Correspondence to Marcel E. Janson: mjanson{at}mail.med.upenn.edu; or P.T. Tran: tranp{at}mail.med.upenn.edu
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Introduction |
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The rod-shaped fission yeast Schizosaccharomyces pombe organizes a relatively simple linear MT array in interphase independent of the spindle pole body (SPB), which is the yeast analogue of the centrosome (Hagan, 1998). This array comprises about four distinct MT bundles that align along the long axis of the cell (Drummond and Cross, 2000; Tran et al., 2001). Each bundle, in its simplest form, contains two antiparallel oriented MTs that overlap slightly at their minus ends. The plus ends point to the cell ends and occasionally undergo catastrophes, i.e., they switch to a state of depolymerization. Depolymerization is halted at the region of MT overlap near the cell middle and is followed by MT regrowth, hence the region of overlap has become known as interphase MT organizing center (iMTOC). The minus ends that are embedded within the iMTOCs are believed to have no dynamics. Interphase bundles share a common feature with radial MT arrays in higher eukaryotes because MT minus ends are brought together in both cases. Furthermore, we can look upon interphase bundles as a very simple bipolar MT array: two plus ends or poles are separated by a region of MT overlap. Investigation of interphase bundles may therefore provide mechanistic insight into the formation of other bipolar structures, such as the mitotic spindle.
Recent work has characterized the interphase arrays, but many questions remain to be answered. It was shown that iMTOCs can bind to the nuclear membrane (Tran et al., 2001) and contain the well-conserved -tubulin complex (
-TuC; Vardy and Toda, 2000; Zimmerman et al., 2004) that nucleates MTs and caps MT minus ends (Wiese and Zheng, 2000). New iMTOCs are continuously generated during interphase (Sawin et al., 2004), but we do not understand the mechanism nor do we know how the number of bundles is controlled or how MT bundling activity is prevented from bundling all MTs into one big bundle.
Here we identify a novel fission yeast protein mto2p as a protein involved in iMTOC formation. Mto2p associates with cytosolic -TuCs and is essential for MT nucleation from non-SPB sites in interphase. By direct observation of MT nucleation from
-TuCs in wild-type cells we revealed the sequence of events that generate a region of MT overlap. Key are: (a) MT nucleation from
-TuCs bound along the length of existing MTs and (b) selective bundling of antiparallel MTs. These events can efficiently transform single MTs into bipolar structures. Cells that lacked mto2p had primarily a single MT bundle during interphase and we used this phenotype to further investigate the known role of MT bundles in cell morphogenesis (Hagan, 1998) and nuclear positioning (Tran et al., 2001).
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Results |
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Mto2 cells have primarily a single MT bundle
To investigate the role of mto2p in MT organization we generated an mto2 deletion strain, which yielded viable but slightly bent cells (Fig. 3 A). In mto2 cells we observed alp4-GFP at the SPBs in both interphase and mitotic cells, but did not observe any non-SPB localization (Fig. 3 B and Fig. S3 B, available at http://www.jcb.org/cgi/content/full/jcb.200410119/DC1), suggesting that non-SPB
-TuCs are absent. Consistent with this interpretation, we found important defects in the MT cytoskeleton of interphase mto2
cells but no defects in the mitotic spindle (Fig. S3 A). To observe MTs in relationship to the SPB, we performed time-lapse imaging of mto2
cells expressing GFP-tubulin and the SPB marker alp4-GFP (Fig. 3, C and D). The average number of MT bundles in mto2
cells (n = 1.3 ± 0.7 SD; Fig. 3 E) was significantly lower than in wild-type cells (3.6 ± 0.9). The bright medial section of these bundles indicated a region of MT overlap and was reminiscent to, but often longer than the iMTOC region in wild-type cells (Fig. 1 A). Similar to wild-type cells, alternating periods of MT growth from and shrinkage to the overlapped regions were observed in mto2
cells. Bundles in mto2
cells often contained more than two MTs (Fig. 3 C, first plane) and appeared to have more MT mass than wild-type bundles. The SPB in interphase mto2
cells was connected to an MT bundle in 49.7% of the cells (Fig. 3 C, n = 143). This connection was occasionally observed to break. Nonconnected SPBs (Fig. 3 D) had a low nucleation activity (0.0051 min1; n = 5 observed nucleations), corresponding to approximately one event per interphase cycle. The SPB is therefore not the main source of MTs in interphase mto2
cells. The majority of bundles in mto2
cells (60.3%) was not connected to a SPB, suggesting that most bundles form independently of the SPB in mto2
cells. Nonconnected SPBs were not observed in wild-type cells (PT546; Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200410119/DC1).
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Nuclear positioning defects in mto2 cells
Disorganized interphase MT arrays have been linked to nuclear positioning defects (Sawin et al., 2004; Zimmerman et al., 2004) and therefore septation defects (Chang and Nurse, 1996; Tran et al., 2000). Forces, generated by interactions between growing interphase MTs and the cell wall, were hypothesized to push the nucleus toward the middle in wild-type cells (Tran et al., 2001). Deformations of the nuclear membrane at sites of MT-membrane connections were observed in response to MT growth, but it is not known whether these forces are large enough to move the nucleus. To investigate nuclear positioning, we imaged the nuclear membrane in interphase mto2 cells that express the nuclear pore marker nup107-GFP and the SPB marker alp4-GFP (Fig. 4 A). Strong oscillations of the SPB, caused by growth of attached MTs (Fig. 3 C), generated rather large nuclear membrane extensions in comparison to wild-type cells (not depicted). Interestingly, the complete nucleus moved into the direction of these membrane extensions, showing that polymerization forces indeed displaced the nucleus. Large membrane extensions occurred in 60.9% of the cells (n = 87), but 11.3% of this subset showed no SPB oscillations. Extensions were therefore primarily mediated by MT attachments to the SPB, but also occurred by direct binding of MTs to the membrane. We tracked and projected the motion of the nucleus along the long axis of the cell for wild-type and mto2
cells (Fig. 4 B). SDs were calculated for positional traces that lasted 3050 min. The distribution of SDs (Fig. 4 C) was narrow for wild-type cells (average SD is 0.21 ± 0.01 µm; ± SEM) but extended to both smaller and larger values for mto2
cells. Smaller values were primarily caused by mto2
cells that did not show large membrane extensions and likely had no MTs attached to the nucleus (Fig. 4 C, average value of this subset is 0.15 ± 0.01 µm). Larger motion was seen in mto2
cells that did show large membrane extensions (0.34 ± 0.03 µm). This comparison showed that nuclear oscillations in cells that had primarily a single MT bundle attached to the nucleus were larger than in wild-type cells, in which multiple bundles are attached. This observation is predicated by models of pushing-based positioning (Dogterom and Yurke, 1998; Tran et al., 2001). These show that multiple bundles generate partly counteracting forces that cause only small nuclear displacements but maintain a more precise central nuclear position.
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Nucleation of new MTs upon MBC washout is mto2p dependent
To investigate the stability of overlapped regions in MT bundles, we treated cells with the MT depolymerizing drug methyl-benzidazole-carbamate (MBC). As described previously (Tran et al., 2001), MBC addition caused MTs in wild-type cells to depolymerize to short stubs, corresponding to iMTOCs (Fig. 6 A). Stubs maintained a nuclear-bound median position in the cell. MT depolymerization in mto2 cells yielded significantly longer stubs (Fig. 6 B), in agreement with the longer regions of MT overlap in unperturbed mto2
bundles (Fig. 3 C). The average number of stubs per cell after 10 min of MBC treatment in both wild-type (2.9 ± 0.7 SD; n = 11 cells) and mto2
cells (1.09 ± 0.67; n = 34 cells) was in reasonable agreement with the number of bundles shown in Fig. 3 E. Regions of MT overlap in mto2
and wild-type cells were thus similarly resistant to MBC treatment. Treadmilling MTs in mto2
cells quickly disassembled during MBC treatment confirming they are single MTs with no stabilized medial region (Fig. 6 B).
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MT nucleation of single MTs during steady state
The appearance of novel MT bundles was previously reported in unperturbed interphase cells (Sawin et al., 2004). We reasoned that these bundles may originate from the apparent single MTs that were observed in steady-state wild-type cells (Fig. 3 E). These MTs appeared in two distinct manners: (1) short MT fragments separated from existing MT bundles during catastrophes (Fig. S4 B, available at http://www.jcb.org/cgi/content/full/jcb.200410119/DC1) or appeared near iMTOC regions shortly after catastrophes; and (2) MTs appeared independently of other MTs (Fig. 7 A). Rates of observations were 0.054 min1 (31 class A events in 579 min of total cell observation time) and 0.064 min1 (37 class B events). MTs were initially faint and a bright region of MT overlap sometimes became visible at a time scale of 1 min (Fig. 7 A). We measured the length increase after initial appearance for class B MTs and often (11 out of 37 events) observed a kink in the growth curve after 1 min (Fig. 7 B). The average elongation rate after kinks was about two times higher as before kinks (3.80 µm/min vs. 2.07 µm/min; Table I), suggesting that bundles indeed start as single MTs. The transformation to bundles occurred at a rate of at least 0.16 min1 (11 kink events in 68.5 min of length measurements on 37 class B MTs), but apparently did not involve bundling to a separate second MT as observed earlier in mto2
cells (Fig. 5 C). A catastrophe on 17 out of the 37 new MTs did not lead to a complete disassembly of the MT (as in six other cases), but was rescued suggesting that in total 17 MTs made the switch to a bundle. The fate of the remainder 14 MTs could not be observed because these MTs moved close to an existing bundle and often seemed to fuse with them. Class A MTs behaved similarly (unpublished data).
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Mto2-dependent MT nucleation along existing MTs
The MT bundle depicted in Fig. 7 D initially grew only to the right, but later (>216 s) also to the left. This switch coincided with the appearance of additional mto2-GFP dots along the initially single MT. One of these dots might have nucleated a secondary MT in a direction opposing initial growth, thereby forming a bundle. MT nucleation was observed along existing MT bundles in wild-type cells (Fig. 8, A and B). In these cells, dots of GFP fluorescence occasionally moved along MT bundles. The fluorescence intensity at the dot location was about twice that of the underlying MT, suggesting they were very short single MTs. In agreement, these dots were observed to elongate at an average velocity of 2.25 µm/min while sliding (Table I). In both Fig. 8 (A and B), sliding motion stopped or slowed down when the iMTOC region of the underlying MT bundle was reached. We observed 52 inbound (with respect to the iMTOC) sliding events and no MTs that sled toward MT plus ends (outbound). The average velocity of these sliding events, analyzed before motion ceased, was 5.50 ± 0.25 µm/min (± SEM). Additionally we observed four MTs that were nucleated but grew without sliding. The nucleation rate of new MTs along bundles was 0.10 min1 per bundle (56 events in almost 10 h of observation time spread over 32 cells, in which on average one bundle was in focus), which compares well to the rate at which single MTs are transformed to bundles (0.16 min1/MT; Fig. 7). This strongly suggests that bundle formation occurs by nucleation of a second MT along the length of a single MT. In addition, nucleation along MTs likely caused the appearance of class A MTs (0.054 min1/cell or 0.015 min1/bundle), which separated from bundles after they were nucleated along MTs (Fig. S4 B). Note that all rates may be under estimated because we are likely to miss events.
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Observations in mto2 cells showed that mto2p is required for active nucleation along MTs and the transformation of single MTs to bundles; treadmilling MTs (single MTs) were not observed to form bundles (apart from bundling with existing MTs) within a total observation time of 104 min. 17 transitions were expected if transitions would have occurred at wild-type rate (0.16 min1).
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Discussion |
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Observations in other organisms show that -TuCs along MTs and membranes may be conserved sites for nucleation of noncentrosomal MTs: MT nucleation from membrane associated
-TuCs was observed for plant nuclei (Canaday et al., 2000), myotubes (Tassin et al., 1985), nuclear membrane fragments during spindle assembly in Drosophila spermatocytes (Rebollo et al., 2004), and Golgi membranes in mammalian cells (Chabin-Brion et al., 2001; Rios et al., 2004). Nucleation along MTs was recently reported in plant cells (Van Damme et al., 2004), in which
-tubulin is found along MT arrays (Canaday et al., 2000; Drykova et al., 2003). Other findings show that MT nucleation may in general be more dispersed: in animal cells
-tubulin is found along MTs in mitotic spindles (Lajoiemazenc et al., 1994) and punctuated
-tubulin staining was observed along in vitro generated MT asters from egg extracts (Stearns and Kirschner, 1994). In mammalian cells, a majority of
-TuCs does not associate with the centrosome but is cytosolic (Moudjou et al., 1996). New studies, furthermore, show that noncentrosomal MTs contribute to mitotic spindle formation, and aspects of MT nucleation need to be addressed (Tulu et al., 2003; Rebollo et al., 2004).
Our results show an association of mto2p with non-SPB -TuCs, which may be rather large and well-regulated protein complexes involving
-tubulin, mto1p, the J-domain protein rsp1p, alp4p (hGCP2), alp6p (hGCP3), and alp16p (hGCP6; Vardy and Toda, 2000; Fujita et al., 2002; Sawin et al., 2004; Venkatram et al., 2004; Zimmerman et al., 2004).
-Tubulin itself may have a role in binding
-TuCs to MTs (Llanos et al., 1999), but another candidate is the centrosomin-related protein mto1p, which in a very recent study was found to localize all along MTs and the nuclear membrane when overexpressed (Samejima et al., 2005). Mto1p was furthermore shown to bind mto2p directly and associate with
-TuCs in a strongly mto2p-dependent manner. Here, we found several clues on mto2p's function: In mto2
cells, non-SPB
-TuCs were absent during interphase, but
-TuCs at the SPB nucleated a normal mitotic spindle (Fig. S3 A). This excludes a function for mto2p in intranuclear
-TuCs at the SPB. Furthermore, mto2-mRFP did not clearly localize to interphase SPBs but was at the SPB during mitosis. Therefore, a subset of
-TuCs may shuttle between the SPB and the cytoplasm during the cell cycle. Mto2p may play a role in this process or alternatively may be more structurally involved in the assembly of non-SPB
-TuCs.
In agreement with an absence of non-SPB -TuCs in mto2
cells, we observed the steady depolymerziation of MT minus ends in interphase. Fission yeast seems well suited for further studies on the regulation of free MT minus ends, which in general depolymerize in vivo (Dammermann et al., 2003). The deletion of mto2 also increased the polymerization rate of interphase MTs plus ends by 50% compared with wild-type cells (Table I) and caused MTs to curve around cell tips and break occasionally. A possible explanation lies in an increased concentration of free tubulin (Walker et al., 1988) caused by the decreased number of MTs in mto2
cells. However, contradicting this trend, we did not observe very fast polymerization in cells that almost completely lacked MTs (Fig. 5 C). Therefore, a structural change of MTs, related to their nucleation, may form an alternative explanation. All MTs in
-TuCdeficient interphase cells may originate from
-TuCs at the SPB, either from weak nucleation during interphase and mitosis (astral MTs) or as spindle MTs escaping from the nucleus (Sawin et al., 2004; Fig. S3 A). Such "centrosomally" derived MTs were shown to have less protofilaments compared with noncentrosomal MTs in developing wing epidermal Drosophila cells (Tucker et al., 1986).
Based on our observations of MT nucleation, we propose a model for the generation of iMTOCs in fission yeast cells (Fig. 9): nucleation of single MTs from nuclear-membrane-associated -TuCs is followed by nucleation of secondary MTs from MT-bound
-TuCs to form regions of bipolar MT overlap. Key to this model is the observation that iMTOC-like regions can self-assemble out of two MTs. In mto2
cells this occurs when two MTs by chance align in the cytoplasm and in wild-type cells the second MT is directly nucleated along the first MT. The latter mechanism is much more efficient for bundle formation and in fact increases the ratio between bundles and single MTs in cells by a factor of 20 (Fig. 3 E). In terms of MT stabilization and MBC resistance the iMTOCs in wild-type and mto2
cells are quite similar (Fig. 5 B and Fig. 6).
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Our model suggests that the number of MT bundles in cells may be regulated by a tubulin-dependent nucleation rate of -TuCs. For this we note the massive increase in MT nucleation after MBC washout in wild-type cells (32 events in 11 cells within 24 s after MBC washout corresponds to a nucleation rate of 7.3 min1/cell vs. 0.064 min1/cell during steady state), caused by a momentarily increased concentration of free tubulin upon MT disassembly. Similar effects were found after cold treatment (Sawin et al., 2004). In steady-state,
-TuCs may be de-activated when there are too many MT bundles in the cell, and vise-versa. The accumulation of too many bundles may furthermore be regulated by the observed fusion of MT bundles during steady state (unpublished data), which could start near the nucleus. Only there MTs from two different bundles can bundle in an antiparallel manner. In this way, iMTOCs that move too close to each other on the nuclear membrane may be actively combined, making iMTOCs on average well dispersed over the nuclear membrane. After fusion, single MTs may get expelled from the bundle and be removed by catastrophes similar to class A MT appearance. MT contacts in iMTOCs with more than two MTs (Sagolla et al., 2003; Fig. 8 C) may be unstable because nonbundling parallel MT contacts become possible within the iMTOC.
Our analysis has shown how the generation of multiple and well-separated bipolar MT bundles in fission yeast is optimized by MT nucleation along MTs and selective bundling of antiparallel MTs. Similar mechanisms could be important for the generation of linear MT bundles in other cell types, e.g., the cortex of plant cells (Shaw et al., 2003; Van Damme et al., 2004), neurons and myotubes. Based on the existence of large pools of cytoplasmic -TuCs (Moudjou et al., 1996), we further hypothesize that the described mechanisms may act in more complex bipolar arrays such as mitotic spindles. Of particular interest are chromosomal MTs, which are important for the formation of robust bipolar spindles (Karsenti and Nedelec, 2004). They are randomly nucleated around mitotic chromosomes but are later bipolarly organized. MT-plus-enddirected motors would be required to overlap MT plus ends in this instance. The full spindle is more complex and MTs bundling occurs both parallel and antiparallel (Chakravarty et al., 2004). In general, the ability to discriminate between parallel and antiparallel may be key to forming morphological different MT networks, and regulation of bundling activity may give cells the ability to dynamically change MT structures. Further analysis of the fission yeast system should yield key proteins involved in bundling/sliding, and provide insight into the molecular mechanism of maintaining a stable region of MT overlap. This may prove to be instrumental to understanding aspects of MT organization in higher eukaryotic cells.
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Materials and methods |
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COOH (pPT73) and NH2 (pPT76) termini nmt1-based mRFP tagging vectors were constructed by replacing GFP by mRFP (Campbell et al., 2002) in pSGP572 (NotISalI) and pSGP573 (XhoINotI; Siam et al., 2004). Mto2 (XhoINotI) and tub1 (NotISalI) were then subcloned to create mto2-mRFP (pPT79) and mRFP-tub1 (pPT77) vectors.
Cell preparations
All cells containing nmt-based plasmids were grown on EMM + 5 µg/ml thiamine agar plates for 12 d at 30°C. Non-plasmids cells were grown on YE5S. Cells were then grown overnight in a 2-ml shaking liquid culture of the same media at 25°C. Cells in mid-log phase were transferred to an agar pad for imaging (Tran et al., 2001). Exceptions to this procedure were made for cells containing mRFP and CFP plasmids for which pads were used that contained 50 µM n-propyl gallate (Fluka) to minimize photobleaching (Sagolla et al., 2003). Furthermore, for time-lapse imaging of CFP-tubulin (pRL71; Glynn et al., 2001) we transferred very fresh cells directly from plate to pad, which yielded a higher fluorescence signal. The mRFP-fluorescence signal in mid-log growing cells in liquid was very weak, possibly as a result of a delayed folding of mRFP (Campbell et al., 2002). To circumvent this problem, cells were overgrown for an additional day in liquid, which slowed down cell growth and increased the fluorescence signal. These cells were transferred to fresh medium 3 h before imaging. Thiamine suppression (5 µg/ml) was used for pPT79 but not for pPT80.
MT depolymerization and regrowth experiments were conducted on yeast cells that were bound to the bottom of a flow chamber (Browning et al., 2003; Zimmerman et al., 2004). To maximize binding we incubated cells with Con A (Sigma-Aldrich; 5 min in 5 mg/ml in EMM). Cells were washed (three times in EMM) and bound to a poly-L-lysine (P-1274; Sigma-Aldrich)coated coverslip. To depolymerize MTs, 25 µg/ml MBC (Sigma-Aldrich) was flown into the chamber.
For cell growth initiation studies, we grew a liquid mid-log cell culture for an additional 36 h to reach stationary state. Cells were directly transferred to an agar pad for imaging.
Microscopy
Microscopy was performed using a wide-field microscope (model Eclipse TE2000; Nikon; equipped with 100x/1.45 NA Plan Apo objective, Nikon CY GFP and EN GFP HQ filter sets, and Hamamatsu ORCA-ER camera) or a spinning disk confocal scanner (Perkin Elmer combined with a Nikon eclipse E600, equipped with 100x/1.45 NA Plan Apo objective, electronically controlled filter wheels [Sutter Instruments] and Hamamatsu ORCAII-ERG camera). When noted, images were deconvoluted with Softworx (Applied Precision). In confocal mode, the 488- and 568-nm laser lines of an Argon/Krypton laser (Melles Griot) were used for excitation of GFP and mRFP in combination with a triple band pass dichroic mirror. Microscope control, image acquisition and image analysis were done using Metamorph software (Universal Imaging). All imaging was done at room temperature (close to 23°C).
For MT speckle analysis we used wide-field microscopy because of its large depth of field in comparison to confocal microscopy. This allowed for the observations of speckles on MTs that were not completely in focus over their full length. Especially in mto2 cells MTs buckled out of plane.
Data analysis
Velocities of MT growth and motion were obtained using linear least square fits to distance versus time data. Statistical means and errors (Table I) were obtained by weighting data with the total distance covered in individual events. Outbound growth of MTs from regions of MT overlap in wild-type and mto2 cells were analyzed by measuring distances between MT tips and bright central regions. Curved lines were used to measure lengths of buckled MTs. Plus-end polymerization and minus-end depolymerization rates of treadmilling MTs were obtained by measuring distances between MT tips and individual speckles. Only speckles were selected that were visible for at least six frames. Elongation of MTs bundles after MBC treatment was measured over the full length of the bundle (from plus end to plus end) between the first two frames taken after MBC washout (24-s time interval). Here, lengths were not measured from maximum projected images but in the full three-dimensional stacks. In this way, we minimized errors due to the rotation of small MTs in space. The same procedure was applied to the length of short MTs directly after their nucleation (Fig. 7 A). Sliding motion of nucleated MTs (Fig. 8) was analyzed with respect to the bright central region of the underlying MT bundle. In cases were MTs growth could be analyzed while sliding, we measured the velocity of the MT side that pointed away from the central region of the underlying bundle.
Tea1p patches at cell tips were quantified using ImageJ (http://rsb.info.nih.gov/ij/). All images were rotated such that the central cell axis was horizontal. A rolling balltype background subtraction (radius 0.5 µm) was used to isolate the patch from background fluorescence. Additional background subtraction with a fixed value was applied to make every pixel outside the patch zero. We calculated the center of gravity over a 5- by 2.5-µm area that was centered on the cell tip and measured its vertical position relative to the cell middle (estimated from a simultaneously acquired differential interference contrast [DIC] image). The sign of this height was chosen positive in the direction of future cell growth (up or down) or was chosen such that it was positive at the moment of cell growth initiation for the case of straight cell growth.
Displacements of nuclei (Fig. 4) were quantified using Metamorph's track objects algorithm. As a reference image, a cropped image of a nucleus was selected at a time point at which no nuclear deformations were visible. Tracked positions in other frames largely corresponded to the main mass of the nucleus and were not skewed toward deformations.
Online supplemental material
Fig. S1 shows localization of mto2-GFP along the nuclear membrane in interphase cells. Fig. S2 shows immunoprecipitation of alp4-3HA with mto2-GFP and motion of alp4pmto2p complexes along MTs. Fig. S3 displays defects in MT organization and alp4-GFP localization in mitotic mto2 cells. Fig. S4 A shows additional evidence for selective anti-parallel MT bundling after MT nucleation along existing MT bundles and Fig. S4 B shows the detachment of a class A MT from an existing bundle along which it was nucleated. Used cell strains are listed in Table S1 and a supplemental text lists DNA primers. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200410119/DC1.
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Acknowledgments |
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This work is supported by the ADF funds from the University of Pennsylvania to P.T. Tran.
Note added in proof. Mto2 cells largely lack post-anaphase array MTs (Fig. S3). A recent study (Venkatram et al., 2005. Mol. Biol. Cell. 10.1091/mbc.E04-12-1043) shows that mto2
cells consequently have a defective anchoring of the cytokinetic actin ring to the medial region of the cell.
Submitted: 25 October 2004
Accepted: 10 March 2005
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