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Genes to Cells (2006) 11, 337-352. doi:10.1111/j.1365-2443.2006.00947.x
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The carboxy-terminus of Alp4 alters microtubule dynamics to induce oscillatory nuclear movement led by the spindle pole body in Schizosaccharomyces pombe

Hirohisa Masuda*, Rumi Miyamoto, Tokuko Haraguchi and Yasushi Hiraoka

Cell Biology Group and CREST/JST, Kansai Advanced Research Center, National Institute of Information and Communications Technology, Kobe, 651-2492, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Alp4 is an essential component of the S. pombe{gamma}-tubulin complex. Overproduction of the carboxy-terminus of Alp4 induces oscillatory nuclear movement led by the spindle pole body (SPB). The movement is not dependent on cytoplasmic dynein dhc1, or kinesin-related proteins pkl1 and klp2. Rates of SPB movement correlate with elongation rates of microtubules (MTs) extending backwards from the moving SPB (backward-extending MTs), showing that pushing forces exerted by backward-extending MTs move the nucleus via the SPB. These backward-extending MTs are more stable than those of control cells and, thus, are able to push the SPB further towards the cell end, inducing nuclear oscillation with larger amplitudes than in control cells. SPB movement is biased towards the new end of the cell where levels of the CLIP170 homolog Tip1 increase, suggesting that the movement is related to MT-mediated cell polarity control. These results demonstrate that the carboxy-terminus of Alp4 alters MT dynamics and induces nuclear oscillation by modulating a nuclear positioning mechanism based on the balance of MT pushing forces, and suggest that regulation of {gamma}-tubulin complex activity is important for controlling MT dynamics and nuclear positioning.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Nuclear positioning is an active process that moves and maintains the cell nucleus in the correct cellular position, and is crucial for many processes including cell division, fertilization, and development (reviewed in Reinsch & Gonczy 1998). For instance, movement of male and female pronuclei towards each other, following fertilization, is essential for zygote formation. In Drosophila, the position of the oocyte nucleus determines the anterior/posterior and dorsal/ventral axes of symmetry. Nuclear positioning in many cells depends on microtubules (MTs), although in some cases it involves actin filaments or both MTs and actin filaments (reviewed in Reinsch & Gonczy 1998; Starr & Han 2003). Since the nucleus physically associates with the microtubule-organizing center (MTOC), known as the centrosome in animal cells and the spindle pole body (SPB) in fungi, MT-dependent nuclear positioning in many cells is tightly linked to positioning of the MTOC. The nucleus is moved via the MTOCs by pushing forces exerted by MT polymerization and/or by pulling forces exerted by MT motor proteins. In S. cerevisiae, re-positioning of the nucleus from the mother cell to the bud neck is achieved via the SPB by MT pushing forces that move the nucleus away from the mother cell cortex and by MT motor protein pulling forces that move the nucleus towards the daughter cell cortex.

Nuclear positioning in S. pombe cells is dependent on cytoplasmic arrays of MTs. S. pombe cells grow cylindrically and cytoplasmic MTs are organized parallel to the long axis of the cell and extend from iMTOCs (MTOCs for interphase MTs), which are located at the SPB and a few other sites on the nuclear envelope (Drummond & Cross 2000; Tran et al. 2001; Sagolla et al. 2003; Sawin et al. 2004; Zimmerman et al. 2004). Tubulin mutants show defects in both nuclear positioning and cytoplasmic MT organization (Toda et al. 1983; Umesono et al. 1983; Radcliffe et al. 1998). S. pombe cells grow both at the old end (the end inherited from the parent cell) and at the new end created by septation, but in an asymmetric manner. After cytokinesis, the cell grows initially at the old end. At some point in the G2 phase of the cell cycle, the cell switches from monopolar to bipolar growth, growing at both ends (Mitchison & Nurse 1985). This event is referred to as new-end take-off (NETO). The nucleus, however, maintains its position at the cell center, showing that nuclear positioning is an active process that moves and maintains the nucleus at the cell center. Live cell observation of MTs, the SPB, and the nuclear envelope shows that MTs elongate from iMTOCs on the nuclear envelope toward the cell ends, exert pushing forces against the cell ends, and then disassemble (Tran et al. 2001). A balance of pushing forces of MTs extending from iMTOCs towards both cell ends provides a mechanism to position the nucleus at the middle of the cell. Positioning of the mitotic nucleus also seems to be regulated by pushing forces exerted by astral MT polymerization (Tolic-Norrelykke et al. 2004).

When S. pombe cells switch from the mitotic cell cycle to the process of meiosis, nuclear movements and cytoplasmic MT organization characteristic of each stage of the premeiotic process are observed (Ding et al. 1998). Spatial reorganization of chromosomes in the nucleus is also observed during this switch, which involves centromere detachment from the SPB and telomere attachment to the SPB (Chikashige et al. 1994, 1997). During mating, cells form their mating projection towards the pheromone source. After cell fusion, the two nuclei approach each other while displaying oscillatory movements, and a bundle of MTs extending from the two SPBs are observed between the nuclei. Later, MTs extending from the SPBs converge at the center to form an X-shaped array. During meiotic prophase following karyogamy, oscillatory nuclear movement called "horsetail movement" is observed. This movement facilitates paring of homologous chromosomes and is important for homologous recombination (Yamamoto et al. 1999; Ding et al. 2004). The horsetail movement is led by the SPB, from which astral MTs extend, and is mainly induced by cytoplasmic dynein located at the cell cortex pulling MTs extending from the SPB (Ding et al. 1998; Yamamoto et al. 1999, 2001). In addition to cytoplasmic dynein, Pkl1 and klp2, kinesin-like proteins of the KAR3/kinesin-14 family (Lawrence et al. 2004) that may function for MT shortening, are implicated in karyogamy (Troxell et al. 2001). These observations indicate that S. pombe cells apply MT pushing forces for maintaining the nucleus at the middle of the cell and MT pulling forces for generating nuclear movement.

As shown in the accompanying paper, in this issue of Genes to Cells (Masuda et al. 2006), overproduction of the carboxy-terminus of Alp4 (Alp4C), an essential component of the S. pombe{gamma}-tubulin complex, induces oscillatory nuclear movement. The movement is dependent on MTs and resembles the horsetail movement induced in premeiotic phases. Alp4C and NES-tagged Alp4C, but not NLS-tagged Alp4C, induces oscillatory nuclear movement, showing that the movement is induced by cytoplasmic Alp4C (Masuda et al. in this issue). In S. pombe, the cytoplasmic {gamma}-tubulin complex is required for organizing cytoplasmic MT arrays and is recruited to the cytoplasmic face of the nuclear envelope via Mto1/Mod20/Mbo1 (Sawin et al. 2004; Venkatram et al. 2004; Zimmerman & Chang 2005), and Mto2 (Janson et al. 2005; Samejima et al. 2005; Venkatram et al. 2005). To elucidate mechanisms of oscillatory nuclear movement induced by Alp4C overproduction and its relation with meiotic nuclear movement and interphase nuclear positioning, we examined the behavior of cytoplasmic MTs. We show here that Alp4C overproduction alters MT dynamics and modulates a nuclear positioning mechanism to induce extensive nuclear oscillation driven by MT pushing forces, and that Alp4C-induced nuclear oscillation is influenced by MT-mediated cell polarity control.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Alp4C-induced oscillatory nuclear movements are led by the SPB

We have shown in the accompanying paper (Masuda et al. in this issue), that overproduction of the carboxy-terminus of Alp4 (566–784 amino acids; termed Alp4C) induces MT-dependent oscillatory nuclear movement. Using Sid4-mRFP as an SPB marker (Chang & Gould 2000) and Hht2-GFP (Wang et al. 2002) as a chromosome marker, we examined the behavior of the SPB and nucleus (Fig. 1). In control cells (average cell length = 9.9 ± 1.3 µm, n = 35), the SPB was positioned towards the long axis of the cell to within 2–3 µm of the nucleus (2.8 ± 0.4 µm, n = 35), which was located at the cell center (Fig. 1C). In contrast, in most of the Alp4C-overproducing (Alp4C-OP) cells the nucleus exhibited oscillatory movement. In Alp4C-OP cells that elongated to > 13 µm (average cell length = 16.2 ± 2.3 µm, n = 38) the nuclei were deformed and enlarged while normal-sized Alp4C-OP cells (11.1 ± 1.5 µm, n = 25) had a normal sized nuclei; in both cases, the nucleus exhibited significant movement and the SPB was located at the leading edge of the oscillating nucleus. In the larger Apl4C-OP cells the SPB moved as much as 5–13 µm (7.1 ± 2.8 µm) at rates of 1.46 ± 0.87 µm/min (n = 126) (Fig. 1A), and in normal sized Apl4C-OP cells SPB movement had amplitudes of 4.0 ± 1.3 µm (Fig. 1B). These observations indicate that the SPB leads nuclear oscillation mediated by MTs.


Figure 1
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  		Figure 1  SPB leads Alp4C-induced nuclear movements. Chromosomes and SPBs were observed for about 14 min in hht2-GFP sid4-mRFP cells overproducing Alp4C (Alp4C-OP) or harboring pREP1 (Control). Merged images of GFP and mRFP signals are shown. Schematics of the movements of chromosomes (green and light green) and the SPB (red) are also shown. Regions with lower intensity of GFP fluorescence are shown in light green and presumably correspond to the nucleolus. Bar, 10 µm.

 
Alp4C-induced oscillatory nuclear movements are not dependent on dynein Dhc1 or kinesins Pkl1 and Klp2

We explored the possibility that the nuclear oscillation is caused by ectopic induction of a premeiotic stage, since it resembles the "horsetail" nuclear movement observed in meiotic prophase (Chikashige et al. 1994). The "horsetail" movement is mainly dependent on pulling forces exerted by cytoplasmic dynein located at the cell tip via MTs extending forward from the SPB (Ding et al. 1998; Yamamoto et al. 1999, 2001). Deletion of dhc1, the gene encoding the heavy chain of cytoplasmic dynein, suppresses most premeiotic nuclear movement (Yamamoto et al. 1999). In addition to Dhc1, a dynein light chain, Dlc1 (Miki et al. 2002), and kinesin-like proteins, Pkl1 and Klp2 (Troxell et al. 2001), are also implicated in nuclear movement. We found that nuclear movement was still induced by Alp4C overproduction in cells with single deletions of dhc1, dlc1, pkl1 or klp2, with double deletions of dhc1 and dlc1, dhc1 and klp2, or pkl1 and klp2, and with triple deletions of dhc1, pkl1 and klp2 (Fig. 2A). These results suggest that nuclear movements observed in Alp4C-OP cells are induced by a mechanism other than that which is responsible for premeiotic nuclear movements.


Figure 2
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  		Figure 2  SPB-led nuclear movements induced by Alp4C overproduction are not linked to activation of meiotic processes. (A) Dhc1, Pkl1, and Klp2 are not required for oscillatory nuclear movement. The behavior of the chromosomes of Alp4C-OP cells deleted for pkl1, klp2, and dhc1 was visualized with Hoechst33342. A schematic of the movement is shown on the right. (B) A centromere protein, Mis6, is localized at the moving SPB. Centromeres and the SPB were visualized in mis6-GFP sid4-mRFP cells overproducing Alp4C. A time-lapse sequence of approximately 6.5 min is shown. (C) Telomeres disperse in the moving nucleus. Telomeres were visualized in taz1-GFP cells. A time-lapse sequence of approximately 9 min is shown. (D) The centromere protein Nuf2 is reduced at the SPB. Nuf2 was visualized in nuf2-GFP cells overproducing Alp4C or harboring pREP1. (E) Quantification of the reduction in the levels of Nuf2-GFP and Mis6-GFP in Alp4C-OP cells. Error bars represent standard deviation. *P < 0.0001 and **P = 0.01 vs. control cells (two-tailed Student's t-test). (F) The cen2 locus is localized close to the moving SPB. The centromere locus of Chromosome II (cen2) was visualized with GFP using a lacO/lacI recognition system. The SPB was visualized with Sid4-mRFP. A time-lapse sequence of about 22 min is shown. Bars, 10 µm.

 
Behavior of centromeres and telomeres during nuclear oscillation

To confirm that Alp4C-OP cells are not in a premeiotic stage, we examined the positions of centromeres and telomeres during nuclear movement. Induction of meiotic prophase is accompanied by spatial reorganization of chromosomes in the nucleus, which involves centromere detachment from the SPB and telomere attachment to the SPB (Chikashige et al. 1994, 1997). Mis6-GFP (Saitoh et al. 1997) and Nuf2-GFP (Nabetani et al. 2001; Wigge & Kilmartin 2001), and Taz1-GFP (Chikashige & Hiraoka 2001) were used to study the behavior of centromeres and telomeres. We found that in alp4C-OP cells centromeres remained attached to the SPB (Fig. 2B) and telomeres did not cluster at the SPB but remained dispersed (Fig. 2C), showing that chromosome organization in the nucleus of Alp4C-OP cells is typical of the mitotic cell cycle (Funabiki et al. 1993). Interestingly, however, many of the elongated cells had less intense Nuf2-GFP or Mis6-GFP signals at the SPB than control cells (Fig. 2D,E), suggesting that levels of centromere proteins at the SPB were reduced. To examine whether some of the centromeres detached from the SPB in Alp4C-OP cells, the centromeric locus of chromosome II (cen2-proximal locus) was visualized by GFP using a lac operator/repressor recognition system (Yamamoto & Hiraoka 2003). Cen2 signals were exclusively localized close to the SPB (Fig. 2F) indicating that despite the reduction in centromere protein levels at the SPB, the centromeres remained attached to the SPB. We concluded that nuclear oscillation in Alp4C-OP cells is not related to meiotically induced events.

Rates of SPB movement correlate with elongation rates of MTs extending backwards from the moving SPB

Next we sought the possibility that nuclear oscillation is related to a nuclear positioning mechanism. Nuclear positioning in S. pombe interphase cells has been shown to be regulated by a balance of pushing forces of anti-parallel MTs organized from the SPB and other iMTOCs on the nuclear envelope through plus-end polymerization against the two cell ends (Tran et al. 2001). We reasoned that changes in the dynamics of MTs organized from the SPB in Alp4C-OP cells might affect nuclear positioning in a way that induces nuclear oscillatory movement. To examine the dynamics of MTs extending from the SPB, the behaviors of both the MTs and the SPB were monitored simultaneously in GFP-atb2 sid4-GFP or GFP-atb2 mis6-GFP cells overproducing Alp4C. The SPB, visualized with Sid4-GFP or Mis6-GFP, was observed as a particularly bright spot on GFP-labeled MTs (Fig. 3A). Figure 3B,C show the positions of the SPB in a GFP-atb2 mis6-GFP cell and the lengths of MTs extending from the SPB during one full cycle of SPB movement (10.5 min). MTs extending backward from the moving SPB (termed backward-extending MTs) remained in contact with the cell end and elongated continuously (shown as red lines in Fig. 3C). In contrast, MTs extending forward from the moving SPB (termed forward-extending MTs) shrunk (3.8 min; 36% of one full cycle period) or grew (1.9 min; 18%) while remaining attached to the cell ends (blue lines). They also detached and shrunk back from the cell ends and then resumed growing towards the cell ends (4.8 min; 45%) (green lines). Growth rates of backward-extending MTs (1.28 ± 0.64 and 1.30 ± 0.76 µm/min for the first and the second half of the cycle) correlated with the SPB movements (1.28 ± 0.38 and 1.30 ± 0.60 µm/min for the first and the second half of the cycle) (Fig. 3C). These results suggest that pushing forces exerted by polymerization of backward-extending MTs move the nucleus via the SPB, and that nuclear oscillation is induced by a mechanism similar to that for nuclear positioning in interphase cells.


Figure 3
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  		Figure 3  Behavior of the SPB and MTs during one full cycle of SPB movemet in Alp4C-OP cells. (A) Visualization of the SPB and MTs. The SPB and MTs were visualized in GFP-atb2 mis6-GFP (a) or GFP-atb2 sid4-GFP (b, c) cells overproducing Alp4C (a, b) or harboring pREP1 (c). SPBs can be seen as bright dots attached to or on MTs. The parameters of MT dynamics and SPB movements in three cells in b (arrows) and two cells in c (arrows) are shown in Tables 1 and 2. (B, C) Elongation of backward-extending MTs pushes the SPB forward. The behavior of the SPB and MTs in a mis6-GFP GFP-atb2 cell overproducing Alp4C (arrow in A-a) was examined during one full cycle of SPB movement. Time-lapse image sequences taken for 10.5 min at 30 s intervals were recorded. Images of 13 different focal planes at 0.35 µm spacing were obtained at each time point. Positions of MTs extending forward and backward from the moving SPB were determined by tracing MTs in different focal planes for measurement of their lengths. (B) Projection images reconstituted at 5 time points (time 0, 2.5, 5.5, 8.5, and 10.5 min). Positions of the SPBs (arrows) and ends of forward-extending MTs (arrowheads) are shown. (C) Positions of the SPB (red circle) and lengths of MTs extending from the SPB towards the left (L) and right (R) cell ends during one full cycle of SPB movement. (red) Backward-extending MTs; (blue) forward-extending MTs in contact with the cell ends; and (green) forward-extending MTs with free ends. Bars, 10 µm.

 
Backward-extending MTs are more stable in Alp4C-OP cells than in control cells

To confirm that Alp4C increases the stability of backward-extending MTs that push the SPB towards the cell end, we examined the dynamics of MTs extending from the SPB in living cells expressing GFP-Atb2 and Sid4-GFP (Table 1). Three Alp4C-OP cells (arrows in Fig. 3A-b) were followed during four full cycles of SPB movement (25.8 min for cell 1, 30.3 min for cell 2 and 16.8 min for cell 3), and two control cells (arrows in Fig. 3A-c) were followed for 18.0 min for cell 1 and 20.2 min for cell 2. In Alp4C-OP cells, the length of time that the MT ends remained in contact with the cell ends (dwell time of MTs at the cell ends) was significantly longer than in control cells (6.0 ± 4.2 min (n = 14) vs. 1.5 ± 0.9 min (n = 25), P < 0.001), and catastrophe of MTs (defined here as rapid shrinkage of MTs with rates of > 4 µm/min) was observed less frequently than in control cells (0.19 ± 0.04/min (n = 6) vs. 0.33 ± 0.06/min (n = 4), P = 0.002). We then compared the behavior of backward-extending MTs in Alp4C-OP cells and in control cells (Table 1). In control cells, the backward-extending MTs (n = 21) elongated for 1.2 ± 0.8 min at 1.6 ± 0.8 µm/min, and then 90% of them (19/21 MTs) showed catastrophe at a frequency of 0.71/min (19 events/26.9 min) when the SPB changed its direction of movement. In contrast, backward-extending MTs in Alp4C-OP cells (n = 24) elongated for 2.9 ± 1.5 min at 1.8 ± 0.9 µm/min. When the SPB changed its direction of movement, catastrophe was observed in only 29% of the former backward-extending MTs (7/24 MTs) at 0.1/min (7 events/71.8 min). Thus, backward-extending MTs in Alp4C-OP cells grew for longer periods at the cell end and showed catastrophe at lower frequencies than in control cells. This increase in stability of backward-extending MTs in Alp4C-OP cells explains the larger amplitude of SPB movement than was observed in control cells.


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  		Table 1 Parameters of MT dynamics and SPB movement in Alp4C-OP cells and in control cells
 
Dynamic behavior of forward-extending MTs

We next examined the behaviors of forward-extending MTs (Fig. 4). In control cells, forward-extending MTs had free ends for most of the observation time (77%), whereas in Alp4C-OP cells, forward-extending MTs remained attached to the cell end for more than half of the time (57%). Both in Alp4C-OP cells and in control cells, forward-extending MTs exhibited both shrinkage and growth phases. In control cells, they showed only either a growth or shrinkage phase, or alternated a shrinkage phase and a growth phase only once, before the SPB changed its direction of movement (Fig. 4C, lower panel). In contrast, about half of forward-extending MTs in Alp4C-OP cells (11/24 MTs) alternated a shrinkage phase and a growth phase more than once, and therefore had multiple shrinkage phases, before the SPB changed its direction of movement (Fig. 4A,B, lower panels).


Figure 4
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  		Figure 4  Dynamics of forward-extending MTs in Alp4C-OP cells and in control cells. (A and B) Dynamics of forward-extending MTs in GFP-atb2 sid4-GFP cells overproducing Alp4C. Live images of MTs and the SPB observed during a half cycle of SPB movement are shown in the upper panels. Arrowheads point to ends of MTs, shown here to extend upwards from the SPB (*). Lengths of MTs extending upwards from the SPB in the upper panels are plotted in the lower panels. Backward-extending MTs (B1) became forward-extending MTs (F1) at the start of a half cycle of SPB movement. The forward-extending MTs (F1–F5 in A, F1–F9 in B) showed shrinkage (S), growth (G), or pause (P) before they became backward-extending MTs (B2). Note that F7 in B curled around the cell end. (C) Dynamics of forward-extending MTs in control GFP-atb2 sid4-GFP cells. Live images of MTs and the SPB observed during one and a half cycles of SPB movement are shown in the upper panels. Arrowheads mark the ends of MTs extending from the SPB (*). Lengths of MTs extending upwards from the SPB in the upper panel are plotted in the lower panel. The MTs extending upwards from the SPB behaved as backward-extending MTs (B1-B3) at time 0, 2 : 48, and 4 : 29, and as forward-extending MTs (F1–F6) at the other time points. These MTs showed shrinkage (S) and growth (G). Bars, 10 µm.

 
We found that the average length of one shrinkage phase was longer in Alp4C-OP cells than in control cells (1.15 ± 0.67 min (n = 35) vs. 0.74 ± 0.53 min (n = 25), P = 0.008), whereas the average length of one growth phase was similar between Alp4C-OP cells and control cells (0.76 ± 0.30 min (n = 31) vs. 0.74 ± 0.53 min (n = 22), P = 0.93). We then compared the behavior of forward-extending MTs during the shrinkage phase. Forward-extending MTs in control cells showed rapid shrinking (> 4 µm/min) and had unattached free ends for 91% of the shrinkage phase. In contrast, forward-extending MTs in Alp4C cells showed rapid shrinking for only 37% of the shrinkage phase. For 64% of the phases, they exhibited slow shrinkage, both while remaining attached to the cell end (44%) and while having free ends (19%). These results indicate that in Alp4C-OP cells forward-extending MTs are in an unstable state and exhibit long and multiple shrinkage phases at the cell end, while backward-extending MTs with increased stability elongate continuously.

The SPB movement is biased towards the new end

We found that SPBs in Alp4C-OP cells do not move equally towards both ends of the cell but exhibit a bias towards one end. We tested the possibility that this biased movement is related to cell polarity control, i.e. whether the bias is towards the new or the old end of the cell. Calcoflour was used to distinguish the new from the old ends as it stains newly inserted wall material; the new end is identified as the end close to the birth scar, which is created by septum formation (Mitchison & Nurse 1985). Using the data obtained from three GFP-atb2 sid4-GFP cells overproducing Alp4C which were used for measuring MT dynamics (arrows in Fig. 3A-b), we examined SPB positions relative to the cell center during four full cycles of SPB movement. We found that both the average SPB position and the center of SPB tracks were not located at the cell center but were biased towards the new end (Table 2). Figure 5 shows the positions of the center of SPB tracks visualized with an SPB marker, Sad1-GFP, over a 20-min period in Calcoflour-stained Alp4C-OP cells (n = 63). In control cells, the tracks were short (2.8 ± 0.4 µm, n = 35) and were found at the cell center. The average center of the tracks was less than 0.1 µm away from the cell center (0.05 ± 0.3 µm, n = 35) (Table 2, Fig. 5B). In contrast, the tracks in many Alp4C-OP cells were long (5.9 ± 2.8 µm, n = 63) and were not equally distributed from the cell center towards the two ends along the long axis of the cell (Fig. 5B). Fifty-nine per cent (38/63) of Alp4C-OP cells had the center of the SPB track biased towards the new end by more than 0.25 µm from the cell center (1.17 ± 1.13 µm), 33% (21/63) of the cells had the track center less than 0.25 µm away from the cell center (0.07 ± 0.13 µm), and 8% (6/63) of the cells had the track center biased towards the old end by more than 0.25 µm (0.6 ± 0.24 µm). These results show that SPB movement in Alp4C-OP cells is biased towards the new end.


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  		Table 2 SPB positions relative to the cell center in Alp4C-OP cells and in control cells
 

Figure 5
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  		Figure 5  Movement of the SPB is biased towards the new end. (A) Calcoflour staining and visualization of Sad1-GFP tracks. sad1-GFP cells overproducing Alp4C (Alp4C-OP) or harboring pREP1 (Control) were stained with Calcoflour and observed for 24 min at 1.2 min intervals. The new end of the cell was identified as the end with a birth scar (arrowheads) by Calcoflour staining. Projection images from each time point were superimposed as Sad1-GFP tracks. Red bars represent the cell centers. N, new end; and O, old end. Bar, 10 µm. (B) Sad1-GFP tracks in Alp4C-OP cells are biased towards the new end of the cell. Distances from the center of Sad1-GFP tracks to the cell center were measured from merged images (n = 63 for Alp4C-OP cells; n = 35 for control cells). Positive distance represents displacement of the center of Sad1-GFP track from the cell center towards the new end, and negative distance towards the old end.

 
Comparison of dynamic behaviors of MT ends at the new and old ends

We compared the parameters of the dynamics of MTs extending from the SPB towards the new end with those extending towards the old end (Table 1). Although most of the parameters, including growth rates of backward-extending and forward-extending MTs in contact with the cell end, were similar at both ends we found a difference in catastrophe frequency of backward-extending MTs; catastrophe frequency was lower at the new end (0.06/min (2 events/32.0 min)) than at the old end (0.13/min (5 events/39.8 min)). We also found that growth and curling of forward-extending MTs, which were not normally observed in control cells, were observed more frequently at the new end (24% of the total observation period) than at the old end (11%). These observations suggest that the aberrant behavior of MTs was more prominent at the new end of Alp4C-OP cells than at the old end. The difference in behavior of MTs may be linked to the difference in efficiency of the coordination of force production with MT growth, and account for the biased movement of SPBs towards the new end.

Accumulation of Tip1 at the new ends

We reasoned that the biased movement of the SPB towards the new end, which was observed in Alp4C-OP cells, might result from differences in the protein composition of the two cell ends. These differences could affect interactions between the cell ends and the MT ends, and the coordination of force production with MT growth. Levels of Tip1, a MT-associated protein that binds to the ends of growing MTs and is required for restricting MT catastrophe to the cell end (Brunner & Nurse 2000), increases and levels of Bud6, an actin-binding protein (Glynn et al. 2001; Jin & Amberg 2001), decreases at the cell ends of Alp4C-OP cells (Masuda et al. in this issue). Since the intensity of the signals was not uniform between the two cell ends, we compared Tip1-YFP and Bud6-GFP intensities between the new and the old ends. In control tip1-YFP cells, 68% (n = 34) of the cells had > 2x brighter Tip1-YFP signals at the new end compared with the old end, 21% of the cells brighter at the old end, and 12% had similar intensities at the two ends. Altogether, the intensity of Tip1-YFP signal was 2.4x higher on average at the new end compared to the old end (Fig. 6). Thus, Tip1 localization in control cells, showing no biased SPB movement, was biased towards the new end. In Alp4C-OP cells at an early stage of overproduction when biased SPB movement is observed, intensities of Tip1-YFP signals also increased significantly at the new ends, 63% (n = 38) of the cells had > 2x brighter signals at the new end, 13% of the cells brighter at the old end, and 24% had similar intensities at the two ends. Overall, however, the Tip-YFP signal at the new end was significantly higher in Alp4C-OP cells than in control cells, ~1.8x higher on average than in control cells (Fig. 6B). The intensity at the old end, on the other hand, did not increase to significantly higher levels than seen in control cells (Fig. 6B). We also compared the extent of reduction in Bud6 signals between the new and the old ends. We did not observe a biased reduction in Bud6 levels at either end (data not shown), indicating that the reduction in Bud6 levels is not linked to biased SPB movement. These results suggest that Tip1 accumulation at the new end in Alp4C-OP cells may be related to SPB movement being biased towards the new end, although it remains to be determined whether Tip1 accumulation is directly linked to biased SPB movement.


Figure 6
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  		Figure 6  Tip1 accumulation biased towards the new end. (A) Tip1 localization is biased towards the new end of the cell. Tip1 localization was examined in tip1-YFP cells overproducing Alp4C (Alp4C OP) or harboring pREP1 (Control). The new (N) and old (O) ends of the cells were determined by Calcoflour staining. Arrowheads indicate Tip1 accumulation at the new end. Bar, 10 µm. (B) Tip1 accumulates at the new end of Alp4C-OP cells. Tip1-YFP levels at the cell ends were quantified in tip1-YFP cells overproducing Alp4C (Alp4C-OP) or harboring pREP1 (Control). *P = 0.02, **P < 0.0001, and ***P = 0.007 (two-tailed Student's t-test).

 

   Discussion
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 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
We have shown here that the SPB-led oscillatory nuclear movement induced by Alp4C overproduction are caused by disturbance of a nuclear positioning mechanism based on MT-pushing forces. Analysis of MT dynamics suggests that increased stability of MTs extending backwards from the moving SPB results in nuclear oscillation with larger amplitudes than in wild-type cells. Nuclear oscillation is biased towards the new end of the cell, suggesting the biased movement may be related to cell polarity control.

Oscillatory nuclear movement driven by MT pushing forces

We have found that elongation rates of backward-extending MTs in Alp4C-OP cells correlate with SPB movement, suggesting that the oscillatory nuclear movement is caused by MT pushing; a mechanism that is similar to that used for nuclear positioning in interphase and mitotic cells (Fig. 7A) (Tran et al. 2001; Tolic-Norrelykke et al. 2004). In a model for nuclear positioning in interphase cells, anti-parallel MTs extending from the SPB and other iMTOCs on the nuclear envelope towards the two cell ends exert transient forces at the cell ends that are produced by plus-end MT growth pushing the nucleus. The balance between the pushing forces at the two cell ends provides a mechanism for positioning the nucleus in the middle of the cell, and the kinetics of the opposing forces result in a rocking movement with small amplitude (2–3 µm). This autonomous positioning mechanism relies on the coordination of MT dynamics and organization and MT behavior at the cell ends. Changes in these parameters are expected to affect nuclear positioning. An increase in the stability of backward-extending MTs combined with a decrease in the stability of forward-extending MTs could result in the larger amplitudes of nuclear movement exhibited by Alp4C-OP cells compared to control cells. Although we have shown that the meiotic motor proteins Dhc1, Pkl1, and Klp2 are not involved in the SPB movement, we cannot exclude the possibility that a MT end-tracking protein captures the plus-ends of MTs at the cell end and coordinates force generation with MT growth as observed in kinetochore-MT interaction (Dickinson et al. 2004). In wild-type cells MTs do not curl around the cell end but terminate at the cell end, suggesting the presence of a MT end-tracking protein at the cell end. The tendency of MTs to curl around the cell end, especially around the new end, in Alp4C-OP cells suggests that the levels of putative end-tracking proteins are reduced at the new end. The reduction in the level of end-tracking proteins at the new end would disrupt the coordination of force production with MT growth, which could result in biased SPB movement. Accumulation of Tip1 at the new end may be linked to reduction of end-tracking proteins.


Figure 7
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  		Figure 7  Models for oscillatory nuclear movement. (A) Nuclear positioning in wild-type cells. MTs extending from the SPB and iMTOCs provide pushing forces that position the nucleus at the middle of the cell. Nuclear oscillation with small amplitudes is observed in these cells. (B) Nuclear positioning in mutant cells deleted for Mto2. iMTOCs other than the SPB are lost from the nuclear envelope. A single MT bundle is assembled from the SPB. Nuclear oscillation with larger amplitudes than in wild-type cells is observed. (C) Horsetail nuclear movement observed at meiotic prophase. Astral MTs emanating from the SPB are organized by the action of Hrs1/Hcp6. Oscillatory nuclear movement is mainly driven by MT pulling forces that are exerted by cytoplasmic dynein localized at the cell ends. (D) Oscillatory nuclear movement with larger amplitudes induced by Alp4C overproduction. Pushing forces exerted by backward-extending MTs move the nucleus via the SPB. Backward-extending MTs are more stable than in control cells, whereas forward-extending MTs are in an unstable state, showing long and multiple shrinkage phases. Backward-extending MTs therefore push the SPB further in Alp4C-OP cells than in wild-type cells (A) or in mto2-deleted cells (B). MTs that do not extend from the SPB may detach from the nuclear envelope.

 
To induce SPB-led nuclear oscillation, forces causing movement must be applied exclusively through MTs extending from the SPB. The horsetail movement, SPB-led nuclear oscillation observed during a premeiotic stage, is preceded by reorganization of MTs from interphase cytoplasmic arrays into premeiotic astral arrays in which MTs exclusively originate from the SPB (Fig. 7C) (Ding et al. 1998). Overproduction of Hrs1/Mcp6, a protein that remodels MTs into astral arrays induces oscillatory nuclear movement in interphase cells (Tanaka et al. 2005). Cells deleted for Mto2, a protein required for nucleation of cytoplasmic MTs from iMTOCs other than the SPB, have only one MT bundle, and some of them show SPB-led nuclear oscillation with larger amplitudes than in wild-type cells (Fig. 7B) (Janson et al. 2005). These phenotypes could be explained by a nuclear positioning model based on pushing forces of MTs extending from several iMTOCs; such a model predicts larger amplitudes of oscillation for a nucleus having only one iMTOC (Tran et al. 2001). Alp4C overproduction induces SPB-led nuclear oscillation; however, the region of the nucleus distal to the SPB exhibits little movement (Fig. 1). This indicates that MTs extending from the SPB, but not from other iMTOCs, are responsible for the movement. Nevertheless, assembly of cytoplasmic MTs in Alp4C-OP cells is not restricted to the SPB region, and several cytoplasmic MTs are observed that are not connected to the moving SPB (Cell 1 and 3 in Fig. 3A-b), suggesting that these MTs do not produce forces used for nuclear positioning. One possibility is that Alp4C overproduction may affect formation or function of iMTOCs other than the SPB, which could result in loss of MTs connected to the nucleus (Fig. 7D).

Nuclear movement similar to that elicited by Alp4C is induced by a carboxy-terminal form of Cut1 (Cut1C) (Nakamura et al. 2002), although the efficiency of induction of nuclear oscillations by Cut1C is low and the amplitude of oscillation is small compared to that of Alp4C. Cut1/separase, which is localized to the cytoplasm during interphase, moves on to the spindle and the SPB at mitosis and cleaves cohesin at the metaphase/anaphase transition to allow for chromosome separation. The Cut1 carboxy-terminus is required for MT binding (Kumada et al. 1998) and NLS-tagged Cut1C does not induce nuclear movement (Nakamura et al. 2002). It is therefore likely that Cut1C induces nuclear movement by affecting MT dynamics in the cytoplasm. This movement seems analogous to those induced by Alp4C, since cytoplasmic Alp4C, but not nuclear Alp4C, induces nuclear movement accompanying changes in MT dynamics.

Alp4C-induced nuclear oscillation is biased towards the new end of the cell, and the aberrant behavior of MTs is more significant at the new end of Alp4C-OP cells than at the old end. The two ends of the S. pombe cells are not equivalent for cell growth; the old end is the site for cell growth before NETO. Cell growth at the cell end is dependent on cytoplasmic MTs and cell polarity factors, such as tea1 that is transported on the tip of MTs to the cell end (reviewed in Chang & Peter 2003). Although the behavior of cytoplasmic MTs at the cell end seems to be directly linked to cell growth at the cell end, it is not clear whether MTs behave differently at the two ends before and after NETO. Our results suggest that the two ends are not equivalent for MT-end behavior.

The {gamma}-tubulin complex and MT dynamics

Alteration of MT dynamics seems to be induced by disturbing the function of the {gamma}-tubulin complex. In S. pombe, some {gamma}-tubulin mutants (Paluh et al. 2000; Tange et al. 2004) and temperature-sensitive mutants of alp4 and alp6 (Vardy & Toda 2000) have long, curved cytoplasmic MTs. Cytoplasmic MT assembly is enhanced in some Aspergillus{gamma}-tubulin mutants (Jung et al. 2001), and in S. cerevisiae{gamma}-tubulin and Spc97 (Alp4 homolog) mutants (Sobel & Snyder 1995; Marschall et al. 1996; Spang et al. 1996; Knop et al. 1997), suggesting that the {gamma}-tubulin complex has a role in regulating MT dynamics and organization (Jung et al. 2001). In Alp4C-OP cells, altered MT dynamics induce oscillatory nuclear movement as well as MTs curved or curled around the cell end. This phenotype is not specific to Alp4C; similar movements are observed in a temperature-sensitive alp4 mutant and in some of the cells overproducing full length Alp4 and Alp6, or fragments of these proteins (Masuda et al. in this issue). Taken together, these data lead us to suggest that cytoplasmic Alp4C alters MT dynamics and organization by affecting the function of the cytoplasmic {gamma}-tubulin complex.

We have shown that cytoplasmic MTs in Alp4C-OP cells are less dynamic than in control cells and that some parameters of MT dynamics are specifically affected by Alp4C overproduction. In particular, the catastrophe frequency of backward-extending MTs that produce the pushing force against the cell ends is reduced to 14% of that in control cells (0.1/min vs. 0.71/min). Although we do not have any data that explain how Alp4C alters MT dynamics at a molecular level, the behavior of MTs may be affected by changes in levels of MT-binding proteins (MAPs) such as Tip1, a CLIP170 homolog that accumulates at the cell end in Alp4C-OP cells (Masuda et al. in this issue) (Fig. 6). Changes in the levels of MAPs and the behavior of MTs may be directly induced by modulation of {gamma}-tubulin complex function. Recently it has been reported that the "satellite" of the {gamma}-tubulin complex shuttles along MTs and sometimes localizes to the MT plus end (Zimmerman et al. 2004). Localization of the {gamma}-tubulin complex at the plus end of MTs may be involved in regulating the behavior of MT ends, and overproduction of Alp4C may affect the localization and function of the {gamma}-tubulin complex and MAPs at the MT plus end.

Alternatively, modulation of {gamma}-tubulin complex function by Alp4C may indirectly affect the behavior of cytoplasmic MTs through alteration of the MT structure. EM studies have shown that MTs nucleated from the MTOCs have 13 protofilaments, whereas most MTs spontaneously assembled in vitro have 14 protofilaments (Evans et al. 1985). It is therefore likely that the {gamma}-tubulin complex localized at the MTOCs specifies the exact structure of MTs. Cytoplasmic MTs may be assembled from the {gamma}-tubulin complex, the function of which is modulated by Alp4C, or without nucleation by the {gamma}-tubulin complex. A change in MT structure may alter the dynamic behavior of MTs. In S. cerevisiae, expression of mutated {alpha}-tubulin lacking the carboxy-terminal phenylalanine (Glu-tubulin) reduces the amount of Bik1, a CLIP170-like MT plus-end binding protein, bound to the ends of cytoplasmic MTs and suppresses nuclear oscillation (Badin-Larcon et al. 2004). A change in MT structure induced by Alp4C overproduction may affect the behavior of MT ends through accumulation of Tip1. It remains to be investigated whether Tip1 accumulation at the cell end is directly linked to changes in MT dynamics, and how Alp4C induces these phenoytpes at a molecular level.

In conclusion, we have shown that oscillatory nuclear movement induced by Alp4C overproduction is driven by MT-pushing forces via the SPB. This movement is caused by modulation of a nuclear positioning mechanism found in wild-type cells. Alteration of MT dynamics and regulation of the behavior of MT ends could be a mechanism for positioning the nucleus in the cell.


   Experimental procedures
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 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Cell culture and strains

The S. pombe strains used in this study are shown in Table 3. The S. pombe cells harboring pREP vectors were maintained in EMM2 media containing appropriate amino acids, supplements, and 10 µM thiamine. pkl1-D25 and kpl2-D25, and dlc1-d strains were generously provided by J. Richard McIntosh and Osami Niwa, respectively. The GFP-atb2 strain was created by integrating the GFP-atb2+ gene under the control of the nda3 promotor at the lys1 locus using the pYC36 vector (Chikashige et al. 2004). Construction of mis6-GFP and sad1-GFP strains was previously described (Chikashige et al. 2004). Sid4-GFP, sid4-mRFP, and bud6-GFP and tip1-YFP strains were generously provided by Kathleen L. Gould, Yuji Chikashige, and Fred Chang, respectively.


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  		Table 3 Strains used for this study
 
Live cell imaging

Cells grown to late-exponential phase in EMM2 in the presence of thiamine were washed three times with thiamine-free EMM2. The cells were then added to thiamine-free EMM2 at a density of 2 x 105 cells/mL and incubated at 30 °C for 18–20 h. One hundred microlitre of cells were transferred to a glass-bottom dish (MatTek Corp., Ashland, MA, USA) that had been coated with 0.2% (w/v) concanavalin A and incubated for 30 min or more at 25–26 °C for cell adhesion. Live cell imaging was carried out at 25–26 °C using a DeltaVision microscope system (Applied Precision Inc., Seattle, WA, USA), a computer-operated microscope system with a cooled CCD camera, as previously described (Chikashige et al. 1994; Ding et al. 1998; Haraguchi et al. 1999). Images of 10–13 different focal planes at 0.3–0.35 µm were collected at each time point, and projection images were reconstituted from these. For quantification of levels of cell end markers, Tip1-YFP and Bud6-GFP, projection images of summed intensity were reconstituted. Fluorescence intensity over the background intensity (intensity in the cytoplasm) was integrated per each cell end and used for statistic data analysis. For Calcoflour staining, 100 µL of cells were incubated with 0.2 mg/mL Calcofluor in H2O for 15 min and washed once with H2O before adding to a glass-bottom dish with thiamine-free EMM2. For Hoechst 33342 staining, 500 µL of cells were washed twice with H2O, incubated for 30 min with 5 µg/mL Hoechst 33342 in 100 µL H2O, and then resuspended in 500 µL of EMM2 without thiamine. One hundred microlitre of cells were added to a glass-bottom dish.


   Acknowledgements
 
We thank Takashi Toda for stimulatory discussion, Fred Chang, Yuji Chikashige, Osami Niwa, Kathleen L. Gould, Richard McIntosh, and Iain Hagan for providing strains and vectors, and Takashi Toda and David Alexander for critical reading of the manuscript. This work was supported by grants from the Japan Science and Technology Corporation to T.H. and Y.H. and Human Frontier Science Program to H.M.


   Footnotes
 
Communicated by: Masayuki Yamamoto

* Correspondence: E-mail: hmasuda{at}nict.go.jp


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 Discussion
 Experimental procedures
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Received: 14 September 2005
Accepted: 22 December 2005




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