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Vol. 17, Issue 5, 2212-2222, May 2006
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* Cell Dynamics Group, Temasek Life Sciences Laboratory, 117604 Singapore, Singapore;
Department of Biological Sciences, National University of Singapore, 117543 Singapore, Singapore; and
The Boulder Laboratory for 3-D Electron Microscopy of Cells, Department of MCD Biology, University of Colorado, Boulder, CO 80309
Submitted August 30, 2005;
Revised January 12, 2006;
Accepted February 6, 2006
Monitoring Editor: Tim Stearns
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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cells. Mia1p was required for stability of microtubule bundles and persistent use of nucleation sites both in interphase and postanaphase array dynamics. The 
-tubulin–rich material was not organized in large perinuclear or microtubule-associated structures in mia1 cells. Interestingly, absence of microtubules in dividing wild-type cells prevented appearance of large -tubulin–rich MTOC structures in daughters. When microtubule polymerization was allowed, MTOCs were efficiently assembled de novo. We propose a model where MTOC emergence is a self-organizing process requiring the continuous association of microtubules with nucleation sites. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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), formation of such arrays is temporally and spatially restricted in living cells where MTOCs concentrate microtubule nucleation, attachment, and bundling factors at specific locations. Thus, positioning of microtubule arrays within a cellular space represents an important aspect of MTOC function.
The fission yeast Schizosaccharomyces pombe is an excellent model for study of MTOC and microtubule dynamics. A relatively large cell size allows detailed dynamic observation of cellular components tagged with fluorescent proteins, its genetics is straightforward, and the genome is fully sequenced and annotated (Wood et al., 2002).
The cellular locations of MTOCs and consequently, architecture of microtubule arrays change in a cell cycle-specific manner (for review, see Hagan, 1998). The spindle pole bodies (SPBs) nucleate and organize spindle microtubules during mitosis. On mitotic exit, the equatorial MTOC (eMTOC) nucleates the postanaphase array (PAA), a specialized symmetrical microtubule structure extending from division site toward the tips of a dividing cell. The eMTOC constricts with the actomyosin ring and eventually disassembles at the end of septation. In interphase, the microtubule-nucleating activity is concentrated at the several nuclear envelope (NE)-bound interphase MTOCs (iMTOCs), the SPB, and satellite sites on microtubules (Sawin et al., 2004; Janson et al., 2005; Zimmerman and Chang, 2005). Establishment of the iMTOCs proceeds synchronously with the disassembly the eMTOC (Zimmerman et al., 2004), but the precise origin of the iMTOCs remains obscure.
A number of S. pombe mutants exhibit microtubule defects that are consistent with abnormalities in MTOC function, such as a decreased number of interphase microtubules and low frequency of microtubule nucleation events. In these cases, the existing microtubules are often longer, probably due to a larger pool of soluble tubulin and microtubule accessory factors. The majority of these mutations are in core components of -tubulin ring complexes (-TuRCs) (Paluh et al., 2000; Vardy and Toda, 2000; Zimmerman and Chang, 2005) or in -tubulin accessory factors (Sawin et al., 2004; Venkatram et al., 2004, 2005; Janson et al., 2005; Samejima et al., 2005; Zimmerman and Chang, 2005) and therefore show pronounced defects in microtubule nucleation.
The S. pombe mutant lacking the homologue of the transforming acidic coiled coil (TACC) protein Mia1p/Alp7p (thereafter referred to as Mia1p) shows multiple microtubule abnormalities. The previous studies concentrated on Mia1p functions in mitosis. It was shown that aster microtubules were either absent or unbalanced (Oliferenko and Balasubramanian, 2002), and the TOG family protein Alp14p was not loaded on spindles and the SPBs resulting in spindle abnormalities (Sato et al., 2004). Interestingly, interphase mia1 cells exhibit a reduced number of microtubule bundles that are longer than usual and curve around cell tips, suggesting a possible involvement of Mia1p in interphase MTOC function.
Here, we show that unlike previously described nucleation-deficient mutants, mia1 cells can and do nucleate microtubules. However, we find that Mia1p is required for sustaining the attachment of microtubules to nucleation sites and for emergence of the iMTOCs. We propose that Mia1p ensures the persistent use of nucleation sites and allows the existence of stable microtubule bundles. In light of our results, we suggest and test a dynamic model for MTOC assembly.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Genetic crosses and sporulation were performed on YPD agar plates. The homologous recombination-based method was used to tag endogenous Alp4p with green fluorescent protein (GFP) at its C terminus. The Mto1p-GFP and Rsp1p-GFP strains were gifts from Drs. K. Gould (Vanderbilt University, Nashville, TN) and F. Chang (Columbia University, New York, NY), respectively. For protein purification, Escherichia coli BL21-CodonPlus(DE3)-RIL competent cells (commercially available from Strategene, La Jolla, CA) was used. Dr. Y. Hiraoka (Kansai Advanced Research Center, Kobe, Japan) kindly provided us with the pREP1-
-tubulin-GFP construct. The anti--tubulin antibody, TAT-1, was a gift from Dr. K. Gull (University of Oxford, Oxford, United Kingdom). Polylysine used for coating was from Sigma-Aldrich (St. Louis, MO), and the microtubule-destabilizing drug methyl-1-(butylcarbamoyl)-2-benzimidazolecarbamate (Carbendazim; MBC) was from Aldrich Chemical (Milwaukee, WI).
Time-Lapse Fluorescent Microscopy
Time-lapse images were generated on a Zeiss Axiovert 200M microscope equipped with UltraView RS-3 confocal system: CSU21 confocal optical scanner, 12 bit digital cooled Hammamatsu Orca-ER camera (OPELCO, Sterling, VA), and krypton-argon triple line laser illumination source (488, 568, and 647 nm). Still images were collected on a Zeiss Axiovert 200M microscope using Cascade:650 camera (Photometrics/Roper Scientific, Trenton, NJ) and Uniblitz shutter driver (Photonics, Rochester, NY) under the control of MetaMorph software package (Universal Imaging, Sunnyvale, CA). For imaging of microtubule dynamics, S. pombe cells expressing -tubulin-GFP were grown in appropriate selective medium and placed in sealed growth chambers containing 2% agarose media. For three-dimensional time-lapse imaging, each image stack consisted of 13 sections of 0.5-µm spacing and 15-s intervals between stacks. For single-section time-lapse analyses, images were collected at 5-s intervals. Experiments were carried out at room temperature.
Live Cell Chamber Experiments
Poly-lysine (2 mg/ml) was used to fix cells (which were washed with EMM medium) on the Fisher cover glass with two parallel pieces of double-stick tape mounted on it. The flow chamber was created by overlaying the cover glass with a Matsunami coverslip. Flow-through could be achieved by adding medium on one side and absorbing the liquid from the opposite side by tissue paper. Microtubules were typically depolymerized by 50 µg/ml MBC.
Immunofluorescence Techniques
Cells were fixed with 3.7% formaldehyde and spheroplasted using lysing enzymes and Zymolyase in 1.2 M sorbitol in phosphate-buffered saline (PBS). Permeabilization was performed in 1% Triton X-100 in PBS. PBAL (1 mM sodium azide, 50 µg/ml carbenicillin, 1% bovine serum albumin, and 100 mM lysine hydrochloride in PBS) was used for blocking and for incubation with primary and secondary antibodies. Imaging was done on a Zeiss Axiovert 200M microscope with appropriate sets of filters, and images were generated using Cascade:650 camera and MetaMorph software. Image processing was done in Adobe Photoshop 7.0.
Electron Microscopy Techniques
Cells were rapidly frozen by high-pressure freezing (BAL-TEC HPM-010; Technotrade International, Manchester, NH) and freeze-substituted at –90°C in 0.2% glutaraldehyde plus 0.01% uranyl acetate in acetone for 96 h in an EM-AFS device (Leica, Vienna, Austria). The cells were warmed over 25 h to –40°C and then infiltrated with HM20 (Electron Microscopy Sciences, Hatfield, PA) resin over a period of 5 d. The cells were embedded under UV light at –40°C in HM20 for 3 d and then warmed to room temperature over a 6-h period. Embedded cells were sectioned and immunostained as follows: sections on gold grids (Electron Microscopy Sciences) were floated on blocking buffer (0.02% Tween 20, 0.8% bovine serum albumin, and 0.1% fish gelatin in PBS) for 1 h, immunostained with anti-GFP antibodies overnight at 4°C as described previously, rinsed with PBS plus 0.1% Tween 20 three times, incubated with 10-nm gold goat-anti-rabbit secondary antibodies (BB International, Cardiff, United Kingdom) for 2 h at room temperature, rinsed and fixed with 1.0% glutaraldehyde for 5 min, stained with aqueous uranyl acetate and lead citrate, and imaged in a Philips Tecnai TF20 FEG electron microscope operating at 80 keV.
Mia1p/Microtubule Association Experiments
E. coli BL21 was used to express glutathione S-transferase (GST)-Mia1p. Pelleted bacterial cells were dissolved in the SDS-PAGE sample buffer, separated on SDS-PAGE gel, and then transferred to nitrocellulose filter and incubated in the PEMG buffer [100 mM piperazine-N,N'-bis(2-ethanesulfonic acid), pH 6.9, 2 mM EGTA, 1 mM MgSO4, and 1 mM GTP] containing 5% milk and then with taxol-stabilized bovine brain 3X microtubules (50 µg/ml) in the same buffer, followed by washing and immunoblotting with TAT1 anti-tubulin antibodies.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Interestingly, when microtubules were depolymerized by Carbendazim (MBC) treatment, Mia1p-GFP was visualized as several distinct dots around the NE (Figure 1B) colocalizing with -tubulin and short microtubule "stubs." Far Western analysis using prepolymerized taxol-stabilized microtubules suggested that the recombinant Mia1p-GST could physically associate with microtubules (Figure 1C).
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mia1 Cells Exhibit Disorganized and Heterogeneous Microtubule Bundles
Intensity scan measurements along the length of microtubule bundles in wild-type cells revealed distinct medial zones of overlap (Figure 2A) as published previously (Tran et al., 2001; Loiodice et al., 2005). These double intensity zones are usually positioned near the nucleus and often overlap with the iMTOCs. Using similar techniques, we found no evidence of well defined or properly positioned zones of microtubule overlap in mia1 cells (Figure 2B). Indeed, these measurements suggested extreme heterogeneity in architecture of microtubule bundles in the absence of Mia1p. Time-lapse analyses of microtubule and the SPB dynamics in mia1 cells revealed presence of free microtubules, not associated with antiparallel bundles and the nuclear surface (Figure 2C and Supplemental Movie 1). When encountering cell tips, microtubules often continued growth and curved around them (Figure 2C and Supplemental Movie 1). We also detected instances of the SPB detachment from the associated microtubule bundles (Figure 2C, star).
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mia1 Cells Are Defective in Attachment of Microtubules to the Nucleation Sites
To investigate the phenotypes described above, we followed the SPB behavior and microtubule dynamics in wild-type and mia1 cells expressing Sid2p-GFP and -tubulin-GFP. In wild type, the SPB periodically oscillated along the long axis around the geometric cell center (100%, n = 20 cells, observed for total of 3.5 h; see also Figure 3A and Supplemental Movie 2). This behavior is due to the balanced pushing forces that are exerted by plus ends of microtubules arranged in antiparallel bundles anchored at the nuclear surface (Tran et al., 2001). When microtubules in wild-type cells were depolymerized by MBC, the SPB remained stationary (Figure 3C). In mia1 cells, the SPB was either oscillating on much broader scale or abruptly stopped for periods of time (Figure 3B and Supplemental Movie 3). We found that the SPB laterally detached from the associated microtubule bundle during the periods of rest (26%, n = 19 cells, observed for a total of 9.5 h; also see Figure 3B and Supplemental Movie 3).
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Sid2p-GFP -tubulin-GFP cells (100%, n = 20 cells, observed for total of 3 h) (Supplemental Figure 1A and Supplemental Movie 4). However, we found that the SPBs hardly oscillated, suggesting that microtubules did not exert sufficient pushing force (Supplemental Figure 1B).
Time-lapse analyses of microtubule dynamics in -tubulin-GFP expressing mia1 cells showed instances of microtubule nucleation at the NE (as shown in Figure 3D and Supplemental Movie 5). In this case, a growing microtubule was ejected from the nucleation site and continued to grow at its plus end, pushing the stable minus end across the cell (Figure 3, D–F). Subsequently, we observed a second nucleation event, as judging by a sudden increase in fluorescence intensity in the vicinity of the minus end (as marked by a cross in Figure 3, D and E). This nucleation event was followed by antiparallel bundling, and the resulting bundle now grew at both plus ends (Figure 3, E and F).
Our analyses so far suggested that nucleation and bundling could occur in mia1 cells. However, Mia1p was required for microtubule attachment to the nucleation sites.
Mia1p Is Not Required for Microtubule Nucleation but Is Required for Maintenance of Microtubule Bundles
We followed -tubulin-GFP–expressing wild-type and mia1 cells and performed fast (every 5 s) time-lapse analyses of single focal planes. We found that in wild-type cells, microtubule bundles were relatively stable, and we rarely observed the disappearance of preexisting bundles (1.2 events per bundle per hour, n = 8, total time = 3.5 h) (Figure 4A and Supplemental Movie 6). However, in mia1 cells the lifetime of microtubule bundles was significantly reduced (bundles disappeared with the rate of 13.6 events per bundle per hour, n = 10, total time = 3.5 h) (Figure 4B and Supplemental Movie 7). We also found that microtubule nucleation in wild-type cells was mainly restricted to preexisting MTOCs (we observed only 4.7 de novo nucleation events per cell per hour, n = 6, total time = 2.5 h). In contrast, microtubules were frequently nucleated from different sites around the NE in the absence of Mia1p (23 events per cell per hour, n = 11, total time = 3.3 h).
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cells. We decided to perturb the microtubule cytoskeleton by treating cells with MBC and observe microtubule dynamics upon washout of the drug. We performed time-lapse analyses of -tubulin-GFP Sid2p-GFP–expressing wild-type and mia1 cells in a perfusion chamber mounted on a microscope stage. In wild-type cells, microtubules rapidly depolymerized upon addition of 50 mg/ml MBC (within 45 s), leaving stable microtubule "stubs" around the NE (Figure 4C and Supplemental Movie 8). When the drug was washed out with fresh medium, microtubules quickly repolymerized from these "stubs" in a bidirectional manner (Figure 4C and Supplemental Movie 8). MBC-induced depolymerization of microtubules in mia1 cells proceeded somewhat slower (
90 s), but eventually most microtubules disassembled (Figure 4D and Supplemental Movie 9). On washout with fresh medium, we observed microtubule nucleation from several sites around the NE (Figure 4D and Supplemental Movie 9). These microtubules could eventually detach, and we documented one of them undergoing catastrophe and shrinking across the cell body exhibiting a plus-end depolymerization rate of 12 mm/min (later time points in Figure 4D and Supplemental Movie 9).
We confirmed that microtubule polymerization could be initiated from the nuclear surface by returning ice-treated wild-type and mia1 cells expressing the NE marker Uch2p-GFP and -tubulin-GFP to the room temperature and performing the fluorescent time-lapse analyses of microtubule regrowth. We found that microtubules could be polymerized from the NE in both wild-type (Supplemental Figure 2A and Supplemental Movie 10) and mia1 (Supplemental Figure 2B and Supplemental Movie 11) cells. However, microtubules eventually detached from the NE in cells lacking Mia1p (later time points in Supplemental Figure 2B and Supplemental Movie 11).
Mia1p Is Required for Sustaining Proper Postanaphase Array Dynamics
Dividing fission yeast cells assemble a specialized microtubule arrangement at the division site, called PAA. Microtubules are nucleated symmetrically at the eMTOC with their plus ends reaching toward mother cell tips. eMTOC constricts together with the actomyosin ring and eventually disassembles. We found that Mia1p-GFP localized in the vicinity of the eMTOC both on light microscopy (Figure 5A) and ultrastructural (Figure 5B) levels. We thus set out to examine whether any aspects of the eMTOC/PAA dynamics could be affected in mia1 cells. -Tubulin-GFP–expressing wild-type cells started assembling the medial microtubule foci in late anaphase (Figure 5C, marked with stars in 1'30" time point, and Supplemental Movie 12). These foci persisted and eventually fused into a single ringlike structure that constricted together with the actomyosin ring (Figure 5C and Supplemental Movie 12). We tracked paths of these focal structures over the course of cell division and combined them on a single superimposed image (Figure 5C, C'). Our analysis confirmed that once assembled, these microtubule-nucleating structures remained fairly stable. In contrast, although we detected microtubule nucleation events in the medial region of mia1 cells (Figure 5D and Supplemental Movie 13), the resulting microtubule structures were unstable (Figure 5D and Supplemental Movie 13; note the ejection event of a focal structure between 1 min 30 s and 2 min, marked with arrow). Tracking analysis suggested that each structure had a fairly short lifetime and that cells proceeded initiating multiple nucleation events from different regions of the eMTOC (Figure 5D, D'). This behavior resulted in a poorly organized PAA and a precocious loss of microtubules from the medial region (Figure 5D and Supplemental Movie 13).
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cells was comparable with wild-type cells (Figure 5E). The eMTOC constriction and disappearance of the equatorial ring material proceeded similarly in mia1 and wild-type cells (our unpublished data). We concluded that although Mia1p was not required to localize the -tubulin complexes to the actomyosin ring, it was necessary to sustain the continuing use of nucleating sites through the course of cell division.
There Are No Detectable -Tubulin–rich Interphase MTOC Structures in Cells Lacking Mia1p
Because microtubules could be nucleated from around the NE in mia1 cells, it could be expected that Mia1p executed its function downstream of MTOC components and that those localized normally in the absence of Mia1p. In steady-state interphase, wild-type cells Mto1p-GFP and Alp4p-GFP localized to the SPB and to several bright dots around the nucleus and along microtubules (Figure 6A), likely representing the MTOCs. In mia1 cells, we normally detected only the SPB signal together with very faint and even staining along microtubules and around the NE in Mto1p-GFP (Figure 6A).
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-tubulin (Figure 1B). We did not detect iMTOCs as visualized by these markers in MBC-treated mia1 cells; instead, they only localized to the SPB (Figure 6B).
Because Alp14p was shown to interact with Mia1p (Sato et al., 2004), we addressed the question of -tubulin complex distribution in cells lacking this protein. Interestingly, Mto1p-GFP in alp14 cells exhibited a strong diffuse signal around the nuclear envelope, in addition to its SPB localization, both in steady-state cells and upon pretreatment with MBC (Supplemental Figure 3, top). This was in marked contrast with cells lacking a distinct microtubule-stabilizing protein, Mal3p (Beinhauer et al., 1997), where Mto1p-GFP could be found in bright puncta around the NE, similar to the wild-type situation (Supplemental Figure 3, bottom).
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cells, they were not organized into discernible iMTOC structures, probably reflecting the lack of microtubule/NE attachment sites. Thus, it seemed that mia1 cells were deficient in establishing the iMTOC structures at the NE.
Microtubules Are Required for Emergence of Interphase MTOC Structures Upon Entry into the New Cell Cycle
We hypothesized that lack of large MTOC structures was related to the deficient microtubule anchorage in the absence of Mia1p. There is a considerable amount of microtubule-nucleating material traveling along microtubules in both plus- and minus-end–directed manner, and it was recently proposed that -tubulin complexes nucleate new microtubules as they travel along the preexisting microtubules (Janson et al., 2005). These microtubules could later slide toward the cell center (Carazo-Salas et al., 2005) and eventually get anchored at the NE. We proposed a model where fluctuations in initial distribution and/or activity of -tubulin complexes are positively reinforced when additional material is delivered to the nucleating sites via attached microtubules (Figure 7D). Thus, the resulting large structures could serve as dominant microtubule-organizing centers. This positive feedback loop would be disrupted in mia1 cells due to the lack of microtubule attachment to the nucleation sites, leading to a defect in -tubulin complexes' coalescence into large NE-bound MTOCs (Figure 7D).
This model predicts that emergence of interphase MTOCs in each cell cycle should depend on microtubules. We made use of the cold-sensitive 
-tubulin mutation nda3-KM311 and followed the fluorescent MTOC marker Mto1p-GFP through the cell cycle. To allow cell division, we introduced a deletion mutation of the spindle assembly checkpoint gene, mad2 (Li and Murray, 1991) into this genetic background. We found that when Mto1p-GFP mad2 nda3-KM311 cells underwent septation at the restrictive temperature of 18°C, only one daughter cell inherited the undivided nucleus. Mto1p-GFP was uniformly distributed around the NE and also localized to non-separated SPBs (Figure 7A; 85%; n = 66 cells). In contrast, upon division of control Mto1p-GFP mad2D cells, we detected distinct bright fluorescent structures around the NE of both daughter cells (Figure 7A; 95%; n = 62 cells). This experiment suggested that microtubules were required for enrichment of -tubulin complex material in distinct NE-bound MTOCs in each new cell cycle.
To follow this process in real time, we immobilized Mto1p-GFP–expressing wild-type cells in a perfusion chamber mounted on a fluorescence microscope and searched for cells completing mitosis where Mto1p-GFP was localized exclusively to the SPBs and the nascent eMTOC (Figure 7B). We depolymerized microtubules by perfusing medium containing 25 mg/ml MBC and observed the localization of Mto1p-GFP. We found that although eMTOC disassembly and septation proceeded normally, the majority of MBC-treated cells (75%; n = 38 cells) did not exhibit any noticeable MTOC structures around the nuclear envelope; instead, Mto1p-GFP localized exclusively to the SPB (Figure 7B). We also found that when MBC was added just after the onset of the eMTOC/actomyosin ring constriction, we could detect nascent MTOCs around the NE (Supplemental Figure 4), suggesting that iMTOC establishment proceeded simultaneously with the eMTOC disassembly.
Our model also assumes that as long as -tubulin complexes and microtubules are present in cells with no previous iMTOCs, these structures should be able to readily self-organize (Figure 7D). We prepared Mto1p-GFP cells lacking iMTOCs as described above (Figure 7B) and allowed microtubule polymerization by washing out MBC. We found that 20 min after MBC washout, 100% of cells (n = 20) exhibited Mto1p-GFP–positive aggregates at the nuclear envelope and spread along the microtubules. We then briefly depolymerized microtubules in these cells to allow a better visualization of the fluorescent material at the NE (Figure 7C). We concluded that -tubulin complexes could efficiently self-organize into large interphase microtubule-organizing centers only in the presence of microtubules.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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There are several clusters of experimentally described mutant phenotypes leading to abnormalities in the fission yeast microtubule cytoskeleton. For example, shorter interphase microtubules might indicate deficiencies in microtubule-stabilizing factors (Beinhauer et al., 1997; Brunner and Nurse, 2000), and disorganized microtubules without obvious overlapping regions could result from the lack of bundling proteins (Loiodice et al., 2005). A separate class of mutants exhibits fewer microtubule bundles that on average are longer than usual. Examples include mutations in -tubulin (Gtb1p) (Paluh et al., 2000), the core -TuRC components Alp4p and Alp6p (Vardy and Toda, 2000; Zimmerman and Chang, 2005), or -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). Microtubule nucleation and therefore MTOC function are suppressed in cells lacking either of these factors.
Similarly, we found a decreased number of microtubule bundles in interphase mia1 cells (Figure 1, D and E). Microtubules were disorganized (Figure 2B) and curved around cell tips (Figures 1D and 2C); mia1 cells were often bent (Radcliffe et al., 1998; Oliferenko and Balasubramanian, 2002). However, unlike previously described mutants, cells lacking Mia1p were not deficient in microtubule nucleation and indeed nucleated microtubules, both in steady state (Figures 3, D and E, and 4B, and Supplemental Movies 5 and 7), and upon experimental perturbations of the microtubule cytoskeleton (Figure 4D, Supplemental Figure 2B, and Supplemental Movies 9 and 11). Indeed, the main abnormality in mia1 cells seems to be a faulty attachment of microtubule minus ends to the nucleation sites (Figures 2C, 3, B and D, Supplemental Figure 2B, and Supplemental Movies 1, 3, 5, and 11). We detected instances of lateral loss of microtubule bundles from the NE and the SPBs (Figures 2C and 3B), and microtubule ejection events when minus ends of nucleated microtubules were displaced from the NE before antiparallel bundling (Figure 3, D–F).
Mia1p is related to a large family of TACC proteins implicated in sustaining mitotic centrosome function and spindle integrity and dynamics. Misregulation of TACC gene expression has been linked to development of human cancers (for review, see Raff, 2002). The fruit fly D-TACC participates in spindle pole focusing and organization during both anastral meiotic (Cullen and Ohkura, 2001) and mitotic divisions (Lee et al., 2001). Lack of TAC-1 in one-cell embryos of Caenorhabditis elegans leads to very short, unstable astral and spindle microtubules (Bellanger and Gonczy, 2003; Le Bot et al., 2003; Srayko et al., 2003). TACC family members usually associate with the XMAP215 family of microtubule-stabilizing proteins, and this interaction is functionally significant: mutations in these genes to a certain extent phenocopy each other (Cullen and Ohkura, 2001; Lee et al., 2001; Bellanger and Gonczy, 2003; Le Bot et al., 2003; Srayko et al., 2003).
It was reported that temperature-sensitive allele of the fission yeast XMAP215 protein Alp14p caused shortening of interphase microtubules (Sato et al., 2004), which is consistent with its function in regulating microtubule stability. We found that microtubules remained attached to the SPBs in alp14 cells (Supplemental Figure 1A and Supplemental Movie 4), unlike in mia1 genetic background. However, it is possible that lack of SPB oscillations in alp14 cells could mask the potential detachment phenotype (Supplemental Figure 1B). Detailed studies of the microtubule cytoskeleton in alp14 cells will require development of additional tools because only very low levels of -tubulin expression are tolerated in this genetic background (our unpublished data). In principle, it is possible that Mia1p could function in microtubule attachment to the nucleation sites independently of Alp14p, because 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.
Another exciting aspect of Mia1p function has been illuminated by lack of large interphase MTOCs in mia1 cells. Although microtubule-nucleating activity is present at the NE (Figures 3D and 4D) and along microtubules (Figure 3, E and F) of mia1 cells, the -tubulin complexes are not organized into discernible structures, unlike in the wild-type case (Figure 6). Interestingly, we noticed that components of the -tubulin complex were enriched at the nuclear surface in alp14 cells (Supplemental Figure 3). It could be either due to their inability to load on microtubules or due to the general scarcity of microtubules in this genetic background. Interphase alp14 cells have very few microtubules, even compared with cells deficient in other microtubule-stabilizing protein, Mal3p (EB1) (Supplemental Figure 1 and Supplemental Movie 4; our unpublished data). It is worth mentioning that mal3 cells assembled functional nuclear-bound iMTOCs (Supplemental Figure 3), suggesting that lack of these structures in mia1 and alp14 cells did not result from general problems with plus end microtubule stability.
We proposed that absence of the iMTOCs in mia1 cells could result from a molecular defect of microtubule attachment to the nucleation sites (see model in Figure 7D). In our model, the interphase MTOCs are dynamic structures that are established de novo in each cell cycle after the disassembly of the eMTOC. First, microtubules are nucleated randomly due to variations in density or activity of nascent -tubulin complexes at the nuclear surface. However, when newborn microtubules remain anchored, more -tubulin complexes together with microtubules nucleated by them (Janson et al., 2005) can be delivered to the original sites of nucleation, resulting in growth of the nucleating structures and eventually restricting the MTOC number. This positive feedback loop would be disrupted in mia1 cells due to lack of microtubule attachment to the nucleating sites leading to a defect in coalescence of -tubulin complexes into larger iMTOCs (Figure 7D).
These large structures at the NE are best revealed upon depolymerization of microtubules; however, we 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). Although nucleation of microtubules can occur elsewhere, either on preexisting microtubules or in cytosol, the resulting microtubules slide toward 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.
Interestingly, we observed that although we detected instances of microtubule loss from the eMTOC (Figure 5, C and D), all -tubulin complex components that we looked at localized to the equatorial MTOC in mia1 cells (Figure 5E). This suggested that Mia1p and microtubule attachment were not required for their localization, which is not surprising given that microtubules are not required for the eMTOC formation (Heitz et al., 2001) and Mia1p does not seem to be an integral component of the nucleating complex.
Based on our model for emergence of the interphase MTOCs, we went ahead to show that microtubules are required for their assembly in wild-type cells (Figure 7). When microtubules were absent in dividing mother cells, we could not detect the interphase MTOCs in daughters (Figure 7, A and B). In contrast, when we allowed microtubule polymerization in daughter cells with no previous history of the interphase MTOCs, these structures readily occurred (Figure 7C).
Noncentrosomal 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 -tubulin satellites 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 toward 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 do 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 -tubulin complexes seemed necessary for the birth of centrioles (Dammermann et al., 2004).
In conclusion, self-organization of -tubulin–containing material into large MTOCs could facilitate efficient nucleation, bundling, and intracellular positioning of cytoskeletal arrays.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
Address correspondence to: Snezhana Oliferenko (snejana{at}tll.org.sg).
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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