Roles of TACC related protein, mia1p in MTOCs and microtubule dynamics in schizosaccharomyces pombe 2

Chapter I Introduction 1.1 A general introduction to microtubules 1.1.1 Properties of microtubules Microtubules mediate a wide variety of cellular functions. Chromosome segregation requires assembly and function of the microtubule-based mitotic spindle (for review, see Musacchio and Salmon, 2007); Establishment and maintenance of proper cell morphology relies on microtubules to deposit cell growth components to precise sites (for review, see Chang and Peter, 2003). Properties of microtubules allow them to successfully perform these tasks. They consist of 13 protofilaments which associate laterally to form hollow, cylindrical structures with ~25nm diameter. Each protofilament is composed of α/β tubulin heterodimers that are longitudinally linked. However microtubules are not static structures but are highly dynamic, undergoing repeated transitions between growth and shrinkage, a phenomenon called dynamic instability (for review, see Dammermann et al., 2003). In an in vitro situation, during polymerizing, growing state, α/β-tubulin subunits are added to both ends of the microtubule with different rates: the slow growing end terminated by α-tubulin is defined as “minus end”, while the fast growing end capped by β-tubulin is termed “plus end” (for review, see Dammermann et al., 2003). As tubulin heterodimers bind to the growing ends of microtubules, β-tubulin guanosine triphosphate (GTP) within the heterodimer subsequently hydrolyzes to guanosine diphosphate (GDP), leaving GDP bound form of tubulin subunits in the lattice. Hydrolysis of GTP causes conformation changes in the tubulin subunits but the resulting curved protofilaments are stabilized by the lateral bonds within the microtubule wall, especially in the vicinity of the stable cap of GTP-containing subunits at microtubule plus end. During shrinking state, the GTP cap is lost from the plus end of microtubule and the unstable curved protofiliments peel back from the microtubule wall. Eventually, the protofilaments disassemble into free tubulin heterodimers (for text book, see Molecular Biology of the cell, Third Edition). The dynamic instability of microtubules is shown on the following cartoon (for review, see Wiese and Zheng, 2006). However in an in vivo environment lacking a relatively high tubulin concentration, microtubule nucleation requires another member of the tubulin family, γtubulin. γ-tubulin is found in a large protein complex called γ-tubulin ring complex (γTuRC) that is present both at the centrosome and non-centrosomal microtubule nucleation sites to promote assembly of microtubules (Raynaud-Messina and Merdes, 2007) 1.1.2 Centrosome The centrosome is thought to nucleate the majority of microtubules in many animal cells. In the fluorescence microscopy analyses, the microtubules are seen in greatest density around the nucleus and radiate out into the cell periphery during interphase. This ordered microtubule network is organized by one key player—the centrosome. Centrosome consists of a pair of centrioles surrounded by an amorphous cloud of pericentriolar material (Bornens, 2002). Functions of centrioles within the centrosome are still poorly understood, although centrioles are thought to serve as the organizer of the pericentriolar material based on the observation of pericentriolar material dispersion upon microinjection of antibody to disassemble the centrioles (Bobinnec et al., 1998). Nevertheless, there is now little doubt that γ-tubulin ring complexes (γ-TuRCs), identified within the pericentriolar material, are responsible for nucleating microtubules (Bobinnec et al., 1998; Wiese and Zheng, 2006). The detailed function of γ-TuRC will be discussed later in this thesis. 1.1.3 Non-centrosomal microtubule Non-centrosomal microtubules generated by centrosome-independent mechanisms are also found in neurons and epithelial cells as well as skeletal muscle cells (Bartolini and Gundersen, 2006). How the non-centrosomal microtubules are formed is poorly understood. Recent data from studies of non-centrosomal microtubules propose that there are several general mechanisms that cells might employ to nucleate non-centrosomal microtubules. Firstly, existing microtubules may be released from the centrosome probably through action of some severing proteins such as Katanin (for review, see Bartolini and Gundersen, 2006; McNally and Vale, 1993). Secondly, microtubules are nucleated from the free γ-TuRCs present in the cytoplasmic form (for review, see Bartolini and Gundersen, 2006). Thirdly, microtubules can be nucleated from distinct non-centrosomal sites such as trans- Golgi network in human cells (Efimov et al., 2007). Forth, existing microtubules could break into two parts to generate free microtubule under mechanical stress (for review, see Bartolini and Gundersen, 2006). 1.1.4 Schizosaccharomyces pombe (S. pombe) as a model for studying MT dynamics The unicellular model organism, fission yeast Schizosaccharomyces pombe (S. pombe) is an attractive model system to investigate various aspects of microtubule dynamics. Straightforward genetic analyses and a fully sequenced and annotated genome (Wood et al., 2002) are useful in dissecting molecular mechanisms of microtubule organization. A relatively large cell size allows detailed dynamic observation of cytoskeletal filaments and cellular components tagged with fluorescent proteins. Importantly, fission yeast cells exhibit simple but distinct types of microtubule arrays depending on the cell cycle stage. So far four fission yeast tubulin genes have been identified: γ tubulin (gtb1/tug1), two α tubulin (nda2, atb2) and a β tubulin (nda3) (Yanagida, 1987). Among these, α- tubulin and β-tubulin are components of microtubules, while γ-tubulin, identified within the microtubule organizing centers, acts as a microtubule nucleating template (for review, see Hagan, 1998). 1.2 MTOCs and microtubule cytoskeleton in Schizosaccharomyces pombe (S. pombe) The vegetative cell cycle of S. pombe consists of interphase (including G1, S and G2 phases) and mitosis. To perform their functions at different cell stage, microtubules are organized into complex arrays by MTOCs. In S. pombe cells, other than the spindle pole body (SPB) which is equivalent to the mammalian centrosome, two other transient, cell-cycle-regulated nucleation sites (defined here as non-centrosomal MTOCs) are known: the interphase MTOCs (iMTOCs) and the equatorial MTOC (eMTOC) (Hagan and Petersen, 2000; Tran et al., 2001). 1.2.1 Microtubule Nucleating sites—MTOCs in S. pombe 1.2.1.1 Spindle pole body (SPB) The fission yeast spindle pole bodies (SPBs) are functionally analogous to centrosomes and undergo a duplication and separation cycle, correlated with the cell division cycle. Like the centrosome of vertebrate cells, the SPB of S. pombe spends most of interphase in the cytoplasm, immediately next to the nuclear envelope (NE) (Ding et al., 1997; Uzawa et al., 2004). Currently, it is still a controversial issue about the duplicating time of SPBs: Some researchers think that it occurs in the late G2 phase as shown in the following cartoon (from Ding et al., 1997). Another opinion is that SPBs are duplicated at the G1/S boundary and matured in the G2 phase (Uzawa et al., 2004). Unlike other vertebrate cells undergoing open mitosis in which the NE disassembles in early prophase, fission yeast undergoes “closed mitosis”: The NE remains intact throughout the cell cycle. To ensure the access of microtubules to the hereditary material, a portion of the NE underlying the SPB pair breaks down to form an opening called “fenestra” once the cell enters mitosis. Then the duplicated SPBs settle into fenestra to nucleate intranuclear microtubules. During the elongation of the spindle, SPBs are always localized at the leading edge of the NE. As anaphase proceeds, the nuclear fenestrae close, and the SPBs are extruded back into the cytoplasm to nucleate interphase microtubules. The summary of dynamics of the NE and the SPBs through out the cell cycle is shown in the following cartoon (Ding et al., 1997). Diagram summarizing dynamics of the NE and the SPBs throughout the cell cycle. The SPB (shaded ellipse with line) is associated with an appendage (small solid ellipse) and lies close to the NE. Late in G2 phase, the SPB duplicates and matures, daughter SPBs are connected by a bridge derived from the appendage. Upon mitotic entry, the NE invaginates, perforates to form fenestra and the SPB settle into it. The two halves of the structure separate as the spindle forms, such that each SPB occupies its own fenestra. At the end of mitosis, the fenestrae close and extrude the SPB back into the cytoplasm for the next interphase. 1.2.1.2 Non-centrosomal MTOCs Compared to the SPBs which exist throughout the cell cycle, the iMTOCs and the eMTOC are assembled at different cell cycle stages: The eMTOC localizes to the cell center to nucleate the post anaphase array (PAA) at the end of mitosis (Venkatram et al., 2005). On the other hand, the iMTOCs are only present in interphase cells and disassemble once the cells enter mitosis. So far, all components found in the eMTOC are also present in the iMTOCs. It is believed that disassembly of the eMTOC is synchronous with the establishment of the iMTOCs (Zimmerman et al., 2004a). 1.2.1.2.1 Interphase microtubule organizing centers (iMTOCs) The definition of iMTOCs is still largely controversial to date. Some researchers refer to iMTOCs as “satellites” of γ-tubulin complex proteins (Janson et al., 2005). In my opinion, the iMTOCs are the sites on the NE where interphase microtubules are nucleated. It is hard to observe the iMTOCs structures in live cells at steady state due to low fluorescence intensity and distribution of γ-TuRC components along microtubule bundles. However, after drug-induced microtubule depolymerization, γ-TuRC components aggregate at microtubule nucleation sites and the iMTOCs can be observed as several dots around the NE by tagging the components of γ-TuRC with green fluorescent protein (GFP). Repolymerization of microtubules will be initiated from such dots by washing out the depolymerizing agent, demonstrating the microtubule-nucleating capability of the iMTOCs. It is known that the establishment of the iMTOCs is linked to the disassembly of the eMTOC (Zimmerman et al., 2004a), but the precise origin of the iMTOCs remains elusive. 1.2.1.2.2 Equatorial microtubule organizing center (eMTOC) At the end of anaphase, the mitotic spindle breaks down and microtubules originate from the middle of the cell to create the PAA (Hagan and Hyams, 1988). The PAA is nucleated from a distinct MTOC called the eMTOC. In fluorescence microscope analyses, the eMTOC appear as a ring structure co-localizing with the actomyosin ring in the middle of the dividing cell. Assembly of the eMTOC requires the activity of a GTPase signaling cascade known as the septation initiation network (SIN) that regulates the onset of cytokenesis (Heitz et al., 2001). In addition, integrity of the eMTOC relies on the integrity of the Factin but not the microtubules: the dispersal of the eMTOC in the absence of the actomyosin ring indicates the essential role of actomyosin ring in eMTOC formation (Heitz et al., 2001). Recent studies found that the eMTOC breaks down into small pieces defined as satellites that can move along the microtubules (Zimmerman et al., 2004a; Sawin and Tran, 2006). During the eMTOC-breakdown process, the DnaJ domain protein, Rsp1p, functions in disassembly of the eMTOC, and possibly in assembly of the iMTOCs, probably through interacting with the cytoplasmic hsp70 protein, Ssa1p (Zimmerman et al., 2004a). It’s believed that Rsp1p may stimulate the ATPase activity of Ssa1p, which then exerts an ATP-dependent conformational change on its substrate, causing the protein-protein interaction to weaken and resulting in the breakdown of the eMTOC (Zimmerman et al., 2004a). The establishment of the iMTOCs is believed to proceed synchronously with the disassembly of the eMTOC since γ-TuRC components were found to move from eMTOC to the iMTOCs as satellites when the eMTOC ring constricts (Zimmerman et al., 2004a). Failure to disassemble the eMTOC in the rsp1-1 mutant leads to a defect in the organization of interphase cytoplasmic microtubules (Zimmerman et al., 2004a). 1.2.1.2.3 γ-TuRC satellites In addition to the MTOCs mentioned above, when fused with GFP, the γ-TuRC components are also seen as weakly fluorescent dots traveling along the interphase microtubules in a bi-directional manner (Zimmerman et al., 2004a). Such satellites are capable of nucleating microtubules on the preexisted microtubule in interphase cells (Janson et al., 2007). 1.2.2 Components of MTOCs 1.2.2.1 The γ-TuRC Microtubule polymerization at the MTOCs requires the action of a large, conserved multisubunit protein complex—γ-TuRC. Although there are two different forms of the γ-tubulin complex present in higher eukaryotes, centrosomal γ-TuRC and cytosolic γ-tubulin small complex (γ-TuSC), it seems that only the γ-TuRC possesses microtubule nucleating capacity on its own in vitro [(Oegema et al., 1999) 10 Model of interphase microtubules bundling in fission yeast. Model includes the following steps: (1) The γ-TuRC satellites were recruited to the lattice of mother MTs to nucleate daughter MTs. (2) Ase1p bundles and stabilizes the antiparallel arrangement of daughter-mother MTs. (3) Klp2p is recruited to the plus-end of the daughter MT, where it pulls the daughter MT toward the minus end of the mother MTs. 17 1.2.4 Arrangement of microtubule cytoskeleton in S. pombe Since MTOCs alter their microtubule-nucleating activity in a cell-cycle specific manner, the architecture of microtubule network is regulated dynamically, allowing it to perform specific functions at different stages of the cell cycle. 1.2.4.1 Interphase microtubule cytoskeleton 1.2.4.1.1 Architecture and dynamics of interphase microtubule cytoskeleton During interphase, 4-5 bundles of microtubules run along the long axis of a cylindrical cell. One of them is attached to the SPB and the rest to the multiple iMTOCs at the nuclear envelope. Thus the minus ends of microtubules are anchored / organized at the MTOCs while the plus ends extend to the cell tips (Drummond and Cross, 2000; Tran et al., 2001). Such an anti-parallel arrangement can be detected in the cells with α-tubulin tagged with GFP under the fluorescence microscope: higher intensity of fluorescence appears in the cell center where minus ends of microtubules overlap around the nuclear envelope. Interphase microtubules are highly dynamic. Generally, microtubules are nucleated from the MTOCs on the nuclear envelope and continue to grow until they hit the cell tip, pausing there for a while (Brunner and Nurse, 2000), then undergo depolymerization. The transition from polymerization to depolymerization is defined as “catastrophe”. Like in higher eukaryotic cell, such interphase dynamic microtubule arrays play an important role in establishment and maintenance of cell polarity and overall intracellular organization. 18 1.2.4.1.2. Functions of interphase microtubule cytoskeleton 1.2.4.1.2.1 Maintaining cell morphology Fission yeast cells grow longitudinally by restricting the growth at two opposite cell tips (Nurse, 1994). To maintain its rod-shape morphology, fission yeast has developed a mechanism to position polarity factors at precise location. The role of a dynamic microtubule array is to provide tracks for transporting those polarity factors to the sites where they are needed (Chang and Peter, 2003). Polarity of microtubules is required in this process: microtubule plus ends need to be facing the cell tips to ensure polarized growth at the cell tips by depositing the kelch-repeat protein, Tea1p, at the cell tips. There, Tea1p functions in polarity establishment by recruiting a complex, forminbinding protein—Bud6p and the formin, For3p, to assemble actin cables (Feierbach et al., 2004). Cells with abnormal microtubules often face difficulties in delivering the polarity factors to the precise sites, causing misplaced growth and producing bent, branched, or Tshaped cells (Beinhauer et al., 1997; Brunner and Nurse, 2000). A model for establishment of cell polarity based on current knowledge has been generated: the cell end marker, Tea1p, plays a critical role in maintaining cell morphology by binding to the plus ends of growing interphase microtubules in a Tip1p and Tea2p-dependent manner (Chang and Peter, 2003). When the growing microtubules reach the cell tips and dwell there for a while, the microtubule plus end associated proteins such as Tea1p are released to the cell tips (Behrens and Nurse, 2002). On the other hand, a complex at the cell tips is waiting to dock the released Tea1p. One candidate of this complex is a novel membrane-anchoring protein—Mod5p. This protein contains a prenylation site (CaaX motif) responsible for its plasma membrane localization 19 (Snaith and Sawin, 2003). Coincident with microtubule shrinkage, Tea1p is released from microtubules and stabilized at the plasma membrane in a Mod5p-dependnent manner. Once in the cell tip, Tea1p functions to regulate cell polarity by recruiting other polarity factors (Feierbach et al., 2004). This model for microtubule regulation of cell polarity in fission yeast is shown in the following cartoon (Chang and Peter, 2003). 20 Role of microtubules in regulating cell polarity in fission yeast. a, Tea1p binds to the growing plus end of MTs (green) in a Tip1p and Tea2p dependent manner. b, Tea1p are released to the cell tip when MT has shrunk back. c, Tea1p recruits a large protein complex of cell polarity factors to the cell tips. d, The complex includes For3p and Bud6p, which function to assemble actin cables (red) at the cell tip. 21 1.2.4.1.2.2 Controlling central localization of nucleus The interphase microtubules are also involved in positioning the nucleus at the cell center, which in turn provides a critical cue for depositing the components of the cell division plane later mitosis (Tran et al., 2001). Since minus ends of interphase microtubules are attached to the nuclear envelope, transient forces are transduced back to the nucleus where the minus ends of microtubules are anchored, hence pushing the nucleus to move away from the cell tips (Tran et al., 2001). Owing notably to the symmetrical longitudinally arrangement of microtubules in S. pombe cells, a balance of the pushing forces is achieved by the polymerizing microtubules, centering the nucleus between the two cell tips. Mutants with only one or two microtubule bundles often exhibit misplaced a nucleus due to the reduced ability in maintaining the balance of the pushing forces (as showed in my own data). As a consequence, the cell division plane in these mutants is often misplaced (Sato et al., 2004; Zimmerman and Chang, 2005). 1.2.4.2 Mitotic microtubule cytoskeleton 1.2.4.2.1 Architecture of mitotic microtubule cytoskeleton The interphase microtubules together with large iMTOCs are disassembled once cells enter mitosis. The duplicated SPBs separate and nucleate microtubules to form mitotic spindle, which is composed of three kinds of microtubules: kinetochore microtubules that emanate from SPBs to capture kinetochores, polar microtubules from opposite SPBs that overlap each other in the cell center, and astral microtubules that extend outward from the SPBs to the cell periphery (Ding et al., 1993). 22 To ensure faithful segregation of chromosomes, the kinetochore microtubules capture the kinetochores and eventually move the chromosomes to the metaphase plate (McCully and Robinow, 1971; Uzawa and Yanagida, 1992). Subsequently they shorten and pull the separated chromosomes to the poles. The polar microtubules then extend and slide apart from each other at the anti-parallel region to separate the genomes (for text book, see the molecular biology of Schizosaccharomyces pombe; Uzawa and Yanagida, 1992). At the end of anaphase, the mitotic spindle disassembles and the eMTOC appears at the medial cell division site to nucleate microtubules that form the PAA (Hagan, 1998). Microtubules are symmetrical at both side of the eMTOC with their plus ends reaching toward cell tips in two radial arrays. Eventually, as the eMTOC constricts, the nucleation sites of the PAA fuse together, causing microtubules to divide into two parts to each daughter cell (Zimmerman et al., 2004a). 1.2.4.2.2 Functions of mitotic microtubule cytoskeleton The function of mitotic spindle is to ensure efficient and high-fidelity chromosome segregation, which requires the concerted effort with the spindle assembly checkpoint machinery (Zhou et al., 2002). Before the transit from metaphase to anaphase, chromosomes need to be lined up in the center of the cell with both sister kinetochores attached by the microtubules from the two opposite SPBs (Zhou et al., 2002). As the spindle elongates, DNA masses are equally separated into two daughter cells. If the microtubules within the spindle fail to attach to the kinetochores, a component of the spindle check-point, Mad2p, will localize to the unattached kinetochore to prevent downstream protein separase from cleaving cohesin links between sister chromotins, thus 23 arresting cells in metaphase (He et al., 1997). Therefore, the order and the timing of dynamic changes in chromosomal morphology and behavior must be tightly controlled to coincide with the cytoskeletal changes, ensuring the faithful distribution of genome in both daughter cells. Changes in microtubule distribution and MTOCs during cell cycle progression in S. pombe are summarized in the following cartoon. 24 iMTO SPB eMTOC * * * * * * satellite * * * * Model of MTs and MTOCs organization throughout the cell cycle in fission yeast. During interphase, 4-5 MT bundles ran along the long axis of cells. One of them is nucleated by the SPB, the rest are nucleated from the iMTOCs. Upon mitotic entry, the interphase MT bundles disassemble and two SPBs (one of them is duplicated in interphase) separate to form the mitotic spindle, then spindle elongates. At the end of mitosis, the eMTOC appear in the middle of cell to nucleate the PAA. As the eMTOC constricts with the actomyosin ring, it breakdown to small pieces called satellites that can move along the MTs. Subsequently, cell divides into two daughter cells. 25 1.2.5 Self-organization of fission yeast microtubules Although it is well known that organization of microtubule network relies on MTOCs, interestingly, interphase microtubules are capable of self-organizing into ordered microtubule arrays in anucleated fission yeast cells (Carazo-Salas and Nurse, 2006; Daga et al., 2006). Anucleated cells were generated by centrifugation to displace nuclei and spindles during mitosis. Then the subsequent cell division proceeds normally and produces different daughter cells: one with two nuclei and one without nuclei. The microtubule bundles in the anucleated cells exhibit normal organization, dynamics and orientation with minus ends overlapping in the cell center in Mto1p and Ase1p, Klp2p and Tip1p-dependend manner. The discovery indicates that microtubules can be nucleated de novo without the MTOCs (Carazo-Salas and Nurse, 2006; Daga et al., 2006). Self-organizing ability of microtubules gives rise to further questions: How are microtubules in anucleated cells nucleated without MTOCs? What is the role of MTOCs during interphase? Are there any differences between the microtubules nucleated by the SPB and the iMTOCs or not? Although the microtubule-nucleating function of the MTOCs is a universally accepted concept, other functions are waiting to be further investigated. It is presently unknown how the iMTOCs occur in interphase S. pombe cells and how minus ends of microtubules are precisely attached to the MTOCs around the nuclear envelope. Below we will discuss one microtubule associated protein called— Mia1p (or Alp7p), a homologue of TACC family, which, as I have shown, functions in anchoring minus ends of microtubule at the nuclear envelope in interphase fission yeast. 26 1.3 Mia1p as a TACC-related protein Although the centrosome has fascinated cell biologists for over hundred years, the molecular mechanisms responsible for its functions remain unclear. Recently, a novel family of centrosomal proteins, the transforming acidic coiled-coil-related (TACC) proteins, has been identified in a wide variety of organisms. These closely related proteins are enriched in centrosomes. They have shown to be involved in regulating microtubule stabilization (Lee et al., 2001), translation (Stebbins-Boaz et al., 1999), acentrosomal spindle assembly (Cullen and Ohkura, 2001) and cancer progression (Still et al., 1999a). 1.3.1 TACCs in human The first “TACC” gene was identified in the process of studying human breast carcinomas. Amplification of one chromosome region, 8p11, was found in the tumor sample, resulting in cellular transformation and anchorage independent growth (Still et al., 1999b), characteristics of oncogenesis. The gene in this region was defined as TACC1, the first human TACC gene. Currently, the mammalian TACC family consists of at least three genes: TACC1, TACC2/AZU-1/ECTACC and TACC3/ARNT/ERIC1 (Still et al., 1999a; Sadek et al., 2000; McKeveney et al., 2001). Since over-expression of TACC1 might contribute to cancer and both TACC2 and TACC3 map to chromosomal regions that are disrupted in some cancers, TACC genes are suggested to play a role in oncogenesis. However, recently TACC2 has been identified as a potential tumor suppressor (Still et al., 1999a; Still et al., 1999b). How can TACC proteins function as both transforming and tumor-suppressing protein? What are 27 the normal functions of TACC proteins? A clue to this question came with the discovery that Drosophila TACC (D-TACC) is a centrosomal protein which is localized to the centrosome throughout the cell cycle (Gergely, 2000). 1.3.2 TACC in Drosophila Although only one TACC protein was found in Drosophila, studies in this system identified a partner of D-TACC protein, another centrosomal protein---Minispindles (Msps), making a breakthrough in understanding normal function of TACC protein (Lee et al., 2001). Msps belongs to the highly conserved XMAP215/ch-TOG family which is well known to stabilize microtubules by promoting their polymerization. Interestingly, these proteins are also enriched at the centrosome (Charrasse et al., 1998). D-TACC and Msps colocalize at the centrosome to stabilize microtubules in mitotic spindle (Lee et al., 2001). During female meiosis, Msps is delivered by the minus-end directed motor—Ncd kinesin (homologue of Klp2p) to the acentrosomal pole where DTACC functions to anchor Msps to stabilize minus ends of microtubules at the acentrosomal pole. Importantly, efficient localization of Msps to the centrosome requires D-TACC. A model of how cooperation between D-TACC and Msps contributes to stabilization of centrosomal microtubules during mitosis and the acentrosomal spindle in female meiosis is shown in the following cartoon (Gergely, et al.,2002). 28 Model of D-TACC and Msps function in Drosophila. During mitosis, D-TACC and Msps colocalize at the centrosome to stabilize centrosomal MTs in two ways. First, in additional to motor proteins that function to keep the release MTs focused at the spindle poles, D-TACC and Msps also stabilize the minus ends of the MTs that have been released from their nucleation sites. Second, the concentration of D-TACC and Msps at the centrosome ensures that they bind to the plus ends of the centrosomal MTs as they grow out from the centrosome. In this way, centrosomal MTs are stabilized by D-TACC and Msps. During meiosis, Msps is transported by the minus-end directed motor protein, Ncd, to the poles where it is anchored by D-TACC. Msps then stabilizes MT ends at the acentrosomal spindle poles. 29 Family members of TACC proteins contain a homologous coiled-coil region at their C terminus that is highly conserved throughout evolution not only on the structural but also on the amino acid sequence level. Previously, no plant or yeast TACC homologues have been found by BLAST searches (Gergely, 2002). However, a microtubule associated protein, Mia1p, was revealed as a coiled-coil protein which is predicted to be the fission yeast homologue of TACC, as a functional and binding partner of Alp14/ch-TOG (Sato et al., 2004). 1.3.3 Current knowledge about Mia1p in S. pombe The gene Mia1+ (ORF SPAC890.02, allelic to alp7+) was identified through a visual screen for temperature-sensitive mutations showing microtubule and spindle pole body abnormalities in the fission yeast. The Mia1+ open reading frame encodes a protein of 474 amino acids with a predicted relative molecular mass of 53,100 (Mr 53.1 KDa). The name of Mia1p (microtubule associated protein 1) came from its ability to associate with microtubules. In interphase fission yeast cells, Mia1p-GFP was found to localize to microtubule organizing centers (MTOCs) and along microtubules, and was concentrated in microtubule overlap regions. During mitosis, Mia1-EGFP was observed on both SPBs and along the length of the metaphase and early anaphase spindles (Oliferenko and Balasubramanian, 2002). Translocation of Mia1p from the cytoplasm to the nucleus is a Ran-dependent process (Sato and Toda, 2007). Mia1 is not an essential gene. Cells lacking Mia1p are viable but exhibit multiple microtubule abnormalities: first, astral microtubules are either diminished from both SPBs or emanate from only one of the SPBs (Oliferenko and Balasubramanian, 2002). 30 Second, spindle assembly checkpoint, Mad2p-GFP, localized to the kinetochores in mia1Δ cells, indicating microtubules couldn’t properly attach to the kinetochores (Sato et al., 2003). Consequently, cells were arrested at the metaphase stage (Oliferenko and Balasubramanian, 2002). This could properly due to the defects either in kinetochore attachment (Sato et al., 2003) or in the interaction between astral microtubules with actomyosin ring (Oliferenko and Balasubramanian, 2002). These data suggest that Mia1p was required for both astral microtubule function and bipolar spindle formation. Later, it was found that the functions of Mia1p in organizing bipolar spindle required its partner Alp14/TOG (Sato et al., 2004). They form a complex that does not fluctuate during the cell cycle. Both Mia1p and Alp14p localize along the cytoplasmic microtubules during interphase. Once cells enter mitosis, this complex is transported into the nuclei where they colocalize to the SPBs and mitotic spindle to organize bipolar spindles (Sato and Toda, 2007). The SPB and mitotic spindle localization of Alp14 is fully dependent on Mia1p. Conversely, in the absence of Alp14, Mia1p localizes to the SPBs but not mitotic spindle (Sato and Toda, 2007). This complex formation between TOG and TACC is evolutionarily conserved (Lee et al., 2001). Being a TACC-related protein, the role of Mia1p in fission yeast is characterized through addressing the relationship with Alp14p in mitosis, but functions of Mia1p in supporting microtubule dynamics are still poorly understood. One obvious defect of mia1Δ cells is the presence of only one or two long curved microtubule bundles during interphase. My work during my Ph.D. project has concentrated on uncovering the molecular functions of Mia1p and understanding how it might be involved in supporting microtubule dynamics. 31 1.4 Aim and objectives of this thesis Microtubule-organizing centers (MTOCs) concentrate microtubule nucleation, attachment and bundling factors and thus restrict formation of microtubule arrays in a spatial and temporal manner. Isolation and characterization of mutants that are defective in microtubule dynamics have significantly contributed to the understanding of the mechanism of the MTOCs in organizing the microtubule cytoskeleton. However, how MTOCs form remains an exciting question in cell biology. The aim of this study is to investigate the role of MTOCs in organizing the microtubule cytoskeleton in S. pombe using genetic, cellular and molecular biology means. Mia1p, a TACC-related protein, is known to be a microtubule-associated protein. Cells lacking Mia1p exhibit an aberrant interphase microtubule cytoskeleton similar to the mutants of γ-TuRC components: reduced number of microtubule bundles with existing longer microtubules curved around the cell tips. The objective of this thesis was to examine whether Mia1p has roles in supporting microtubule dynamics. 1.5 Significance of this study: Although a lot of γ-TuRC components have been identified to function in establishing anti-parallel linear microtubule array, how MTOCs form remains poorly understood. In this study, we attempt to investigate the role of Mia1p in forming an ordered interphase microtubule network and the establishment of the iMTOCs during interphase in S. pombe cells. We anticipate that our studies will shed light on novel aspects of MTOCs assembly and function. 32 [...]... proteins are involved in this process One of them is the minus-end-directed protein—Klp2p 1 .2. 3.1 Minus-end-directed motor protein—Klp2p Klp2p is a kinesin of the KAR3 subfamily in fission yeast In mitotic cells, it localizes to kinetochores to regulate elongation of the anaphase spindle and to control appropriate disassembly of the spindle at the completion of mitosis (Troxell, 20 01) During interphase,... how MTOCs form remains an exciting question in cell biology The aim of this study is to investigate the role of MTOCs in organizing the microtubule cytoskeleton in S pombe using genetic, cellular and molecular biology means Mia1p, a TACC- related protein, is known to be a microtubule- associated protein Cells lacking Mia1p exhibit an aberrant interphase microtubule cytoskeleton similar to the mutants of. .. functions of Mia1p in supporting microtubule dynamics are still poorly understood One obvious defect of mia1Δ cells is the presence of only one or two long curved microtubule bundles during interphase My work during my Ph.D project has concentrated on uncovering the molecular functions of Mia1p and understanding how it might be involved in supporting microtubule dynamics 31 1.4 Aim and objectives of this... cargo of Klp2p to be delivered to the minus end of mother microtubule In the absence of Klp2p, sliding of newly nucleated microtubules on pre-existing microtubules is compromised, leading to defects in focusing of microtubules at the overlapping regions (Carazo-Salas et al., 20 05) This suggests that Klp2p functions as a microtubule slider to transport newly nucleated microtubules to the overlapping region,... attachment and bundling factors which coordinate each other in effectively organizing interphase microtubules into ordered anti-parallel linear arrays Recent studies in MTOCs discover more and more components of the γ-TuRC Their functions in organizing microtubules have been characterized, providing more clues and leading to form a model of organizing the microtubule bundles (Carazo-Salas and Nurse, 20 07)... plays a critical role in maintaining cell morphology by binding to the plus ends of growing interphase microtubules in a Tip1p and Tea2p-dependent manner (Chang and Peter, 20 03) When the growing microtubules reach the cell tips and dwell there for a while, the microtubule plus end associated proteins such as Tea1p are released to the cell tips (Behrens and Nurse, 20 02) On the other hand, a complex at the... Firstly, microtubule nucleators, MTOCs, need to maintain the right amount of microtubules and localize them at precise sites Secondly, crosslinkers are required to bundle microtubules Thirdly, motor proteins mediate microtubule sliding within the microtubule bundles to facilitate the maintenance of microtubule polarity (Carazo-Salas and Nurse, 20 07) In interphase S pombe cells, in addition to microtubules... of this thesis Microtubule- organizing centers (MTOCs) concentrate microtubule nucleation, attachment and bundling factors and thus restrict formation of microtubule arrays in a spatial and temporal manner Isolation and characterization of mutants that are defective in microtubule dynamics have significantly contributed to the understanding of the mechanism of the MTOCs in organizing the microtubule cytoskeleton... (Busch and Brunner, 20 04), other factors that regulating microtubule plus-end dynamic need to be loaded at the microtubule minus ends during nucleation: One sample is kinesin Teap2p that is loaded onto the microtubules in close proximity to the nucleus and then travels using its intrinsic motor activity primarily at the tips of polymerizing microtubules (Browning et al., 20 03; Sawin and Tran, 20 06)... contributing to maintenance of the antiparallel linear microtubule arrays in interphase cells 1 .2. 3 .2 Bundling protein—Ase1p Another protein which functions in organizing antiparallel microtubule arrangement is Ase1p The yeast Ase1p belongs to the conserved ASE1/PRC1/MAP65 14 family of microtubule bundling proteins found at the mitotic spindle midzone (Chan et al., 1999) Ase1p functions in both interphase and . the minus end of mother microtubule. In the absence of Klp2p, sliding of newly nucleated microtubules on pre-existing microtubules is compromised, leading to defects in focusing of microtubules. center and plus-ends facing toward the cell tips. Model of maintaining ordered microtubules (from Sawin and Tran, 20 06) is shown in the following cartoon. 17 Model of interphase microtubules. precise origin of the iMTOCs remains elusive. 1 .2. 1 .2. 2 Equatorial microtubule organizing center (eMTOC) At the end of anaphase, the mitotic spindle breaks down and microtubules originate from