95 4.3.1 Morphology defects of spc24-9 ...............................................................95 4.3.2 spc24 mutants have actin defects .............................................................96 4.3.3 Septin localization in mutants of the Ndc80 complex .............................98 4.3.4 spc24 alleles might have more actin patches ...........................................99 viii  4.3.5 Genetic interactions between spc24 alleles and actin mutants ...............100 4.3.6 Formin mutants rescue the ts of spc24 alleles ........................................101 4.3.7 Hyperactivation of Cdc42 pathway might rescue the ts of spc24-9 .......102 4.4 Discussion ............................................................................................................103 Chapter 5  Summary and Perspectives.................................................................................125 Bibliography ..........................................................................................................................132 Appendices .............................................................................................................................154 Appendix A. spc24-9 arrests in G1 after metaphase release. .......................................154 Appendix B. Analysis of Ndc80 localization. ..............................................................159   ix  List of tables Table 1.1 Homologs of Ndc80 complex in multiple organisms .................................. 16 Table 2.1 HCS screen of spc24-9 HU lethality ........................................................... 51 Table 2.2 List of Yeast Strains ..................................................................................... 52 Table 3.1 List of strains ............................................................................................... 82 Table 4.1 List of strains ............................................................................................. 107    x  List of figures  Figure 1.1  Life cycle of Saccharomyces cerevisiae ...........................................................17 Figure 1.2 Mitotic cell cycle stages of budding yeast ........................................................18 Figure 1.3 A simplified view of the cell-cycle control system ..........................................19 Figure 1.4 Centromere of Saccharomyces cerevisiae ........................................................20 Figure 1.5 Protein architecture of the Saccharomyces cerevisiae kinetochore ..................21 Figure 1.6 The molecular machinery of kinetochore–microtubule attachment .................23 Figure 2.1 spc24-9 mutants are sensitive to HU due to inappropriate spindle expansion .54 Figure 2.2  Ndc80 CEN association is disrupted in spc24-9 mutants ..................................55 Figure 2.3  Ndc80 is mislocalized in spc24-9 mutants ........................................................56 Figure 2.4 spc24-9 mutants are capable of establishing bipolar attachment .....................57 Figure 2.5  Spindle expansion in spc24-9 mutants depends on active Stu2 ........................58 Figure 2.6  Stu2∆N-VFP localization overlaps with endogenous Stu2-CFP ......................59 Figure 2.7  Stu1 localizes to kinetochores and the spindle midzone ...................................60 Figure 2.8  Stu2 CEN binding is abolished in spc24-9 mutants whereas Stu1 is still able to associate with CEN DNA...............................................................................................61 Figure 2.9 Time-lapse analysis of Stu2-VFP .....................................................................62 Figure 2.10  Increased spindle length correlates with Stu2 mislocalization and reduction .63 Figure 2.11  Decreased Stu2 in the spc24-9 mutant results in oscillation of spindle length xi  ........................................................................................................................................64 Figure 2.12  Overexpression of STU2∆N does not affect wild type spindle length in an unperturbed cell cycle ....................................................................................................65 Figure 2.13  spc24-9 mutants display spindle expansion early in the cell cycle but a delay in anaphase .....................................................................................................................66 Figure 2.14  spc24-9 mutants accelerate through S phase and delay at anaphase at restrictive temperature ...................................................................................................67 Figure 3.1 The cAMP-PKA pathway in yeast (A) and its physical effects (B) .................84 Figure 3.2 Genetic interactions between spc24-9 and the components of the Ras2/cAMP/PKA pathway.............................................................................................85 Figure 3.3 Growth curves (A-C) and trehlaose levels (D-F) in spc24 cells.......................86 Figure 3.4 Kinetic analysis of Msn2 phosphorylation upon glucose starvation ................87 Figure 3.5 Cell dilution assay of kinetochore mutants on various carbon sources ............88 Figure 3.6 Cell dilution assay of indicated strains grown on YPD for 3 days ...................88 Figure 4.1 Cell polarity in budding yeast is established by a polarized actin cytoskeleton throughout the cell cycle .............................................................................................. 111 Figure 4.2  A variety of cellular components are polarized through interactions with actin cables and the cell cortex ............................................................................................. 112 Figure 4.3 Septins in budding yeast ................................................................................. 113 Figure 4.4 Cell cycle-regulated organization of actin cytoskeleton and Cdc42 localization xii  in budding yeast ........................................................................................................... 114 Figure 4.5 Swe1-dependent morphology checkpoint in budding yeast ........................... 115 Figure 4.6 The Swe1 dependent morphology checkpoint is activated in spc24-9 ........... 117 Figure 4.7  Actin defects and Bud6 mislocalization in spc24-9 mutants........................... 118 Figure 4.8  spc24 mutants have normal actin ring formation ............................................ 119 Figure 4.9  Analysis of septin function in spc24 mutants ..................................................120 Figure 4.10  spc24 mutants have increased numbers of actin patches ...............................121 Figure 4.11  Genetic interactions between spc24 and actin mutants .................................122 Figure 4.12  Suppression of spc24 mutant growth defect by formin mutants ...................123 Figure 4.13  Genetic interaction between spc24 and cdc42-117 and gic mutants .............124 Figure A.1 spc24-9 mutants delay in G1 after a metaphase arrest ...................................158 Figure B.1 Ndc80 is scattered along the spindle in anaphase ...........................................162 Figure B.2 Localization of Ndc80 to a non-kinetochore foci ...........................................163 Figure B.3 Non-kinetochore pool of Ndc80 co-localizes with Tub1................................164    xiii  List of symbols and abbreviations ∆      deletion (symbol for a gene knockout) α-factor     alpha-factor APC     anaphase promoting complex bp      base pair cAMP     cyclic AMP Cdc      Cell division cycle Cdk     cyclin-dependent kinase CFP     Cyan Fluorescent Protein CH      calponin-homology ChIP     chromatin immunoprecipitation cMT(s)     cytoplasmic microtubule(s) DAPI     4’,6-diamino-2-phenylindole FACs     fluorescence activated cell sorting FH      formin-homology GAP     GTPase activating protein GEF     GDP-GTP exchange factor GFP     Green Fluorescent Protein GPCR     G protein coupled receptor xiv  HCS     high copy suppressor IP      immunoprecipitation kb      kilobase pair(s) kD      kilodaltons LatA     latrunculin A min.     minute(s) MT(s)     microtubule(s) NPCS     non-preferred carbon source PAGE     polyacrylamide gel electrophoresis PCR     polymerase chain reaction PKA     protein kinase A SAC     spindle assembly checkpoint S. cerevisiae    Saccharomyces cerevisiae SL      synthetic lethal SPB     spindle pole body SS      synthetic sick ts      temperature sensitive VFP     Venus Fluorescent Protein xv  Nomenclature Wild-type alleles in Saccharomyces cerevisiae are represented by italicized capital letters (e.g. SPC24), while mutant recessive alleles are denoted in lower case italics (e.g. spc24-9). Gene products are written with the first letter capitalized (e.g. Spc24). xvi  Acknowledgements I want to greatly thank Dr.Vivien Measday for her striking particularity and for giving me the opportunity to come to Canada and work in her lab. Thanks for waiting for me for over two hours in the airport on the first day I landed in Canada. Thanks for teaching me all the methods that I needed to learn. Thanks for reading all my proposals, committee meeting reports and thesis and making precise comments every time. Thanks for helping me prepare for my comprehensive and listening to my practice before each presentation. Thanks for prolonging my vacation every time when I visited China. Thanks for all the sweet gifts for the birth of my baby and each Christmas. More importantly, I am grateful that she always saw the end of the tunnel, especially when I didn’t. Her talent always sheds light on my research and accelerates my pace for publishing a paper.  I also want to thank Liz Conibear, Jim Kronstad and Geoffrey Wasteneys for agreeing to be members of my thesis committee, squeezing in time to attend my committee meetings and sharing suggestions whenever we met.  A very special thanks goes to my program director Hugh Brock for introducing me to the world of genetics, telling me what a real scientist is and what the life will be, and giving me the self-confidence to start a PhD. And thanks the program secretary Monica Deutsch for all the information and official letters.  The Measday lab is a great place to work and the support from everybody at any given time is more than one could expect. We are a small team but super warm. Thanks Nina Piggott, for ordering all the reagents I need and making the lab so neat and organized, and thanks for making all the delicious cakes when celebrating everyone’s birthday. Thanks Jennifer McQueen, for flying to Montreal and working day and night on the live imaging for my paper. Thank Angel Chang for all the nice biscuits. Thanks Krystina Ho for our delightful chats and help for my writing. Thanks Mike Anderson for teaching me the tips for powerpoint. Thanks Ravi, Nick, Linda, Steve and Dheva for making the media.  I also want to thank Hieter lab especially the following people: Thanks Irene for all the reagent help, thanks Jan, Kirk and Ben for sharing ideas and the help with microscope, thanks Karen and Jessica for teaching me how to use the FACs machine.  I want to thank my husband Hongwei Song, for doing as much housework as possible and taking care of Bryan when I was busy. Thanks to my parents, for agreeing to let their only xvii  child going abroad, suffering their missing and holding the longing for the short reunion every two or three years. Thanks to my son Bryan, your smile, sweet and smart always makes me happy and feel that all my effort is deserved. 1 Chapter 1  General Introduction  1.1 Cell division Life has the ability to pass on its genetic material to the daughter cell by cell division. Cell division involves the division of the nucleus (mitosis) and of the cytoplasm (cytokinesis) of a cell. Errors in chromosome transmission lead to aneuploidy (an abnormal number of chromosomes) and even cell death. Therefore, highly conserved machineries and mechanisms orchestrate cell cycle events to ensure proper chromosome transmission and promote genome stability.  1.1.1 Life cycle in the budding yeast The budding yeast Saccharomyces cerevisiae is a small, unicellular eukaryote that provides a perfect tool for studying mitotic events. It is easy to utilize genetic and biochemical experimental methods in budding yeast. Budding yeast grows rapidly when supplied with full nutrients and is able to alternate between a haploid and diploid state. Haploid cells have two mating types, MATa and MATα, which can mate with each other and generate diploid zygotes. In response to starvation diploid cells can sporulate and germinate to form four haploid cells (Figure 1.1) (Morgan, 2007). 2 1.1.2 Mitotic cell cycle During cell division, chromosomes undergo a cycle of replication and segregation that is synchronized with the cell cycle.  The mitotic cell cycle is divided into four phases, named G1, S (DNA replication), G2 and M (mitosis) (Figure 1.2).  M phase has two substages: metaphase and anaphase. In metaphase, duplicated chromosomes (sister chromatids) achieve bipolar attachment to the mitotic spindle via the kinetochore, which is a multiprotein complex connecting centromere DNA and spindle microtubules.  In anaphase, the sister chromatids are pulled apart from each other and segregated into the mother cell and the daughter cell.  The key machinery to define the boundaries between these phases is the cyclin dependent kinase (CDK) complex, which is activated by cyclin binding. The levels of Cdks are constant but the levels of cyclins oscillate during the cell cycle, therefore Cdk/cyclin complexes perform different and specific tasks depending on the cyclin partner. In budding yeast, there are nine cyclins (Cln1-3, Clb1-6) but only one CDK, called Cdc28 (Cdk1) (Figure 1.3A). Successive activation and inactivation of Cdc28/cyclin is required for ensuring proper progress during the cell cycle (Figure 1.3B) (Morgan, 2007).  Budding yeast must achieve a critical cell size in G1 prior to entering a new cell cycle. The G1/S cell cycle transition, termed START, occurs upon initiation of budding, replication of the 16 chromosomes and spindle pole body (SPB) duplication. SPBs are embedded in the nuclear envelope throughout the cell cycle, which is different from higher eukaryotes whose nuclear envelope is broken down during centrosome duplication (animal cells) or after spindle pole 3 formation (plant cells). SPBs nucleate microtubules both inside and outside of the nucleus. Cytoplasmic microtubules project towards the cell cortex where they perform an important function by generating forces required for spindle orientation. Intranuclear microtubules are composed of 32 kinetochore microtubules which each attach to one kinetochore, and 8 overlapping polar microtubules (Tanaka, et al., 2005; Morgan, 2007).  Prometaphase is defined as the stage when replicated chromosomes are transported to the spindle through the interaction between kinetochores and microtubules (Tanaka, et al., 2005). In yeast an accurate boundary between S phase and prometaphase is hard to define (McAinsh, et al., 2003; Tanaka, et al., 2005). During metaphase cells prepare the duplicated sister chromatids for a proper division. Unlike in higher eukaryotes, where chromosomes align at the metaphase plate between the spindle poles, yeast chromosomes are distributed along the metaphase spindle. The reason for this is precocious splitting of the sister chromatids around the centromere region while chromatid arms are still held together. This process is also known as biorientation, because kinetochores of two sister chromatids have to attach to spindle microtubules emanating from opposite SPBs (Straight, 1997; Tanaka, et al., 2005). Once the checkpoints monitoring the correct attachment between kinetochore and microtubules are satisfied, the cell is ready to transit from metaphase to anaphase and to separate the sister chromatids. The anaphase promoting complex (APC) is activated by Cdc20 and thereafter ubiquitinates proteins which are subsequently targeted for degradation by the 26S proteasome. One target of APC Cdc20  is securin which inhibits the protease separase. Ubiquitination of securin releases separase to cleave Scc1, 4 which is one of the four subunits of the cohesin complex bundling sister chromatids (Morgan, 2007; Peters, et al., 2008). After release from cohesin, sister chromatids rapidly move to opposite spindle poles in anaphase A. Anaphase B is characterized by the subsequent separation of the poles and elongation of the mitotic spindle (Straight, et al., 1997). Telophase is defined as the stage when the spindle reaches its maximum length and is subsequently disassembled, followed by the separation of the cell and the nucleus into two during cytokinesis (Tanaka, et al., 2005; Morgan, 2007) (Figure 1.2).  1.2 Kinetochore The kinetochore is defined as the complex of proteins that binds centromeric DNA and provides a platform for microtubule attachment that mediates chromosome movement and checkpoint signaling (Maney, et al., 2000). The budding yeast kinetochore is the best characterized and kinetochore architecture is highly conserved among higher eukaryotes (Kitagawa & Hieter, 2001; Przewloka & Glover, 2009; Santaguida & Musacchio, 2009).  1.2.1 Centromere Kinetochore proteins are required to assemble on a specific region of the chromosomes to ensure correct segregation. The region for assembly can be restricted to one site (monocentric chromosomes) or extended over the whole chromosome (holocentric chromosomes, e.g. 5 Caenorhabditis.elegans) (Madddox, et al., 2004). In budding yeast a relatively short stretch of 125 base pairs (bp), which is referred to as the centromere (Figure 1.4), is necessary and sufficient to direct the assembly of a kinetochore. By comparison, in fission yeast and vertebrates centromeres are spread over several kilobases or megabases (regional centromeres). Furthermore, the centromeric DNA sequence in budding yeast is conserved whereas no conserved centromere sequence is determined in other eukaryotes (Santaguida & Musacchio, 2009). These differences make the centromere of budding yeast much more accessible for genetic manipulations.  1.2.2 Kinetochore assembly The budding yeast kinetochore consists of more than 65 proteins which assemble in a hierarchical order as several multiprotein subcomplexes.  According to their relative position between centromere and spindle microtubules, they are often grouped into three categories — inner kinetochore, centre kinetochore and outer kinetochore protein complexes. Inner kinetochore proteins interact directly with centromeric DNA and provide a platform for the assembly of other kinetochore proteins. Outer kinetochore proteins bind directly to the spindle microtubules, and central kinetochore proteins are linkers for inner and outer kinetochore layers (Westermann, et al., 2007). This classification is consistent with a model that suggests that the kinetochore is built up from the centromere towards the microtubule attachment site in an inside-out manner (Santaguida & Musacchio, 2009). However, these categories only provide an approximate position and sometimes it is difficult to assign protein complexes strictly to one of 6 these layers. The core protein complexes are described in more detail in the following paragraphs and a simplified scheme of the kinetochore architecture is given in Figure 1.5 (Bloom, et al., 2010). In budding yeast, the inner kinetochore is composed of the centromere binding factor 3 (CBF3) complex and the Cse4-nucleosome and is essential for the recruitment of all other kinetochore proteins (Santaguida & Musacchio 2009). The CBF3 complex, which consists of Ndc10, Cep3, Ctf13 and Skp1, interacts directly with CDEIII (see Figure 1.4) and is the primary determinant for kinetochore construction since all analyzed budding yeast kinetochore proteins depend on CBF3 for association with the centromere (Westermann, et al., 2007). Although the CBF3-like complex seems to be restricted to fungi with a sequence-defined centromere, Skp1 is functionally conserved between yeast and humans (Kitagawa & Hieter, 2001). In addition, Ndc10 alone can also bind to CDE II and probably serves as a foundation for recruiting other kinetochore proteins to the centromere (Espelin, et al., 2003). The relatively uncharacterized Ctf19 complex consists of more than 10 proteins and four of these proteins, Ctf19, Okp1, Mcm21 and Ame1, copurify as a subcomplex called COMA. Only Okp1 and Ame1 are required for viability and Mcm21 is essential for pericentromeric cohesion in ensuring kinetochore biorientaion (Westermann, et al., 2007; Santaguida & Musacchio, 2009; Ng, et al., 2009). The Spc105 complex consists of Spc105 and Kre28 (YDR532C) and is poorly described in budding yeast (De Wulf, et al., 2003; Necrasov, et al., 2003). Homologs in different 7 eukaryotes are known as Spc7, KNL-1 or Blinkin. It was shown that this complex tightly interacts with two other complexes, the MIND (Mtw1-Nnf1-Nsl1-Dsn1) and Ndc80 complexes, and with components of the spindle assembly checkpoint. Studies of the D. melanogaster Spc105 protein suggest a role in providing a platform within the outer kinetochore upon which various other kinetochore proteins can assemble (Schittenhelm, et al., 2009). In budding yeast, Spc105/Kre28 was suggested to have essential roles in both bipolar attachment and recruitment of spindle checkpoint components (Pagliuca, et al., 2009). The budding yeast proteins Mtw1, Dsn1, Nnf1 and Nsl1 form the MIND-complex. Mutants in complex subunits result in chromosome missegregation and insufficient tension between sister kinetochores. The MIND complex interacts with Cse4, Mif2 and Ndc80 and is conserved from yeast to humans (Euskirchen, et al., 2002). One of the best characterized kinetochore components is the Ndc80 complex, which I will describe in more detail later (see 1.2.4). In budding yeast, the MIND, Ndc80 and Spc105 complexes likely interact tightly to form the highly conserved KMN network (KNL1, Mis12, Ndc80) as a core kinetochore microtubule binding complex (Cheeseman and Desai, 2008). The combined protein network was shown to bind with higher affinity to microtubules than the single complexes and likely forms a microtubule binding site for attaching kinetochores to dynamic microtubules (Tanaka & Dasai, 2008). The budding yeast Dam1 complex is a fungal-specific outer kinetchore complex, that consists of ten proteins: Dam1, Ask1, Dad1, Dad2, Dad3, Dad4, Duo1, Spc19, Spc34 and Hsk3 8 (Hofmann, et al., 1998; Enquist-Newman, et al., 2001; Cheeseman, et al. 2001; Cheeseman, et al., 2002; De Wulf, et al., 2003; Janke, et al., 2002; Li, et al., 2002) . The complex has an important role in mediating kinetochore-microtubule interactions and chromosome segregation. Dam1 is a target of the Ipl1 kinase, which plays an important role in the correction of improper kinetochore-microtubule attachments (Cheeseman, et al., 2001; Li, et al., 2002; Pinsky, et al., 2006; Westermann, et al., 2007). Biochemical analysis and in vitro studies revealed the formation of a ring around a microtubule formed by 16 single heterodecameric Dam1 complexes. The ring diffuses along the microtubule lattice and moves processively with a depolymerizing end of a microtubule (Westermann, et al., 2005; Wang, et al., 2007; Grishchuk, et al., 2008; Lampert, et al., 2010; Tien, et al., 2010). Microtubule associated proteins (MAPs) and motor proteins are also present at kinetochore structures and play additional roles in chromosome attachment and segregation. These MAPs include Stu2 (XMAP215), Bik1 (CLIP170) and Bim1p (EB1), which recognize and bind to plus ends of microtubules and regulate microtubule dynamics (Berlin, et al., 1990 ; Schwartz, et al., 1997 ; Wang and Huffaker, 1997; Tirnauer, et al., 1999 ; Kosco, et al., 2001; Hwang, et al., 2003 ; Miller, et al., 2006 ; Wolyniak, et al., 2006 ; Al-Bassam, et al., 2006; Gardner, et al., 2008 ; Zimniak ,et al., 2009et al; Blake-Hodek, et al., 2010). Motor proteins like Kar3, Kip1, Kip3 and Cin8 play a role in chromosome segregation by crosslinking spindle microtubules or organizing chromosome movements, but their exact function during mitotic processes is not fully understood (McAinsh, et al., 2003; Westermann, et al., 2007; Gardner, et 9 al., 2008).  1.2.3 Regulation of kinetochore-microtubule attachment During metaphase, sister chromatids must attach to the microtubules from opposite SPBs in order to ensure equal distribution of DNA to both cells. Mature, bipolar attachments generate tension across kinetochores. Errors in attachment or tension are recognized by two mechanisms. Aurora B/Ipl1 and its partner INCENP/Sli15 respond to the absence of tension by stimulating kinetochore-microtubule detachment which liberates kinetochores to attach to the opposite pole (Biggins and Murray, 2001; Tanaka, et al., 2002; Pinsky & Biggins, 2005; Liu & Lampson, 2009; Kelly & Funabiki, 2009). However, Aurora B/Ipl1 does not repair all the attachment errors and the spindle assembly checkpoint (SAC) is required to block the metaphase-anaphase transition until all the chromosomes have attained bipolar attachment to the mitotic spindle (Taylor and McKeon, 1997; Gillett & Sorger, 2001; Warren, et al., 2002). Even though the function of the SAC is well established, it is unclear whether the checkpoint responds only to chromosome detachment or to the loss of tension as well. In budding yeast, the key components of the SAC pathway are Mad1, Mad2, Mad3 (BubR1 in humans), Bub1, Bub3 and Mps1 (Hoyt, et al., 1991; Li & Murray, 1991; Weiss, et al., 1996). This checkpoint is extremely sensitive. One unattached kinetochore is sufficient to activate the checkpoint (Ault, et al., 1991; Ault, et al., 1992). In higher eukaryotes, the SAC is essential and all the checkpoint proteins localize to kinetochores in prometaphase cells. In 10 budding yeast the BUB and MAD genes are non-essential however other genes involved in the spindle checkpoint, such as Mps1 and Ipl1 are essential (reviewed in Lew & Burke, 2003). Bub1 and Bub3 associate with kinetochores until metaphase. However, Mad2 is only recruited to unattached kinetochores upon microtubule depolymerization or kinetochore disruption, but does not associate with the kinetochore in a normal cell cycle (Hoyt, et al., 1991; Li, et al., 1991; Gillett, et al., 2004; Musacchio & Salmon, 2007; Santaguida, et al., 2009). Kinetochores are required for SAC activation. The complete inactivation of CBF3 leads to a failure of checkpoint control and prevents Mad and Bub proteins from associating with kinetochores (Goh & Kilmartin, 1993; Gardner, et al., 2001). However, hypomorphic mutations in CBF3 subunits which causes a partial loss of gene function engage the checkpoint (Doheny, et al., 1993). Therefore, the checkpoint is sensitive to partially inactive kinetochores but the signaling complexes cannot form in the absence of the kinetochore.  1.2.4 The Ndc80 complex The Ndc80 complex contains four proteins – Ndc80, Nuf2, Spc24 and Spc25 – all of which are essential and localize exclusively to kinetochores based on fluorescence microscopy imaging (Wigge & Kilmartin, 2001; He, et al., 2001; Janke, et al., 2001). All the Ndc80 complex components are evolutionarily well conserved and are also required for chromosome segregation in human cells, Xenopus and other organisms (Table 1.1) (Zheng, et al., 1999; Howe, et al., 2001; 11 Janke, et al., 2001; Wigge, et al., 2001; DeLuca, et al., 2002; Le Masson, et al., 2002; Hori, et al., 2003). The structure of Ndc80 complex was first determined by electron microscopy (Wei, et al., 2005). It forms a long dumbbell shape with globular domains at the ends and an elongated coiled-coil domain in between (Figure 1.6A) (Ciferri, et al., 2005; Wei, et al., 2005). The Spc24/Spc25 subcomplex forms a dimer that interacts with Ndc80/Nuf2 with their N-terminal coiled-coil domains and interacts with other kinetochore proteins via their C-terminal globular domains (Joglekar, et al., 2009; Wan X, et al., 2009). The Ndc80/Nuf2 subcomplex binds microtubules directly with their N-terminal globular domains (Cheeseman, et al., 2006; Wei, et al., 2007; Ciferri, et al., 2008) whereas their C-terminal coiled-coil domains interact together and form a tetramerization domain that meets the Spc24/Spc25 dimer (Ciferri, et al., 2005; Wei, et al., 2005) (Figure 1.6A,C). In budding yeast, there are approximately 8 copies of the Ndc80 complex on each microtubule attachment site (Joglekar, et al., 2006). A recent study with Ndc80 complex coated beads proposed that the Ndc80 complex undergoes biased diffusion towards the minus end of a depolymerizing microtubule (Powers, et al., 2009) (Figure 1.6E). However, how the Ndc80/Nuf2 subcomplex binds the microtubule remains unclear. Structural analyses reported that the microtubule binding domain of Ndc80/Nuf2 consists of one unique unstructured tail in Ndc80 and two calponin-homology (CH) domains, which have high affinity with the acidic C-terminus of tubulin (Cheeseman, et al., 2006; Wei, et al., 2007; Ciferri, et al., 2008; Powers, et al., 2009), 12 whereas other studies argue that the CH domain is not necessary for microtubule binding (Wilson-Kubalek, et al., 2008; Guimaraes, et al., 2008; Miller, et al., 2008). Besides directly binding microtubules, the Ndc80 complex plays a role in the regulation of kinetochore-microtubule attachment as a target of the Aurora B/Ipl1 kinase (DeLuca, et al., 2006; Cheeseman, et al., 2006; Pinsky, et al., 2006; Akiyoshi, et al., 2009). The Aurora B/Ipl1 mediated phosphorylation of Ndc80, probably in the N-terminal unstructured region of the protein, reduces Ndc80 microtubule binding ability (DeLuca, et al., 2006; Cheeseman, et al., 2006; Wei, et al., 2007). Preventing Ndc80 from Aurora B/Ipl1 phosphorylation leads to overstretched kinetochores due to the stabilization of the kinetochore-microtubule attachments, high frequency of merotelic kinetochores (a sister chromatid attached to both poles) and overstabilized plus end microtubules (Cimini, et al., 2006; DeLuca, et al., 2006). In addition, the Ndc80 complex is also involved in the Ipl1 dependent corrective detachment of kinetochores in an indirect manner. Tien et al. (2010) have demonstrated that the Dam1 complex promotes Ndc80 complex tracking along the microtubules and that this interaction is abolished by Ipl1 phosphorylation of Dam1 when the kinetochores lack tension. A second role of the Ndc80 complex is signaling attachment defects to the spindle checkpoint. The Ndc80 complex is essential for recruiting the SAC proteins Mad1, Mad2 and Mps1 to unattached kinetochores (Martin-Lluesma, et al., 2002; DeLuca, et al., 2002; Hori, et al., 2003; DeLuca, et al., 2003; McCleland, et al., 2003; Bharadwaj, et al., 2004; Meraldi, et al., 2004; McCleland, et al., 2004; Gillett, et al., 2004).  Depleting Ndc80 or Nuf2 in mammalian 13 cells dramatically reduces the protein levels of Mad1 and Mad2, but a Mad2-dependent mitotic arrest is still triggered probably due to the residual kinetochore recruitment of the checkpoint proteins (DeLuca, et al., 2002; Hori, et al., 2003; McCleland, et al., 2003; Bharadwaj, et al., 2004; Martin-Lluesma, et al., 2002; DeLuca, et al., 2003). Complete depletion of Ndc80 complex subunits or interference with their function abolishes the SAC (McAinsh, et al., 2003; McCleland, et al., 2004; Meraldi, et al., 2004; Guimaraes, et al., 2008). Overexpression of Ndc80 in a mouse model leads to hyperactivation of the SAC and tumor formation (Diaz-Rodríguez, et al., 2008). It remains unknown how checkpoint proteins associate with kinetochores and sense attachment problems, but two-hybrid interactions have been detected between yeast Spc25 and Mad1 (Newman, et al., 2000) and between human Ndc80 and Mad1 (Martin-Lluesma, et al., 2002) and synthetic growth defects between mad1∆ and spc24 ts alleles (Montpetit B, et al., 2005). Moreover, recruitment of Bub1 and Bub3 to centromeres requires some but not all members of the Ndc80 complex, indicative of a requirement of the Ndc80 complex in SAC (Gillett, et al., 2004).  1.3 Summary Kinetochores are large, complicated structures whose function is vital to the fidelity of chromosome segregation. Although much is known about the structure of the Ndc80 complex, all its known activities are restricted to kinetochore-microtubule attachment and spindle checkpoint functions. The major goal of my thesis work was to investigate novel roles of the Ndc80 complex 14 by utilizing spc24 temperature sensitive (ts) alleles in budding yeast. Objective 1: Since the spc24-9 kinetochore mutant prematurely elongates its spindle in response to hydroxyurea (HU) which stalls DNA replication, a potential role for Spc24 in the DNA replication checkpoint and spindle stability was studied. In Chapter 2, I performed a high copy suppressor (HCS) screen to identify genes that when overproduced would rescue the HU sensitivity of spc24-9.  I further studied one of the genes identified in this screen, the Stu2 microtubule binding protein, and characterized the roles of Spc24 and Stu2 in maintaining spindle stability by quantitative and time-lapse fluorescence analysis. Objective 2: Previous studies from the Measday lab have shown a genetic interaction between spc24-9 and two components of cAMP/PKA pathway, and two other studies have demonstrated an interaction between kinetochore proteins and cAMP/PKA pathway (Dubacq, et al., 2002; Li, et al., 2005). Therefore, the relationship between the Ndc80 complex and the cAMP/PKA pathway was of interest to study. In Chapter 3, I analyzed cAMP/PKA pathway activity through two of its targets, trehalose levels and Msn2 phosphorylation, in spc24 mutant strains and compared the growth of different kinetochore ts mutants on different carbon sources by cell dilution assay. Objective 3: Some of spc24-9 mutant cells have an elongated bud at the non-permissive temperature which suggested that the Swe1-dependent morphogenesis checkpoint was activated in these mutants.  Indeed I found that Swe1 levels were stabilized in spc24-9 cells at restrictive temperature.  The objective of this chapter was to characterize why the morphogenesis 15 checkpoint is activated in spc24-9 mutants.  I focussed my analysis on genetic interactions with mutants in actin patch and cable formation and key polarity determinants and fluorescence imaging of the septin scaffold and the actin cytoskeleton. 16  Table 1.1 Homologs of Ndc80 complex in multiple organisms.  S. cerevisiae S. pombe C. elegans Xenopus H. sapiens  Ndc80 complex Ndc80 Ndc80 Ndc-80 xNdc80 HsNdc80 Nuf2 Nuf2 Jim-10 xNuf2 HsNuf2 Spc24 Spc24 Kbp-4 xSpc24 HsSpc24 Spc25 Spc25 Kbp-3 xSpc25 HsSpc25  17    Figure 1.1  Life cycle of Saccharomyces cerevisiae (Cited from D. Morgan, 2007, by permission © Oxford University Press, 2010)  18   Figure 1.2 Mitotic cell cycle stages of budding yeast (Modified from Tanaka et al., 2005, by permission © Nature Publishing Group)  19   Figure 1.3  A simplified view of the cell-cycle control system A. Cyclins in budding yeast. B. Oscillations of cyclins (top) and cyclin-Cdk complexes (bottom) drive cell-cycle events. (Cited from D. Morgan, 2007, by permission © Oxford University Press, 2010) 20  Figure 1.4 Centromere of Saccharomyces cerevisiae The centromere of budding yeast is divided into three regions: CDEI, recruits a dimer of the Cbf1 transcription factor (Bram and Kornberg, 1987); CDEII surrounds the Cse4-nucleosome (Meluh et al, 1998; Keith and Fitzgerald-Hayes, 2000); CDEIII, which is bound by the CBF3 complex(Lechner and Carbon, 1991). (Cited from Santaguida & Musacchio, 2009, by permission © Nature Publishing Group)   21  Figure 1.5 Protein architecture of the Saccharomyces cerevisiae kinetochore A. The S. cerevisiae kinetochore image derived from in vivo super-resolution microscopy (Joglekar et al., 2009). The left part: yellow strands: DNA; dark blue: histone; dark pink: CBF3 complex; light pink sphere: centromeric DNA. The right part: central green structures: microtubules; purple ring: DASH complex; orange rods: Ndc80 complex. The linker complexes: light blue: Spc105 complex; green: MIND complex; small purple: Ctf19 complex. B. A schematic model of the kinetochore structure in A. (Cited from Bloom & Joglekar, 2010, by permission © Nature Publishing Group) 22    23 Figure 1.6 The molecular machinery of kinetochore–microtubule attachment (A) Topology of the Ndc80 complex. aa, amino acids. N and C indicate the N- and C-termini, respectively. The image is reproduced from Ciferri et al (2008). (B) Electron microscopy images of negatively stained Ndc80 complexes. The arrowheads point to the kink along the shaft. Scale bar: 10nm. The images are reproduced from Wang et al (2008). (C) A model of the full length Ndc80 complex. (D) The Ndc80 complex forms rod-like projections on the microtubule lattice. Left: the control microtubules stabilized with a non-hydrolysable GTP analogue GMPCPP. The middle image is the negatively stained GMPCPP microtubules in the presence of 5 mM Ndc80 complex (C. elegans). Right: traces of the EM images representing the angled rod-like complexes bound to the lattice. Scale bars:200 nm. The images are reproduced from Cheeseman et al (2006). (E) A biased diffusion model of the Ndc80 complex proposed by Powers et al (2009). Kinetochores (showed as red hollow discs) diffuse along the lattice by the interaction with five microtubule-binding elements. ―+‖ and ―−‖ refer to the plus end and minus end, of the microtubules, respectively. (Adapted from Santaguida & Musacchio, 2009, by permission © Nature Publishing Group)      24 Chapter 2  Spc24 and Stu2 Promote Spindle Integrity when DNA Replication is Stalled 1  2.1  Introduction Preserving the integrity of the genome is a fundamental requirement for eukaryotic cell viability.  DNA replication must be completed prior to segregation of the chromosomes to prevent the transmission of partially replicated chromosomes to daughter cells.  In the budding yeast Saccharomyces cerevisiae, cells undergo a closed mitosis and the MT organizing centres, which in yeast are called spindle pole bodies (SPBs), are embedded in the nuclear envelope. SPB duplication begins at G1 and spindle formation begins during S phase when duplicated SPBs separate from each other (Adams and Kilmartin, 1999; Jaspersen and Winey, 2004). Because chromosomes remain attached to kinetochore MTs throughout the cell cycle, spindle expansion must be restrained until all sixteen chromosomes have duplicated and kinetochores on sister chromatids have formed bipolar MT attachments.  When DNA replication is stalled by HU treatment, cells arrest with a large bud, an undivided nucleus is positioned at the mother-bud neck and a short bipolar spindle forms (Allen et al., 1994).  Maintaining a short spindle is   1  Chapter 2 is a modified version of a published paper: Ma L., McQueen J., Cuschieri L., Vogel J., Measday V, Spc24 and Stu2 Promote Spindle Integrity When DNA Replication is Stalled, Molecular Biology of the Cell, 18(8): 2805-2816 (2007). By permission © The American Society for Cell Biology. See details in Preface. 25 crucial for cell survival during HU induced arrest, which activates the DNA replication checkpoint effectors Mec1 and Rad53 (Kolodner et al., 2002).  When mec1 and rad53 mutants are treated with HU, the DNA replication checkpoint is not activated and as a result replication forks are not stabilized, the spindle expands and unequal division of incompletely replicated nuclear material occurs – all of these events contribute to cell lethality (Allen et al., 1994; Weinert et al., 1994; Lopes et al., 2001). In human cells, stalled replication forks activate the ATM (ataxia telangiectasia mutated) and Chk2 kinases, which are Mec1 and Rad53 homologs respectively, and arrest the cell cycle by inhibiting mitotic entry (Canman, 2001). Until recently, it was presumed that mec1 and rad53 mutants enter mitosis prematurely upon HU treatment.  However, two recent studies have shown that this is not the case, suggesting that spindle expansion is actively restrained when DNA replication is stalled (Krishnan et al., 2004; Bachant et al., 2005; Krishnan and Surana, 2005).  Two mechanisms, which are not mutually exclusive, have been proposed for how spindle expansion is prevented during the DNA replication checkpoint.  One mechanism suggests that spindle associated proteins are regulated in a Mec1/Rad53-dependent manner (Krishnan and Surana, 2005).  Spindle expansion and nuclear division of mec1-1 mutants is reduced in cells carrying mutations of the kinesin-5/BimC ortholog Cin8 and XMAP215 ortholog Stu2 (Krishnan et al., 2004).  The second mechanism proposes that tension imposed by the bipolar attachment of kinetochores to MTs emanating from opposite SPBs is responsible for maintaining a short spindle upon inhibition of DNA replication (Bachant et al., 2005). 26 In budding yeast, each kinetochore, a multi-protein complex that resides on centromere (CEN) DNA, attaches to a single MT (McAinsh et al., 2003).  After chromosome replication, kinetochores on sister chromatids must attach to MTs emanating from opposite SPBs in order to achieve bipolar attachment.  The SPB pulling force opposes the cohesion complex holding sister chromatids together and creates tension that physically separates CEN regions during metaphase (Goshima and Yanagida, 2000; He et al., 2000; Pearson et al., 2001).  The kinetochore not only attaches to spindle MTs but is also capable of regulating MT dynamics and spindle stability before and during mitosis.  Firstly, MT associated proteins such as Stu2 and kinesin-related motor proteins localize to and function at kinetochores (He et al., 2001; McAinsh et al., 2003; Tanaka et al., 2005; Tytell and Sorger, 2006).  Secondly, the Dam1 outer kinetochore complex encircles MTs and mutations in Dam1 components severely affect MT dynamics (Cheeseman et al., 2001; Miranda et al., 2005; Westermann et al., 2005; Shimogawa et al., 2006).  Thirdly, a group of kinetochore proteins called chromosome passenger proteins relocalize from kinetochores to the spindle midzone during anaphase and regulate spindle stability and cytokinesis (Bouck and Bloom, 2005). During her postdoctoral studies in Dr. Hieter’s lab, Dr. Measday identified an HU sensitive spc24-9 kinetochore mutant that prematurely elongates its spindle upon HU treatment. By performing a high copy suppressor (HCS) screen, I have identified ten genes that when over-expressed rescue the HU sensitivity and spindle expansion defect of the spc24-9 mutant strain.  I characterized the rescue function of two of these genes – Stu1, a MT associated protein 27 that shares a region of similarity to the CLASP/Mast/Orbit sub-family of MT plus-end tracking proteins and Stu2 which is a member of the conserved Dis1/XMAP215 family of MT plus end binding proteins (Inoue et al., 2000; Yin et al., 2002; Gard et al., 2004).  I demonstrate that both Stu1 and Stu2 are localized to the kinetochore early in the cell cycle and that Stu2 kinetochore binding depends on Spc24.  By performing quantitative and time-lapse analysis of Stu2 fluorescence on spindles during HU treatment, we show that spindle expansion in spc24-9 cells correlates with mis-localization of Stu2.  I propose that localization of Stu2 to the kinetochore in cells when DNA replication is stalled is imperative for maintaining a short spindle and preventing separation of incompletely replicated DNA.  2.2  Materials and methods 2.2.1 Strain construction Standard methods for yeast culture and transformation were followed (Guthrie and Fink, 1991).  Rich medium (YPD), and supplemental minimal medium (SC) were used (Kaiser et al., 1994) as well as FPM (minimal media supplemented with adenine and 6.5g/L sodium citrate) for microscopy analysis (Pot et al., 2005).  Yeast strains used in this study are described in Table 2.2.  Genes were deleted or epitope tagged using standard yeast methods (Longtine et al., 1998).  28 2.2.2 HCS screen I transformed a 2μ yeast genomic DNA library carrying 6-8kb genomic DNA fragments (Connelly and Hieter, 1996) into the spc24-9 strain and plated 40,000 colonies onto SC-URA plates to select for the presence of the library plasmid.  I then replica plated the colonies to 0.1M HU (Sigma) SC-URA plates and incubated at 30 o C to identify colonies that could rescue the HU lethality of spc24-9 mutants.  Library plasmids were rescued from colonies growing on the 0.1M HU SC-URA plates and transformed back into spc24-9 mutants to confirm the HU rescue phenotype.  Plasmids were then sequenced using T3 and T7 primers to identify the flanking sequences of the genomic insert.  2.2.3 Plasmid construction - subcloning of HCS genes The coordinates of the genomic DNA identified in the HCS screen rescue plasmids and their subsequent subclones to confirm identity of the gene are as follows: STU1∆N: Chr.II, 151363-158219; STU1∆N subclone: Chr.II, 153753-158219; STU2∆N: Chr.XII, 230442-237078; STU2∆N subclone: Chr.XII 233569-237078; KIP2: Chr.XVI, 252390-259933; KIP2 subclone: Chr.XVI, 257172-259933; GIC1: Chr.VIII, 220246-228530; GIC1 subclone: Chr.VIII, 220246-222771; RCK2: Chr.XII, 634230-640397; RCK2 subclone: Chr.XII, 634230-636611; HCM1: Chr.III, 224203~231351; MCK1: Chr. XIV, 52447-58767; DMA1; Chr.VIII, 337353-344031; DMA1 subclone: Chr. VIII, 337353-342008.  DMA1 was confirmed as the gene responsible for rescue by digesting the DMA1 subclone with EcoRI followed by Klenow 29 treatment to create a frameshift in the DMA1 gene.  Full length MCK1 was a gift from Dr. Phil Hieter (Shero and Hieter, 1991), full length HCM1 was a gift from Dr. Trisha Davis (Zhu et al., 1993), and full length STU1 and STU2 were gifts from Dr. Tim Huffaker (Pasqualone and Huffaker, 1994; Wang and Huffaker, 1997).  2.2.4 Microscopic analyses Immuno-fluorescence Cells shown in Figures 1, 3, 4 and 5 were  imaged using a Zeiss Axioplan 2 microscope equipped with a CoolSNAP  HQ camera (Photometrics, Tucson, AZ) and Metamorph (Universal  Imaging, West Chester, PA) software.  The indirect immunofluorescence microscopy studies in Figures 1 and 4 were performed as described previously (Hyland et al., 1999) with the following modifications.  Cells were synchronized in G1 at 25 oC using α-mating factor (5 μg/ml) (BioVectra), released into 0.2M HU for 3 hours at 30˚C and fixed with a final concentration of 3.7% formaldehyde for 1 hour.  Spindles were visualized by staining with Yol  1-34 rat antitubulin antibody (1:50) (Serotec, Oxford) followed by fluorescein-conjugated goat anti-rat secondary antibody (1:2000).  Single focal plane images were acquired with a 100X objective.  Analysis of GFP-centromeres, Stu1-VFP and Stu2-VFP localization in fixed cells CEN15-green fluorescent protein (GFP)-tagged [Figure 2.4, (Goshima and Yanagida, 2000)], cells were synchronized with α−mating factor and LacI-GFPHIS 30 LacO::URA3-CEN15(1.8) was activated with 30mM 3-aminotriazole in SC–HIS media.  Cells were released into indicated concentrations of HU in FPM media for 3 hours.  Cells were washed and fixed in a total concentration of 2% formaldehyde for 15 min.  Image stacks were acquired with a 100X objective at a step of 0.2 μm to span the entire cell.  Stu1-VFP fluorescence (Figures 5) was imaged as described above with the following alterations.  Cells were grown in FPM media at 30 oC, synchronized with α-mating factor and released to 30oC. After 30 min., samples were taken every 15 min. and fixed in 70% ethanol.  Stu2-VFP in fixed cells (Figure 2.10) was imaged with a WaveFX spinning disc confocal microscope (Quorum Technologies) as previously described (Cuschieri et al., 2006) without agar pads. Optical sections (0.5 µm) were acquired through a ±2.5 µm z-plane (total of 5.0 µm stack) using Volocity 3DM acquisition software (Improvision, UK).  2.2.5 Live cell analysis For live-cell imaging of Stu2-VFP fluorescence intensities and spindle length measurements (Figure 2.11 and Figure 2.9), overnight cultures were grown in YPD (containing 2X adenine sulfate) at 25˚C to a cell density of ~0.3-0.4 OD600 units ml -1 . Cultures were then diluted to 0.2 OD600 units ml -1 and grown for an additional generation. Cells were arrested with 5 μg/ml of alpha factor for 1.5 hr at 25˚C, washed and then released into media containing 0.2 M HU for 1.5 hours at 25˚C. 1 ml samples of each strain were taken and re-suspended in ~ 50 µl of 31 30˚C  pre-warmed media containing 0.2 M HU. Cells were mounted on a pre-warmed 30˚C heated stage and allowed to equilibrate for 15 min prior to imaging. Multi-channel 4D imaging of Spc29-CFP and Stu2-VFP fluorescent fusion proteins was performed using a WaveFX spinning disc confocal system (Quorum Technologies, Guelph ON) as previously described (Cuschieri et al., 2006). A Tokai Hit stage warmer was used to shift cells from 25˚C to 30˚C. Image acquisition commenced 15 min. after the stage reached 30˚C. Optical sections (0.5 µm) were acquired through a ±2.5 µm z-plane (total of 5.0 µm stack) at 2 min. intervals for 30 min. using Volocity 3DM acquisition software (Improvision, UK).  2.2.6 Spindle length measurements Calculation of spindle lengths in fixed and live cell analyses shown in Figures 7 and 8 was performed using Volocity Classification (Improvision, UK). Spindle lengths (in µm) were measured in triplicate for each time point and the average value and standard error of the mean determined.  Lengths were determined by measuring the linear distance (µm; in x, y, z) between the mid-point of one SPB (Spc29-CFP channel) to the midpoint of the opposite SPB. All spindle lengths were measured in the XYZ plane view using the line length measurement tool.  Average spindle lengths and standard errors were calculated using Excel software.  2.2.7 Stu2-VFP fluorescence measurement For Stu2-VFP fluorescence measurements shown in Figures 7 and 8, image stacks were 32 acquired using an exposure of 91 msec/frame (providing an un-saturated image).  For both fixed and live cell analyses, fluorescence intensity was measured by rastering a 4x4 voxel volume along the length of the long axis of the spindle (4x4 voxel: spindle fluorescent unit), including both SPBs. Background subtraction as performed as follows: the fluorescence in a 4x4 voxel volume positioned in the cytoplasm was measured, and subtracted from each fluorescence units acquired along the spindle resulting in corrected spindle fluorescence units (arbitrary units). For live cell analyses, background fluorescence was determined for each time point.  The corrected fluorescence per unit length (fluorescence/µm) was calculated. Background subtractions, corrected fluorescence values and standard deviations were calculated using Excel software.  2.2.8 ChIP assays (Chromatin immunoprecipitation) ChIP experiments and primers used for PCR analysis were performed as described previously (Measday et al., 2002; Pot et al., 2003).  The linear range for PCR analysis was determined and dilutions used for Figure 2.2A were T (total chromatin,1:200), immunoprecipitation (IP, 1:1); Figure 2.2B, T (1:200), IP (5:1); Figure 2.2C, T (1:200), IP (5:1). Dilutions used for Figure 2.8A were T (1:780), IP (1:6); Figure 2.8B, T (1:780), IP (1:2.5); Figure 2.8C and 6D T (1:125), IP (1:1).  33 2.3  Results 2.3.1 Spc24 is required for viability and preventing spindle expansion during HU arrest The budding yeast Ndc80 central kinetochore complex, which is composed of the four coiled-coil proteins, Ndc80, Nuf2, Spc24 and Spc25, is required for proper attachment of chromosomes to spindle MTs and activation of the spindle checkpoint in the presence of defects in attachment (Janke et al., 2001; Wigge and Kilmartin, 2001; Le Masson et al., 2002; Montpetit et al., 2005; Pinsky et al., 2006).  It has recently been shown that the kinetochore has a role in maintaining a short (1.5-2 μm) spindle when DNA replication is stalled by treatment of cells with HU (Bachant et al., 2005).  Previously, Dr. Measday created two mutants in the coiled-coil region of Spc24 (spc24-8 and spc24-10) and one mutant in the C-terminus of Spc24 (spc24-9) (Montpetit et al., 2005).  Dr. Measday tested these alleles for viability in the presence of HU at semi-permissive temperature (30 o C) and found that the growth of the spc24-9 mutant is sensitive to levels of HU that do not inhibit the growth of spc24-8 and spc24-10 mutants and the wild type strain (Figure 2.1A).  Next, Dr. Measday monitored the spindle length and bulk segregation of DNA in spc24 mutants arrested in G1 and released into 0.2M HU media.  This analysis revealed that 100% of wild type and 98% of spc24-8 and spc24-10 mutants maintain a short spindle and undivided nuclei after 3 hours exposure to HU, however the majority (76%) of spc24-9 mutants displayed elongated spindles and segregated nuclei (Figure 2.1B, C).  Her data suggests that the spc24-9 mutation results in a defect in the function of the Ndc80 complex in preventing expansion of the spindle in cells with partially replicated DNA. 34 2.3.2 Characterization of the kinetochore in spc24-9 mutants The Ndc80 complex is composed of two subcomplexes – Nuf2/Ndc80 and Spc24/Spc25 – that are linked via their coiled-coil domains (Wei et al., 2005).  The C-terminal mutation in spc24-9 lies within the Spc24 globular domain (Montpetit et al., 2005; Wei et al., 2005).  To determine the state of the kinetochore and the Ndc80 complex in spc24-9 mutants, I performed chromatin immunopreciptation (ChIP) assays using a member of the inner kinetochore CBF3 complex, Ndc10 and the Ndc80 protein.  I found that Ndc10 was able to interact with centromere (CEN) DNA in spc24-9 cells at both restrictive temperature (37 o C) and after three hours of 0.2M HU treatment at 30 o C, suggesting that the core kinetochore is still intact in spc24-9 mutants (Figure 2.2A, lanes 10 and 12).  Ndc80, however, showed a clear defect in its ability to associate with CEN DNA in spc24-9 cells both at 37 o C and after three hours of 0.2M HU treatment at 30 o C (Figure 2.2B, lanes 10 and 12).  In corroboration with our ChIP data, I found that Ndc80-VFP localization was also perturbed in spc24-9 mutants at 37 o C and is even more affected in HU arrested cells (Figure 2.3).  80% of HU treated spc24-9 mutants showed diffuse and weak Ndc80-VFP staining suggesting that the Ndc80 complex is disrupted when spc24-9 cells are exposed to 0.2M HU (Figure 2.3).  2.3.3 Bipolar attachment is not a requirement for maintaining a short spindle in HU treated cells Previous studies have suggested that specific kinetochore mutants display inappropriate 35 spindle expansion during HU exposure due to their inability to establish kinetochore-MT bipolar attachment and thus appropriate tension on the spindle (Bachant et al., 2005).  Using a CEN15-GFP marked strain, Jennifer McQueen found that a similar percentage of wild type, spc24-9 and spc24-10 cells displayed bipolar attachment in cells released from a G1 block to 30 o C (Figure 2.4A).  Thus, spc24-9 kinetochores are capable of bipolar attachment to spindle poles during a normal cell cycle at the same temperature (30 o C) that results in spc24-9 HU lethality.  Whether or not kinetochores attain bipolar attachment in a wild type strain in the presence of partially duplicated DNA (as a result of treatment with HU) is unclear.  We reasoned that exposing cells to increasing concentrations of HU would increase replication fork stalling (.05M-0.3M HU) and impact the number of CENs that were replicated (Clarke et al., 2001).  Jennifer monitored CEN15 separation as a sign of bipolar attachment using CEN15-GFP and Spc29-CFP tagged wild type and spc24-9 mutant strains.  Wild type cells treated with the lowest concentration of HU (.05M HU) exhibited CEN15 bipolar attachment in 60% of cells after 3hours (Figure 2.4B).  Importantly, spc24-9 mutants displayed the same percentage of separated CEN15 foci suggesting once again, that spc24-9 mutants are capable of bipolar attachment (Figure 2.4B).  Similar to previous studies, Jennifer found that separated CEN15 foci were detected in 23% of wild type cells exposed to 0.1M HU yet only 4% of separated CEN15 foci were seen in 0.2M HU treated wild type cells (Goshima and Yanagida, 2000; Krishnan et al., 2004).  The majority of wild type cells treated with the highest concentration of HU (0.3M HU) contained one CEN15 focus that colocalized with one SPB suggesting either that 36 CEN15 had not yet replicated or had replicated but had not yet established bipolar attachment (Figure 2.4C).  Previous work has demonstrated that unreplicated monocentric minichromosomes also remain in the close vicinity of one SPB (Dewar et al., 2004).  Thus the ability of CEN15 to attain bipolar attachment is not correlated with maintaining a short spindle during the DNA replication checkpoint.  2.3.4 Identification of HCS genes that rescue spc24-9 HU lethality To understand why Spc24 is required for viability when DNA replication is stalled, I performed a HCS screen to identify genes that, when overexpressed, could suppress the lethality of spc24-9 cells exposed to HU.  Ten genes were identified in our HCS screen (Table 2.1). Multiple isolates of SPC24 and its interacting partner SPC25 were recovered (Janke et al., 2001; Wigge and Kilmartin, 2001).  I also identified four genes encoding proteins that regulate spindle dynamics – MT associated proteins Stu1 and Stu2, the kinesin-related motor protein Kip2 and a protein involved in spindle positioning called Dma1 (Roof et al., 1992; Pasqualone and Huffaker, 1994; Wang and Huffaker, 1997; Fraschini et al., 2004).  Two protein kinases were isolated – Mck1, which has a role in chromosome segregation and Rck2 which has a role in the osmotic stress response pathway (Neigeborn and Mitchell, 1991; Shero and Hieter, 1991; Bilsland-Marchesan et al., 2000).  I identified Gic1 which has roles in cell polarity and mitotic exit (Brown et al., 1997; Chen et al., 1997; Hofken and Schiebel, 2004).  Finally, I identified the Hcm1 transcription factor, which has also been isolated in a variety of synthetic lethal screens 37 pertaining to the cell division cycle and chromosome segregation (Zhu and Davis, 1998; Horak et al., 2002; Sarin et al., 2004; Montpetit et al., 2005; Daniel et al., 2006).  Interestingly, Hcm1 has recently been shown to activate expression of spindle and chromosome segregation proteins specifically in S phase (Pramila et al., 2006).  All the HCS genes were able to rescue the HU lethality of two other alleles of the Ndc80 complex, ndc80-1 and spc25-1, however, none of them was able to rescue the inviability of a mec1 mutant on HU plates (data not shown).  Therefore, the HCS genes suppress the HU specific defects of Ndc80 complex mutants but do not suppress mec1, a key effector of the DNA replication checkpoint.  2.3.5 HCS rescue occurs through restraining spindle expansion I next determined if the HCS genes rescued spc24-9 HU lethality by restraining spindle expansion and thus premature chromosome segregation.  spc24-9 mutants carrying the HCS rescue plasmids were synchronized in G1 phase, released into HU for 3 hours and immunofluorescence was performed to analyze chromosome segregation and spindle morphology.  All of the HCS genes were able to restore a single nuclei phenotype to spc24-9 HU treated cells (Figure 2.5D).  The STU1 and STU2 rescue clones that I identified lacked the N-terminal 97 and 252 amino acids of Stu1 and Stu2, respectively (herein referred to as STU1∆N and STU2∆N).  I tested if high copy full length STU1 or STU2 expression plasmids were capable of rescuing spc24-9 HU lethality.  Expression of full length STU2 was clearly not able to rescue either the HU lethality or chromosome segregation defects of spc24-9 mutants 38 suggesting that the N-terminal truncation is an important feature of the STU2∆N rescue activity (Figure 2.5A, D).  Expression of full length STU1 was able to rescue spc24-9 HU lethality and chromosome separation at levels above vector alone, but not as well as the STU1∆N clone (Figure 2.5A, D). I reasoned that expression of STU2∆N might be rescuing the spindle expansion defect in HU treated spc24-9 mutants by destabilizing MTs.  Consistent with this hypothesis, STU2∆N clone lacks the entire TOG1 domain of Stu2 which binds tubulin heterodimers (Al-Bassam et al., 2006; Al-Bassam et al., 2007).  Previous work has shown that Stu2 promotes microtubule polymerization whereas expression of Stu2 lacking its TOG1 domain, which still binds MTs, results in decreased mitotic spindle length and slows down anaphase spindle elongation (Al-Bassam et al., 2006).  I asked if Stu2∆N localization is similar to endogenous Stu2 by tagging Stu2∆N with VFP (which still retains its spc24-9 HU rescue activity) and endogenous Stu2 with CFP.  Indeed I found that Stu2∆N-VFP localization overlapped with Stu2-CFP in both wild type and spc24-9 mutants in log phase or HU treated cells, consistent with previous data demonstrating that Stu2∆TOG1-GFP still binds MT plus ends (Figure 2.6, (Al-Bassam et al., 2006)).  To test if depletion of Stu2 activity using another mutant form of Stu2 is also capable of preventing spindle expansion in spc24-9 cells, I combined spc24-9 with the stu2-10 Ts mutation (Severin et al., 2001) and tested the double mutant for separation of nuclei upon HU treatment.  Only 8% of stu2-10 spc24-9 mutants segregated nuclei after 3 hours of HU treatment compared to 59% of spc24-9 mutants (Figure 2.5E).  Therefore functional Stu2 is 39 required for the spindle expansion defect in spc24-9 mutants.  Although stu2-10 spc24-9 double mutants maintained a short spindle during HU treatment, they were lethal on HU plates at 30 o C (Figure 2.5B) suggesting that the defects in both Stu2 and Spc24 prevent cell cycle recovery after HU exposure.       Stu2 interacts with two other MT plus-end tracking proteins, Bim1 and Bik1 (Chen et al., 1998; Lin et al., 2001; Wolyniak et al., 2006).  Since we had previously shown that bim1 spc24-9 mutants have a synthetic growth defect (Montpetit et al., 2005), I deleted BIK1 in spc24-9 cells and analyzed growth phenotypes.  The bik1 spc24-9 double mutant rescued the nuclei separation defect of HU treated spc24-9 mutants and both the HU (at 30 o C) and Ts (at 33 o C) lethality of spc24-9 mutants (Figure 2.5C, E).  Therefore the activity of Stu2 and Bik1 is responsible for the spindle expansion and subsequent nuclei separation and lethality of spc24-9 cells upon HU exposure. We also determined if Stu1 is required for the spindle expansion activity in spc24-9 HU treated cells by creating a stu1-5 spc24-9 double mutant (Yin et al., 2002).  The stu1-5 spc24-9 mutant behaved in a similar manner to spc24-9 mutants and elongated their spindles when treated with HU suggesting that, unlike Stu2 and Bik1, Stu1 activity is not required for spindle expansion in HU exposed spc24-9 mutants.  Although both spc24-9 and stu1-5 individual mutants grow well at 30 o C on rich media (YPD), the double mutant is synthetically lethal at 30 o C (Figure 2.5D).  In addition, the spc24-9 stu1-5 double mutant is viable at 25 o C in rich media but inviable when grown on HU plates (Figure 2.5D).  The sensitivity of spc24-9 stu1-5 40 double mutants to HU and the synthetic lethal interaction between spc24-9 and stu1-5 mutants suggests that Stu1 and Spc24 have a joint or parallel role in restraining spindle expansion during the DNA replication checkpoint.  2.3.6 Stu1 localizes to kinetochores prior to anaphase Stu1, which was originally isolated as a suppressor of a tub2 (β-tubulin) mutation, interacts with Tub2 and localizes to the spindle midzone in anaphase spindles (Pasqualone and Huffaker, 1994; Yin et al., 2002).  However, the localization of Stu1 in relation to a SPB marker has not been assessed.  I imaged Stu1 fused to VFP in relation to the Spc29-CFP SPB protein by synchronizing cells in G1 phase with mating pheromone, then releasing into the cell cycle and fixing cells every 30min.  The budding yeast spindle reaches a length of 1.5-2 μm prior to entering anaphase (Pearson et al., 2001).  Prior to anaphase, I detected three evenly distributed patterns of Stu1-VFP localization in fixed cells - a bilobed distribution pattern in between the Spc29-CFP foci which is a hallmark localization pattern for a kinetochore protein (Figure 2.7, top row) (He et al., 2001; Measday et al., 2002), a single focus located closer to one of the SPBs (Figure 2.7, second row) and a continuous signal inbetween SPBs (Figure 2.7, third row).  In agreement with previous results, I also found that Stu1-VFP localized to the midzone of anaphase spindles (Figure 2.7, fourth row) (Yin et al., 2002).  Finally, in telophase, I observed a dispersed Stu1 signal near the SPBs (Figure 2.7, bottom row). Our localization data suggests that Stu1 may interact with the kinetochore.  I tested if 41 Stu1 localizes to the kinetochore by performing Stu1-Myc chromatin immunoprecipitation (ChIP) assays from logarithmically growing cells.  Stu1-Myc specifically associated with CEN1 and CEN3 DNA but not with a non-CEN locus, PGK1 (Figure 2.8A, lane 4).  I performed a Stu1-Myc ChIP assay in a spc24-9 mutant strain at both permissive (25 o C) and restrictive (37 o C) temperature (Figure 2.8B).  Stu1-Myc is still able to associate with CEN DNA at restrictive temperature suggesting that Stu1 does not require Spc24 to bind kinetochores (Figure 2.8B, lane 4). In summary, our localization and ChIP data demonstrate that Stu1 localizes to kinetochores early in the cell cycle in an Spc24 independent manner and relocalizes to the spindle around the time of the metaphase to anaphase transition.  2.3.7 Stu2 localizes to kinetochores early in the cell cycle In anaphase cells, Stu2-GFP localizes to the cytoplasmic side of the SPB and along the spindle MTs as determined by immunoelectron microscopy (Kosco et al., 2001).  In addition, Stu2 has been shown to colocalize with kinetochores and SPBs and bind CEN DNA in metaphase cells (He et al., 2001).  We analyzed Stu2-VFP localization in short (<1.5µm) spindles (acquired at the time of bud emergence) and during normal spindle expansion using time-lapse microscopy (Figure 2.9).  We observed that Stu2-VFP signal displayed a bilobed pattern between Spc29-CFP foci in short spindles (Figure 2.9, image 10b).  Time-lapse microscopy using longer exposures (which saturate Stu2-VFP spindle fluorescence) revealed that Stu2-VFP also tracks on astral microtubules and transiently associates with SPBs as astral MT shorten (data 42 not shown). Our imaging data is consistent with Stu2 localizing primarily to kinetochores and/or the nuclear spindle prior to metaphase.  Once spindles had lengthened (>2 μm), we detected co-localization of Stu2-VFP with Spc29-CFP as well as Stu2-VFP at the spindle midzone as previously described (Figure 2.9, image 0c) (He et al., 2001; Kosco et al., 2001). The localization of Stu2 to kinetochores early in the cell cycle suggests that defects in kinetochore function when DNA replication is stalled by HU treatment may significantly affect Stu2 activity.  2.3.8 Stu2 is mislocalized in HU treated spc24-9 cells We tested if Stu2 localization to the kinetochore depends on functional Spc24 by using both ChIP and microscopy analyses.  Stu2-Myc displayed a decreased ability to interact with CEN DNA in spc24-9 cells as I increased the temperature from permissive (25 o C) to semi-permissive (30 o C) conditions (Figure 2.8D, lanes 8 and 10).  Stu2-Myc did not co-precipitate with CEN DNA in spc24-9 mutants shifted to restrictive temperature (37 o C) suggesting that Stu2 requires Spc24 to interact with the kinetochore (Figure 2.8D, lane 12). Although Stu2 requires Spc24 for proper CEN localization in logarithmically growing cells, this does not necessarily reflect the situation when cells are exposed to HU.  I performed a Stu2-Myc ChIP assay in wild type and spc24-9 cells after treating cells with HU for three hours at 30 o C.  Stu2 interaction with CEN DNA was highly reduced in spc24-9 mutants compared to wild type cells (Figure 2.8C, compare lanes 2 and 4).  Thus Spc24 is required for Stu2 to efficiently interact with CEN DNA in both log phase and HU treated cells.  To test if Stu2 43 localization is perturbed in spc24-9 cells, Jennifer McQueen, Lara Cuschieri and Dr. Jackie Vogel performed a quantitative analysis of Stu2-VFP fluorescence during HU exposure.  Cells were released from a G1 pheromone block into HU at 25 o C, shifted to 30 o C and fixed after 60min of incubation.  At this time point, spindle expansion had clearly begun in spc24-9 cells as spindle lengths averaged 2.5 μm in mutant cells compared to 1.8 μm in wild type cells (Figure 2.10B).  Analysis of individual cells revealed that Stu2 remained as bilobed foci in wild type cells whereas Stu2 signal was clearly mis-localized along the spindle midzone (cs) or next to one pole (monopolar) in spc24-9 cells (Figure 2.10A, 3-dimensional render, rotated). Analysis of Stu2-VFP fluorescence on the spindle indicated that Stu2-VFP fluorescence intensity decreased significantly in the spc24-9 mutant relative to wild-type (Figure 2.10C). Stu2-VFP also re-distributed from discrete foci to diffuse fluorescence along the length of the spindle (Figure 2.10A); thus Stu2-VFP fluorescence on the spindle per unit length (µm; see Materials and Methods for details) was used for the comparison of Stu2-VFP in wild type versus spc24-9 cells. In general, this analysis revealed that spindles in the spc24-9 mutant had decreased Stu2-VFP fluorescence and were longer, suggesting that spindle expansion correlates with mis-localization of Stu2 (Figure 2.10C).  However, low levels of Stu2-VFP fluorescence were found on both long and short spindles in spc24-9 cells suggesting that the relationship between spindle length and Stu2 levels was not absolute.  More specifically, it was not clear if the observed spindle expansion observed in spc24-9 cells was permanent or represented oscillations in spindle length. To further explore the dynamics of Stu2 interaction with the spindle and spindle 44 expansion, Jennifer McQueen, Lara Cuschieri and Dr. Jackie Vogel  analyzed dynamic changes in Stu2-VFP fluorescence and spindle length in living cells using time-lapse microscopy.  For this analysis, HU-arrested wild type and spc24-9 mutant cells were shifted to 30 o C on the microscope stage and the HU arrest maintained throughout the time-lapse by mounting the cells in FP medium supplemented with HU.  This analysis revealed that spindle length remains relatively static in wild-type cells, with an average net change (either shrinking or elongating) in length of 0.40±0.125 µm during the time-lapse for all cells (n=4) analyzed (Figure 2.11A and 2.11C).  Spindle length at the start of the time-lapse was not significantly different between wild-type and spc24-9 cells, and was similarly static in spc24-9 cells (n=4 cells/strain) during the first 5 min. of the time-lapse (Figure 2.11B). In contrast with wild-type cells, spindle length increased significantly (1.45±0.638 µm) in the spc24-9 mutant over time (Figure 2.11B and 2.11C). At 18 min. after the shift to 30˚C, a net increase in spindle length occurred in all four spc24-9 cells; in each cell spindle length had increased (0.9 – 2.3 µm; mean length increase of 1.3.± 0.66 µm) relative to length at the start of the time lapse and relative to all four wild-type cells (Figure 2.11B and 2.11D).  For this reason, we chose to compare the intensity of Stu2-VFP fluorescence on the spindle at time 0 and at 18 min. Stu2-VFP fluorescence was significantly decreased in spc24-9 cells compared to wild type cells (Figure 2.11E), suggesting that mis-localization of Stu2 is correlated with the spindle expansion observed (Figure 2.11D). Finally, we detected significant oscillation of spindle length between 10- 28 min. in spc24-9 cells (Figure 2.11B), suggesting that mis-localization of Stu2 results in transient spindle expansion. 45 The transient nature of this defect is consistent with the observation of a sub-population of spc24-9 cells with short spindles and mis-localized or low Stu2-VFP levels.  2.4  Discussion The kinetochore is required to restrain spindle expansion in budding yeast when DNA replication is stalled however the mechanism by which kinetochores maintain spindle length in this state is not well understood.  I identified genes that when over-expressed, rescue the lethality and spindle expansion defects of the spc24-9 kinetochore mutant when exposed to HU. Two MT plus end binding proteins, the CLASP related protein Stu1 and XMAP215 homologue Stu2, were identified and their interactions with the kinetochore were explored further.  I demonstrated that Stu1 localizes to kinetochores early in the cell cycle and relocalizes to the spindle midzone after metaphase and that Stu2 binds to the kinetochore in a Spc24-dependent manner.  In addition, inappropriate spindle expansion in HU-treated spc24-9 cells can be prevented by inhibiting Stu2 activity.  I propose that mislocalization of Stu2 in spc24-9 cells enables spindle expansion during the DNA replication checkpoint.  Kinetochore-MT bipolar attachment and the DNA replication checkpoint. DNA microarray studies have suggested that most CENs are replicated upon exposure to HU (Yabuki et al., 2002; Feng et al., 2006).  Thus budding yeast CENs may be capable of attaining bipolar attachment during HU treatment.  However, the data presented in Figure 2.4 of this Chapter 46 suggests that bipolar attachment may not be the only mechanism by which kinetochores maintain short spindles during HU arrest.  Firstly, wild type and spc24-9 cells display similar percentages of CEN15-GFP bipolar foci when treated with different concentrations of HU yet only spc24-9 spindles expand (Figure 2.4B).  Secondly, when wild type cells are treated with high concentrations of HU, a single CEN15-GFP focus is detected that clearly colocalizes with one SPB (Figure 2.4C).  These data are similar to previous studies demonstrating that a GFP marked unreplicated minichromosome colocalizes with one SPB after SPB separation (Dewar et al., 2004).  Although the data do not distinguish between replicated and unreplicated CEN15, CEN15 is clearly attached to one pole, yet the spindle remains short.  Thus the ability to attain bipolar attachment during HU treatment is not correlated with restraining spindle expansion.  Stu2 activity enables spindle expansion in spc24-9 HU treated cells. I isolated a truncated version of the XMAP215 homologue, STU2 (STU2∆N), in my spc24-9 HCS screen that lacks the N-terminal 252 amino acids of Stu2 (Table 2.1).  I propose that over-expression of STU2∆N rescues spc24-9 HU lethality by restraining MT dynamics induced by mis-localization of Stu2.  STU2∆N lacks the N-terminal TOG1 domain that binds tubulin heterodimers but retains the TOG2 domain that binds MT plus ends (Al-Bassam et al., 2006).  Previous studies have shown that Stu2 lacking its TOG1 domain binds MT plus ends but cannot promote plus end MT growth suggesting that STU2∆N inhibits spc24-9 HU spindle expansion via the same mechanism (Al-Bassam et al., 2006).  The spc24-9 mutant and the resultant mislocalization of 47 Stu2 (see next section) is an important feature of STU2∆N’s rescue function because overexpression of STU2∆N does not inhibit spindle expansion in a wild type cell cycle (Figure 2.12).  Another possible Stu2∆N rescue mechanism, which is not mutually exclusive with the previous mechanism, is that over-expression of STU2∆N is titrating out a Stu2 interacting protein that is mediating spindle expansion in spc24-9 HU cells.  Stu2 interacts with the CLIP-170 ortholog, Bik1, at the C-terminus of Stu2 (Wolyniak et al., 2006).  I find that deletion of the Stu2 interacting protein Bik1 rescues the spc24-9 spindle expansion defects and HU lethality at 30 o C and that the bik1 spc24-9 double mutant grows at a higher temperature than the spc24-9 mutant alone (Figure 2.5C, 2.5E).  Thus, inhibition of Stu2 or Bik1 plus end MT activity prevents spindle expansion when spc24-9 mutants are under HU arrest.  Stu2 retention at the kinetochore is important for maintaining a short spindle when DNA replication is stalled. My data suggests that Stu2 activity is required for spindle expansion in spc24-9 HU treated cells.  The stu2-10 spc24-9 double mutant no longer displays inappropriate spindle expansion when exposed to HU (Figure 2.5E).  Why is Stu2 able to promote spindle expansion in HU treated spc24-9 cells but not wild type cells?  I propose that the inability to recruit and retain Stu2 at the kinetochore in spc24-9 mutants enables Stu2 to promote MT dynamics.  My ChIP data suggest that the interaction of Stu2 with CEN DNA is perturbed in spc24-9 mutants both in log phase cells and during HU treatment (Figure 2.8C and 6D).  In agreement with my studies, Stu2 does not associate with CEN DNA at restrictive 48 temperature in an ndc80-1 Ts mutant (He et al., 2001).  Thus the Ndc80 complex is required for recruitment of Stu2 to the kinetochore.  Jennifer McQueen, Lara Cuschieri and Jackie Vogel performed a detailed analysis of Stu2-VFP fluorescence and spindle length in both live and fixed spc24-9 HU treated cells.  Shortly after shift to restrictive temperature (30 o C for spc24-9 cells exposed to HU), they detected spindle expansion and mis-localization of Stu2-VFP as well as its diffusion along the axis of the spindle (Figure 2.10).  Stu2-VFP also displayed variable localization patterns including movement to one pole and to the spindle midzone in these cells (Figure 2.10A).  The time-lapse analysis revealed that HU treatment induces spc24-9 mutants to undergo oscillations in spindle length, unlike wild type cells where relatively little change in spindle length is detected (Figure 2.11).  A window of time was identified when the spindle was at an average maximum length in these analyses and Stu2-VFP fluorescence levels were quantified at this time. Stu2-VFP fluorescence was significantly reduced compared to wild type cells suggesting a correlation between reduction in Stu2-VFP fluorescence and spindle expansion (Figure 2.11E). The oscillations observed also explain why short spindles with decreased Stu2 are observed in populations of spc24-9 cells.  Finally, oscillations in spindle length were also detected in HU exposed rad53 mutants, suggesting that activation of the DNA replication checkpoint regulates spindle dynamics and in the absence of the checkpoint this restraint is compromised (Bachant et al., 2005).  Our studies have uncovered the role of an effector of the checkpoint, Stu2, in restraining spindle dynamics while localized at the kinetochore.  49 The role of Stu2 during the DNA replication checkpoint. Does Spc24 participate in regulating spindle expansion only during the DNA replication checkpoint or also during an unperturbed cell cycle?  To address this question we measured spindle length in a wild type versus spc24-9 mutant after release from a G1 block to restrictive temperature (Figure 2.13). We found that early in the cell cycle, spc24-9 mutants had longer spindles than wild type cells consistent with a defect in the S phase checkpoint during a normal cell cycle.  As cells progressed, spc24-9 spindle expansion lagged behind wild type cells suggesting a delay in anaphase.  These data are consistent with our analysis of DNA content during a synchronous cell cycle at restrictive temperature which demonstrated that spc24-9 cells progress more rapidly through S phase than wild type cells (compare 60 min. time point between wild type and spc24-9, Figure 2.14).  However, once DNA has replicated in spc24-9 cells, a 2N content of DNA is maintained for two hours before 1N DNA content is once again detected [Figure 2.14, (Montpetit et al., 2005)].  Thus spc24-9 cells accelerate through S phase but are delayed in anaphase.  Mechanism of spindle expansion in rad53 and mec1 mutants. The results shown here suggest that the kinetochore regulates spindle integrity during an HU induced DNA replication checkpoint by sequestering proteins such as Stu2 that regulate MT dynamics.  Why then do mec1 and rad53 mutants elongate their spindles during the DNA replication checkpoint?  A previous study used a CEN transcription read through assay to demonstrate that the kinetochore is still capable of blocking access to the transcription machinery in a rad53-21 strain suggesting 50 that the inner CBF3 kinetochore complex that binds DNA is still intact (Bachant et al., 2005). Ndc10, a CBF3 component, is also present on CEN DNA in spc24-9 cells suggesting that the spindle expansion is not due to defects in inner kinetochore assembly (Figure 2.2A).  Not all central kinetochore mutants display CEN transcription read through thus the central kinetochore may be compromised in rad53 or mec1 mutant strains (Doheny et al., 1993).  mec1-1 HU treated cells display upregulation of STU2 and CIN8 mRNA and protein levels suggesting that increased levels of MT regulatory proteins may contribute to spindle expansion in mec1-1 cells (Krishnan et al., 2004).  Our data suggest that mis-localization of Stu2 by disruption of a central kinetochore complex also causes spindle expansion during the DNA replication checkpoint. By using HU as a method to stall cells in the process of DNA replication, I have uncovered a role for the kinetochore in regulating spindle dynamics in S phase.  I have discovered a role for Spc24 in recruiting Stu2 to the kinetochore to mediate MT dynamics prior to metaphase.  Mutation of Spc24 results in mis-localization of Stu2 and deregulation of spindle dynamics when DNA replication is stalled and likely during an unperturbed S phase as well.  I propose that the kinetochore regulates spindle integrity during an HU induced DNA replication checkpoint by sequestering proteins such as Stu2 that play central roles in controlling spindle MT dynamics.  51 Table 2.1 HCS screen of spc24-9 HU lethality HU rescue a  Gene Name ORF Biological Process d  + DMA1 YHR115C Spindle position and orientation + RCK2 YLR248W Oxidative and osmotic stress signaling + STU1 truncated b  YBL034C MT dynamics + + GIC1 YHR061C Cell polarity + + + KIP2 YPL155C Mitotic spindle positioning + + +  MCK1 YNL307C Mitotic and meiotic chromosome segregation + + + + HCM1 YCR065W Transcription + + + + + STU2 truncated c  YLR045C MT dynamics + + + + + SPC24 YMR117C Chromosome Segregation + + + + + SPC25 YER018C Chromosome Segregation  a growth of spc24-9 mutant carrying rescue clone struck on .05M HU plates at 30 o C from weak (+) to strong (+++++) growth b the STU1 rescue clone is missing the N-terminal 97 amino acids c the STU2 rescue clone is missing the N-terminal 252 amino acids d GO Annotation from Saccharomyces Genome Database  52 Table 2.2 List of Yeast Strains Strain Number Genotype Source CU1000 MATa stu1-5 his3-∆200 leu2-3,112 ura3-52 (Yin et al., 2002) CUY1088 MATa stu2-10::URA3 his3-∆200 leu2-3,112 ura3-52 (Kosco et al., 2001) TWY308 MATα mec1-1 ura3 trp1 (Weinert et al., 1994) YLM79  MATa ura3-52, lys2-801, ade2-101, his3-∆200, leu2-∆1, trp1-∆63 STU1-MYC::TRP1 This study YLM185  MATa ura3-52, lys2-801, ade2-101, his3-∆200, leu2-∆1, trp1-∆63 STU1-VFP::kanMX6 SPC29-CFP:: hphMX4 This study YLM187  MATa ura3-52, lys2-801, ade2-101, his3-∆200, leu2-∆1, trp1-∆63 STU2-VFP::kanMX6 SPC29-CFP:: hphMX4 This study YLM288  MATa ura3-52, lys2-801, ade2-101, his3-∆200, leu2-∆1, trp1-∆63 STU2-VFP::kanMX6 SPC29-CFP:: hphMX4 spc24-9::natMX4 This study YLM400  MATa ura3-52, lys2-801, ade2-101, his3-∆200, leu2-∆1, trp1-∆63 STU2-MYC::TRP1 spc24-9::natMX4 This study YLM451  MATa ura3-52, lys2-801, ade2-101, his3-∆200, leu2-∆1, trp1-∆63 STU1-MYC::TRP1 spc24-9::natMX4 This study YLM533 MATa ura3-52, lys2-801, ade2-101, his3-∆200, leu2-∆1, trp1-∆63 NDC80-VFP::kanMX6 SPC29-CFP:: hphMX4 This study YLM535 MATa ura3-52, lys2-801, ade2-101, his3-∆200, leu2-∆1, trp1-∆63 NDC80-VFP::kanMX6 SPC29-CFP:: hphMX4 spc24-9::natMX4 This study YLM542 MATa ura3-52, lys2-801, ade2-101, his3-∆200, leu2-∆1, trp1-∆63 NDC80-13MYC::His3MX6  SPC24::natMX4 This study YLM544 MATa ura3-52, lys2-801, ade2-101, his3-∆ trp1-∆63 NDC80-13MYC::His3MX6  spc24-9::natMX This study YLM557 MATa ura3-52, lys2-801, ade2-101, his3-∆200, leu2-∆1, trp1-∆63 NDC10::13MYC::kanMX spc24-9::natMX4 This study YLM609 MATa ura3-52, lys2-801, ade2-101, his3-∆200, leu2-∆1, trp1-∆63 STU2-CFP:: hphMX4 pSTU2∆N-VFP::kanMX  (To be continued) 53 Table 2. 2  List of Yeast Strains (continued) Strain Number Genotype Source YLM610 MATa ura3-52, lys2-801, ade2-101, his3-∆200, leu2-∆1, trp1-∆63 STU2-CFP:: hphMX4 spc24-9::natMX4 -VFP::kanMX This study YM234 MATa ura3-52, lys2-801, ade2-101, his3-∆200, leu2-∆1, TRP1+ stu2-10::URA3  spc24-9::kanMX6 This study YM406  MATa ura3-52, lys2-801, ade2-101, his3-∆200, leu2-∆1, trp1-∆63 STU2-MYC::TRP1 This study YM480 a  MATa LacI-GFP::HIS3 LacO::URA3-CEN15(1.8) Spc29-CFP::kanMX6 SPC24::natMX4 This study YM482 a  MATa LacI-GFP::HIS3 LacO::URA3-CEN15(1.8) Spc29-CFP::kanMX6 spc24-9::natMX4 This study YM487 a  MATa LacI-GFP::HIS3 LacO::URA3-CEN15(1.8) Spc29-CFP::kanMX6 spc24-10::natMX4 This study YM836 MATa ura3-52, lys2-801, ade2-101, his3-∆200, leu2-∆1, trp1-∆63 bik1::kanMX6 This study YM903 MATa ura3-52, lys2-801, ade2-101, his3-∆200, leu2-∆1, TRP1+ stu1-5 spc24-9::kanMX6 This study YM935 MATa ura3-52, lys2-801, ade2-101, his3-∆200, leu2-∆1, trp1-∆63 bik1::kanMX6 spc24-9::natMX4 This study YPH499 MATa ura3-52, lys2-801, ade2-101, his3-∆200, leu2-∆1, trp1-∆63 P. Hieter YPH1734 MATa ura3-52, lys2-801, ade2-101, his3-∆200, leu2-∆1, trp1-∆63 NDC10::13MYC::kanMX6 (Measday et al., 2002) YVM1363 MATa ura3-52, lys2-801, ade2-101, his3-∆200, leu2-∆1, trp1-∆63 spc24-10::kanMX6 (Montpetit et al., 2005) YVM1370 MATa ura3-52, lys2-801, ade2-101, his3-∆200, leu2-∆1, trp1-∆63 SPC24::kanMX6 (Montpetit et al., 2005) YVM1380  MATa ura3-52, lys2-801, ade2-101, his3-∆200, leu2-∆1, trp1-∆63 spc24-9::kanMX6 (Montpetit et al., 2005) YVM1448 MATa ura3-52, lys2-801, ade2-101, his3-∆200, leu2-∆1, trp1-∆63 spc24-8::kanMX6 (Montpetit et al., 2005) YVM1591 MATa ura3-52, lys2-801, ade2-101, his3-∆200, leu2-∆1, trp1-∆63 spc24-9::natMX4 This study a These strains were created by mating a W303 strain derivative (Goshima and Yanagida, 2000) with an S288C strain derivative hence the precise genotype is not known.  54   Figure 2.1 spc24-9 mutants are sensitive to HU due to inappropriate spindle expansion  (A)  Cell dilution assay of indicated strains grown on YPD and .05M HU at 30 o C for 3 days. (B)  Immunofluorescence analysis of wild type (SPC24), spc24-8, spc24-9 and spc24-10 cells synchronized in G1 phase with α-factor, then released into 0.2M HU for 3 hours at 30oC. Shown are representative cells after 3 hours HU treatment imaged for DNA (DAPI), MTs (Tubulin) and cell morphology (DIC).  White arrowheads point to separated nuclei in the spc24-9 mutant.  (C) Percentage of cells (100 cells counted) described in (B) displaying single (grey bars) or separated (black bars) nuclei.  (D) Schematic of point mutations in spc24 mutants. The predicted coiled-coil domain is shaded. The spc24-9 V179D substitution is in the Spc24 C-terminal globular domain. (Cited from Montpetit et al., Genetics 2005, by permission © Genetics Society of Amarica.)   55   Figure 2.2  Ndc80 CEN association is disrupted in spc24-9 mutants Multiplex PCR analysis of CEN1, PGK1 and CEN3 loci was performed with total chromatin (T) or immunoprecipitate (IP) as PCR templates.  Strains were grown at 25 o C to log phase, then either shifted to 37 o C for three hours, or incubated in 0.2M HU at 30 o C for three hours.  (A) Ndc10-Myc wild type (lanes 1-6) or spc24-9 cells (lanes 7-12).  (B)  Ndc80-Myc wild type (lanes 1-6) or spc24-9 cells (lanes 7-12).  (C) Wild type strain carrying no epitope tag (No Tag, lanes 1-6) shown as a control.  An untagged spc24-9 mutant was also used as a control and showed similar results (data not shown). 56   Figure 2.3  Ndc80 is mislocalized in spc24-9 mutants (A)  Ndc80-VFP Spc29-CFP wild type (SPC24) and spc24-9 cells were grown at 25 o C to log phase, then incubated at 37 o C for three hours, fixed in 70% ethanol and imaged.  Four types of Ndc80 localization patterns were detected in spc24-9 mutants:  Type I:  Wild type Ndc80, 44% of cells; Type II:  Ndc80 localized to one SPB (14% of cells); Type III: Multiple Ndc80 foci (20% of cells); Type IV:  Little or no Ndc80-VFP signal (22% of cells).  (B) Ndc80-VFP Spc29-CFP wild type (SPC24) and spc24-9 cells were grown at 25 o C to log phase, then incubated at 30 o C in 0.2M HU for three hours, fixed in 70% ethanol and imaged.  In spc24-9 cells, Ndc80-VFP localization either resembled wild type cells (20% of cells) or Ndc80-VFP levels were very low and barely detectable (80% of cells).  In the overlay, green is VFP signal and red is CFP.  Scale bar is 2μm for all images. 57   Figure 2.4 spc24-9 mutants are capable of establishing bipolar attachment  (A) Wild type (SPC24), spc24-9 and spc24-10 strains carrying LacO repeats integrated 1.8kb from CEN15, LacI-GFP and Spc29-CFP were synchronized in G1 phase with α-factor and released at 30 o C.  Samples were taken every 15 min. and imaged using fluorescence microscopy for the presence of the CEN15-GFP and Spc29-CFP signal.  Shown are the time points (105 min. onwards) at which the cells began to display bipolar attachment (separation of CEN15-GFP signals).  Duplicate experiments were performed with similar results.  Shown is the result of one experiment in which 100 cells containing a spindle of 0.5 μm or larger were counted for each time point. (B) Wild type (SPC24) and spc24-9 CEN15-GFP Spc29-CFP strains were synchronized in G1 phase with α-factor, split into four cultures and indicated concentrations of HU were added for 3 hours at 30 o C.  Similar results were seen with duplicate experiments thus data from one experiment is shown (100 cells counted).  (C) Example of a wild type (SPC24) cell from (B) after 3 hours of 0.3M HU treatment that displays monopolar CEN15 attachment.  In the overlay, CEN15-GFP is green and Spc29-CFP is red.  58   Figure 2.5  Spindle expansion in spc24-9 mutants depends on active Stu2 (A) Cell dilution assay of spc24-9 mutants carrying the following 2μ plasmids: vector (pRS202), STU1∆N-library (HCS clone identified in screen), STU1∆N-subclone (subclone of STU1∆N-library containing only the STU1 gene), STU1 full length (gift of T. Huffaker), full length SPC24, STU2∆N-library (HCS clone identified in screen), STU2∆N-subclone (subclone of STU2∆N-library containing only the STU2 gene), STU2 full length (gift of T. Huffaker) were grown on -URA plates at 30 o C for 4 days or –URA .05M HU at 30oC for 5 days.  (B) and (C) Cell dilution assay of indicated strains grown on YPD at 25 o C (2 days), 30 o C and 33 o C (3 days) and .05M HU at 25 o C, 30 o C and 33 o C (3 days).  (D) Immunofluorescence analysis of spc24-9 mutants carrying the indicated HCS plasmids synchronized in G1 phase with α-factor, then released into 0.2M HU for three hours at 30 o C.  Cells were counted (100 per sample) for single nuclei (grey bars) or separated nuclei (black bars) by DAPI staining.  Duplicate experiments were performed with similar data and shown is the result of one experiment.  (E) Immunofluorescence analysis of indicated strains treated and analyzed as described in (D). 59   Figure 2.6  Stu2∆N-VFP localization overlaps with endogenous Stu2-CFP Wild type (SPC24) and spc24-9 strains carrying Stu2∆N-VFP on a 2μ plasmid and endogenous Stu2-CFP were either grown at 30 o C to log phase or incubated in 0.2M HU for three hours, fixed in 70% ethanol and imaged.  In the overlay, green is VFP signal and red is CFP.  Scale bar is 2μm for all images. 60   Figure 2.7  Stu1 localizes to kinetochores and the spindle midzone Wild type Stu1-VFP Spc29-CFP cells were synchronized in G1 phase with α-factor and released into the cell cycle at 30 o C.  Cells were fixed in 70% ethanol every 15 min. for 90 min. and imaged as described in the Material and Methods.  In the overlay, green is VFP signal and red is CFP.  Scale bar is 2μm for all images. 61   Figure 2.8  Stu2 CEN binding is abolished in spc24-9 mutants whereas Stu1 is still able to associate with CEN DNA Multiplex PCR analysis of CEN1, PGK1 and CEN3 loci was performed with total chromatin (T) or immunoprecipitate (IP) as PCR templates.  Strains were grown at 25 o C to log phase, then either kept at 25 o C or incubated at 30 o C or 37 o C for three hours. (A) Wild type log phase cells grown at 30 o C and carrying no epitope tag (No Tag), (lanes 1-2), Stu1-Myc (lanes 3-4) and Stu2-Myc (lanes 5-6).  (B) spc24-9 mutants carrying Stu1-Myc (lanes 1-4) and a wild type strain with no tag at 37 o C (lanes 5-6).  (C)  Stu2-Myc in a wild type (lanes 1-2), spc24-9 (lanes 3-4) and an untagged wild type strain (NoTag, lanes 5-6).  Strains were grown to log phase at 25 o C, HU was added to a final concentration of 0.2M HU and cells were shifted to 30 o C for three hours.  (D) Stu2-Myc in a wild type (lanes 1-6) and spc24-9 (lanes 7-12) strains.  No Tag strain (lanes 13-18) is an untagged wild type strain.  For all ChIP assays where the spc24-9 mutant was used, I included both a wild type and spc24-9 untagged control and saw similar results thus only the wild type untagged control is presented. 62   Figure 2.9 Time-lapse analysis of Stu2-VFP Stu2-VFP Spc29-CFP cells were imaged using time-lapse microscopy at 30 o C as described in the Materials and Methods.  Stu2-VFP is represented in greyscale and Spc29-CFP in red.  Arrows in Image 10,b point to Stu2-VFP bilobed foci on the nuclear side Spc29-CFP.  Arrows in Image 0,c point to Stu2-VFP localization on the central spindle (cs) and poles (p).  63   Figure 2.10 Increased spindle length correlates with Stu2 mislocalization and reduction Wild type (SPC24) and spc24-9 cells expressing Stu2-VFP Spc29-CFP were synchronized in G1 phase with pheromone, released into 0.2M HU at 25 o C for 1.5h and shifted to 30 o C in HU for 60 min., then fixed.  (A) Representative images (extended focus and 3-dimensional render) of Stu2-VFP (greyscale) and Spc29-CFP (red) fluorescence in wild type and spc24-9 cells at 60 min. after shift to 30 o C are shown; mp indicates monopolar localization (near the SPB), whereas cs indicates localization to the central spindle.  (B) Average (ave) spindle length of wild type (SPC24) and spc24-9 cells after a 60 min. incubation in 0.2M HU at 30 o C (n=15).  (C) Quantitative analysis of Stu2-VFP fluorescence plotted as a function of spindle length. 64   Figure 2.11  Decreased Stu2 in the spc24-9 mutant results in oscillation of spindle length Wild type (SPC24) and spc24-9 cells carrying Stu2-VFP Spc29-CFP were synchronized in G1 phase with pheromone, released into 0.2M HU at 25 o C for 1.5h, mounted in FP supplemented with HU,  shifted to 30 o C on a heated stage and time-lapse microscopy performed.  Time zero is time in HU at 30 o C after equilibration on the stage at 30 o C for 15 min. Spindle length is plotted for four cells of each strain (A, SPC24; B, spc24-9) as a function of time, each depicted with a different colour, and shows oscillation with a net increase in length observed in all four spc24-9 cells at 18 min. (C) In contrast with wild-type cells, spindle length increases in spc24-9 cells.  (D) Spindle length is significantly increased in all four spc24-9 cells at 18 min. relative to length at t=0.  (E) At 18 min., Stu2-VFP fluorescence on the spindle is significantly decreased in all spc24-9 mutant cells relative to wild-type cells. 65   Figure 2.12  Overexpression of STU2∆N does not affect wild type spindle length in an unperturbed cell cycle Vector control (pRS326) and STU2∆N were expressed in cells carrying Stu2VFP Spc29CFP. Cells were grown to log phase at 30 oC, arrested with α-factor for 1.5hours at 30oC, released to 30 o C and time points taken every 15min.  Spindle length was calculated as described in the Materials and Methods.  Thirty cells were counted for each time point. 66   Figure 2.13 spc24-9 mutants display spindle expansion early in the cell cycle but a delay in anaphase Wild type (SPC24) and spc24-9 cells carrying Stu2VFP Spc29CFP were grown at 25 o C to log phase, incubated with α-factor for 1hour at 25oC, resuspended in prewarmed 37oC media with new α-factor and incubated for 2 hours at 37oC.  Cells were then released from the G1 arrest into 37 o C and time points taken every 10min.  Spindle length was measured by calculating the distance from Spc29-CFP foci to Spc29-CFP foci.  Fifty cells were counted per time point. 67   Figure 2.14  spc24-9 mutants accelerate through S phase and delay at anaphase at restrictive temperature Wild type (SPC24) and spc24-9 cells were grown at 25 o C to early log phase, arrested in G1 phase with pheromone, released to 37 o C and time points taken every 15 min.  DNA content was measured by flow cytometry as described in the Materials and Methods.  68 Chapter 3  Determination of a Novel Role for the Ndc80 Kinetochore Complex in the Ras2/cAMP/PKA Pathway  3.1 Introduction Budding yeast Saccharomyces cerevisiae is able to use various carbon sources but prefers glucose.  High concentrations of glucose represses the transcription of a large set of genes involved in utilization of alternate carbon sources, respiration and peroxisomal functions resulting in cells switching from respiratory growth to fermentative growth (Perlman & Mahler, 1974; Gancedo & Serrano, 1989; Ronne, 1995; De Winde, et al., 1997). Glucose repression is derepressed when cells are carbon starved or grown on a non-fermentable carbon source like glycerol or ethanol.  The addition of glucose or other rapidly fermentable sugars to derepressed cells triggers rapid activation of transcription and translation to enable cells to adapt to the fermentative carbon source.  One of the best understood pathways mediating the glucose signaling cascade is the cyclic AMP (cAMP) /Protein Kinase A (PKA) pathway (Figure 3.1). Adenylate cyclase, Cyr1, which catalyzes the synthesis of cAMP from ATP, is a key component of the PKA signal transduction pathway.  In budding yeast, the activity of adenylate cyclase is stimulated by the small G proteins Ras1 and Ras2, and a G protein coupled receptor (GPCR) system.  The GPCR system includes the membrane glucose receptor Gpr1 and Gα protein Gpa2, and glucose activation by the GPCR system is strictly dependent on glucose phosphorylation 69 (Rolland, et al., 2000; Rolland, et al., 2001).   Cdc25 and Sdc25 are two guanine nucleotide exchange factor proteins (GEF) for Ras that switch GDP-bound Ras to GTP-bound Ras, whereas Ira1 and Ira2 are redundant GTPase activating proteins (GAP) that inactivate Ras by hydrolysis of the bound GTP to GDP. PKA, which is composed of the Tpk1, 2, 3 catalytic subunits and the inhibitory Bcy1 regulatory subunit, is the major target of cAMP.  Once cAMP binds Bcy1, Tpk1, 2, 3 are released from Bcy1 and become active to phosphorylate multiple target proteins including the Msn2 and Msn4 transcription factors (Figure 3.1). High activity of the PKA pathway causes immobilization of storage carbohydrates (trehalose and glycogen), high sensitivity to heat and nutrient starvation, low expression of stress response genes and high expression of ribosomal protein genes.  Low activity of the PKA pathway causes the opposite phenotype, such as high levels of trehalose and glycogen and high expression of stress response genes.  cAMP accumulation in yeast is under strong feedback inhibition by PKA (Nikawa, et al., 1987; Mbonyi, et al., 1990). The Pde1 low-affinity cAMP phosphodiesterase, which hydrolyzes cAMP to AMP, is activated by PKA (Ma, et al., 1999).  Ras2, Cdc25 and the Ira proteins have also been suggested as targets of the feedback-inhibition mechanism (Gross, et al., 1992; Tanaka, et al., 1989; Colombo, et al., 2004). Two published articles have demonstrated a potential interaction between kinetochore proteins and the Ras2/cAMP/PKA pathway: 1) Sgt1, which is required for the assembly of the CBF3 inner kinetochore complex, physically interacts with Cyr1 and upregulates the activity of 70 the cAMP/PKA pathway (Dubacq, et al., 2002); 2) the function of the outer kinetochore Dam1 complex might be repressed by the Ras2/PKA pathway during a normal cell cycle (Li, et al., 2005).  In this chapter, I extend these studies to demonstrate that the Ndc80 kinetochore complex might play a role in the cAMP/PKA pathway in addition to its well-known functions in chromosome segregation and the spindle checkpoint.  3.2 Materials and methods 3.2.1 Strains, plasmids and media Yeast strains and plasmids used in this study are described in Table 3.1.  The liquid media were rich medium (YPD) or supplemental minimal medium (SC) (Kaiser, et al., 1994). The solid plates for spot assays were YPD (2% glucose), YPR (2% raffinose), YPGal (2% galactose) and YPGly (2% glycerol).  3.2.2 Trehalose determination The trehalose measurement was modified from a published trehalose quantitation method (Parrou & Francois, 1997).  Cells were grown into stationary phase with 5-10ml YPD at 25°C and collected by centrifugation (3min at 3500rpm). Cell pellets were washed once with distilled water, resuspended in 0.5ml of 0.25 M Na2CO3, and incubated at 95°C for 1 h. The mixture was centrifuged (1min at 5000rpm) and the supernatant was removed into a new 1.5ml eppendorf tube. 50ul of supernatant was taken out and brought to pH5.2 with 30μl 1M acetic acid and 120μl 71 0.2M Na-acetate (pH5.2). The mixture was incubated overnight with 2.5μl trehalase (0.01unit) (Sigma, Cat.No. T8778) at 37°C under constant agitation and glucose was determined with a glucose assay kit (Sigma GAGO20). Each sample was measured in triplicate.  3.2.3 Msn2 Western blot spc24-8 (YVM1448), spc24-9 (YVM1380) and spc24-10 (YVM1363) cells were grown to mid-logarithmic phase in 50ml YPD at semi-permissive temperature for each strain (33°C, 30°C, 30°C, respectively). Wild type (YVM1370) cells were grown at both 33°C and 30°C. One quarter of the culture was harvested and the pellet was washed once with cold dH2O and kept on ice or at -80°C for future use. The rest of the culture was washed once with prewarmed YP media, resuspended in prewarmed YP and kept shaking in the incubator at 33°C or 30°C. 10ml culture was collected for each indicated time point. The cell pellet was lysed and 40μg protein was loaded per lane.  Msn2 phosphorylation was detected with α-P-CREB (Görner, et al., 1998) (Cell Signaling No. 9196, Beverly, MA, USA) (1:1000), and with α-Msn2 (kind gift from Dr. Estruch, Valencia, Spain) (1:5000) as a loading control.  3.3 Results 3.3.1 The temperature sensitivity of spc24-9 is rescued by reducing cAMP/PKA activities The Measday lab previously reported a synthetic genetic analysis (SGA) performed using 72 spc24-9 as the query strain (Montpetit, et al., 2005), which identified a synthetic lethal (SL) interaction with ira2∆ and a synthetic sick (SS) interaction with pde2∆ (Figure 3.2A). Consistently, I found that overexpressing PDE2 rescues the temperature sensitivity (ts) of spc24-9 at 33°C (Figure 3.2B). Both Ira2 and Pde2 are negative regulators of Ras/cAMP/PKA pathway (Colombo, et al., 2004; Park, et al., 2005) (Figure 3.2C). I predicted that if the SS/SL interaction with spc24-9 is due to hyperactivation of Ras/cAMP/PKA pathway, deletion of Ras1 or Ras2 would rescue the ts of spc24-9.  I tested both ras1∆ spc24-9 and ras2∆ spc24-9 double mutants at a series of different temperatures. As shown in Figure 3.2D, spc24-9 was fully rescued by ras2∆ at 33°C and partially at 35°C which is a semi-permissive temperature for ras2∆, whereas ras1∆ partially rescued spc24-9 at 33°C. Although Ras1 and Ras2 are two Ras homologs, there are several differences in their functions. For example, Ras2 activates adenylyl cyclase more efficiently than Ras1 (Broek, et al., 1985), ras2∆ cells have less cAMP levels compared to ras1∆ cells (Toda, et al., 1985), and Ras2 but not Ras1 activates the pseudohyphal pathway (Mösch & Fink, 1997; Mösch, et al., 1996). Since I did not observe pseudohyphal growth of spc24-9 cells (data not shown), the different rescue ability of ras1∆ and ras2∆ probably relies on their effects on cAMP:  ras2∆ cells have less cAMP levels than ras1∆ cells, therefore ras2∆ may rescue spc24-9 ts lethality by reducing cAMP levels in spc24-9 cells. Interestingly, I found that two other spc24 ts alleles, spc24-8 and spc24-10, were also rescued by ras2∆ (Figure 3.2 E), suggesting that compromising the function of the Ndc80 complex, by mutation of Spc24, might induce increased cAMP levels. Alternatively, the Ras cAMP pathway 73 may negatively regulate Ndc80 kinetochore complex function.  3.3.2 spc24-9 cells have less trehalose compared to wild type The level of trehalose is a good marker for evaluating PKA activities because trehalose accumulation is downregulated by PKA via a combination of transcriptional repression of the genes encoding the trehalose synthase subunits and PKA-dependent phosphorylation of the neutral trehalase which degrades trehalose (Figure 3.1B). Accumulation of trehalose begins at the diauxic shift and continues until cells enter stationary phase.  Degradation of trehalose begins once cells have entered stationary phase (Werner-Washburne, et al., 1996). Therefore, before testing trehalose levels in spc24 alleles, it was important to analyze the growth curve of spc24-8, spc24-9 and spc24-10 strains at 25°C and their semi-permissive temperature.  At 25°C all three spc24 alleles have the same growth rate as wild-type cells (Figure 3.3A). At the semi-permissive temperature, spc24-8 and spc24-9 cells have a lower OD600 than the wild type in diauxic phase and sustain lower cell density during stationary phase (Figure 3.3B-C), but the growth curve of spc24-10 is highly similar to wild type (Figure 3.3B).  Subsequently, I tested the trehalose levels of spc24 alleles grown in stationary phase.  At 25°C no dramatic difference is detected between spc24 mutant cells and wild type cells (data not shown). At the semi-permissive temperature, the trehalose levels of spc24-8 and spc24-10 are both similar to the wild type, whereas spc24-9 cells have lower trehalose levels compared to wild type (Figure 3.3D). Since PKA activity is associated with reduced trehalose levels, the spc24-9 strain might have higher 74 PKA activity compared to the other two spc24 alleles.  The addition of glucose to the stationary cells induces trehalase activity and trehalose mobilization (Thevelein, 1984). Therefore, I asked if spc24 alleles have defects in glucose-induced trehalose mobilization. As showed in Figure 3.3E-F, all the three spc24 mutant cells response to glucose in a similar manner to the wild type by reducing trehalose levels, but spc24-8 and spc24-9 cells have lower overall trehalose levels.  Therefore, the glucose response machinery is not affected in stationary phase spc24 mutant cells but the lower trehalose levels in spc24-8 and spc24-9 strains suggest that PKA activity may be higher in these mutants compared to the wild type.  3.3.3 Msn2 dephosphorylation is compromised in spc24 upon glucose depletion The Msn2 transcription factor is one of the targets of the PKA pathway.  Activation of the PKA pathway causes phosphorylation of the Msn2 nuclear localization sequence on serine residues, prevention of Msn2 nuclear import and restoration of Msn2 to the cytoplasm (Görner, et al., 1998; Görner, et al., 2002; Jacquet, et al., 2003). In response to glucose starvation, the serine phosphorylation of Msn2 is acutely decreased due to PKA downregulation, however, this dephosphorylation is transient and is rapidly followed by re-phosphorylation in a PKA dependent manner (Görner, et al., 2002; Trott, et al., 2005). As shown in Figure 3.4, Msn2 phosphorylation was dramatically decreased in wild type cells within 10min (30 o C) or 5min (33 o C), and Msn2 rephosphorylation was established by 20min.  Prior to glucose starvation, the phosphorylation 75 of Msn2 in spc24-8, spc24-9 and spc24-10 was similar to the wild type (Figure 3.4). However, the dephosphorylation of Msn2 was attenuated in all the three spc24 mutant cells: spc24-9 and spc24-10 cells had higher levels of phosphorylated Msn2 at 10min compared to wild type cells; spc24-8 cells also had more phosphorylated Msn2 than wild type at both 5min and 10min.  In addition, these mutant cells showed less phosphorylation compared to wild-type cells at 20min, suggesting that the reestablishment of Msn2 phosphorylation was delayed.  The above alleviated, instead of acute, response to glucose starvation and the delay of Msn2 rephosphorylation implies defects in glucose sensing and PKA signaling in spc24 mutant cells.  3.3.4 Non-glucose carbon sources rescue the ts of the mutants of Ndc80 complex components Fermentable carbon sources increase PKA activity whereas non-fermentable carbon sources lower PKA activity (Thevelein, et al., 2008).  Since our data suggest that PKA signaling is perturbed in spc24 mutants, I asked if growth on non-fermentable carbon sources would rescue the ts of spc24 mutants.  I tested growth of spc24-8, spc24-9 and spc24-10 mutants by plate assay on a non-fermentable carbon source (glycerol) versus fermentable carbon sources (glucose, raffinose and galactose) as the sole carbon source. Interestingly, all the three spc24 alleles were rescued by both the glycerol non-fermentable carbon source and the raffinose and galactose fermentable carbon sources (Figure 3.5B). Even though raffinose and galactose are both fermentable carbon sources, they are known as non-preferred carbon sources (NPCS) because 76 they cannot trigger a strong cAMP/PKA response (Rolland, et al., 2000; Rolland, et al., 2001). Therefore, raffinose, galactose and glycerol rescue the ts growth defect of spc24 alleles possibly due to inability to fully activate the cAMP/PKA pathway, which further implies that spc24 mutants have higher levels of PKA activity.  Another possibility is that spc24 alleles have growth defects on glucose media due to glucose repression of a gene important for kinetochore function.  I tested this possibility by deleting REG1, which is the regulatory subunit of type I protein phosphatase complex and required for maintaining glucose repression (Dombek, et al., 2004). As shown in Figure 3.6, reg1∆ does not rescue the ts of spc24-9, on the contrary, the reg1∆ spc24-9 double mutant grows slightly more slowly compared to spc24-9 at 33 oC. Next I asked if the glucose growth defect is unique to spc24 alleles or universal to the ts alleles of other kinetochore components.  All the tested kinetochore ts alleles are shown in Figure 3.5 (C-E).  Dramatically, all the ts alleles of the components of Ndc80 complex, spc24-1, spc24-12, spc24-13, spc25-1, ndc80-1 and nuf2-61, were rescued by NPCS (Figure 3.5 C).  In contrast, growth of other kinetochore mutants on NPCS did not show such a consistent phenotype.  Figure 3.5D demonstrates that the inner kinetochore mutants: cse4-1, ctf13-30 and ndc10-42 were mildly rescued by NPCS, whereas ndc10-1 and skp1-3 (not shown) had the same growth rate on different carbon sources. Notably the ndc10-1 mutant, which eliminates kinetochore assembly and attachment of chromosomes to microtubules resulting in all chromosomes remaining in the mother cell (Goh, et al., 1993), did not show any growth difference on different carbon source. Therefore, the rescue by NPCS may require a partially 77 assembled kinetochore.  ame1-4 and okp1-5, two ts mutants of the COMA complex which bridges the inner and outer kinetochore, showed opposite sensitivity to NPCS (Figure 3.5 E). Three alleles of Spc34, a component of the Dam1 outer kinetochore complex that circles microtubules, were tested on NPCS.  spc34-5 was mildly rescued by NPCS, spc34-6 did not show any difference on alternate carbon sources, and spc34-7 was slow growing on NPCS. Taken together, the dramatic rescue by NPCS was only consistent within the mutants of Ndc80 complex but not other kinetochore complexes, suggesting that the Ndc80 complex might have a separate role in the PKA pathway or cellular response to glucose.  3.4 Discussion spc24 alleles were rescued by decreasing PKA activity such as by deleting ras2 or overproducing PDE2 (Figure 3.2B, D and E).  In addition, spc24-9 is SS with pde2 and SL with ira2 (Figure 3.2A), suggesting that the cAMP/PKA pathway might antagonize the function of the Ndc80 complex, which is similar to the model predicted for the Dam/Dash kinetochore complex and Ras2/PKA pathway (Li, et al., 2005). Li et al proposed two models for why decreasing PKA activities rescued the ts of the ask1 kinetochore mutant.  The first model is based on the fact that newly replicated centromeres attach to the old SPB because the new SPB is not yet competent for kinetochore attachment, therefore the mono-polar attachments of both centromeres to one SPB must be corrected.  A reduction of PKA activity may either delay the replication of centromeres or enable more rapid maturation of the SPB so that the number of mono-polar 78 attachments might be rescued.  The second model suggests that PKA negatively regulates the kinetochore via phosphorylation of Dam/Dash components.  To further characterize the relationship between the Ndc80 complex and the PKA pathway one approach could be to analyze the phosphorylation status of the Ndc80 complex in wild type versus PKA mutants using a mass spectrometry approach. The rescue of spc24, spc25, ndc80 and nuf2 mutant growth by NPCS might occur because of at least two possibilities: 1) NPCS induce low PKA activities which diminish the increased PKA activities in the ts mutants of the Ndc80 complex; 2) the transcription of the genes essential for kinetochore function when the Ndc80 complex is compromised is repressed by glucose but derepressed on NPCS.  My data suggest that the first possibility is more likely because compromising glucose depression by deleting REG1 does not rescue the ts of spc24-9 (Figure 3.6). Unlike Li et al’s data where some, but not all the ts mutants of Dam/Dash complex were rescued by decreasing PKA activity, I found that all the ts mutants of the Ndc80 complex, but not the other kinetochore mutants, were dramatically rescued by NPCS (Figure 3.5).  My data suggest that the Ndc80 complex might also have a role in regulation of the PKA pathway. spc24 alleles demonstrate cellular phenotypes consistent with a hyperactivated cAMP/PKA pathway, such as decreased trehalose accumulation and alleviated Msn2 dephosphorylation in response to glucose depletion (Figure 3.3, 3.4).  I therefore propose that spc24 mutants have increased PKA activity and the Ndc80 complex may both be a target of the PKA pathway and 79 have a role in downregulation of the PKA pathway as well. Since NPCSs rescue the glucose growth defects of ts alleles of Ndc80 complex components more dramatically than they do for other kinetochore complexes (Figure 3.5), I propose that the Ndc80 complex has a unique function in the PKA pathway or glucose response. Some mutants, such as cse4-1, ctf13-30 and ame1-4, (Figure 3.5D) were mildly rescued by NPCS probably because the cell cycle is slowed down, which may help defects like monopolar attachment get repaired. On the other hand, the severe growth on NPCS of some mutants, like spc34-7 (Figure 3.5E), might be due to the exacerbated defects induced by prolonged cell cycle. I did not analyze the dam1 or ask1 mutants from the Li et al paper by spot assays on NPCS, therefore, I cannot exclude the possibility that Dam1 and Ask1 might also play a role similar to the Ndc80 complex. Mutants defective in the anaphase-promoting complex/cyclosome (APC/C) are also rescued by downregulation of the Ras2/PKA pathway or growth on non-glucose carbon sources (Irniger, et al., 2000; Bolte, et al., 2003).  Activation of the spindle checkpoint results in inhibition of the APC, stabilization of securin (Pds1) and arrest at the metaphase to anaphase transition.  Therefore, if spc24 mutants activate the spindle checkpoint, the genetic interactions with the Ras2/PKA pathway could be due to APC inhibition.  A previous researcher in the Measday lab, Harvey Su, tested Pds1 levels in spc24 alleles throughout the cell cycle after releasing from a G1 block to the non-permissive temperature. Pds1 levels in spc24-8 were stabilized, suggesting that the spindle checkpoint is activated, whereas in spc24-10 mutants Pds1 80 levels were quickly reduced and the spindle extended with an undivided nucleus remaining in the mother cell indicative of a defect in the spindle checkpoint (data not shown and Montpetit et al., 2005). Since both spc24-8 and spc24-10 were rescued by ras2∆ or non-glucose carbon sources (Figure 3.1), activation of the spindle checkpoint cannot explain the genetic interaction detected between the Ndc80 complex and the PKA pathway. In budding yeast, the PKA holoenzyme mostly localizes in the nucleus (Griffioen, et al., 2000). The Bcy1 regulatory subunit almost specifically localizes in the nucleus in the presence of glucose, whereas the activated Tpk1 (and probably Tpk2, Tpk3 as well) is able to enter the cytoplasm presumably to phosphorylate cytoplasmic substrates. Upon depletion of glucose, Bcy1 has equal distribution in both nucleus and cytoplasm, probably for downregulating cytoplasmic PKA activities (reviewed in Griffioen, et al., 2002).  At this point we do not know if the Ndc80 complex directly interacts with components of the PKA pathway. Several questions remain: 1) Is Ndc80 one of the PKA targets? While the components of the Ndc80 complex were not listed as candidate PKA phosphorylation targets (Budovskaya, et al., 2005), we cannot exclude the possibility that Ndc80 components might be phosphorylated by PKA because Budovskaya et al. did not test Ndc80 in vivo. In addition, Ndc80 might be indirectly regulated through a chaperone protein like Sgt1 (Dubacq, et al., 2002). 2) How does the Ndc80 complex negatively regulate the PKA pathway?  The Measday lab has identified Ndc80 complex-interacting proteins by mass spectrometry and detected some components of cAMP-PKA pathway including Gpa2, Asc1 and Sdc25.  However, I did not successfully 81 confirm the physical interaction by co-IP between the PKA components (Gpa2, Asc1 and Sdc25) and Ndc80 components (Ndc80 and Spc24), possibly because Gpa2 and Sdc25 are localized at the cell cortex and Asc1 is in cytoplasm, whereas the majority of Ndc80 localizes to the nucleus. Thus any physical interaction between the Ndc80 cytoplasmic pool (see Appendix B) and cytoplasmic cAMP-PKA components will be challenging to detect by co-IP.  One possibility is to overproduce Ndc80 in an effort to increase the amount of Ndc80 in the cytoplasm.  82 Table 3.1 List of strains Strain  Genotype Reference YVM1370 MATa ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 SPC24::kanMX6 Montpetit et al. (2005) YVM1380 MATa ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 spc24-9::kanMX6 Montpetit et al. (2005) YVM1448 MATa ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 spc24-8::kanMX6 Montpetit et al. (2005) YVM1363 MATa ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 spc24-10::kanMX6 Montpetit et al. (2005) YLM1565 MATa ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 ras1Δ::LEU2 This study YLM1562 MATa ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 ras1Δ::LEU2 spc24-9::kanMX6 This study YLM1572 MATa ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 ras2Δ::LEU2 This study YLM1568 MATa ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 ras2Δ::LEU2 spc24-9::kanMX6 This study YLM1813 MATa ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 ras2Δ::LEU2 spc24-8::kanMX6 This study YLM1815 MATa ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63ras2Δ::LEU2 spc24-10::kanMX6 This study YVM571 MATa ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 ctf13-30 P. Hieter YVM567 MATa ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 ctf14-42 P. Hieter SBD520-6C MATa ura3-52, leu2-3, ade2-101, trp1Δ901, his3-11,15, cse4-1::RSCL1-1H(HIS3) cse4-1 Stoler et al. (1995) YJL158 MATa ade2-101, trp1-Δ63, leu2-Δ1, ura3-52, his3-Δ200, lys2-801, cyh2-r okp1::okp1-5::TRP1 Ortiz et al. (1999) YPH1175 MATa ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 skp1-3::LEU2 P.Hieter YPH1160 MATa ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 CFIII (CEN3L) HIS3 SUP11 skp1-4::LEU2 P.Hieter YPH1338 MATa ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 CFIII (CEN3L) TRP1 SUP11 sgt1-3::LEU2 P.Hieter   (To be continued) 83 Table 3.1 List of strains (continued) Strain  Genotype Reference YPH1337 MATa ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 CFIII (CEN3L) TRP1 SUP11 sgt1-5::LEU2 P.Hieter YPH1676 MATa ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 ame1-4::TRP1 Pot et al. (2005) YM40 MATa ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 spc34-5::kanMX6 V.Measday YVM1864 MATa ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 spc34-6::kanMX6 Montpetit et al. (2005) YM41 MATa ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 spc34-7::kanMX6 V.Measday PWY473 MATa ade2-1, can1-100, his3-11,15, leu2-3,112, trp1-1 spc24-1 Wigge&Kilmartin (2001) ILM126 MATa ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63  spc24-12 Le Masson et al. (2002) ILM127 MATa ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63  spc24-13 Le Masson et al. (2002) PWY754 MATa ade2-1, can1-100, his3-11,15, leu2-3,112, trp1-1 spc25-1 Wigge&Kilmartin (2001) YKH628 MATa ade2-1, can1-100, his3-11,15, leu2-3,112, trp1-1 ndc80-1 Wigge&Kilmartin (2001) PSY455 MATa ura3-52 leu2-3,11 trp1-∆1 nuf2-61 P. Silver        84 A  B   Figure 3.1 The cAMP-PKA pathway in yeast (A) and its physical effects (B) (A) was modified from Peeters et al., 2007, by permission © 2007 Elsevier Ltd All rights reserved. 85    Figure 3.2 Genetic interactions between spc24-9 and the components of the Ras2/cAMP/PKA pathway (A) SGA data was cited from Montpetit et al., Genetics 2005, by permission © Genetics Society of America. (B) Cell dilution assay of wild type cells and spc24-9 mutants carrying the plasmid vectors pRS326 and pRS326-PDE2. (C) Genetic diagram of the Ras/PKA pathway. Blue arrows represent positive regulatory interactions and red arrows represent negative regulatory interactions. (D) & (E) Cell dilution assay of indicated strains grown on YPD for 2 days.   86    Figure 3.3 Growth curves (A-C) and trehlaose levels (D-F) in spc24 cells (A-C) spc24-8, spc24-9, spc24-10 and wild type cells were grown to logarithmic phase at 25°C, diluted to OD600=0.1, and incubated at 25°C (all four strains), 30°C (spc24-9, spc24-10 and wild type) or 33°C (spc24-8 and wild type). Purple arrow in (B) points the start of the diauxic shift. (D) Trehalose levels in stationary spc24 cells at their semi-permissive temperatures (30°C for spc24-9 and spc24-10, 33°C for spc24-8). wt-33°C is the wild type control for spc24-8 and wt-30°C is the wild type control for spc24-9 and spc24-10. Trehalose levels were measured enzymatically and normalized to total cell density (OD600). (E-F) Glucose induced trehalose mobilization. spc24-8, spc24-9, spc24-10 and wild type cells were grown to stationary phase at 30°C (spc24-9, spc24-10 and wild type in E) or 33°C (spc24-8 and wild type in F). 100 mM glucose was added at time zero. 87   Figure 3.4 Kinetic analysis of Msn2 phosphorylation upon glucose starvation Cells were grown to midlogarithmic phase at the semi-permissive temperature of spc24 mutants (30°C for spc24-9 and spc24-10, 33°C for spc24-8) in YPD medium and shifted to prewarmed YP medium lacking glucose.  A timecourse was performed whereby samples were taken every 5 or 10min and equal amount of protein lysates were loaded on a 12% SDS-PAGE gel. Western blot were probed with antiphospho CREB for detecting the phosphorylation of Msn2 and anti-Msn2 as a loading control.     88   Figure 3.5 Cell dilution assay of kinetochore mutants on various carbon sources Cell dilution assay of indicated strains on YP plus 2% of either glucose, raffinose, galactose or glycerol. Plates were incubated at the indicated temperature for 3 days.   Figure 3.6 Cell dilution assay of indicated strains grown on YPD for 3 days 89 Chapter 4  Identification of Actin Defects in Kinetochore Mutants in the Budding Yeast S. cerevisiae  4.1 Introduction The unicellular yeast Saccharomyces cerevisiae is a good model for the establishment of cell polarity.  Yeast cells grow  by budding, which requires recognizing a specific site of the mother cell cortex and polarizing many cellular components towards this site to promote bud growth.  Figure 4.1 shows the establishment of the cell polarity throughout the cell cycle. Many proteins concentrate in  the incipient bud site to form the polarity cap and facilitate bud emergence and growing.  Actin patches concentrate around the polarity cap and actin cables  align along the mother-bud axis towards the bud tip.  Myosin motors (Myo2p and Myo4p) walk along the cables  towards the polarity cap transporting various cargoes including  secretory vesicles to support bud growth, the plus ends of cytoplasmic  microtubules to position the nucleus for nuclear division, organelles for the daughter cell and RNA-protein complexes to provide daughter cell-specific proteins (Figure 4.2). A cytoskeletal filament ring composed of septin proteins surrounds the polarity cap. The septins are a conserved family of GTP binding proteins that include Cdc3, Cdc10, Cdc11, Cdc12 and Shs1/Sep7 in the budding yeast.  Septins assemble into filaments in a cell cycle regulated pattern (Figure 4.3).  In G1 phase, septins are recruited to the incipient bud site and form a ring 90 in a fluid state.  After bud emergence, septin rings are stabilized and expand like an hourglass at the bud neck.  At the onset of cytokinesis, the hourglass shaped septin collar splits into two rings on mother and bud sides flanked by the actomyosin contractile ring.  The septin ring is disassembled and degraded after cell separation.  The filament ring/collar of septins at the bud neck is a scaffold that recruits various proteins important for cytokinesis, the morphogenesis checkpoint, the spindle assembly checkpoint and bud-site selection.  Additionally, the septins act as diffusion barriers to compartmentalize the mother and daughter cells (see reviews in Kinoshita, 2006 and Douglas, et al., 2005). Filamentous actin in budding yeast has three different structures: patches, cables and rings (Figure 4.4A).  Actin patches undergo rapid turnover and mediate endocytosis and exocytosis, whereas actin cables function as polarized tracks that deliver cargo to build the daughter cell.  Patches are formed by Arp2/3-dependent actin nucleation, whereas cables are assembled by the actin nucleating activity of the formins (Bnr1 and Bni1) and profilin.  Prior to anaphase, actin cables are assembled at two locations, the bud tip (where Bni1 localizes) and the bud neck (where Bnr1 localizes).  At anaphase, Bni1, Bud6 and other components of the polarity cap abruptly relocalize from the bud tip to the bud neck.  Consequently, actin cables are reorganized to polarize towards the bud neck and direct all secretion to the division plane.  The actomyosin contractile ring forms in cytokinesis and constricts in a Myo1-dependent manner to help close the neck (see review in Moseley, et al., 2006). The Rho-family Cdc42 GTPase is essential for polarity establishment and plays a key 91 role in actin organization and septin assembly (see review in Park, et al., 2007).  Cdc42 localizes to the plasma membrane and the sites of polarized growth, to the incipient bud site in G1 cells, the tips of growing buds in S/G2/M cells, and to the bud neck in anaphase and telophase cells (Figure 4.4B).  The Cdc42 effectors Ste20p, Cla4p, Gic1p and Gic2p, and the scaffold proteins Bem1, Boi1 and Boi2 act together with Cdc42 and its GDP-GTP exchange factor (GEF) Cdc24 to establish polarity (Park, et al., 2007). The formins Bni1 and Bnr1 are another set of downstream effecters of Cdc42 for the assembly of actin cables (Evangelista, et al., 2002; Pruyne, et al., 2002; Sagot, et al., 2002a; Sagot, et al., 2002b) and the actomyosin ring (Tolliday, et al., 2002).  The two formins partially overlap in their localization at the bud tip and the bud neck (Figure 4.4B), and in their functions in the assembly of actin cables and the actomyosin ring.  Bni1 is one of the components of the 12S polarisome which is required for apical growth.  The polarisome also includes Spa2, Pea2 and Bud6, which all localize in a similar manner to Bni1 (Tcheperegine, et al., 2005;van Drogen & Peter, 2002).  Bud6 stimulates the activity of Bni1 to promote actin organization, and Bud6 and Bni1 are both suggested to have roles in spindle orientation and organization of the endoplasmic reticulum at the bud neck (Moseley & Goode, 2005). Bud growth in budding yeast is divided into two phases, apical growth and isotropic growth. Apical bud growth occurs in G1 and is restricted to the tip of the bud. Upon entry into mitosis, buds switch to isotropic growth, in which the bud continues growing but growth is no longer restricted to the tip and instead growth occurs uniformly throughout the entire bud surface 92 (Lew & Reed, 1995). One checkpoint related to apical versus isotropic growth is the Swe1 dependent morphology checkpoint (Figure 4.5). In response to depolarization of the actin and/or a delay in bud emergence, the Swe1 kinase stalls the cell cycle in G2/M with an apical bud shape by inactivating the cyclin-dependent kinase (CDK) Cdc28 until the cell has recovered from the stress (see review in Keaton, et al., 2006). This morphogenesis checkpoint pathway starts with septin reorganization from a ring to an hourglass shaped collar during bud emergence. Subsequently, a septin-binding protein kinase, Hsl1, is activated to recruit an adaptor protein Hsl7, which brings Swe1 to the septins at the bud neck to undergo degradation.  As a result of Swe1 degradation, Cdc28 is activated and cells enter mitosis with spindle elongation and isotropic bud growth (Lee, et al., 2005).  Stresses that delay bud formation prevent Swe1 recruitment to the bud neck and block Swe1 degradation which results in mitotic delay.  Even after a bud has formed and Hsl1 is active, a Swe1-dependent cell cycle arrest can be triggered by actin depolarization (McNulty & Lew, 2005). The CBF3 inner kinetochore has been reported to have a novel role in regulating septin assembly and cell polarity (Gillis, et al., 2005) independently of its kinetochore function.  Gillis et al. described a delay in septin ring separation and disassembly and a defect in actin polarization upon inhibiting CBF3 assembly.  However, no septin defects were detected in central or outer kinetochore mutants. Interestingly, I have found that spc24 mutant cells have actin defects.  In addition, spc24 alleles have multiple genetic interactions with actin, formin, and cdc42 mutants and overexpression of the Cdc42 effector, GIC1 rescues spc24-9 temperature 93 sensitivity.  4.2 Materials and methods 4.2.1 Strains, plasmids and microbial techniques Yeast strains and plasmids used in this study are described in Table 4.1.  The CEN plasmid containing the CDC3 promoter followed by the full length CDC3 ORF was a gift from John Pringle’s lab.  The CEN plasmid containing the GFP tag followed by the full length BUD6 ORF was a gift from Charlie Boone’s lab.  The 2μ plasmid containing the full length GIC1 ORF and its own promoter was isolated from the work in Chapter 2 (Ma, et al., 2007). All the GFP tagging was performed by PCR-based homologous recombination (Longtine, et al., 1998). The liquid media used was either rich medium (YPD) or supplemental minimal medium (SC) (Kaiser, et al., 1994).  For spot assays, fivefold serial dilutions were spotted on YPD (2% glucose) plates and grown at 30°C for 2 days.  To assay latrunculin A (Lat A) sensitivity, Lat A was added at the indicated concentration onto the paper discs that had been placed on the YPD plates.  Dimethyl sulfoxide was used in the control spot (0 μg/ml Lat A). Flow cytometry analysis to monitor DNA content was performed as previously described (Haase, et al., 1997).  4.2.2 Timecourse and western blot Cells were grown to early-logarithmic phase in 150 ml of liquid YPD at 25°C, arrested with 5ug/ml α-mating factor (BioVectra, Charlottetown, Prince Edward Island, Canada) for 2 hours at 94 25°C, resuspended with 37°C prewarmed fresh liquid YPD and shifted to 37°C. 10ml of culture was collected for each indicated time point and the cell pellet was lysed using glass beads. Western blot analysis was performed using standard procedures. A mouse monoclonal antibody specific for the MYC epitope (anti-c-MYC) was purchased from Roche Diagnostics Canada (Laval, QB) and used at a 1:5000 dilution.  4.2.3 Fluorescence Microscopy For actin staining, cells were fixed with SC containing 4% formaldehyde for 1 hour, washed with PBS three times, resuspended in 100ul PBS plus 10ul Rhodamine Phalloidin (6.6μM in MeOH) (Molecular Probes Inc.), incubated in the dark at 4°C overnight, washed and resuspended with PBS, and 1-2μl placed on slides for imaging. Cells were imaged at room temperature using a fluorescence microscope (Axioplan 2; Carl Zeiss MicroImaging, Inc.) with a plan-Apochromat 63× NA 1.4 differential interference contrast oil immersion objective (Carl Zeiss MicroImaging, Inc.) with filter sets 38 for GFP and filter set 45 for Rhodamine Phalloidin (Carl Zeiss MicroImaging, Inc.). 3D images (0.2-μm steps) were acquired with a camera (CoolSNAP HQ; Roper Scientific) and analyzed using MetaMorph software (Invitrogen). Images are presented as maximum intensity 2D projections.  95 4.3 Results 4.3.1 Morphology defects of spc24-9 spc24-9 arrests with 2N DNA content (Figure 4.6D) and large budded G2 cells at the non-permissive temperature of 37°C and ~12% of mutant cells have elongated buds when incubated at 37°C for 4hrs (Figure 4.6A). Bud elongation is a characteristic morphological phenotype of cells with stabilized Swe1.  Upon the activation of the Swe1-dependent morphology checkpoint, Swe1 is stabilized and the switch from apical to isotropic growth is blocked, resulting in bud elongation.  Therefore, I tested if the Swe1 dependent morphology checkpoint is activated in spc24-9 by performing a time course with synchronized cells. Wild type and spc24-9 cells were arrested in G1 phase at 25 o C with mating pheromone, then released from the arrest into 37 o C and samples taken every 10 min for a total of 150 min.  Western blot analysis was performed to compare the protein expression levels of Swe1-MYC in wild type versus spc24-9 cells.  As shown in Figure 4.6B, after G1 release, Swe1 levels in wild type cells peak at 60~70min followed by Swe1 degradation and the second peak of Swe1 expression occurs at 100 min followed by degradation at 130min (Figure 4.6B).  However, in spc24-9 cells Swe1 levels first peaked at 100min, a 30min-delay compared to the wild type, and Swe1 was stabilized with high levels after 100min, indicative of activation of the morphology checkpoint (Figure 4.6B). Next, I tested whether Swe1 accumulation is the consequence of the morphology defects of spc24-9 with spc24-9 swe1Δ double mutants.  Even though the elongated bud morphology is 96 partially rescued by the absence of Swe1 (Figure 4.6C), cells still accumulate with a 2N content of DNA in spc24-9 swe1Δ strains at 37oC (Figure 4.6D), and the temperature sensitivity (ts) of spc24-9 is not suppressed by swe1Δ at 33oC (Figure 4.6E). The above observations indicate that the poor viability of spc24-9 at the restrictive temperature is not solely due to a Swe1-dependent G2 block.  4.3.2 spc24 mutants have actin defects Actin defects might contribute to the unsuccessful apical/isotropic switch in the spc24-9 mutant that leads to the elongated bud morphology.  Therefore, I studied actin in spc24-9 versus wild type cells by staining with rhodamine phalloidin after growing cells at 37°C for 3 hrs (Figure 4.7).  In the wild type cells with medium to large buds, the actin patches are concentrated in the daughter cell, whereas in spc24-9 cells with similar bud sizes, more actin patches are present in the mother cell (Figure 4.7A, yellow arrows).  The distribution of actin patches to the spc24-9 mother cell might suggest a defect of the septin diffusion barrier at the bud neck (see Figure 4.4).  Additionally, the actin patches fail to accumulate at the bud neck and no actin rings are visible in spc24-9 cells with large buds (Figure 4.7A, light blue arrows, compare wild type to spc24-9). The actin localization analysis suggests that spc24-9 mutants might have defects in actin ring formation, formin and/or Bud6 function since all of these factors are required for the assembly of the actin cables and rings at the bud neck.  To address these possibilities, I studied the localization of Bni1, Bnr1 and Bud6 fused to a C-terminal GFP tag in 97 spc24-9 versus wild type cells.  While no dramatic localization defects were detected for Bni1 and Bnr1 in spc24-9 (data not shown), Bud6 is mislocalized in the mutant upon shifting to 37°C (Figure 4.7B).  During a normal cell cycle in wild type cells, Bud6 localizes to the prebud site at START, to the bud cortex while the bud is growing and accumulates at the bud neck upon entry into mitosis where it remains for the rest of the cell cycle (Segal, et al., 2000) (Figure 4.7B, upper panel). When spc24-9 cells were incubated at 37°C for 3 hrs, Bud6 is either randomly scattered within the cell or does not form the rings at the mother-bud neck in large budded cells (24% in spc24-9 versus 6% in wild type) (Figure 4.7 B, C).  Additionally, bud6∆ has synthetic growth defect with spc24-9 (Figure 4.7D), indicative of a genetic interaction between BUD6 and SPC24. To determine if there is any interaction between the Ndc80 complex and Bud6, I performed a co-immunoprecipitation with a Ndc80-13MYC Bud6-GFP strain, but no direct interaction was detected (data not shown).  Therefore, the mislocalization of Bud6 in spc24-9 might be induced by the defects of the cytoskeleton or septin scaffold in spc24-9 cells but not due to defects in physical interaction with the Ndc80 complex. The lack of visible actin rings in spc24-9 cells compared to the wild type cells may be because the spc24-9 cells arrest prior to actin ring formation.  Actin ring formation occurs when Cyk1, a protein playing an essential and specific role in cytokinesis, is recruited to the bud neck during late anaphase (Lippincott, et al., 1998).  To distinguish between defects in actin ring formation and spc24-9 cell cycle arrest, I blocked cell entry into mitosis by repressing expression of Cdc20. Cdc20 is an activator of the anaphase-promoting complex/cyclosome (APC/C) and 98 mediates the proteolysis of securin (Pds1).  Degradation of Pds1 allows the Esp1 separase (Esp1) to cleave the Scc1 cohesin subunit and initiate sister chromatid separation.  Inhibition of Cdc20 leads to cell cycle arrest in metaphase due to stabilization of Pds1.  In order to regulate CDC20 expression, a GAL1-CDC20 strain was created whereby growth on galactose enables expression of CDC20 and growth on glucose turns off CDC20 expression (Hwang, et al., 1998). I mated the GAL1-CDC20 strain with spc24-9, spc24-8 and spc24-10 mutants and isolated meiotic recombinants that contained both the spc24 mutation and GAL1-CDC20.  The strains were grown in galactose, arrested in metaphase with glucose treatment and released into mitosis with galactose at 37 o C.  As shown in Figure 4.8 when released from a metaphase block to restrictive temperature, none of the three spc24 alleles have defects in actin ring formation. Therefore, the lack of actin rings in spc24-9 cells (Figure 4.7A) is likely due to the stage of the cell cycle arrest and not due to a specific defect in actin ring assembly.  4.3.3 Septin localization in mutants of the Ndc80 complex Septins are a family of conserved proteins that assemble into filaments at the site of bud growth and are membrane associated scaffolds important for separating the mother/bud compartments and cytokinesis.  Gillis et al. studied the ndc80-1, spc25-7 and dam1-1 outer kinetochore mutants and found that none of them had septin defects (Gillis, et al., 2005). Since ndc80-1 and spc25-7 might have different defects than our spc24 mutants, and I have shown that spc24-9 has more actin patches in the mother cell and mislocalized Bud6 (see 4.3.2), I analyzed 99 septin localization in our spc24 mutants.   I constructed both Cdc10-GFP and Cdc12-GFP endogenously tagged strains but the Cdc12-GFP strain grew slowly at 33°C and both strains had a filamentous phenotype suggesting that the C-terminal GFP tag interfered with septin function. Therefore, I transformed the GFP-CDC3 plasmid (gift from Brenda Andrews) into the spc24-8, spc24-9, spc24-10 mutants plus another two alleles of the Ndc80 complex, spc25-1 and nuf2-61, to study septin localization.  I found that none of these mutants have septin defects upon shifting to 37°C (data not shown).  However, when I looked at spc25-1 mutants in another strain background (W303) , 70% of spc25-1 cells were not able to form septin rings after incubation at restrictive temperature for 3 hours, suggesting that defects in kinetochore function dramatically affect septin localization in the W303 strain background but not our lab strain background (S288C) (Figure 4.9).  Interestingly, spc24-9 is synthetically lethal when combined with the septin mutant cdc12-1 at 30°C (Figure 4.9D), suggesting that spc24-9 might have subtle septin defects. I did not analyze dynamic septin defects in spc24 mutants because the cells were fixed before imaging. Therefore, septin defects may be revealed in spc24-9 cells by performing live imaging.  4.3.4 spc24 alleles might have more actin patches During the analysis of actin ring formation using a GAL-CDC20 block/release method (described in 4.3.2), I also analyzed actin by staining large budded cells with Rhodamine Phalloidin.  As shown in Figure 4.10B, 40min after release from arrest due to Cdc20 depletion, 100 wild type cells have actin patches mostly around the cell cortex, but spc24-8 and spc24-9 cells have more actin patches throughout the cell, which is consistent with the scattered actin patches in spc24-9 shown in Figure 4.7A. Since brighter or even chunky actin patches are associated with defects of actin turnover (Lappalainen, et al., 1997), I speculated that the actin patches in spc24 cells might not undergo rapid turnover. Studying the cell sensitivity to Latrunculin A (Lat A), an actin sequestering drug (Ayscough, et al., 1997), is a method for evaluating the stability of the actin cytoskeleton.  The double mutant tpm1-2 tpm2∆, which has mutations in two isoforms of the tropomyosin (Tpm) actin cable components (Pruyne, et al., 1998), is sensitive to Lat A due to the defects in actin polymerization (Figure 4.10C). Since spc24-8, spc24-9 and spc24-10 mutants may have stabilized actin structures, I expected the Lat A halo to be reduced in size compared to the wild type.  However, no dramatic difference was detected between the halo size of spc24 mutants and the wild type (Figure 4.10D), probably because the actin defects in spc24 cells at 33°C are not as dramatic as 37°C, or the Lat A concentration is too high to visualize the difference.  4.3.5 Genetic interactions between spc24 alleles and actin mutants To further confirm the interaction between spc24 alleles and actin, I crossed spc24-8 and spc24-9 strains into a series of actin mutants, generated double mutants and analyzed their growth by spot assay (Figure 4.11).  The actin mutants used for these genetic interaction studies are pfy1-111, a ts allele of the PFY1 profilin gene (Haarer, et al., 1993), cof1-5 and cof1-22, both 101 ts alleles of the COF1 cofilin gene, and a null mutation of the TWF1 twinfilin gene.  Cof1 and Twf1 are both actin filament severing proteins and facilitate actin cable turnover (Moseley, et al., 2006). Although the activity of profilin remains unclear, it is proposed to have a function in accelerating actin turnover (Moseley, et al., 2006). As shown in Figure 4.11A, cof1-5 and cof1-22, which both have more actin patches compared to wild type (Lappalainen, et al., 1997), have a severe synthetic growth phenotype when combined with spc24-9 or spc24-8 mutants. Additionally, spc24 mutants are synthetic sick with pfy1-111 (impaired in binding actin (Haarer, et al., 1996)).  The genetic interactions between cof1, pfy1-111 and spc24 are consistent with the hypothesis that spc24 mutants have actin turnover defects (described in 4.3.4). Surprisingly, twf1∆, which causes reduced rates of actin patch turnover (Moseley, et al., 2006), suppresses the ts of spc24-9 at 33°C and spc24-8 at 35°C (Figure 4.11B).  4.3.6 Formin mutants rescue the ts of spc24 alleles In budding yeast, formins are required for actin cable nucleation. Since spc24 mutants have more actin patches, I was curious to determine if there are also actin cable defects in these mutants. If spc24 mutants have more stabilized actin cables, they might be rescued by compromising the nucleation function of formins.  bni1-11 has two amino acid mutations (D1511G, K1601R) in the second formin-homology domain (FH2) which is thought to have an essential role in actin cable nucleation (Figure 4.12A) (Evangelista, et al., 2002). I crossed spc24 alleles with bnr1∆, bni1∆ and the bni1-11 ts allele and generated double mutant strains. 102 spc24-10 does not have a dramatic growth phenotype when combined with any of the three formin alleles, however both spc24-8 and spc24-9 were rescued by bnr1∆ and bni1-11 (Figure 4.12B), suggesting spc24 mutants might have excess actin filaments.  Interestingly, the ts of spc24 mutants were not rescued by bni1∆ (Figue 4.12B).  Since Bni1 but not Bnr1 plays an additional role in microtubule dependent nuclear migration through its Rho-binding domain (RBD) (Fujiwara, et al., 1999), I speculate that bni1∆ does not rescue spc24 due to the defects of the Bni1 in nuclear migration.  4.3.7 Hyperactivation of Cdc42 pathway might rescue the ts of spc24-9 The Cdc42 GTPase is the key component in polarity establishment.  Since spc24-9 is rescued by formin mutants and the formins are major targets of Cdc42 in actin cable nucleation, I determined the phenotype of combining a cdc42 mutant with the spc24-9 mutant. cdc42-117 has mutations in the GTPase activation protein (GAP) binding domain (Figure 4.13A), suggesting that cdc42-117 may fail to interact with one of its GAPs and thus be in a hyperactivated state (Kozminski, et al., 2000).  I found that cdc42-117 is synthetic lethal with spc24-9 (Figure 4.13B), suggesting that the actin defects of spc24-9 might be induced by hyperactivation of the cellular polarization signal transduction pathway.  Gic1 and Gic2, two effectors of Cdc42, were first identified as homologs with redundant functions in cell polarization (Brown, et al., 1997), septin recruitment (Iwase, et al., 2006) and mitotic exit (Höfken, et al., 2004). Even though no distinct function of Gic1 or Gic2 has been 103 reported, Gic1 is well-known to be stable throughout the cell cycle (Chen, et al., 1997), whereas Gic2 is partially degraded upon bud emergence probably to restrict cytoskeleton polarization and thereby contribute to efficient bud emergence (Jaquenoud, et al., 1998). Since Gic1 and Gic2 play positive roles in Cdc42 signal transduction pathway (Chan, et al., 1997) and the hyperactive allele cdc42-117 is synthetic sick with spc24-9, I tested if deletion of Gic proteins would rescue the ts of spc24-9 mutants. As showed in Figure 4.13C, gic2∆ and gic1∆ gic2∆ double mutant, but not gic1∆, rescues spc24-9 at 33°C. Since Gic2 is involved in recruiting Bud6 and Bni1 to activated Cdc42 to facilitate actin polarization (Jaquenoud, et al., 2000), the rescue of spc24-9 mutants by deletion of GIC2 perhaps occurs due to the downregulation Cdc42 signaling or a decrease in efficiency of actin nucleation. gic1∆ does not rescue spc24-9 mutants probably because the adapter function linking Cdc42 and actin nucleation is specific to Gic2 but not Gic1. Interestingly, overexpressing GIC1 suppresses the ts of spc24-9 at 33°C (Figure 4.13D), suggesting overproducing GIC1 might antagonize the adapter function of Gic2.  4.4 Discussion Swe1 protein accumulation is periodic during the cell cycle, rising at the time of bud emergence and declining before nuclear division (McMillan, et al., 1998), so that Clb2-Cdc28 can be activated to initiate mitosis. When spc24-9 cells are synchronized in G1 phase with mating pheromone and released to restrictive temperature of 37°C, cells are arrested in G2 with 2N DNA content and Swe1 is not degraded after its appearance at 100min, (Figure 4. 6B), 104 suggesting that the Swe1-dependent morphology checkpoint is activated. The activation of the morphology checkpoint, and subsequence defects in Swe1 degradation, inhibits isotropic bud growth and induces an abnormal elongated bud morphology.  However, elongated bud growth is not a major defect in spc24-9 mutants (only ~12% cells), possibly because our S288C strain background does not tend to display highly elongated buds compared to other S. cerevisiae strain backgrounds (Enserink, et al.,  2006). Nevertheless, the minor defects in morphology of bud elongation in spc24-9 is suppressed by swe1∆ (Figure 4.6C).  However, swe1∆ does not suppress either the G2 arrest or the ts of spc24-9 (Figure 4.6D, E), suggesting the activation of the morphology checkpoint is not the only reason for the inviability of spc24-9 at the non-permissive temperature.  Interestingly, before 90min in Figure 4.6D, the cell cycle progression in spc24-9 is similar to the wild type, as indicated by the DNA content, but the abundance of Swe1 is lower in spc24-9 than the wild type for all the indicated timepoints (0~90min) (Figure 4.6B), suggesting a delay of Swe1 expression in spc24-9. One possibility is that SWE1 is transcribed during S-phase, and spc24-9 cells have defects at S-phase (see Chapter 2), which compromises the transcription of SWE1.  Alternatively, Spc24 or Ndc80 complex might be required for the expression of Swe1 during normal cell cycle. The following data presented in this chapter suggest that spc24 cells might have defects in actin turnover: (1) spc24 cells have brighter, or bigger actin patches thoughout the cell; (2) spc24 mutants are synthetic sick with cof1 mutants which have more stable actin structures; (3) the ts of spc24 cells is suppressed by compromising the actin nucleation functions of formins. 105 However, the fact that spc24-9 ts growth is suppressed by twf1∆ (Figure 4.10B) conflicts with the above conclusion.  As stated above, Twf1 is an actin filament severing protein that facilitates actin cable turnover (Goode, et al., 1998; Moseley, et al., 2006).  The defects in actin turnover in twf1∆ mutants are not nearly as severe as cof1-22 mutants (Moseley, et al., 2006). Perhaps having highly stabilized actin structures is lethal for spc24 mutants as suggested by the synthetic lethality of spc24 cof1-22 double mutants, however slightly stabilized actin is beneficial for spc24, as suggested by the twf1 spc24 rescue.  The lack of LatA sensitivity suggests that spc24 mutants do not have a deficiency in the organization of actin cytoskeleton. Activation of Cdc42 by its exchange factor Cdc24 is required to organize the actin cytoskeleton and establish cell polarity. Since the viability of spc24-9 is severely compromised when combined with the cdc42-117 dominant allele (Kozminski, et al., 2000), spc24-9 might have a highly activated Cdc42 signaling cascade, which is consistent with the data that depleting the downstream effectors of Cdc42, such as bni1-11, bnr1∆ and gic2∆, suppresses the ts of spc24-9.  Why a nuclear kinetochore protein has genetic interactions with the actin cytoskeleton and cytoplasmic/cell cortex elements is still mysterious.  I have detected a minor pool of Ndc80 in the cytoplasm, likely at the plus end of cytoplasmic microtubules or in the vicinity of cell cortex (see Appendix 2).  Therefore, the cytoplasmic pool of Ndc80 might be able to mediate the crosstalk between kinetochore and the cell cortex, possibly at the polarity cap.  Alternatively, mutation of the nuclear Ndc80 complex might affect a transcription factor and cause defects in 106 expression of genes involved in polarity establishment or actin dynamics.  Future work will distinguish between a direct role for the Ndc80 complex in regulating actin dynamics versus an indirect role in regulating transcription of polarity genes.  107 Table 4.1 List of strains Strain  Genotype Reference YVM1370 MATa ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 SPC24::kanMX6 Montpetit et al. (2005) YVM1380 MATa ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 spc24-9::kanMX6 Montpetit et al. (2005) YVM1448 MATa ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 spc24-8::kanMX6 Montpetit et al. (2005) YVM1363 MATa ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 spc24-10::kanMX6 Montpetit et al. (2005) YLM1165 MATa ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 SWE1-13MYC::HIS3 This study YLM1166 MATa ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 SWE1-13MYC::HIS3 spc24-9::kanMX6 This study YJM125 MATa ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 swe1Δ::LEU2 Measday lab YLM1133 MATa ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 swe1Δ::LEU2 spc24-9::kanMX6 This study YJM79 MATa ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 hsl1Δ::HIS3 Measday lab YJM121 MATa ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 hsl1Δ::HIS3 swe1Δ::LEU2 Measday lab YLM1131 MATa ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 hsl1Δ::HIS3 spc24-9::kanMX6 This study YLM1129 MATa ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 swe1Δ::LEU2 hsl1Δ::HIS3 spc24-9::kanMX6 This study YJM87 MATa ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 hsl7Δ::HIS3 Measday lab YJM134 MATα ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 hsl7Δ::HIS3 swe1Δ::LEU2 Measday lab YLM1135 MATa ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 hsl7Δ::HIS3 spc24-9::kanMX6 This study YLM1137 MATa ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 swe1Δ::LEU2hsl7Δ::HIS3 spc24-9::kanMX6 This study                                         (To be continued)  108 Table 4.1   List of strains (Continued)  Strain  Genotype Reference ABY944 MATa his3Δ-200 leu2-3,112 lys2-801 trp1-1 ura3-52 tpm1-2::LEU2 tpm2::HIS3 This study BHY46 MATa ura3 his3 leu2 ade2 ade3 pfy1-111::LEU2 Haarer et al. (1993) YLM1424 MATa ura3 his3 leu2 ade2 ade3/ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 pfy1-111::LEU2spc24-8::KanMX6 This study YLM1414 MATa ura3 his3 leu2 ade2 ade3/ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 pfy1-111::LEU2spc24-9::KanMX6 This study DDY1254 MATα ura3-52, his3Δ200, leu2-3,112, lys2-801 cof1-5::LEU2 Lappalainen et al. (1997) DDY1266 MATα ura3-52, his3Δ200, leu2-3,112, lys2-801 cof1-22:LEU2 Lappalainen et al. (1997) YLM1485 MATa ura3-52, his3Δ200, leu2-3,112, lys2-801/his3-∆200 ade2-101 ura3-52 lys2-801 leu2-∆1 trp1-∆63 cof1-5::LEU2 spc24-9::KanMX6 This study YLM1481 MATa ura3-52, his3Δ200, leu2-3,112, lys2-801/his3-∆200 ade2-101 ura3-52 lys2-801 leu2-∆1 trp1-∆63 cof1-5::LEU2 spc24-8::KanMX6 This study YLM1497 MATa ura3-52, his3Δ200, leu2-3,112, lys2-801/his3-∆200 ade2-101 ura3-52 lys2-801 leu2-∆1 trp1-∆63 cof1-22::LEU2 spc24-9::KanMX6 This study YLM1493 MATa ura3-52, his3Δ200, leu2-3,112, lys2-801/his3-∆200 ade2-101 ura3-52 lys2-801 leu2-∆1 trp1-∆63 cof1-22::LEU2 spc24-8::KanMX6 This study DDY1434 MATa ade2-1, his3200, leu2-3,112, ura3-52 twf1::URA3 Goode et al. (1998) YLM1473 MATa ade2-1, his3-∆200, leu2-3,112, ura3-52/his3-∆200 ade2-101 ura3-52 lys2-801 leu2-∆1 trp1-∆63 twf1::URA3 spc24-8::KanMX6 This study YLM1475 MATa ade2-1, his3-∆200, leu2-3,112, ura3-52/his3-∆200 ade2-101 ura3-52 lys2-801 leu2-∆1 trp1-∆63 twf1::URA3 spc24-9::KanMX6 This study YLM1285 MATa his3Δ1 met15Δ0 leu2Δ0 ura3Δ0/his3-∆200 ade2-101 ura3-52 lys2-801 leu2-∆1 trp1-∆63 bni1::KanMX6 This study  (To be continued) 109 Table 4.1   List of strains (Continued)  Strain  Genotype Reference YLM1289 MATa his3Δ1 met15Δ0 leu2Δ0 ura3Δ0/his3-∆200 ade2-101 ura3-52 lys2-801 leu2-∆1 trp1-∆63 bni1::KanMX6 spc24-8::KanMX6 This study YLM1314 MATa his3Δ1 met15Δ0 leu2Δ0 ura3Δ0/his3-∆200 ade2-101 ura3-52 lys2-801 leu2-∆1 trp1-∆63 bni1::KanMX6 spc24-9::KanMX6 This study YLM1217 MATa his3Δ1 met15Δ0 leu2Δ0 ura3Δ0/his3-∆200 ade2-101 ura3-52 lys2-801 leu2-∆1 trp1-∆63 bnr1::KanMX6 This study YLM1266 MATa his3Δ1 met15Δ0 leu2Δ0 ura3Δ0/his3-∆200 ade2-101 ura3-52 lys2-801 leu2-∆1 trp1-∆63 bnr1::KanMX6 spc24-8::KanMX6 This study YLM1268 MATa his3Δ1 met15Δ0 leu2Δ0 ura3Δ0/his3-∆200 ade2-101 ura3-52 lys2-801 leu2-∆1 trp1-∆63 bnr1::KanMX6 spc24-9::KanMX6 This study YLM1188 MATa his3Δ1 met15Δ0 leu2Δ0 ura3Δ0/his3-∆200 ade2-101 ura3-52 lys2-801 leu2-∆1 trp1-∆63 bni1-11::URA3 This study YLM1276 MATa his3Δ1 met15Δ0 leu2Δ0 ura3Δ0/his3-∆200 ade2-101 ura3-52 lys2-801 leu2-∆1 trp1-∆63 bni1-11::URA3 spc24-9::KanMX6 This study YLM1274 MATa his3Δ1 met15Δ0 leu2Δ0 ura3Δ0/his3-∆200 ade2-101 ura3-52 lys2-801 leu2-∆1 trp1-∆63 bni1-11::URA3 spc24-8::KanMX6 This study YLM1404 MATa ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 gic2::ClonNAT This study YLM1406 MATa ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 gic1::TRP spc24-9::KanMX6 This study YLM1402 MATa ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 gic2::ClonNAT spc24-9::KanMX6 This study YLM125 MATa ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 gic1::TRP This study YLM1408 MATa ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 gic1::TRP gic2::ClonNAT This study YLM1400 MATa ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 gic1::TRP gic2::ClonNAT spc24-9::KanMX6 This study (To be continued) Table 4.1   List of strains (Continued) 110  Strain  Genotype Reference DDY1324 MATa his3-∆200 ura3-52 lys2-801 leu2-∆1 trp1-∆63/ura3-52 leu2-3,112 his3Δ200 lys2-801am cdc42-117::LEU2 Kozminski et al. (2000) YLM1347 MATa his3-∆200 ura3-52 lys2-801 leu2-∆1 trp1-∆63/ura3-52 leu2-3,112 his3Δ200 lys2-801am cdc42-117::LEU2 spc24-9::KanMX6 This study YLM56 MATa ade2-1 can1-100 his3-3-11,15 leu2-3,112 trp1-1 spc25-1 Measday lab YLM1381 MATa his3-∆200 ura3-52 lys2-801 leu2-∆1 trp1-∆63 ade2-101 spc25-1 This study YLM1424 MATa ura3 his3 leu2 ade2 ade3/ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 pfy1-111::LEU2 spc24-8::KanMX6 This study YLM1414 MATa ura3 his3 leu2 ade2 ade3/ura3-52, lys2-801, ade2-101, his3Δ200, leu2Δ1,trp1Δ63 pfy1-111::LEU2 spc24-9::KanMX6 This study YLM860 MATa his3-∆200 ura3-52 lys2-801 leu2-∆1 trp1-∆63 ade2-101 bud6::TRP1 This study YLM921 MATa his3-∆200 ura3-52 lys2-801 leu2-∆1 trp1-∆63 ade2-101 bud6::TRP1 spc24-9::KanMX6 This study  111   Figure 4.1 Cell polarity in budding yeast is established by a polarized actin cytoskeleton throughout the cell cycle Cell polarity (arrows) is established by a cluster of regulatory and cytoskeletal proteins (blue). Brown: actin cables and actin patches. Pink: actin ring. (Cited from Pruyne D & Bretscher A. J Cell Sci. 2000, 113 (Pt 4):571-85, reproduced with permission of the Company of Biologists©)  112  Figure 4.2 A variety of cellular components are polarized through interactions with actin cables and the cell cortex (A) During early bud growth, Myo2p transports secretory vesicles (a) and vacuolar membranes (b) from the mother cell to the bud tip along actin cables. (c) Myo4p delivers mRNA encoding the transcriptional repressor Ash1p along cables. (d) Mitochondria migrate along actin cables by an unknown mechanism.  (e) Cytoplasmic microtubules emanating from one spindle pole enter the bud and attach to the bud tip. (B) During later bud growth, (a) Myo2p continues to deliver secretory vesicles into the bud along actin cables in the mother cell, but (b) in the bud actin cables form a meshwork that randomizes Myo2p motions, which permits isotropic bud growth. (c) Proteins secreted at the start of bud emergence remain anchored to the bud neck by a scaffold of septin neck. (d) A cortical anchor that was established at the bud tip during earlier apical bud growth immobilizes ASH1 mRNA, mitochondrial membranes and cytoplasmic microtubules at the bud tip. (e) Cortical anchors in the mother retain mitochondria within the mother and anchor cytoplasmic microtubules emanating from the other spindle pole. (Cited from Pruyne D & Bretscher A. J Cell Sci. 2000 Feb;113 ( Pt 4):571-85, reproduced with permission of the Company of Biologists©) 113    Figure 4.3 Septins in budding yeast (A) Model for septin filament assembly. (Cited from Douglas LM, et al., Eukaryot Cell. 2005 September; 4(9): 1503–1512, by permission © American Society for Microbiology) (B) A scheme showing the dynamicity of the septin organization during the cell cycle. (Cited from Kinoshita M. Curr Opin Cell Biol. 2006 Feb;18(1):54-60, by permission © Elsevier Ltd. All rights reserved.) 114    Figure 4.4 Cell cycle-regulated organization of actin cytoskeleton and Cdc42 localization in budding yeast (A) Cell cycle-regulated organization of the budding yeast actin cytoskeleton (Cited from . Moseley JB and Goode BL. Microbiol Mol Biol Rev. 2006 Sep;70(3):605-45, by permission © American Society for Microbiology) (B) Localization of Cdc42 (green) and Cdc42-controlled actin (red) and septin organization. (Actin cables are nucleated by the formins Bni1 (brown) and Bnr1 (yellow). (Cited from HO Park and E Bi, Micro Mol Bio Reviews. 2007 71(1):48-96, by permission © American Society for Microbiology) 115 A   Figure 4.5 Swe1-dependent morphology checkpoint in budding yeast (A)  A scheme showing transition from apical to isotropic growth of the bud. (B) Swe1 localization and degradation in yeast. Swe1 is firstly accumulated and phosphorylated by Cdc28 in the nucleus in unbudded cells. Upon bud growth, Hsl1 is activated and recruits Hsl7, which recruits Swe1 and Cdc5 to the septin. Cdc5 phosphorylates Swe1 leading to Swe1 degradation. Stresses that delay bud emergence prevent activation of Hsl1 and degradation of Swe1. (Cited from Keaton MA & Lew DJ. Curr Opin Microbiol. 2006 Dec;9(6):540-6, by permission © Elsevier Ltd. All rights reserved.) 116     117 Figure 4.6 The Swe1 dependent morphology checkpoint is activated in spc24-9 (A) Example of the bud elongation morphology in spc24-9 mutants (B) Periodic Swe1 accumulation during the cell cycle. Wild type cells and spc24-9 cells containing an integrated SWE1-13MYC were synchronized in G1 with mating pheromone at 25°C and released to 37°C. Time points were taken at 0min, 30min and every 10min thereafter for 2.5 hours. Lysates were separated by SDS-PAGE and immunoblotted with the monoclonal anti-myc antibody, or monoclonal anti-ACT1 antibody as a loading control. (C) Bud elongation in spc24-9 is suppressed by swe1∆. Asynchronous wild type cells, spc24-9 cells and spc24-9 swe1∆ double mutant cells were grown at 37°C for 4 hours. The percentage of the cells with elongated buds is shown. (D) Cells were grown in YPD at 25 o C, synchronized in G1 with mating pheromone at 25°C and released to 37°C.  Time points were taken at 0min, and every 15min for 135min. FACs data is shown at indicated times. (E) (F) Spot assays of the indicated mutants and wild type. Plates were incubated at the indicated temperatures for 2 days. 118  Figure 4.7 Actin defects and Bud6 mislocalization in spc24-9 mutants Log phase wild type and spc24-9 cells were incubated at 37 o C for 3 hours and then fixed with formaldehyde.  (A) Cells were stained with Rhodamine Phalloidin to visualize actin (B)(C) Cells carrying the pBud6-GFP plasmid were imaged (B) and percentage of the cells with mislocalized Bud6 is shown (C). Scale bar, 2μm. (D) Spot assay of the indicated mutants and wild type. Plates were incubated at the indicated temperature for 2 days. 119  Figure 4.8 spc24 mutants have normal actin ring formation GAL-CDC20 cells were grown in galactose at 25 o C, arrested in metaphase with 2% glucose (YPD) for 2hrs at 25°C, washed and resuspended with YPG and released to 37°C.  Time points were taken at indicated time. Actin was stained with Rhodamine Phalloidin to enable visualization of actin ring formation. 120   Figure 4.9 Analysis of septin function in spc24 mutants (A) -(C) Cells harboring the pCDC3-GFP plasmid were fixed with formaldehyde after an asynchronous block at 37ºC for 3 hrs.  scale bar, 2μm. (A) Septin localization in wild type cells. (B) Septin disorganization in spc25-1 (W303 background) (C) Comparison of septin defects in spc25-1 with two different strain backgrounds, W303 and S288C. (D) Spot assays of the indicated mutants and wild type. Plates were incubated at the indicated temperatures for 2 days. 121  Figure 4.10 spc24 mutants have increased numbers of actin patches (A) GAL-CDC20 cells were grown in galactose at 25 o C, arrested in metaphase with 2% glucose (YPD) for 2 at 25°C, washed and resuspended with YPG and released to 37°C for 40min. Actin was stained with Rhodamine phalloidin. Scale bar, 2μm. (B) (C) Lat-A halo sensitivity assay was carried out as described in the Materials and Methods. Round white filter paper with Lat-A (with indicated concentration) is in the center of each image. Grown colonies are pink or white and the dark halos are due to the absence of cell growth. Plates were incubated at 30 o C for 2 days. 122  Figure 4.11 Genetic interactions between spc24 and actin mutants Spot assays of the indicated mutants and wild type. spc24-8 and spc24-9 are synthetic sick with cof1 mutants (cof1-5 and cof1-22) (A) and pfy1-111 (B), whereas spc24-8 and spc24-  are rescued by twf1∆ (B) at the indicated temperature. Plates were incubated at the indicated temperatures for 2 days. 123    Figure 4.12 Suppression of spc24 mutant growth defect by formin mutants (A) Schematic representation of the primary structures of the yeast formin homologue Bni1. Domains include Rho-binding domain (RBD), formin-homology domains (FH1, FH2 and FH3), Dia-autoregulatory domain (DAD), Spa2-binding domain (SBD) and Bud6-binding domain (BBD). Percentage identities between related domains are indicated. (Cited from Evangelista M et al., Nat Cell Biol. 2002 Mar;4(3):260-9.) (B) Spot assays of the indicated mutants and wild type. Plates were incubated at the indicated temperatures for 2 days. 124   Figure 4.13 Genetic interaction between spc24 and cdc42-117 and gic mutants (A) Schematic representation of Cdc42 structure. Mutations of cdc42-117 are in the GAP binding domain as indicated. (Modified from HO Park and E Bi, Micro Mol Bio Reviews. 2007, 71(1):48-96) (B)-(D) Spot assays of the indicated mutants and wild type. Plates were incubated at the indicated temperatures for 2 days.  125 Chapter 5  Summary and Perspectives  In this thesis, I have dissected the cellular function of the Ndc80 kinetochore complex in spindle integrity, the cAMP/PKA pathway and the regulation of polarity.  Firstly, our laboratory demonstrated that a mutation of Spc24 (spc24-9), a component of the conserved Ndc80 kinetochore complex, causes lethality when cells are exposed to the DNA replication inhibitor hydroxyurea (HU) due to premature spindle expansion and segregation of incompletely replicated DNA. Consequently, I performed a high copy suppressor (HCS) screen and identified 10 genes that when overexpressed rescue the HU sensitivity and spindle expansion defect of the spc24-9 mutant strain (Ma et al., 2007). Overexpression of Stu1, a CLASP-related MT-associated protein or a truncated form of the XMAP215 orthologue Stu2 rescues spc24-9 HU lethality and prevents spindle expansion. Truncated Stu2 likely acts in a dominant-negative manner, because overexpression of full-length STU2 does not rescue spc24-9 HU lethality, and spindle expansion in spc24-9 HU-treated cells requires active Stu2. I have shown that Stu1 and Stu2 localize to the kinetochore early in the cell cycle and Stu2 kinetochore localization depends on Spc24. Therefore I propose that mislocalization of Stu2 results in premature spindle expansion in S phase stalled spc24-9 mutants, and anticipate that identifying factors that restrain spindle expansion upon inhibition of DNA replication is likely applicable to the mechanism by which spindle elongation is regulated during a normal cell cycle. Secondly, the Measday lab 126 previously reported a synthetic lethal screen performed using spc24-9 as the query strain and identified a genetic interaction with two mutants (ira2, pde2) that display increased activation of the cAMP/PKA pathway. I extended this study and have shown that the ts of three spc24 mutants, spc24-8, spc24-9 and spc24-10, is suppressed by ras1∆ and ras2∆, which have reduced activation of the cAMP/PKA pathway.  Therefore, my data suggest that spc24 mutants do not tolerate increased cAMP/PKA activity and are rescued by reduction of cAMP/PKA activity.  In wild type cells, the transcription factor Msn2 is rapidly dephosphorylated in response to glucose, however spc24 mutants have defects in Msn2 dephosphorylation in response to glucose depletion. Non-preferred carbon sources like galactose, raffinose and glycerol dramatically rescue the glucose growth defects of all mutant alleles of the Ndc80 complex and specific alleles of other kinetochore complexes. I therefore propose that spc24 mutants have increased PKA activity and the Ndc80 complex may both be a target of the PKA pathway and have a role in downregulation of the PKA pathway as well. Thirdly, I identified actin defects in spc24 mutants and genetic interactions with mutants defective in actin cable nucleation, actin patch formation, and upstream regulators of cell polarity. I found that the Swe1-dependent morphology checkpoint is activated in spc24-9 mutants probably due to actin disorganization, but the checkpoint activation is not the only reason for spc24-9 G2 arrest and inviability at the non-permissive temperature. Since a dominant allele of Cdc42 reduces the restrictive temperature of spc24-9 whereas depletion of downstream targets of Cdc42 suppresses the ts of spc24-9, I speculate that the actin defects in spc24 mutants might be due to hyper activated Cdc42. Below, I discuss new questions raised by 127 my findings and future directions for research on the cellular function of the Ndc80 complex.  What is the specific role of Ndc80 in cAMP/PKA pathway? I have shown evidence that the Ndc80 complex might have a role in the cAMP/PKA pathway but it is still unclear whether the Ndc80 complex is negatively regulated by the PKA pathway or whether the Ndc80 complex is a negative regulator of the PKA pathway.  Firstly, I could test if the Ndc80 complex is phosphorylated by PKA (Tpk1,2,3) using in vitro kinase assays and to use a mass spectrometry approach to identify any PKA phosphorylation sites on the components of Ndc80 complex. If PKA phosphorylation sites are identified on Spc24, Spc25, Nuf2 or Ndc80, I would next proceed by using site-directed mutagenesis to abolish or mimic phosphorylation sites.  The phenotypes of the mutant alleles would be studied by comparing growth conditions under different carbon source, analyzing Msn2 phosphoryation levels and trehalose levels as described in Chapter 3. Secondly, if the PKA pathway negatively regulates kinetochore function, hyperactivation of the PKA pathway, for example by using dominant alleles of the PKA pathway such as ras2val19 or bcy1∆, would promote detachment of kinetochores from MTs which could be detected by GFP-tagged tetracycline operator (tetO) system or monitoring kinetochore proteins fused to fluorescent tags (Michaelis et al., 1997; Fuchs et al., 2002). On the other hand, if the Ndc80 complex negatively regulates the PKA pathway, overproducing Ndc80 may decrease the viability of ras2∆ or rescue ras2val19 or affect intracellular cAMP levels. One intriguing data from recent quantitive mass spectrometry analysis performed by Angel Chang (MSc student in 128 the Measday lab) in collaboration with Dr. Thibault Mayor at UBC is that Ndc80 may interact with Gcr1 which is a transcriptional activator of genes involved in glycolysis, suggesting that the Ndc80 complex might regulate the PKA pathway at the transcription level. Therefore, we propose to confirm the Gcr1-Ndc80 interaction by co-IP and to determine if expression levels of Gcr1 regulated genes, such as ENO1,2 or GAPDH are affected in spc24-9 mutants (Holland, et al., 1987).  Next I propose to perform follow up studies such as ChIP assays to determine if Ndc80 binds to Gcr1 binding sites (Huie, et al., 1992) or if Gcr1 binds to the centromere.  Does Ndc80 accumulate to the plus end of cMT?  In Appendix B, I show that Ndc80-GFP, in addition to its well established kinetochore localization, also localizes to a punctate focus in the cytoplasm.  I speculate that the cytoplasmic localization of Ndc80-GFP is the plus end of cMT, but the co-localization need to be further confirmed. Thereafter, I am interested to know if Ndc80 itself, the Ndc80-Nuf2 dimer, or the entire Ndc80 complex accumulates at the plus end of cMTs. It is reported that Ndc80 components interact by the two-hybrid assay with the dynein-dynactin complex (Jnm1 and Nip100) (Wong, et al., 2007).  The dynein-dynactic complex anchors at the cell cortex and walks along the cMTs towards SPBs, which might provide a mechanism of communication between the cytoplasm and nucleus. Thus it would be interesting to study if Ndc80 is transported by these motor proteins to the plus ends of cMT and if the cMT pool of Ndc80 is translocalized from the kinetochore or from a separate cytoplasmic pool of Ndc80. Next, what are the functions of Ndc80 at the cMT? Since the dynein/dynactin complex 129 facilities spindle orientation and nuclear migration, it is possible that Ndc80 at cMTs are also involved in this process.  To address this further, I propose to determine if spc24 mutants have any defects in spindle orientation or nuclear migration.  The second possibility is that the pool of Ndc80 at cMT reaches the cell cortex and therefore interacts with the components in the PKA pathway (see discussion in Chapter 3), responds to environmental stress (see below), or, as described in Chapter 4, interacts with proteins that function in regulation of actin dynamics (see below).  Why do cells recruit Ndc80 to the cytoplasm? I have some preliminary data with overproduced pNDC80-GFP suggesting that more Ndc80 may accumulate in the cytoplasm at higher temperature (37 o C) versus normal temperature (25 o C) in glucose but less in the cytoplasm at 25 o C in NPCS such as galactose compared to glucose media (25 o C) (data not shown). Different carbon sources might address the role of the Ndc80 complex in the PKA pathway or other nutrient pathway, whereas 37 o C is a mild heat stress belonging to the environmental stress response (ESR). Yeast is a unicellular cell, unlike mammalian cells that have multiple organs to retain a relatively stable internal environment, therefore yeast has evolved autonomous programs for adapting to changes in environmental conditions.  Consequently, the yeast Ndc80 complex might have additional functions compared with its vertebrate homologue. Gasch (2000) and others have reported that the PKA pathway may govern the entire ESR in response to nutritional signals, and both the positive and negative regulators of the PKA pathway are induced by the 130 ESR, presumably to allow sensitive posttranslational control through the pathway. Therefore, I am planning to confirm and study the localization of Ndc80, especially at the cMT, in response to different ESR, such as glucose depletion, switching the media from NPCS to glucose, or mild heat shock. Notably, we need to repeat the experiment with endogenous GFP tagged Ndc80 strains using a better camera or confocal microscope in order to confirm that the phenotypes observed upon different carbon sources or mild heat are not due to the overexpression of Ndc80. The Measday lab is currently setting up a sensitive system on the microscope for these experiments.  What the role of Spc24 in G1? The cellular function of Spc24 in G1 has never been carefully studied. In Appendix 1, I show that when spc24-9 cells are blocked in metaphase and released to restrictive temperature, the cells arrest as unbudded cells with a 1N DNA content, suggesting that Spc24 might have a role in G1. However, since this experiment involves a carbon source shift (from glucose to galactose), the G1 arrest could be occurring because of defects with the spc24-9 strain in adapting to the different carbon source.. Therefore, I propose to repeat this experiment using a different approach such as a MET-CDC20 or telophase ts alleles (e.g. cdc15-2, cdc5-1, cdc14-1) to block the cells in anaphase prior to release.  If the G1 arrest is confirmed, I could characterize the arrest further by analyzing G1 cyclin levels, the position of polarity determinants, and the SPB attachment state of the kinetochore to determine the cause of G1 arrest.  131 What are the cytoplasm interaction partners of Ndc80? The data presented in Chapter 3, Chapter 4 and Appendix 2 all indicate that the Ndc80 complex has a cytoplasmic cellular function - either in the PKA pathway or regulation of actin dynamics. Thus an important question is ―What are the cytoplasmic targets of the Ndc80 complex?‖ A graduate student in Dr. Measday’s lab, Angel Chang, is currently performing quantitative Mass-spectrometry in collaboration with Dr. Thibault Mayor by immunoprecipitating a GFP-tagged version of Ndc80. One caveat to this approach is that the majority of Ndc80 is localized to the nucleus and thus it will be difficult to detect interactions with the minor pool of Ndc80 that is localized to the cytoplasm.  One possibility is to use a kinetochore mutant (such as ndc10-1) to disrupt kinetochore function and possibly increase the chances of detecting an interaction between Ndc80 and its cytoplasmic targets. One interesting candidate to mention here is Srv2, which binds to adenylyl cyclase complex to activate the cAMP/PKA pathway, and also binds to actin monomers to play a central role in regulation of actin dynamics (Gerst, et al., 1991; Kawamukai, et al., 1992; Freeman, et al., 1995; Yu, et al., 1999; Mattila, et al., 2004).  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Mol Cell Biol., 13, 1779-1787.  154 Appendices Appendix A  spc24-9 arrests in G1 after metaphase release A.1  Results and Discussion When spc24 mutants are synchronized in G1 phase with mating pheromone and released to restrictive temperature, a variety of arrest phenotypes are detected.  spc24-8 cells are arrested in metaphase with an undivided nucleus and short spindles due to an active spindle checkpoint; spc24-10 mutants lack a functional spindle checkpoint and as a result the spindle elongates despite the fact that chromosomes are not attached to the spindle and the DNA remains in the mother cell; spc24-9 mutant cells undergo chromosome mis-segregation with elongated spindles which separate the DNA in the mother cell and the bud unequally, and finally have a mixture of large budded and G1 cells (Montpetit, et al., 2005; Ma, et al., 2007). All the above phenotypes are induced after G1 phase and the defect is detected either in metaphase or anaphase. However the Ndc80 complex may also have a role after anaphase but this would not have been detected due to the G1 cell synchrony method.  Therefore, I constructed a GAL-CDC20 fusion (with the CDC20 open reading frame fused to the GAL1 promoter) in spc24 mutant cells (see 4.3.2) so that the cells could be arrested in metaphase upon glucose treatment due to Cdc20 depletion.  In order to compromise Spc24 function after metaphase, spc24 ts mutants were arrested in metaphase by glucose depletion at 25°C and then released into galactose media at the non-permissive temperature (37°C) to allow expression of CDC20 and progression through the 155 cell cycle.  As determined by monitoring cellular DNA content and nuclear position (Figure A.1A,B), spc24-8 cells passed through anaphase at a similar pace to wild type cells then stalled in G1 for about an hour prior to continue the cell cycle, indicative of a delay at START. Combined with our previous data showing the phenotype after G1 release, spc24-8 cells are expected to arrest with a 2N DNA content in metaphase which probably would have occurred if the time course had been extended for longer.  spc24-10 cells entered G1 phase earlier than the wild type probably due to the inactivation of the spindle checkpoint.  At 4 hours spc24-10 cells mostly had 2N DNA content with an undivided nucleus in either small budded or large budded cells whereas the wild type cells had a mixture of 1N and 2N DNA content along with a mix of G1 and budded cells.  When released from a G1 arrest to restrictive temperature for 2 hours, spc24-10 also has a 2N DNA content but upon further incubation at restrictive temperature, the unsegregated DNA is replicated to 4N indicative of a defective spindle checkpoint (Montpetit, et al., 2005).  Future work would be to determine if spc24-10 cells released from the GAL-CDC20 metaphase block eventually bypass the spindle checkpoint as well.  Interestingly, spc24-9 cells were arrested with 1N DNA and ~60% G1 cells after release from the Cdc20 block. Since over 90% of spc24-9 G1 cells have unpolarized actin cables (Figure A.1C), spc24-9 cells might have defects in bud site selection or polarity establishment. Alternatively, spc24-9 cells might not able to pass START.  In budding yeast, small daughter cells do not pass START until they have reached the size of the mother cell.  PKA activity is required for cell growth in G1, but needs to be downregulated at START due to cAMP mediated repression of CLN1,2 transcription (Baroni, 156 et al., 1994).  Therefore, the other possibility for the G1 arrest of spc24-9 is an inability to downregulate the PKA pathway and a lack of CLN1,2 expression due to high cAMP levels. This possibility could be tested by performing a microarray analysis of spc24-9 gene transcripts during a synchronous cell cycle, followed by northern blot analysis of specific transcripts.  A.2 Materials and methods Timecourse, FACs, budding index and microscopy.  Cells were grown to early-logarithmic phase in 150ml YPGal (2% galactose) at 25°C, spun down, resuspended in YPD (2% glucose) and incubated for 2 hours to arrest cells in metaphase. Cells were collected, washed once with YPGal, resuspended in prewarmed YPGal and shifted to 37°C. 10ml of culture was collected for each indicated time point. 5ml cells were fixed with 4% formaldehyde for 1hour, washed with PBS three times, resuspended in 100ul PBS plus 10ul Rhodamine Phalloidin (6.6μM in MeOH) (Molecular Probes Inc.), incubated in the dark at 4°C overnight, then washed and resuspended with PBS/DAPI solution (1:2000) before imaging. See the details of microscopy in Chapter 4.2. FACs analysis was performed as described (Haase, et al., 1997). 157    C  158 Figure A.1 spc24-9 mutants delay in G1 after a metaphase arrest GAL-CDC20 cells were grown in galactose at 25 o C, arrested in metaphase with 2% glucose (YPD) for 2h at 25°C, washed and resuspended with YPG and released to 37°C.  Time points were taken at 0min, and every 20min for 4 hours. FACs (A) and budding index (B) are shown at indicated times. (C) spc24-9 mutants have defects in polarity establishment in G1. Actin was stained with Rhodamine Phalloidin.  159 Appendix B Analysis of Ndc80 localization B.1  Results and discussion The well-known localization of the Ndc80 complex is at the kinetochore, however phenotypic analysis of mutants of this complex suggest that it might have functions at other cellular locations. I imaged Ndc80 tagged with VFP at its endogenous locus and, in addition to the well established kinetochore localization of Ndc80, I found that Ndc80 localizes along the spindle in large budded cells with an extended spindle (Figure B1.A). The spindle localization of Ndc80 has not been reported before probably because the intensity of the dots along the spindle is fairly low and therefore has likely been missed.  To further characterize the minor pool of Ndc80 by amplifying the fluorescence signal, I constructed a high copy plasmid carrying Ndc80-GFP (pNdc80-GFP) and imaging of wild type cells containing this plasmid confirmed that Ndc80 localizes to punctate spots along the spindle (Figure B1.B). Several kinetochore proteins (Ndc10, Slk19), MT plus end-tracking proteins (Bim1, Bik1, Stu1 and Stu2), kinesins (Cin8, Kip3) and the Ipl1 complex have been reported to localize to the spindle midzone during anaphase and be required for anaphase spindle stability (Khmelinskii, et al., 2007).  Therefore, Ndc80 may also have a role in anaphase spindle stability or later events. In addition, I also detected a fainter Ndc80-GFP signal in the vicinity of the cell cortex (Figure B2.A).  With time-lapse imaging, I found that the Ndc80-GFP foci localized to the cortex transiently and then moved away (arrow in Figure B2.B) which is reminiscent of the highly dynamic behavior of the plus end of cMT. Aiming to confirm the precise localization of 160 the non-kinetochore pool of Ndc80, I studied the localization of Ndc80 in mCherry-Tub1 wild type cells harboring pNdc80-GFP.  As shown in Figure B3, the non-kinetochore pool of Ndc80 overlays with the mCherry-Tub1 signal which could be the cMT plus end. However, in some of the cells (Figure B3.C-D) there is a strong GFP foci of non-kinetochore Ndc80 that overlaps with a weaker Tub1 signal and the Tub1 signal is not at the expected location of a cMT plus end. Since Ndc80 is known to bind directly to MTs (Cheeseman et al. 2006; Deluca et al. 2006; Wei et al. 2007; Ciferri et al. 2008; Guimarase et al. 2008; Miller et al. 2008), it is possible that overexpression of Ndc80 produces excess Ndc80 that recruits free cellular tubulin which is why I detect a bright off spindle Tub1 signal colocalized with Ndc80-GFP.  To clarify whether pNdc80-GFP localizes to the cMT plus end or not, I attempted imaging pNdc80-GFP with another cMT marker - Bik1-RFP.  However, the Bik1-RFP signal is extremely weak and the current camera system on our miscroscope requires a 2 to 3 second exposure to detect this signal. It is therefore impossible to study the dynamics of Bik1 localization under these conditions. The lab is currently waiting to purchase a more sensitive cooled CCD camera to enable detection of weak RFP signals so that this experiment can be completed in the future.  B.2  Materials and methods B.2.1 Strains and plasmid The 2μ plasmid containing the NDC80 promoter followed by the full length NDC80 ORF (backbone p5467) was a gift from Charlie Boone’s lab (University of Toronto). pNDC80 was 161 tagged with GFP+ (Scholz, et al., 2000) by PCR-based homologous recombination (Longtine, et al., 1998). pRS406-mCherry-Tub1, which was a gift from Steven. I. Reed (The Scripps Research Institute), was digested with NdeI before homologous recombination in yeast. All endogenous tagging with GFP+, VFP, CFP or RFP was performed by PCR-based homologous recombination (Longtine, et al., 1998).  B.2.2 Microscopy Microscopic analysis of yeast cells was performed with a Zeiss Axio Observer Inverted Microscope equipped with a Zeiss Colibri LED illuminator and a Zeiss Axiocam Ultra High Resolution Monochrome Digital Camera Rev3.0. Cells were grown to mid-log phase in SC medium at 25 °C, resuspended with fresh media and added onto a concanavalin A coated (Nissan, et al., 2008) glass bottom dish (MatTek Corporation #P35G-1.5-14-C) for imaging.  Imaging stacks were acquired with a 40x objective at a step of 0.2 μm to span the entire cell and images were analyzed with Zeiss Axiovision software. 162   Figure B.1 Ndc80 is scattered along the spindle in anaphase Ndc80VFP Spc29CFP cells (A) and mCherry-Tub1 cells harboring the pNdc80-GFP high copy plasmid were grown to mid-log phase in SC media at 25°C and resuspended with fresh media for live imaging. Scale bar, 5μm.  163   Figure B.2 Localization of Ndc80 to a non-kinetochore foci Endogenous Ndc80-GFP cells were grown into mid-log phase in SC media at 25°C and resuspended with fresh SC media for live imaging (A) and live imaging timelapse (B). The white arrow indicates the non-kinetochore Ndc80 foci. Scale bar, 5μm.  164  Figure B.3 Non-kinetochore pool of Ndc80 co-localizes with Tub1 mCherry-Tub1 cells harboring pNdc80GFP were grown to mid-log phase in SC media at 25°C and resuspended with fresh SC media for live imaging. The white arrow indicates the non-kinetochore Ndc80 foci. Scale bar, 5μm.  

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