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Genes to Cells (2006) 11, 815-827. doi:10.1111/j.1365-2443.2006.00980.x
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Schizosaccharomyces pombe homolog of Survivin, Bir1p, exhibits a novel dynamic behavior at the spindle mid-zone

Srividya Rajagopalan, Mithilesh Mishra and Mohan K. Balasubramanian*

Cell Division Laboratory, Temasek Life Sciences Laboratory and the Department of Biological Sciences, National University of Singapore, Singapore


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Members of the BIR-domain containing Survivin family of proteins have been identified in a variety of eukaryotes and are known to play important roles in the regulation of mitosis. The Schizosaccharomyces pombe homolog of Survivin, Bir1p, is essential for chromosome condensation and spindle elongation and integrity. Bir1p, a nuclear protein, resides at the kinetochores in metaphase and anaphase A and spreads to the spindle mid-zone in anaphase B. Here we show that this relocation requires Cdk (Cyclin dependent kinase) inactivation and intact microtubules. With the aid of a kinesin mutant, klp5{Delta}, we also show that completion of anaphase A is vital for effecting Bir1p re-location to the spindle mid-zone. Although minimal exchange of Bir1p sub-units occurs between the spindle and the nucleoplasm, the protein redistributes laterally within the mid-zone region. Bir1p dynamics therefore significantly differs from that of tubulin on an anaphase B spindle, which is loaded at the plus ends of growing microtubules and shows no lateral redistribution within the spindle. Thus, Bir1p, and possibly its associated proteins, might organize a dynamic mid-zone region that helps spindle elongation and maintenance.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Survivin, an Inhibitor of Apotosis protein (IAP) characterized by the presence of the Baculoviral IAP repeat (BIR) domain, is conserved in many eukaryotes ranging from yeasts to humans (Crook et al. 1993; Li et al. 1998; Silke & Vaux 2001). Recently, Survivin-like proteins have been shown also to be involved in more fundamental, cell-cycle related processes (Li et al. 1998; Fraser et al. 1999; Uren et al. 1999; Rajagopalan & Balasubramanian 1999, 2002; Uren et al. 2000; Morishita et al. 2001; Petersen & Hagan 2003). Along with the mitotic kinase Aurora B, the inner centromere protein (INCENP) and a few other proteins, Survivin constitutes a chromosome passenger complex that is thought to coordinate multiple events during the process of mitosis and cytokinesis (Adams et al. 2001; Vernos 2004). This protein complex localizes to kinetochores in early mitosis, the spindle mid-zone in anaphase B and eventually to the telophase mid-body in higher organisms. It has been proposed that the passenger complex coordinates various events based on its timely localization to different structures during the course of mitosis (Adams et al. 2001).

The Schizosaccharomyces pombe (S. pombe) homolog of Survivin, Bir1p (also known as Pbh1p/Cut17p) has previously been shown to be important for chromosome condensation, segregation and spindle elongation during mitosis (Samejima et al. 1993; Rajagopalan & Balasubramanian 1999, 2002; Uren et al. 1999; Morishita et al. 2001; Petersen & Hagan 2003). Bir1p, together with the S. pombe Aurora B kinase, Ark1p, localizes to kinetochores and then to the anaphase B spindle mid-zone in mitotic cells. Bir1p regulates the localization of Ark1p during the course of mitosis (Morishita et al. 2001; Rajagopalan & Balasubramanian 2002). Time-lapse microscopy analysis has suggested that the kinetochore-associated pool of Bir1p appears to redistribute to the spindle when kinetochores reach the spindle poles at the end of anaphase A (Rajagopalan & Balasubramanian 2002).

In order to determine the cell cycle events and molecular components that are required for this striking, timely shift in localization, we have investigated the dynamic behavior of Bir1p through the course of mitosis. With the use of a kinesin mutant, klp5{Delta} (Garcia et al. 2002; West et al. 2002), we have demonstrated that completion of anaphase A (when kinetochores reach the spindle poles) is required prior to re-distribution of Bir1p to the spindle mid-zone. Additionally, we show that kinetochore to spindle re-localization of Bir1p is dependent on Cdk (Cyclin dependent kinase) inactivation and an intact microtubular network. Interestingly, upon localization to the spindle, the dynamic behavior of Bir1p at the mid-zone sharply contrasts with that of tubulin, in that lateral redistribution of the protein is observed within the boundaries of the mid-zone. We discuss these observations in terms of the molecular function of Bir1p during various stages of mitosis.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Completion of anaphase A is required for Bir1p relocation to the spindle mid-zone

Previous time-lapse studies have suggested that Bir1p localizes to the spindle mid-zone at the onset of anaphase B (Rajagopalan & Balasubramanian 2002). It has been hypothesized that Bir1p possibly travels on kinetochores to the spindle pole bodies (SPBs) in anaphase A to eventually move to the spindle mid-zone in anaphase B. Alternatively, it was possible that Bir1p simply re-localized to the mid-zone when the spindle reached a certain length during mitosis. Null mutants of S. pombe kinesins, Klp5p and Klp6p, were used to test these possibilities. klp5{Delta} and klp6{Delta} mutants display multiple defects in early mitosis, although equal segregation of chromosomes eventually occurs later in anaphase, thereby resulting in viable progeny (Garcia et al. 2002; West et al. 2002). One prominent defect in klp5{Delta} and klp6{Delta} cells is the frequent initiation of anaphase A on a spindle that is 4–6 µm in length (West et al. 2002), in contrast to wild-type cells in which anaphase A is normally initiated on spindles that are < 3 µm in length (Nabeshima et al. 1998). If relocation of Bir1p to the spindle mid-zone was indeed dependent on the mitotic stage of the cell, that is, completion of anaphase A, and not simply based on the length of the mitotic spindle, klp5{Delta} and klp6{Delta} mutants would retain Bir1p on kinetochores until anaphase A was completed.

We therefore checked the localization of Bir1p in exponentially growing klp5{Delta} cells at 24 °C that were fixed and stained to visualize chromosomes, microtubules and GFP-Bir1p. In 37 of 40 cells counted with missegregated chromosomes, GFP-Bir1p was detected on kinetochores that co-localized with the nuclear material along the spindle of an average length of 4 ± 0.4 µm (Fig. 1A). Co-localization of Bir1p with Mis6p, a previously characterized centromeric protein (Saitoh et al. 1997), confirmed the kinetochore localization of Bir1p (Fig. 1B upper row). Consistent with the ability of klp5{Delta} cells to eventually segregate their chromosomes and complete mitosis, Bir1p was detected at the spindle mid-zone in all anaphase B cells (n = 200 cells) in which the chromosomes were segregated to the spindle poles (Fig. 1B lower row). Similar results were observed in klp6{Delta} cells expressing GFP-Bir1p (data not shown).


Figure 1
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  		Figure 1  Bir1p moves from kinetochores to the spindle mid-zone upon completion of anaphase A. (A) Exponentially growing klp5{Delta} cells expressing GFP-Bir1p at 24 °C were fixed with formaldehyde and stained with DAPI to visualize chromosomes, anti-GFP antibodies to visualize GFP-Bir1p and tubulin antibodies to visualize microtubules. The "merge" images indicate GFP-Bir1p in red and microtubules in green. (B) Exponentially growing klp5{Delta} cells expressing GFP-Bir1p and Mis6p-13Myc at 24 °C were fixed with formaldehyde and stained with DAPI to visualize chromosomes, anti-GFP antibodies to visualize GFP-Bir1p and anti-myc antibodies to visualize Mis6p stained kinetochores. The "merge" images indicate GFP-Bir1p in green and Mis6p-13Myc in red. (C) Stills from a time-lapse movie analysis of GFP-Bir1p localization in klp5{Delta} cells. Images were captured at 5-second intervals at ~24 °C. Numbers refer to time in seconds.

 
To better visualize the dynamics of Bir1p in klp5{Delta} cells, live imaging analyses were performed (n = 10 cells). Stills from a single time-lapse movie of GFP-Bir1p in klp5{Delta} cells at 24 °C are displayed (Fig. 1C). GFP-Bir1p stained kinetochores exhibited rapid back and forth movement, presumably along the length of the spindle, for a considerable period of time during mitosis (> 16 min in this movie clip; Supplementary Movie S1). Eventually anaphase A movement occurred (Fig. 1C, time points 970 s to 985 s; see Supplementary Movie S1) and the protein spread to the spindle mid-zone. The visualization of Bir1p dynamics in the klp5{Delta} mutant further strengthened the idea that anaphase A movement of kinetochores to the SPBs is required for the relocation of Bir1p from kinetochores to the spindle-mid-zone.

High Cdk activity prevents Bir1p relocation from kinetochores to the spindle mid-zone

A series of biochemical events ensure anaphase progression. A major biochemical event that triggers progression through anaphase is ubiquitin-mediated cyclin B proteolysis (Murray & Kirschner 1989; Glotzer et al. 1991). Time-lapse analyses have previously suggested that cyclin B destruction is initiated soon after inactivation of the spindle assembly checkpoint (Clute & Pines 1999). Since this event temporally, closely coincides with the redistribution of Bir1p from kinetochores to the spindle, we investigated whether the levels of cyclin B in the cell influenced this relocation. In S. pombe, over-expression of truncated cyclin B, Cdc13{Delta}81p (Cdc13p deleted for its first 81 amino acids), which lacks the "destruction box" required for recognition by the Anaphase Promoting Complex (APC), blocked cells in anaphase with high Cdk activity (Yamano et al. 1996). A strain expressing GFP-Bir1p was transformed with a plasmid in which expression of Cdc13{Delta}81p was under control of the medium-strength nmt1 promoter (Basi et al. 1993). Cells were grown in medium lacking thiamine to induce maximal expression of the truncated Cdc13p for 20 h at 24 °C, fixed and stained to visualize DNA, microtubules and GFP-Bir1p. These cells displayed missegregated chromosomes along a spindle of an average length of 3.6 ± 0.6 µm. Interestingly, GFP-Bir1p localized to more than three kinetochores scattered along the length of the spindle in 37 of 40 cells counted with missegregated chromosomes (Fig. 2A). In these cells, kinetochore localization of Bir1p was confirmed by co-localization of Bir1p with Mis6p, and no mid-zone staining of Bir1p was observed (Fig. 2B, right panels "–Thiamine"). In contrast, spindles of comparable lengths in the un-induced control cells displayed normal mid-zone accumulation of GFP-Bir1p (Fig. 2B, left panels "+Thiamine"). Thus it can be concluded that high cyclin B levels, and therefore high Cdk activity, prevent redistribution of Bir1p from kinetochores to the spindle mid-zone.


Figure 2
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  		Figure 2  (A) High Cdk activity prevents spindle localization of Bir1p. gfp-bir1+ cells expressing Cdc13{Delta}81p were exponentially grown at 24 °C in medium lacking thiamine for 20 h, fixed with formaldehyde and stained with DAPI to visualize chromosomes, anti-GFP antibodies to visualize GFP-Bir1p and tubulin antibodies to visualize microtubules. The "merge" images indicate GFP-Bir1p in red and microtubules in green. (B) Co-localization of GFP-Bir1p and the kinetochore marker, Mis6p. gfp-bir1+ cells expressing Cdc13{Delta}81p and Mis6p-13Myc were exponentially grown at 24 °C in medium with or without thiamine for 20 h, fixed with formaldehyde and stained with DAPI to visualize chromosomes, anti-GFP antibodies to visualize GFP-Bir1p and anti-Myc antibodies to visualize Mis6p stained kinetochores. The "merge" images indicate GFP-Bir1p in green and Mis6p-13Myc in red. (C) The plus-end kinesin, Klp5p, localizes to the spindle mid-zone in cells containing high levels of cyclin B. Cells expressing Klp5p-GFP were exponentially grown in YES medium at 24 °C and klp5+-gfp cells expressing Cdc13{Delta}81p were exponentially grown at 24 °C in medium lacking thiamine for 20 h. Both cultures were fixed and stained with DAPI to visualize chromosomes, anti-GFP antibodies to visualize Klp5p-GFP and tubulin antibodies to visualize microtubules. The "merge" images indicate Klp5p-GFP in green and microtubules in red.

 
It remained possible that Bir1p failed to be recruited to the spindle due to the absence of an intact mid-zone structure in cells with high levels of cyclin B. In order to address this question, we analyzed the localization of a previously reported spindle mid-zone protein, the kinesin Klp5p, in these cells. Klp5p localizes to kinetochores in metaphase and moves to the spindle mid-zone in anaphase (Garcia et al. 2002; West et al. 2002). klp5+-gfp cells over-expressing Cdc13{Delta}81p were fixed after 20 h of induction in medium lacking thiamine and stained with DAPI, tubulin and GFP antibodies. Control cells grown in the presence of thiamine were also fixed and stained with the same reagents. In cells with lagging chromosomes, indicative of high cyclin B levels, Klp5p-GFP clearly localized to the mid-zone of the elongated spindle, similar to the pattern of Klp5p localization seen in wild-type cells (Fig. 2C). This result demonstrated the presence of an intact spindle mid-zone in cells with high levels of cyclin B, thus lending support to the observation that high Cdk activity prevents kinetochore to spindle mid-zone re-localization of Bir1p during mitosis.

Microtubules are essential for the removal of Bir1p from kinetochores

Given that Bir1p relocates to the spindle mid-zone at the end of anaphase A, when kinetochores, with the aid of microtubules (MTs), reach the spindle poles, we asked whether its loss from kinetochores might require intact MTs. However, it was also possible that Cdc13p proteolysis and passage through anaphase would result in the loss of the Bir1p signal from the kinetochore even in the absence of MTs. To distinguish between these possibilities, a strain was constructed in which GFP-Bir1p was expressed in the ß-tubulin mutant, nda3-KM311 (Hiraoka et al. 1984). The spindle-assembly checkpoint (SAC) gene, mad2 was also deleted in this strain since nda3-KM311 mad2{Delta} cells, when shifted to the restrictive temperature of 19 °C, fail to arrest at metaphase and proceed through mitosis and cytokinesis in the absence of MTs (He et al. 1997). nda3-KM311 mad2{Delta} cells expressing GFP-Bir1p were fixed after 4 h of incubation at 19 °C and stained to visualize DNA, MTs and GFP-Bir1p. In 38 of 50 cells counted with mis-segregated chromosomes, which were devoid of MTs, it was found that GFP-Bir1p remained localized to kinetochores, even in cells that had undergone septation (Fig. 3A, data not shown). Kinetochore staining of Bir1p was confirmed by co-localization of Bir1p with Mis6p (Fig. 3B). In the remaining 12 out of 50 cells, clear staining of kinetochores was not visible due to the intensity of the nucleoplasmic signal of Bir1p (data not shown). These observations together suggested that the unloading of Bir1p from kinetochores was temporally dependent on cyclin B proteolysis and anaphase A progression and structurally dependent on an intact microtubular cytoskeleton.


Figure 3
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  		Figure 3  (A) Microtubules are essential for the release of Bir1p from kinetochores. Exponentially growing nda3-KM311 mad2{Delta} cells expressing GFP-Bir1p at 32 °C were shifted to 19 °C for 4 h. Cells were then fixed with formaldehyde and stained with DAPI to visualize chromosomes, tubulin antibodies to visualize microtubules and anti-GFP antibodies to visualize GFP-Bir1p. The "merge" images indicate GFP-Bir1p in red and chromosomes in blue. (B) Co-localization of GFP-Bir1p with the kinetochore marker, Mis6p. Exponentially growing nda3-KM311 mad2{Delta} cells expressing GFP-Bir1p and Mis6p-13Myc at 32 °C were shifted to 19 °C for 4 h. Cells were then fixed with formaldehyde and stained with DAPI to visualize chromosomes, anti-Myc antibodies to visualize Mis6p-13Myc and anti-GFP antibodies to visualize GFP-Bir1p. The "merge" images indicate GFP-Bir1p in green and Mis6p-13Myc in red.

 
Bir1p laterally redistributes within the mid-zone and its dynamics is distinct from that of tubulin on the anaphase spindle

Spindle elongation is proposed to occur by sliding apart of over-lapping MTs utilizing kinesin motors and tubulin polymerization at plus-ends (Cande & MacDonald 1985; Masuda et al. 1990; Ding et al. 1993; Mallavarapu et al. 1999). Fluorescence recovery after photobleaching (FRAP) analyses have previously shown that, upon metaphase to anaphase transition, an abrupt switch in microtubule dynamics occurs such that anaphase B spindles are more stable compared to highly dynamic metaphase spindles (Mallavarapu et al. 1999). Photobleaching of the mid-zone region of a GFP-tubulin stained anaphase spindle resulted in minimal fluorescence recovery and the bleached region could be observed to move towards spindle poles indicative of the sliding of over-lapping MT arrays (Mallavarapu et al. 1999). Since the mitotic spindle exhibits such distinct behavior during anaphase B, we investigated if the dynamics and turnover of Bir1p on the spindle-midzone (the region that contains MT plus-ends) coincided with that of MTs.

We first carried out experiments similar to those performed for anaphase B cells expressing GFP-tubulin (Mallavarapu et al. 1999), in that the medial region of the Bir1p stained spindle mid-zone was bleached in gfp-bir1+ cdc25-22 cells at 24 °C (Fig. 4A, n = 8 cells). Interestingly, although Bir1p is a bona fide component of the spindle mid-zone, Bir1p fluorescence, unlike GFP-tubulin, recovered in the bleached region of the spindle mid-zone (Fig. 4A,B), though never up to pre-bleach levels. In fact, turnover of GFP-Bir1p on the mid-zone appeared quite rapid, as fluorescence recovery began to occur in about 1 minute after bleach (Fig. 4A,B). Also, the sliding apart of bleach marks was not observed as has been previously reported for GFP-tubulin (Mallavarapu et al. 1999). Experiments in which one half (n = 5 cells) of the spindle mid-zone was bleached also resulted in rapid recovery of GFP-Bir1p fluorescence, again not matching prebleach levels (Fig. 4A,B). Photo-bleaching both ends of the spindle mid-zone (n = 3 cells) resulted in fluorescence recovery albeit to very low levels (Fig. 4A,B). In all the above scenarios, the recovered signal did not spread beyond the spindle mid-zone. Together, this suggested that Bir1p behaved differently from underlying MTs on the anaphase B spindle mid-zone.


Figure 4
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  		Figure 4  Dynamics of GFP-Bir1p on the spindle mid-zone. (A) Bir1p recovers fluorescence on the spindle mid-zone when the middle, one end and both ends of the mid-zone are bleached. Bir1p fails to recover fluorescence on the spindle mid-zone when the GFP signal on the entire mid-zone is bleached. In all the above cases, fluorescence recovery was monitored by time-lapse analysis of images captured at 30-second intervals. Numbers refer to time in minutes and seconds. The area of bleach is outlined by white boxes. (B) Recovery of fluorescence intensity (in relative units) plotted against time (in minutes) in the bleached area of the mid-zone. GFP-Bir1p signal recovers to varying intensities upon photo-bleaching the middle, one end and both ends of the mid-zone and fails to recover upon photo-bleaching the entire mid-zone. (C) Recovery of fluorescence intensity (in relative units) plotted against time (in min) over the entire mid-zone. The GFP-Bir1p signal over the entire mid-zone upon photo-bleaching the middle, one end and both ends of the mid-zone fails to recover to prebleach intensities. As control, fluorescence intensity of Bir1p over the entire mid-zone was plotted in an unbleached cell, which indicated no significant reduction in GFP-Bir1p signal under identical microscopy conditions. Plots of GFP intensity along spindle length as a function of time, when (D) the middle or (E) one end of the mid-zone region is bleached, indicate that Bir1p redistributes laterally from the unbleached to the bleached areas of the spindle mid-zone.

 
Interestingly, when the total fluorescence recovery along the entire spindle mid-zone was plotted as a function of time after bleaching, we found that post-recovery fluorescence never attained pre-bleach levels in all the above cases (Fig. 4C). In fact, plots of GFP intensity along spindle length as a function of time after bleach strongly indicated that Bir1p redistributed laterally from the unbleached to the bleached regions within the spindle mid-zone (Fig. 4D,E). However, a possible exchange of GFP-Bir1p subunits between the spindle and the nucleoplasm could not be overlooked. To test these scenarios, two approaches were used. First, the entire Bir1p stained mid-zone was bleached (Fig. 4A, n = 7 cells). No observable GFP-Bir1p signal was subsequently recovered (Fig. 4A,B). Second, the fluorescence loss in photobleaching (FLIP) technique was used to test a possible exchange of subunits between the nucleoplasmic and spindle pools of Bir1p in gfp-bir1+ cdc25-22 cells (n = 5 cells). Still images from a single FLIP experiment are depicted in Fig. 5A. The cell displaying mid-zone staining of Bir1p was chosen for active bleaching, while the cells showing nucleoplasmic and kinetochore staining of Bir1p were used as inherent controls for photo-bleaching that occurs during image acquisition. A region of interest, ROI 1, enclosing a part of the nucleoplasmic pool of Bir1p, was subjected to repeated photo-bleaching under low laser power. This level of laser power was sufficient to bleach the GFP-Bir1p signal on the mid-zone when the mid-zone was directly irradiated (data not shown). If active exchange of Bir1p sub-units indeed occurred between the nucleoplasm and the spindle, repeated photo-bleaching of the nucleoplasm would result in a marked decrease in fluorescence of GFP-Bir1p on the spindle mid-zone. We observed that the fluorescence intensity on the mid-zone (ROI 2) did not decrease significantly over a period of ~6.5 min (~400 s in this experiment; Fig. 5A,B). GFP fluorescence eventually reduced and was subsequently lost from the mid-zone presumably due to spindle breakdown (Fig. 5A, time points 429.9 s to 573.2 s). In view of the previous observation that the turnover of Bir1p on the spindle mid-zone is quite rapid (~1.0 min, see Fig. 4), the apparent lack of any significant reduction in GFP-Bir1p fluorescence over time in the FLIP experiment suggested that Bir1p was dynamic at the spindle mid-zone, but its turnover was not related to exchange with nucleoplasmic Bir1p.


Figure 5
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  		Figure 5  Minimal exchange of protein sub-units occurs between the spindle mid-zone pool and the nucleoplasmic pool of Bir1p. (A) Images from a single fluorescence loss in photo-bleaching (FLIP) experiment performed on gfp-bir1+ cdc25-22 cells at ~24 °C. The first image depicts the four different regions of interest (ROIs) that were analyzed. ROI 1 (indicated by a white oval), which includes a portion of the nuclear pool of Bir1p, was subjected to bleach after every two scans. The white asterisk indicates the image scanned just after bleach. Numbers refer to time in seconds. (B) Recovery of fluorescence intensity (in relative units) plotted against time (in seconds) in the four ROIs. Repeated bleaching of a portion of the nuclear pool of Bir1p (ROI 1) failed to affect the fluorescence intensity of Bir1p on the spindle mid-zone (ROI 2, depicted by a white square in (A)), suggesting minimal exchange of protein between these two regions. As inherent control for photo-bleaching, ROI 3 and 4 (depicted as white circles in (A)) were analyzed, which indicated no significant reduction in GFP-Bir1p signal under identical microscopy conditions.

 
An intact anaphase B spindle is essential for mid-zone localization of Bir1p

Previous studies have provided evidence for a putative "spindle matrix" structure whose existence and dynamics are MT-independent (Kapoor & Mitchison 2001). Since the dynamic behavior of Bir1p at the spindle mid-zone is distinct from that exhibited by GFP-tubulin, we checked if the medial retention of the mid-zone Bir1p structure was dependent on polymerized MTs. To test this possibility, a synchronous culture of gfp-bir1+ cdc25-22 anaphase B cells was incubated in an ice/water bath to induce depolymerization of all MTs. Subsequently, Bir1p localization was determined by immunofluorescence microscopy. Whereas the mid-zone Bir1p signal was detected prior to ice/water bath treatment, it was completely lost upon MT depolymerization (Fig. 6). These studies established that although Bir1p at the spindle mid-zone differed from MTs in dynamics, it depended on the presence of the mitotic spindle for its integrity.


Figure 6
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  		Figure 6  Maintenance of Bir1p on the mid-zone requires the presence of an intact anaphase B spindle. Exponentially growing gfp-bir1+ cdc25-22 cells at 24 °C were shifted to 36 °C for 4 h. The cells were synchronously released into mitosis at 24 °C for 45 min. One half of the culture was fixed with formaldehyde at 24 °C. The other half of the culture was subjected to an ice-water bath treatment for 30 min. The cells were then fixed with formaldehyde on ice. The fixed cells were stained with DAPI to visualize chromosomes, anti-GFP antibodies to visualize GFP-Bir1p and tubulin antibodies to visualize microtubules.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
In this study, we have shown that the S. pombe homolog of Survivin, Bir1p, relocates from kinetochores to the spindle mid-zone at the end of anaphase A, in a manner dependent on MTs and cyclin B levels. Upon localization to the spindle mid-zone, Bir1p subunits laterally redistribute within the mid-zone, with no significant exchange occurring between the spindle and nucleoplasmic pools of Bir1p. In this manner, the dynamics of Bir1p differs significantly from that of GFP-tubulin on the anaphase B spindle.

This study has shown that anaphase A movement of kinetochores to SPBs is important for relocation of Bir1p from kinetochores to the spindle mid-zone. Additionally, polymerized MTs are important for unloading of Bir1p from kinetochores. Perhaps, Bir1p arrives at the SPB on kinetochores, with the aid of kinetochore microtubules (kMTs). Upon reaching the spindle pole at the end of anaphase A, the protein then spreads to the spindle mid-zone. Redistribution of Bir1p, possibly as part of the chromosome passenger complex, from kinetochores to the spindle may serve to coordinate the completion of anaphase A with the onset of anaphase B, which is consistent with the multiple defects associated with bir1 mutants during anaphase (Morishita et al. 2001; Rajagopalan & Balasubramanian 2002).

The mechanism by which Bir1p spreads to the mid-zone from the spindle poles remains unclear. One attractive prospect is that Bir1p may bind to microtubule motors that transport it to the mid-zone. In S. pombe, many mitotic kinesins are known to localize to the spindle mid-zone. Cut7p is a member of the BimC family of kinesins and is important for bipolar spindle assembly and elongation (Hagan & Yanagida 1992). Pkl1p and Klp2p are members of the KAR3 subfamily of motors, and are required for establishment and maintenance of the bipolar spindle (Pidoux et al. 1996; Troxell et al. 2001). Interestingly, Klp2p also localizes to kinetochores before moving to the spindle (Troxell et al. 2001). Klp5p and Klp6p kinesins localize to the spindle mid-zone (Garcia et al. 2002; West et al. 2002) but may not be directly involved, as Bir1p was detected at the spindle mid-zone in klp5{Delta} and klp6{Delta} mutants. In the future, it would be interesting to analyze the potential role of microtubule motors in regulating Bir1p movement to the spindle mid-zone. Alternatively, Bir1p could interact with non-motor microtubule associated proteins (MAPs) such as Ase1p (Juang et al. 1997) and CLASP (CLIP associated protein—a member of the Orbit/Mast protein family; Lemos et al. 2000) which localize to the spindle mid-zone. It is also possible that Bir1p directly interacts with polymerized MTs in order to move from the SPBs to the spindle. In vitro studies have previously shown that human Survivin binds to polymerized tubulin (Li et al. 1998). An insight into the mechanism by which Bir1p (and other passenger proteins) localize to the spindle mid-zone should shed further light on the spatial and temporal regulation of mitosis by these proteins.

This study has also shown that high levels of cyclin B in the cell prevent redistribution of Bir1p from kinetochores to the spindle mid-zone. Interestingly, analysis of the protein sequence of Bir1p has revealed seven potential Cdc2p phosphorylation sites (Rajagopalan & Balasubramanian 1999). Perhaps, Cdc2p (Cdk) inactivation during anaphase results in dephosphorylation of Bir1p, causing its redistribution to the spindle, similar to what was shown in the case of relocation of the INCENP-Aurora B complex to the spindle via Cdc14p phosphatase mediated dephosphorylation of the budding yeast INCENP homolog, Sli15p (Pereira & Schiebel 2003). Incidentally, recent studies in fission yeast have established that Clp1p, the S. pombe Cdc14 homolog, functions along with Aurora B kinase at the kinetochore during metaphase (Trautmann et al. 2004).

Our findings on the role of Cdk inactivation and MTs in redistribution of Bir1p from kinetochores to the spindle mid-zone are consistent with the behavior of mammalian aurora B kinase (Murata-Hori et al. 2002), although to our knowledge similar experiments have not been previously performed on Survivin.

This study has shown that depolymerization of MTs by treatment on ice leads to the loss of spindle mid-zone associated Bir1p, suggesting that an intact microtubule cytoskeleton (mitotic spindle) is required for the maintenance of Bir1p at the mid-zone. Surprisingly, notwithstanding this microtubule dependence, Bir1p forms a structure at the spindle mid-zone, whose dynamics and turnover is strikingly different from that of GFP-tubulin. Photo-bleaching of various parts of the GFP-Bir1p stained mid-zone resulted in fluorescence recovery in the bleached areas. The fact that photobleach marks made on the spindle mid-zone did not slide apart suggests that Bir1p is not loaded concomitant with tubulin polymerization at the plus ends of overlapping MTs. Furthermore, FLIP experiments have indicated that Bir1p molecules, once loaded on to the spindle, laterally re-distribute within the mid-zone region without apparent mixing with nucleoplasmic Bir1p. Additionally, the restriction of Bir1p signal to the spindle mid-zone suggests the existence of barriers that prevent further spreading of Bir1p. It is likely that the region of microtubule overlap defines these boundaries. An attractive possibility is that Bir1p might form a structure that overlies the overlapping microtubule arrays and thereby serve to ensure stability of the spindle mid-zone. The lateral redistribution suggests that Bir1p and associated proteins might form a "polymeric" and dynamic structure that is contained within the region of microtubule overlap.

The structure and function of the mitotic spindle is not solely based on the dynamics of MTs, but is a result of a combination of differing dynamics of its various components. For instance, the plus-end kinesin, Eg5, and the MT-bundling mid-zone protein, Ase1p, display very different dynamics on the mitotic spindle as compared to MTs, and have been shown to be part of a putative microtubule-independent "spindle matrix" structure (Kapoor & Mitchison 2001; Schuyler et al. 2003). Probing the dynamic behavior of various spindle components should shed further light on the organization of the mitotic spindle. In the future, it will be interesting to address the physical nature of Bir1p-containing structures at the mid-zone and their interaction with tubulin and other proteins at the mitotic spindle. Of particular interest is the MT-bundling mid-zone protein, Ase1p, which is thought to stabilize overlapping arrays of spindle MTs and generate the forces required for spindle elongation (Juang et al. 1997). Recently, S. pombe Ase1p has been shown to be required for proper organization of the spindle mid-zone, since ase1{Delta} mutants mislocalize Ark1p on the spindle (Yamashita et al. 2005). Understanding the physical nature of spindle mid-zone components, along with its signaling components such as the Ark1p kinase and the Clp1p phosphatase should elucidate the potential regulation of spindle mid-zone function in cell division.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
S. pombe strains, plasmids, antibodies and reagents

Genotype and origin of the S. pombe strains used in this study are listed in Table 1. All gfp-bir1+ and mis6+-13myc containing strains in this study express Bir1p and Mis6p under the control of their respective native promoters (construction of the original gfp-bir1+ strain and the mis6+-13myc strain is described in Rajagopalan & Balasubramanian 2002). Media used for vegetative growth of yeast strains (YES and EMM2) were as previously described (Moreno et al. 1991). Yeast matings were performed on YPD agar plates. Plasmids used in this study are listed in Table 2. The anti-tubulin antibody, {alpha}-TAT1, was a gift from Dr K. Gull (University of Manchester, UK). Anti-GFP antibodies were purchased from Molecular Probes and anti-Myc antibodies from Sigma Aldrich. Drs D. McCollum, S. Oliferenko and J. R. McIntosh (UMass Medical School, Worcester, MA, USA; Temasek Lifesciences Laboratory, Singapore; and University of Colorado, Boulder, CO, USA respectively) kindly provided us with the nda3-KM311 mad2{Delta} strain, the klp5+-GFP strain and the klp5{Delta} & klp6{Delta} strains, respectively.


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  		Table 1 Strains list
 

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  		Table 2 Plasmid list
 
Indirect immunofluorescence microscopy

Exponentially growing (O.D595 0.2 to 0.5) S. pombe cells were fixed at their growth temperature with 7.4% formaldehyde solution for 10–12 min and washed with PBS. Fixed cells were protoplasted in PBS+ 1.2 M Sorbitol using 5 mg/mL lysing enzymes (Sigma) and 3 mg/mL zymolyase (US Biological) and permeabilized with PBS+1% Triton X-100. PBAL solution (PBS + 1% BSA, 100 mM lysine hydrochloride, 50 µg/mL carbenicillin and 1 mM sodium azide) was used for blocking, primary and secondary antibodies incubation steps. Images were captured using an Optronics DEI-750T cooled CCD camera and Leica QWIN software. Image processing was done using Adobe Photoshop 7.0.

Time-lapse microscopy and photo-bleaching experiments

For live-cell imaging of GFP-Bir1p in S. pombe, 1 µL of exponentially growing cells was mounted on a borosilicate glass slide and covered gently with a coverslip (both from Matsunami Trading, Japan). Images were obtained on a Leica DMIRE2 microscope equipped with Uniblitz shutter and CoolSnap HQ CCD camera (Photometrics) driven by MetaMorph 4.6r9 software (Universal Imaging Corporation). Fluorescence recovery after photo-bleaching (FRAP) and fluorescence loss in photo-bleaching (FLIP) experiments were carried out using the Laser Scanning Microscope (LSM) 510 Axiovert inverted light microscope with Zeiss Plan Apochromat 100x/1.4 oil DIC objective, with 100 iterations of the 488 nm argon laser at 100% power for FRAP and 30 iterations at 50% power for FLIP. The region of interest (ROI) was subjected to bleach after every two scans in the time-series. All experiments were carried out at ~24 °C. Intensity of fluorescence recovery was measured using LSM 510 software and graphically plotted using Microsoft Excel.


   Acknowledgements
 
This work was carried out with research funds from the Temasek Life Sciences Laboratory. We thank Drs Keith Gull, Richard McIntosh, Dan McCollum and Snezhana Oliferenko for antibodies and yeast strains. We also thank Dr Suresh Jesuthasan for help with the NIH Image 1.62 software. We thank all members of the yeast and fungal laboratories, especially Dr Suniti Naqvi, for technical help, discussion, encouragement, and excellent suggestions.


   Footnotes
 
Communicated by: Mitsuhiro Yanagida

* Correspondence: E-mail: mohan{at}tll.org.sg


   References
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 Introduction
 Results
 Discussion
 Experimental procedures
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Received: 26 April 2005
Accepted: 14 April 2006





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