Roles of TACC related protein, mia1p in MTOCs and microtubule dynamics in schizosaccharomyces pombe 5

Chapter IV Discussion In the first part of my study, I investigated roles of the TACC-related protein, Mia1p, in organizing interphase microtubule arrays. Subsequently, I proposed and tested a dynamic model of iMTOCs assembly at the nuclear envelope (NE) in interphase fission yeast cells. 4.1 Comparison between mia1Δ cells and other γ-TuRC mutants Mutant phenotypes exhibiting abnormalities in microtubule cytoskeleton organization can be divided into several classes. First, as the length of microtubules is determined by the plus end stabilizing factors, in the absence of those proteins (i.e, Mal3p and Tip1p), microtubules undergo premature catastrophes when they touch cell cortex, producing shorter interphase microtubules (Beinhauer et al., 1997; Brunner and Nurse, 2000). Second, another class of mutants, including mutations in γ-tubulin (Gtb1p) (Paluh et al., 2000), the core γ-TuRC components Alp4p and Alp6p (Vardy and Toda, 2000; Zimmerman and Chang, 2005), or γ-TuRC accessory proteins Mto1p (Sawin et al., 2004; Venkatram et al., 2004; Zimmerman and Chang, 2005) and Mto2p (Janson et al., 2005; Samejima et al., 2005; Venkatram et al., 2005), exhibit fewer microtubule bundles than usual, that normally curve around cell tips. I and others found that mia1Δ cells often displayed bent shape (Radcliffe et al., 1998; Oliferenko and Balasubramanian, 2002). My studies have shown that interphase mia1Δ cells possessed a decreased number of interphase microtubule bundles (see Figure 3.1.1.2A), typical of the γ-TuRC–related phenotypes. However, unlike previously 105 described mutants, cells lacking Mia1p were not overly deficient in microtubule nucleation and bundling (see Figure 3.1.3.1A&B). Indeed microtubules could be nucleated from around the NE, both in steady state and upon experimental perturbations of microtubule cytoskeleton. 4.2 Role of Mia1p in microtubule attachment but not in microtubule nucleation MTOCs concentrate microtubule nucleation, attachment and bundling factors that cooperate to organize ordered microtubule arrays. As a component of the MTOCs (see Figure 3.1.1.1B), Mia1p does not appear to be an integral component of the nucleating complex based on our observation of microtubule dynamics either in interphase or in mitotic cells. For example, microtubules could be nucleated from the NE in mia1Δ cells upon MBC treatment and wash-out experiments (see Figure 3.1.3.2B) and there were no persistent nucleation sites in dividing mia1Δ cells, although microtubules could be nucleated from the eMTOC (see Figure 3.1.5.1C). Furthermore, the finding that new microtubules nucleated by the satellites on the preexisting interphase microtubules were capable of bundling together with mother microtubules in mia1Δ (see Figure 3.1.3.1A&B), indicating that Mialp is not required for the bundling process. Thus, I proposed that the main abnormality in mia1Δ cells could be a faulty attachment of microtubule minus ends to the nucleation sites (see Figure 3.1.2.1A&B2). I detected instances of lateral loss of microtubule bundles from the NE and the SPBs (see Figure 3.1.2.1A), and microtubule ejection events when minus ends of nucleated microtubules were displaced from the NE before antiparallel bundling (see Figure 3.1.3.1A) 106 Therefore, I identified Mia1p as a protein responsible for anchoring of minus ends of microtubules to the nucleation sites at the NE. 4.3 Microtubule attachment sites serve as MTOCs to establish microtubule architecture Currently, “iMTOCs” is still a contentious definition. Some researchers refer to iMTOCs as “satellites” of γ-tubulin complex proteins (Janson et al., 2005). In my studies, the larger iMTOC structures at the NE are best revealed upon depolymerization of microtubules. I prefer to view them as functional entities that represent the sites of attachment of microtubule bundles to the NE. One of the more obvious physiological functions for these structures could be ensuring the proper centering of nuclei (Tran et al., 2001). While nucleation of microtubules can occur elsewhere, either on pre-existing microtubules or in cytosol, the resulting microtubules slide towards the nucleus in a Klp2p-dependent manner (Carazo-Salas et al., 2005; Janson et al., 2005) and are likely captured by the NE attachment sites. These attachment sites are thus likely to serve as dominant microtubule-organizing centers contributing to the establishment and maintenance of overall microtubule architecture. In mia1Δ cells, the γ-TuRC components fail to assemble detectable iMTOCs’ structures. Hence, the nuclei are misplaced and the overall microtubule configuration is disrupted in the absence of proper organizing centers in the cells lacking Mia1p, resulting in altered polarity of microtubules as judged by localization of Tea1p and Klp2p (see Figure 3.3.2B&D). Without stabilizing minus ends of microtubule at the NE, bundling and sliding of daughter microtubules still occur (see Figure 3.1.3.1A and 3.3.2D) but the 107 overall architecture of microtubule arrays is disturbed. Thus, Mia1p function in microtubule anchoring is a prerequisite for establishing the anti-parallel interphase microtubule arrays. 4.4 Establishment of the iMTOCs requires microtubule attachment to the NE It is known that appearance of the iMTOCs is linked to the disassembly of the eMTOC, but the precise origin of the iMTOCs is poorly understood. I was curious to understand how detachment of minus ends of microtubules resulted in the absence of detectable iMTOC structures in mia1Δ cells. I proved the role of microtubules in establishing the iMTOCs in two ways: When microtubules were absent in dividing mother cells by either introducing nda3-KM311 genetic background or treating cells with microtubule depolymerizing drug MBC, I could not detect the interphase MTOCs in daughters (see Figure 3.1.7.2A&B). On the other hand, when I allowed microtubule polymerization in daughter cells with no previous history of the interphase MTOCs, these structures readily appeared (see Figure 3.1.7.2C). Thus, I concluded that microtubule attachment to the NE was essential for iMTOCs assembly and proposed a model of establishment of the iMTOCs where γtubulin complexes distributed initially at the NE to nucleate microtubules. These attached microtubules provide tracks for delivering additional γ-tubulin complexes to the nucleation sites to aggregate prominent iMTOC structures. Thus, the resulting large structures could serve as dominant microtubule-organizing centers. In mia1Δ cells, although initial distribution of γ-tubulin complexes allowed microtubule nucleation from the NE, microtubule bundles disassociated from their nucleation sites, leading to a defect 108 in ability of γ-tubulin complexes to coalescence into large NE-bound MTOCs (see Figure 3.1.7.1). 4.5 Relationship between Mia1p and Alp14p Mia1p belongs to a family of centrosomal proteins, the transforming acidic coiled-coil-related (TACC) proteins characterized by the presence of a C-terminal TACC domain (Gergely, 2002). TACC family members usually associate with the XMAP215/TOG family of microtubule-stabilizing proteins. In fission yeast, Mia1p was revealed as the coiled-coil protein that is predicted to be the homologue of TACC. 4.5.1 Mia1p interacts with Alp14p but not Dis1p In fission yeast cells, two TOG/XMAP215 homologues, Alp14 and Dis1, have been identified (Sato et al., 2004). Both of them localize to interphase microtubule bundles, the SPBs and kinetochores during mitosis. They are required for microtubule stability during interphase and mitosis (Nabeshima et al., 1995; Garcia et al., 2001; Nakaseko et al., 2001). Mia1p is found to be required for specific Alp14p localization to both interphase microtubule bundles and the mitotic spindle. Conversely, in the absence of Alp14p, Mia1p localizes only to the SPB, but not to the spindle or kinetochore vicinity. However, localization of Dis1p is independent on Mia1p and vice verse (Sato et al., 2004). This indicates that Alp14p and Dis1p associate with microtubules in different manners and only Alp14p but not Dis1p functions as a binding partner of Mia1p. 109 4.5.2 Different aspect of Mia1p and Alp14p functions in microtubule attachment to the NE It was reported that the temperature-sensitive allele of Alp14p caused shortening of interphase microtubules (Sato et al., 2004), which is consistent with its function in regulating microtubule stability. I found that microtubules remained attached to the SPBs in alp14Δ cells (see Figure 3.1.2.2A), unlike in mia1Δ genetic background. However, it is possible that lack of SPB oscillations in alp14Δ cells could mask the potential detachment phenotype (see Figure 3.1.2.2B). Detailed studies of the microtubule cytoskeleton in alp14Δ cells will require development of additional tools because only very low levels of a-tubulin expression are tolerated in this genetic background. In principle, it is possible that Mia1p could function in microtubule attachment to the nucleation sites independently of Alp14p, because Mia1p/Alp7p directly interacts with microtubules (see the microtubule overlay assay, Figure 3.1.5.1SA), and localization of Mia1p/Alp7p to the SPBs does not require Alp14p (Sato et al., 2004). However, future studies are needed to address the functional contributions of these two proteins in organizing interphase microtubule arrays. 4.5.3 Lack of Mia1p and Alp14p result in failure of iMTOCs establishment Another striking aspect of Mia1p function has been illuminated by lack of large interphase MTOCs in mia1Δ cells. Although microtubule nucleation does occur at several sites around the NE (see Figure 3.1.3.2B) and also along the pre-existing microtubules (see Figure 3.1.3.1A) of mia1Δ cells, the γ-tubulin complexes are not organized into discernible structures, unlike in the wild-type case (see Figure 3.1.5.2). It could be either 110 due to their inability to load on microtubules or due to the general scarcity of microtubules in this genetic background. Interphase alp14Δ cells have very few and shorter microtubule bundles compared with wild type cells (see Figure 3.1.2.2A). Interestingly, alp14Δ cells exhibited a strong diffuse signal around the NE, in addition to its SPB localization (see Supplementary Figure 4.5.3). It is also observed that microtubule bundles always associate with the SPB in alp14Δ cells although the SPB remains stationary (see Figure 3.1.2.2). Thus it is likely that the lack of iMTOC structures in alp14Δ cells is not due to microtubule dissociation from the SPB as in the mia1Δ cells. One possible explanation was that loading of γ-TuRC components onto the microtubule bundles is compromised in alp14Δ cells and no additional γ-TuRC component could be delivered to the NE for iMTOC formation. 4.6 De novo assembly of the MTOCs is not specific to S. pombe cells Our model proposed that self-organization of γ-tubulin containing material into large MTOCs could facilitate efficient nucleation, bundling and intracellular positioning of cytoskeletal arrays. However, this positive feedback loop would be disrupted in mia1Δ cells due to lack of microtubule attachment to the nucleating sites leading to a defect in γ-tubulin complexes’ coalescence into larger iMTOCs (see Figure 3.1.7.1). Non-centrosomal MTOCs have been reported in many systems ranging from the nuclear surface of higher plants (Stoppin et al., 1994) to the membranes of animal cells (Keating and Borisy, 1999; Rios et al., 2004). Thus, a common self-organizing mechanism could ensure emergence of the membrane-bound MTOCs. Our model also suggested that one or more minus end directed motors transporting γ-tubulin satellites 111 and microtubules nucleated elsewhere could be involved in the process of MTOC assembly. It was recently shown that the Kar3-type kinesin, Klp2p, is involved in sliding of microtubules towards the cell center along preexisting microtubules and focusing of microtubule arrays near the nucleus (Carazo-Salas et al., 2005). Evidence from other systems suggests that the minus end directed transport of the microtubule nucleating machinery contributing to the organization of MTOCs might be a universal occurrence (Kubo et al., 1999; Young et al., 2000). Also, even though the fission yeast iMTOCs not contain centrioles, our findings could, in principle, be extended to explain the de novo establishment of centriole-containing centrosomes in animal cells. It was shown that centrosomes and centrioles could be assembled de novo in mammalian (Khodjakov et al., 2002) and Chlamydomonas (Marshall et al., 2001) cells. Interestingly, assembly of the pericentriolar material as single spots in mammalian cells depended on microtubules (Khodjakov et al., 2002). Similar dense assemblies of the pericentriolar material including γ-tubulin complexes appeared necessary for the birth of centrioles (Dammermann et al., 2004). 4.7 Role of Mia1p in modulating mitotic microtubule dynamics I also showed that S. pombe cells overexpressing the TACC-related protein, Mia1p, were capable of organizing spindle-pole like structures. While formation of intranuclear microtubule bundles in interphase S. pombe cells has previously been observed (Tange et al., 2004), overexpression of Mia1p led to assembly of bipolar and antiparallel spindles (see Figure 3.2.3B&F). These spindles exhibited a well defined spindle midzone and contained the γ-TuRC complexes and the BimC kinesin, Cut7p, at 112 acentrosomal spindle poles (see Figure 3.2.3B). Formation of this bipolar structure required the microtubule-bundling protein, Ase1p (see Figure 3.2.3H). Mia1p-GFP expressed at high levels localized to spindle poles where it formed large fluorescent structures and elsewhere in the cytosol (data not shown). Interestingly, upon overexpression of Mia1p, Ase1p-GFP aggregated at the spindle poles in addition to its normal localization to the spindle midzone (see Figure 3.2.3G). The γ-TuRC component, Alp4pGFP, also localized to these acentrosomal spindle poles (see Figure 3.2.3D) suggesting that they could indeed contain a host of microtubule-associated factors. The fact that some proteins required for proper spindle assembly might be sequestered to these abnormal structures could also explain the reason why the SPBs failed to separate in cells over-expressing Mia1p. TACC proteins contain extensive coiled coil regions and can likely form oligomers. This, in combination with their microtubule binding properties, could provide a framework for clustering and focusing of microtubule minus ends observed in vivo. I envisage that accumulation of the γ-TuRC complexes and microtubule-crosslinking proteins near the SPBs followed by subsequent sliding off through the force produced by the growing spindle could lead to the formation of acentrosomal spindle poles. 4.8Conclusion and future perspectives 4.8.1 Conclusion In summary, I use a combination of cell imaging, cell manipulation and genetics experiments in the fission yeast Schizosaccharomyces pombe, to show that the Transforming Acidic Coiled Coil protein, Mia1p, functions in sustaining proper MTOC 113 and microtubule dynamics, both in interphase and mitosis. Briefly, I show that Mia1p is required for microtubule attachment to the nucleation sites. Furthermore, when overexpressed, Mia1p organized ectopic spindle pole like structures that interfere with normal mitotic spindle assembly. 4.8.2 Future perspectives 4.8.2.1 Microtubules are nucleated by the iMTOCs and the SPB: are they different or similar? In interphase cells, the SPB is a special structure containing its specific proteins (i.e, Pcp1p and Sid2p), whereas the iMTOCs are aggregated by γ-TuRC components. However, the microtubule bundles nucleated by them seem quite similar with respect to both microtubule organization and microtubule dynamics. Interestingly, I found slight differences between microtubules nucleated by the SPB and by a diffuse γ-TuRC-rich material around the NE in mia1Δ cells. Microtubules emanated from the NE are unable to maintain their length after disassociation from nucleation sites and depolymerize from one end to the other, while microtubules initiated from the SPBs tend to maintain their length after detachment and will reattach to the SPBs (see Figure 3.1.2.1A and 3.1.3.2B&D).These data indicate that 1) the microtubule bundles nucleated by the SPBs are anti-parallel whereas those initiated from the iMTOCs are not. 2) The antiparallel configuration facilitates the stability of microtubule bundles. Thus, it raises the question as to why microtubule bundles nucleated by the SPBs tend to maintain their anti-parallel arrangement while those initiated from the iMTOCs are not. It is worth checking functions of microtubule bundling protein, Ase1p, in mia1Δ 114 cells. Unfortunately, Ase1p-GFP functions as a weak allele and is synthetic lethal with mia1Δ mutants, making it difficult to address this question. 4.8.2.2 Distribution of the iMTOCs at the nuclear envelope: random or specific? I proposed that absence of the iMTOCs in mia1Δ cells could result from a molecular defect of microtubule attachment to the nucleation sites (See Figure 3.1.7.1). In my model, the interphase MTOCs are dynamic structures that are established de novo in each cell cycle following the disassembly of the eMTOC. Firstly, nascent γ-tubulin satellites physically bind to the NE to nucleate microtubules. Once nucleated, the microtubules anchor to the NE and provide tracks for more satellites to be delivered to the minus ends of the microtubules. In this way, satellites can aggregate into larger structures that are capable of organizing more stable microtubules. Therefore, the original binding sites of satellites at the NE serve as the site where the iMTOCs localize. One interesting issue in this positive feed-back loop is the distribution of the initiate satellites around the NE: Is it determined by the geometry of the NE and the cell? How are the number and size of the iMTOCs determined in the fission yeast? 3D analyses of distribution of the iMTOCs around the NE in different cell geometry will provide more information to answer this question. 4.8.2.3 What are the partners of Mia1p in terms of anchoring microtubules to the NE? In fission yeast, anchorage of microtubule bundles to their nucleation sites around the NE is found to be important for establishing the persistent iMTOCs during interphase. 115 Since Mia1p is supposed to function in microtubule attachment to the nucleation sites independently of Alp14p (Sato et al., 2004), other proteins might function as partners of Mia1p in term of microtubule attachment. Interestingly, another striking phenotype I observed is that in mia1Δ cells, microtubules initiated from the SPBs were found to maintain their length after detachment and will reattach to the SPBs, while microtubules nucleated from other sites on the NE tend to depolymerize completely after microtubule dissociation, indicating that in addition to Mia1p, other protein(s) is/are function in parallel pathway to anchor microtubule to the SPB during interphase. One possible candidate involving in anchoring minus ends of microtubules to the nuclear surface might be the nuclear rim protein, Amo1p. Amo1Δ cells exhibit very similar phenotype compared with mia1Δ cells: They are bent and they have fewer microtubule bundles that curl around the cell ends (Pardo and Nurse, 2005). Although Mia1p was found to attach microtubules to the NE during interpahse, anchorage of microtubules to the nucleation sites does not depend on Mia1p during mitosis, since mitotic spindles were still capped by the SPBs although assembly of astral microtubules was compromised in mia1Δ cells. It is known that the anchoring of microtubules at the SPB during mitosis requires another fission yeast coiled-coil protein, Msd1p during mitosis (Toya et al., 2007). Although Msd1p was found to localize to the nuclei and around the NE during interphase, interphase function of Msd1p in fission yeast is poorly understood. Time-lapse analyses of microtubule dynamics are due to check whether anchorage of interphase microtubule bundles to the nucleation sites is compromised or not in amo1Δ and msd1Δ cells. Also, biochemical analyses including co- 116 immunoprecipitation and pull down assays can be performed to check whether these proteins interact with each other. 117 Fig 4.5.3 Localization of Mto1p-GFP in alp14Δ cells. Mto1p-GFP exhibits strong nuclear envelope staining, in addition to its SPB localization, in both steady state (left panel) and MBC-treated (right panel) alp14Δ cells. Shown is the maximum projection image of the z-stack obtained by epifluorescence imaging. 118 [...]... the original binding sites of satellites at the NE serve as the site where the iMTOCs localize One interesting issue in this positive feed-back loop is the distribution of the initiate satellites around the NE: Is it determined by the geometry of the NE and the cell? How are the number and size of the iMTOCs determined in the fission yeast? 3D analyses of distribution of the iMTOCs around the NE in different... more information to answer this question 4.8.2.3 What are the partners of Mia1p in terms of anchoring microtubules to the NE? In fission yeast, anchorage of microtubule bundles to their nucleation sites around the NE is found to be important for establishing the persistent iMTOCs during interphase 1 15 Since Mia1p is supposed to function in microtubule attachment to the nucleation sites independently of. .. proteins might function as partners of Mia1p in term of microtubule attachment Interestingly, another striking phenotype I observed is that in mia1Δ cells, microtubules initiated from the SPBs were found to maintain their length after detachment and will reattach to the SPBs, while microtubules nucleated from other sites on the NE tend to depolymerize completely after microtubule dissociation, indicating... dissociation, indicating that in addition to Mia1p, other protein(s) is/are function in parallel pathway to anchor microtubule to the SPB during interphase One possible candidate involving in anchoring minus ends of microtubules to the nuclear surface might be the nuclear rim protein, Amo1p Amo1Δ cells exhibit very similar phenotype compared with mia1Δ cells: They are bent and they have fewer microtubule bundles... cell ends (Pardo and Nurse, 20 05) Although Mia1p was found to attach microtubules to the NE during interpahse, anchorage of microtubules to the nucleation sites does not depend on Mia1p during mitosis, since mitotic spindles were still capped by the SPBs although assembly of astral microtubules was compromised in mia1Δ cells It is known that the anchoring of microtubules at the SPB during mitosis requires... yeast coiled-coil protein, Msd1p during mitosis (Toya et al., 2007) Although Msd1p was found to localize to the nuclei and around the NE during interphase, interphase function of Msd1p in fission yeast is poorly understood Time-lapse analyses of microtubule dynamics are due to check whether anchorage of interphase microtubule bundles to the nucleation sites is compromised or not in amo1Δ and msd1Δ cells... novo in each cell cycle following the disassembly of the eMTOC Firstly, nascent γ-tubulin satellites physically bind to the NE to nucleate microtubules Once nucleated, the microtubules anchor to the NE and provide tracks for more satellites to be delivered to the minus ends of the microtubules In this way, satellites can aggregate into larger structures that are capable of organizing more stable microtubules... analyses including co- 116 immunoprecipitation and pull down assays can be performed to check whether these proteins interact with each other 117 Fig 4 .5. 3 Localization of Mto1p-GFP in alp14Δ cells Mto1p-GFP exhibits strong nuclear envelope staining, in addition to its SPB localization, in both steady state (left panel) and MBC-treated (right panel) alp14Δ cells Shown is the maximum projection image of the... functions as a weak allele and is synthetic lethal with mia1Δ mutants, making it difficult to address this question 4.8.2.2 Distribution of the iMTOCs at the nuclear envelope: random or specific? I proposed that absence of the iMTOCs in mia1Δ cells could result from a molecular defect of microtubule attachment to the nucleation sites (See Figure 3.1.7.1) In my model, the interphase MTOCs are dynamic structures... envelope staining, in addition to its SPB localization, in both steady state (left panel) and MBC-treated (right panel) alp14Δ cells Shown is the maximum projection image of the z-stack obtained by epifluorescence imaging 118 . 1 05 Chapter IV Discussion In the first part of my study, I investigated roles of the TACC- related protein, Mia1p, in organizing interphase microtubule arrays. Subsequently, I proposed and. proteins in organizing interphase microtubule arrays. 4 .5. 3 Lack of Mia1p and Alp14p result in failure of iMTOCs establishment Another striking aspect of Mia1p function has been illuminated. Transforming Acidic Coiled Coil protein, Mia1p, functions in sustaining proper MTOC 114 and microtubule dynamics, both in interphase and mitosis. Briefly, I show that Mia1p is required for microtubule