Molecular Plant Advance Access originally published online on September 26, 2008
Molecular Plant 2008 1(6):950-960; doi:10.1093/mp/ssn054
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Mitotic Spindle Organization by the Preprophase Band
a The Pennsylvania State University, Department of Biology, The Huck Institutes of the Life Sciences, Integrative Biosciences Graduate Degree Program, Plant Physiology Program, University Park, PA 16802, USA
b Present address: Department of Botany, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada
1 To whom correspondence should be addressed, to 208 Mueller Lab at address a. E-mail rjc8{at}psu.edu, fax (814) 865-9131, tel. (814) 863-8618.
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Abstract |
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 TOP Abstract INTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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In higher plants, the preprophase band (PPB) of microtubules (MTs) forecasts the cell division site prior to mitosis and specifies the organization of MTs into a bipolar prophase spindle surrounding the nucleus. However, the mechanisms governing this PPB-dependent establishment of bipolarity are unclear. Here, we present evidence from live cell imaging studies that suggest a role for the MTs bridging the PPB and the prophase nucleus in mediating this function. Results from drug treatments, along with genetic evidence from null kinesin plants, suggest that these MTs contribute to the bipolarity, orientation, and position of the prophase spindle. Specifically, the absence of these bridge MTs is associated with lack of bipolarity, while non-uniform distributions of bridge MTs correlate with prophase spindle migration, deformation, and enhanced bipolarity toward the region of highest bridge MT density. This behavior does not require actomyosin-based forces, and is enhanced by suppressing MT dynamics with taxol. These observations occur during late prophase, and are coincident with the gradual closing of annular spindle poles. Based on these data, we describe a hypothetical mechanism for bridge MT-dependent organization of prophase spindles.
Key Words: preprophase band • mitosis • acentrosomal spindle • microtubule • kinesin • ATK5 • cytokinesis
Received for publication June 11, 2008. Accepted for publication August 9, 2008.
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INTRODUCTION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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In plants, the position and orientation of the cell division plane are established prior to mitosis, and are important in specifying subsequent cell and organ growth axes. The preprophase band (PPB) is a cortical ring of microtubules (MTs) that forms during G2/prophase at the future cell division site (Mineyuki, 1999). The precise roles the PPB plays in division plane determination are unclear, but it appears that the PPB contributes to division plane determination, in part by organizing the prophase spindle, which encapsulates the nucleus during prophase (Bannigan et al., 2008; Lloyd and Chan, 2006; Panteris et al., 2006; Wick and Duniec, 1983). During formation of the prophase spindle, MTs are initially randomly distributed about the nuclear envelope, and later become sorted into two poles perpendicular to the plane of the PPB. In the absence of a PPB, MTs remain randomly distributed about the nuclear surface throughout the duration of prophase, and do not become organized into a bipolar spindle until after nuclear envelope breakdown (NEB) (Chan et al., 2005; Lloyd and Chan, 2006). When present, however, the PPB acts in some way to facilitate the bipolar organization of perinuclear MTs prior to NEB. The establishment of this bipolar configuration during prophase positions MTs to efficiently capture and orient chromosomes later during prometaphase, thereby promoting robust spindle formation (Chan et al., 2005; Yoneda et al., 2005). For these reasons, the role of the PPB is paramount in understanding mitosis in higher plants.
The equatorial region between the two prophase spindle poles typically resides in the plane of the PPB and appears largely devoid of MTs relative to the poles (Marcus et al., 2003; Mineyuki et al., 1991; Wick and Duniec, 1984, 1983). Prior to the establishment of bipolarity, numerous MTs extend between the nucleus and PPB (hereafter referred to as bridge MTs); however, by the time the poles of the prophase spindle are well developed and equatorial clearing appears, the bridge MTs are predominately associated with the polar regions, thereby forming an angle with the PPB plane (Mineyuki et al., 1991). Due to their position connecting the PPB and nucleus, it has been suggested that bridge MTs represent good candidates for the establishment of prophase spindle bipolarity (Granger and Cyr, 2001; Lloyd and Chan, 2006; Wick and Duniec, 1983; Yoneda et al., 2005). In the current study, we sought to test this hypothesis using live cell imaging of bridge MT behavior with respect to prophase spindle formation. Using cytoskeletal drug treatments, along with a kinesin mutant with diminished spindle integrity, we provide evidence in support of the hypothesis that bridge MTs mediate PPB-dependent bipolar spindle formation in cells of higher plants.
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RESULTS |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Lack of Bridge MTs Corresponds with Lack of Prophase Spindle Bipolarity
To test the hypothesis that bridge MTs effect prophase spindle bipolarity, we observed BY-2 cells stably expressing the MT reporter GFP:MBD (Granger and Cyr, 2000). A small proportion of GFP:MBD prophase cells (less than 1%) lack distinct PPBs, and these were observed during prophase/prometaphase spindle formation. In agreement with previously published data (Chan et al., 2005), we observed that cells lacking PPBs display a loss of prophase spindle bipolarity, with the MTs appearing evenly distributed about the nuclear surface immediately prior to NEB (n = 6). Importantly, distinct bridge MTs are also absent in these cells. Figure 1 follows a typical cell lacking both a PPB and bridge MTs during its progression from late prophase (t = 0:00 min:s) into a fully formed metaphase spindle (t = 67:30 min:s). The cell outline is indicated by dotted lines at the start. The sequence starts during late prophase (as indicated by a dense perinuclear MT array), at which time a well developed, narrow PPB normally accompanies the observed perinuclear MTs. NEB occurs at 9 min (1 min after the second time point shown). In agreement with previous studies, we observed that cells lacking PPBs generally required more time to form a clear metaphase spindle (Chan et al., 2005), and that the interceding prometaphase spindle appeared disorganized (as seen at t = 15:30 min:s) The observation that cells lacking distinct bridge MTs also lack prophase spindle bipolarity suggests a functional relationship, although it is also possible that the lack of a PPB itself, or some other process affected by the lack of a PPB, may instead be responsible. To distinguish between these possibilities, we observed prophase/prometaphase spindle formation in cells with fully formed PPBs that lacked a uniform distribution of bridge MTs.
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Prophase Spindle Bipolarity and Behavior Correspond to the Distribution of Bridge MTs
Previous work showed that, in BY-2 cells with an off-centered prophase nucleus, perinuclear MTs in the equatorial region of the nucleus on the side nearest the PPB are less numerous than those on the far side (Granger and Cyr, 2001). We reasoned that this results from an uneven clearing of MTs from the equatorial perinuclear region (i.e. asymmetric prophase spindle bipolarity). Therefore, we sought to observe the behavior of bridge MTs with respect to spindle bipolarity. Observations were made with respect to both spindle and bridge MT behavior, and were compared between cells with uniform and non-uniform distributions of bridge MTs. These differences are best appreciated by first considering cells that have a uniform distribution of bridge MTs.
Figure 2A shows a time series depicting a cell with a centered prophase nucleus that develops a bipolar spindle and enters prometaphase (Supplemental Video 1). At the start of the sequence, the perinuclear MTs show a fairly homogeneous distribution about the nuclear envelope, with a slight build-up at the poles (t = 0 min). At this time, numerous bridge MTs (arrowheads) are seen between both sides of the spindle and the PPB site, and their density in this optical plane is similar on both sides of the nucleus. As prophase proceeds, perinuclear MTs become distributed into two poles oriented along an axis perpendicular to the plane of the PPB, while, at the same time, the MTs become depleted near the equatorial region. Concurrent with this development of bipolarity, the bridge MTs become less numerous, having completely disappeared by early prometaphase (t = 20 min). Just prior to NEB, the well developed bipolar prophase spindle stretches laterally toward the PPB, becoming slightly elliptical (t = 16 min). In the next frame, which is coincident with NEB, the PPB is lost, the spindle has decreased in width, and the MTs of each half-spindle have begun ingression toward the equatorial region (t = 20 min). By late prometaphase, the MTs have fully ingressed to the spindle equator (t = 24 min).
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To better illustrate the spatiotemporal relationship between bridge MTs and prophase spindle development, a kymograph was produced from a transverse slice (indicated by dotted lines) across the equator of the prophase spindle throughout the entire time series (Figure 2B). The bridge MT densities are similar in the left and right regions within the equatorial plane, and become gradually depleted throughout prophase, having completely disappeared by prometaphase. The lateral deformation of the prophase spindle toward the PPB prior to NEB is apparent in the kymograph as a slight bulge in the vertical lines representing the equatorial spindle boundaries (asterisk in Figure 2B). The decrease in width of the spindle after NEB appears as a constriction after the bulge (arrowheads in Figure 2B). Concurrent with the minimum spindle width, MT fluorescence appears in the equatorial region as MTs ingressing from the poles reach the equatorial plane (where, predictably, they will form kinetochore fibers and interpolar bundles).
Figure 2 shows that a uniform distribution of bridge MTs correlates with symmetric prophase spindle bipolarity (i.e. the clearing of MTs from the equatorial region is similar on both sides of the spindle in this focal plane). In order to investigate if the distribution of bridge MTs has an effect on spindle formation, we observed cells with a non-uniform bridge MT distribution to study the resultant bipolarity of the accompanying spindles during the prophase-to-prometaphase transition.
Figure 3 shows a cell with a non-uniform distribution of bridge MTs (Supplemental Video 2). The time frame of observation is similar to that shown in Figure 2. Arrows in Figure 3A serve as a reference point denoting the initial position of the prophase nucleus, and arrowheads indicate bridge MTs. At the start of the sequence, the nucleus is displaced toward the left of the cell, and it exhibits more numerous bridge MTs with the PPB on that side (t = 0:00 min:s). As the spindle develops bipolarity, the bridge MT density remains higher on the left, and the entire nucleus/prophase spindle migrates farther to the left (t = 0:00–23:20 min:s). During this same time period, the perinuclear MTs on the left side of the nucleus stretch out laterally toward the PPB to a greater degree than those on the right side, creating a more pronounced equatorial perinuclear clearing on the left side (i.e. asymmetric spindle bipolarity). As the bridge MTs disappear during late prophase, the prophase spindle MTs ingress to form the prometaphase spindle (t = 23:20–28:00 min:s). Although the MTs have begun ingression to the prometaphase spindle equator by 23 min, many on the left side have retained their oblique orientation from prophase (i.e. they are still oriented toward the PPB site; t = 23:20 min:s). By late prometaphase, however, these oblique MTs have become aligned with the long axis of the spindle (t = 28:00 min:s).
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As in Figure 2B, a kymograph was produced from a transverse slice across the equator of the prophase spindle throughout the entire time series (Figure 3B and Supplemental Video 2). The lateral migration of the nucleus/prophase spindle over time is apparent as a shift to the left of the lines representing the equatorial spindle boundaries. Additionally, due to the enhanced lateral deformation of perinuclear MTs on the left boundary toward the PPB, the shift of the line representing the left equatorial spindle boundary is greater than the corresponding shift of the right equatorial boundary. In this cell, the decrease in spindle width during prometaphase is asymmetrical, with the constriction in the line representing the right equatorial boundary (arrowhead) appearing before the constriction in the left equatorial boundary (open arrowhead). Similarly, the appearance of equatorial fluorescence during MT ingression at early prometaphase is also asymmetrical in this cell, appearing earlier in the right region of the spindle. The delay of both the width decrease and inward congression on the left side of the spindle is notable and indicative of the additional time required for the oblique MTs to realign and straighten along the long axis of the spindle (Supplemental Video 2).
These data show that bridge MTs appear to influence the behavior and morphology of the prophase spindle. Specifically, enhanced bridge MTs precede prophase spindle migration and deformation, and are associated with increased bipolarity (i.e. better sorting of perinuclear MTs to the poles) on the side with denser bridge MTs. The timing and appearance are consistent with a causative role for bridge MTs in mediating these activities.
The Effect of Taxol on Bridge MT Distribution and Prophase Spindle Behavior
The behavior described in Figure 3 was observed in cells that had a persistently higher density of MTs toward the closest region of the PPB. In most cells, however, any deviations from uniformity of bridge MT distribution appear only transiently, and hence do not seem to affect the behavior of the prophase spindle. To further explore the relationship between bridge MT distribution and prophase spindle behavior, cells were treated with taxol, which is known to induce non-uniform bridge MT distributions (Baluska et al., 1996; Panteris et al., 1995). We observed that, within 5 min after the addition of 5 mM taxol, these transient fluctuations in bridge MT distribution become stabilized (i.e. transient fluctuations become dampened and the MTs persist for longer periods), leading to a non-uniform bridge MT distribution in prophase cells. In all cells observed, migration or deformation of the prophase spindle in the direction of the greatest bridge MT density immediately followed the formation of non-uniformity (n = 23). Figure 4 shows a representative cell exhibiting a centered nucleus and uniform bridge MT distribution prior to drug treatment (t = 0 min). Soon after the addition of 5 mM taxol (added approximately 30 s after first frame), the distribution of bridge MTs on the right side of the prophase spindle becomes larger relative to the left side (t = 5 min). During subsequent frames, this imbalance persists and increases, and the nucleus migrates toward the right (t = 10–40 min). By 30 min, the characteristic asymmetric spindle bipolarity (due to greater equatorial clearing on the right side of the prophase spindle compared to the left side) associated with off-centered nuclei is observed. Migration ceases by the beginning of prometaphase, at which point no bridge MTs are seen (t = 35–40 min). The kymograph in Figure 4B (derived from transverse slice along the spindle equator) illustrates bridge MT distribution and nucleus/prophase spindle position over time.
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Taxol-Induced Prophase Spindle Behavior Does Not Require F-Actin
These data suggest that suppressing MT dynamics induces non-uniformity in bridge MT distribution, thereby creating an imbalance in the forces governing prophase spindle behavior. However, an alternative explanation is that the taxol-induced changes in bridge MT density affect F-actin distribution, and that the effects on prophase spindle behavior are not due to MTs, but rather are due to pulling by actomyosin forces. To test this hypothesis, cells were observed for taxol-induced prophase spindle migration in the presence of 20 mM Latrunculin B, which depolymerizes F-actin (Gibbon et al., 1999). As shown by the representative cell in Figure 5, taxol-induced prophase spindle migration and deformation were observed upon treatment of cells with both 5 mM taxol and 20 mM Latrunculin B (n = 8). Prior to drug treatment, the cell exhibits a somewhat off-centered nucleus with a slightly higher bridge MT density on the left side compared to the right (Figure 5A, t = 0 min). Minutes after addition of the drugs (both were added approximately 30 s after first frame), bridge MT distribution shifts, becoming larger on the left relative to the right side, and migration of the prophase spindle to the left occurs over the following frames (t = 3–9 min). Additionally, the asymmetric prophase spindle bipolarity associated with an off-centered nucleus is also observed in this cell (t = 3–12 min). Similar to the situation described in Figure 3, following NEB, many MTs on the left side of the spindle retain their oblique MT orientation, due to the enhanced prophase spindle bipolarity on this side (t = 15 min), but subsequently become aligned predominately with the spindle axis by late prometaphase (t = 18 min). A kymograph was produced (from a transverse slice along the spindle equator) to illustrate bridge MT density and leftward prophase spindle migration/behavior (Figure 5B).
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Bent Spindles of atk5-1 Plants Occur in Cells with Off-Centered Nuclei
The above observations on prophase spindle lateral migration and deformation are consistent with attractive forces between the PPB and prophase spindle. To further understand the nature of these forces, cells were observed during the prophase/prometaphase transition in plants lacking the Kinesin-14, ATK5. Spindles of atk5-1-null mutant plants exhibit diminished structural integrity compared to those of wild-type (Ambrose et al., 2005), and one manifestation of this is bending of prophase/prometaphase spindles in cells with off-centered nuclei (Ambrose and Cyr, 2007). Importantly, the direction of this bending is such that the spindle always bends with its midzone oriented toward the nearest region of the PPB (Figure 6). Shown is a dividing cell from a root tip of an atk5-1 plant expressing GFP-labeled Arabidopsis Tubulin 6 (GFP:TUB6, courtesy of T. Hashimoto). During the time sequence, the cell transitions from prophase into prometaphase, and the spindle axis is bent (arrows indicate angles). This bending was not observed in wild-type spindles with similarly off-centered prophase nuclei (Supplemental Figure 1). It is likely that the decreased structural cohesiveness of atk5-1 spindles makes them more prone to deformation and bending in response to attractive forces between the PPB and spindle.
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Annular Prophase Spindle Poles Close during Late Prophase
During the course of our experiments documenting bridge MT distribution, we observed the annular nature of prophase spindle poles, which has previously been mentioned in the literature (Liu et al., 1993; Marc and Gunning, 1988; Wick and Duniec, 1984). Interestingly, we noticed a gradual decrease in diameter of the polar annuli during late prophase as the cell approached prometaphase. Importantly, we observed that this closing is coincident with the previously reported (Mineyuki et al., 1991) gradual shift in distribution of bridge MTs to exclusively polar connections. Figure 7A shows a time series of 3D reconstructions tilted such that the observer is looking down at the spindle poles. In this cell, which has a non-uniform distribution of bridge MTs, it can clearly be seen that annular closing occurs concomitantly with nuclear migration.
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Figure 7B illustrates annular structure in more detail using 3-D reconstructions of a single half-spindle during early and late prophase. For both time points (0 and 10 min), on the left is a confocal section corresponding to the median optical plane. The sequence on the right is a 3-D reconstruction of the top pole with rotations from 0° to 90°. The inside diameter of the annulus at both time points is denoted by brackets placed at 90° rotation. At 0 min, the prophase spindle has just begun to develop an asymmetry of perinuclear MT fluorescence, which, in this plane, appears as two elongated patches on the sides of the pole. Serial reconstructions reveal that these patches represent cross-sections through the borders of the early annulus. At 10 min, a well developed bipolar prophase spindle is seen and the diameter of the annulus has decreased by about half. Annular poles are also discernable in the cells shown in Figures 2 and 3. Notably, the closing of the annulus is a gradual process that coincides temporally with the appearance of equatorial clearing, the shift in bridge MTs to the poles, as well as with the forces responsible for nuclear deformation and any lateral migration of the nucleus at late prophase (in cells with non-uniform distribution of bridge MTs at pre-prophase, as described in Figures 2 and 3). The online supplementary video corresponding to Figure 2 shows annular formation and closing with greater temporal resolution (Supplemental Video 1).
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DISCUSSION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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The PPB Is an Equatorial Organizer of the Prophase Spindle
In all cases in which the PPB is absent or has been experimentally perturbed, the bipolarity of the accompanying prophase spindle is reportedly affected. Specifically, prophase spindle bipolarity was reduced or absent in the following studies: (1) cells of the Arabidopsis atk1-1 mutant, which exhibit abnormally broad PPBs (Marcus et al., 2003); (2) Arabidopsis suspension cells expressing high levels of EB1:GFP, which frequently fail to form PPBs (Chan et al., 2005); (3) cells of the Arabidopsis tonneau mutant, which lack cortical MTs and PPBs (Camilleri et al., 2002); (4) cells treated with cytochalasin D, which causes narrowed PPBs to broaden (Eleftheriou and Palevitz, 1992; Mineyuki et al., 1991; Mineyuki and Palevitz, 1990); (5) BY-2 cells with double PPBs (Yoneda et al., 2005); (6) cells treated with taxol, which disrupts PPB narrowing and causes bridge MTs to become more numerous and unevenly distributed (Baluska et al., 1996; Panteris et al., 1995); (7) cells that naturally lack PPBs, such as Haemanuthus endosperm cells or meiotic cells (Smirnova and Bajer, 1992; Suzuki and Tanaka, 1999); (8) cells treated with cycloheximide or kinase inhibitors, which inhibit PPB narrowing (Nogami and Mineyuki, 1999; Nogami et al., 1996); (9) caffeine-induced binucleate cells in which one nucleus lacks a PPB (Manandhar et al., 1996); (10) asymmetrically dividing subsidiary cells of Zea mays, where the PPB does not encircle the nucleus, and the accompanying prophase spindle is monopolar (Panteris et al., 2006); and (11) cells from the clasp-1 mutant, which exhibit non-uniform PPB widths (Ambrose et al., 2007).
Although there is a clear correlation between the PPB and prophase spindle bipolarity, the mechanistic underpinnings governing this relationship have remained elusive. Here, we provide evidence that the bridge MTs connecting these two structures play a role in governing the behavior and morphology of the prophase spindle. Most importantly, the lack of distinct bridge MTs corresponds to the absence of prophase spindle bipolarity. Additionally, a non-uniform distribution of bridge MTs corresponds to the following observations: (1) the prophase spindle/nucleus migrates in the direction of more numerous bridge MTs, and this does not require F-actin; (2) the prophase spindle exhibits transient lateral deformations in the direction of the PPB with more extensive bridge MTs; (3) the bipolarity of the prophase spindle is greater (i.e. better sorting into opposing half spindles) on the side with more numerous bridge MTs; (4) the asymmetric bipolarity and lateral deformation of the prophase spindle affect the subsequent direction of ingression of MTs at prometaphase; and (5) the closing of the polar annuli coincides temporally with the lateral deformation and attainment of bipolarity observed during late prometaphase, at which point most bridge MTs are associated with the poles.
A Model for Bridge MT-Based Prophase Spindle Organization
Based on these data, we propose a model wherein bridge MTs transmit tensile forces that facilitate the organization of perinuclear MTs from their initial random distribution, into two halves, oriented perpendicular to the PPB plane. This tension serves to co-align perinuclear MTs in a direction similar to those of the bridge MTs, thus providing a spatial cue for the orientation of the prophase spindle axis (Figure 8). Co-aligning perinuclear MTs with bridge MTs allows kinesin motor proteins to cross-link and slide the MTs past one another, thereby generating attractive forces between PPB and prophase spindle. In cases of uniform bridge MT distribution, these forces are balanced, resulting in symmetrical equatorial clearing of perinuclear MTs and little movement of the nucleus/prophase spindle (Figure 8, left column). Conversely, when bridge MTs become non-uniform, these attractive forces become unbalanced, which leads to nuclear migration and asymmetric equatorial clearing of perinuclear MTs (Figure 8, right column). This model also accounts for the observation that bridge MTs are parallel to the PPB plane early in prophase, but later become associated predominately with the spindle poles by late prophase (Mineyuki et al., 1991). Interestingly, this shift in bridge MT distribution coincides temporally with the closing of the polar annuli, as well as with lateral deformation and formation of equatorial clearing. It is likely that this closing results from classic motor-dependent pole formation, wherein minus ends are tapered to a point by minus end-directed motors (Matthies et al., 1996). Transport of perinuclear MTs to the poles would predictably drag along bridge MTs, resulting in the characteristic equatorial clearing.
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Structural support for this model is provided by electron micrographs revealing that bridge MTs directly connect the prophase spindle to the PPB, which is several MT layers thick; specifically, bridge MTs are actually PPB MTs of which one end curves out into the cytoplasm (both individually and in bundles) toward the nucleus, while the other end remains bundled with other PPB MTs (Bakhuizen et al., 1985; Burgess, 1970). The ends of the bridge MTs that remain inside the PPB are coaligned with PPB MTs, thus providing possible sites for anchorage and/or force generation via MT–MT sliding mechanisms. Indeed, EM studies have shown cross-bridges representing possible cross-linking factors or motors between MTs in PPBs (Hardham and Gunning, 1978). Similarly, interactions between bundled bridge MTs themselves may also provide a scaffold for motor-dependent force generation.
MT Dynamics Maintain Bridge MT Uniformity, thereby Facilitating the Central Positioning of the Prophase Nucleus
In accordance with the findings of Granger and Cyr (2001), we observed here also that, by late prophase in most dividing BY-2 cells, the nucleus typically resides within the plane of the PPB. However, the location of the nucleus within the PPB plane is more variable. In most cells, the distribution of bridge MTs is typically fairly uniform, and any fluctuations in distributions are transient. As has previously been reported, treatment with the MT-stabilizing drug taxol leads to non-uniform bridge MT distribution (Baluska et al., 1996; Panteris et al., 1995). Here, we show that, upon development of bridge MT non-uniformity, the nucleus/prophase spindle migrates and deforms in the direction of the greatest density of bridge MTs in an acto-myosin-independent manner. Based on these findings, we hypothesize that the dynamic properties of MTs serve as a ‘buffering’ system to prevent the formation of non-uniform bridge MT distribution. If left unchecked, the normal stochastic fluctuations in bridge MT distribution can become amplified (i.e. MTs grow longer and more bundled in one region) and lead to imbalances of the forces governing prophase spindle position, morphology, and behavior. Non-uniformity is achieved locally by a feedback mechanism wherein MTs become bundled and stabilized, and any MTs encountering these bundles would subsequently become incorporated into the bundle, thereby leading to local amplification of bundling. In agreement with this hypothesis, the perinuclear MTs of the prophase spindle exhibit increased dynamic properties (Dhonukshe and Gadella, 2003), which would predictably increase the buffering capacity of these MTs.
Other Evidence of Attraction between PPB and Prophase Spindle
Our data provide evidence of attractive forces between the prophase spindle and PPB, and suggest that bridge MTs are a candidate for the transmission of this force. Further evidence for forces between the PPB and prophase spindle comes from the observation that the less structurally cohesive spindles of atk5-1 are prone to deformation toward the close PPB in cells with off-centered prophase nuclei (Figure 6). Other studies have also provided evidence for attractive forces between the nucleus and PPB: (1) laser microsurgery experiments wherein the cytoplasmic strands rapidly recoil upon laser ablation (Goodbody et al., 1991; Hahne and Hoffmann, 1984); (2) prophase nuclei become distorted into an elliptical shape along the plane of the PPB, suggesting the presence of pulling forces acting on the nucleus along the plane of the PPB (Burgess, 1970; Panteris et al., 1991); (3) association of PPBs with isolated prophase nuclei, indicating a physical link between the two (Wick and Duniec, 1984); and (4) reduced organelle motility within mature phragmosomes (Mineyuki et al., 1994; Ota, 1961) and resistance of prophase nuclei to centrifugation, suggesting increased gelation and tensile forces between the PPB and prophase spindle (Mineyuki and Furuya, 1986; Mineyuki and Palevitz, 1990; Pickett-Heaps, 1969). Although these findings provide compelling evidence for tensile forces between the PPB and prophase spindle mediated by bridge MTs, further studies are required to elucidate the molecular players governing the creation of bipolarity along an axis perpendicular to the plane of the PPB.
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METHODS |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Plant Material and Growth Conditions
Suspensions of tobacco BY-2 cells expressing the MT reporter GFP:MBD (Granger and Cyr, 2000) were maintained in BY-2 medium (4.3 g L-1 Murishige and Skoog's Salts (Caisson Lab, Logan UT), 100 mg L-1 inositol, 1 mg L-1 thiamine, 0.2 mg L-1 2,4-D, 255 mg L-1 KH2PO4, 3% sucrose, pH 5.0) and diluted 1:50 into fresh medium at 7-d intervals. Cells were observed in the period of 2–4 d post-sub-culturing.
Plant growth, cell culture maintenance, and Agrobacterium-mediated stable transformations were performed as described previously (Ambrose et al., 2005). Plasmid containing GFP:TUB6 was a gift from T. Hashimoto (Nara Institute of Science and Technology, Ikoma, Japan.), and was stably transformed into wild-type and atk5-1 plants.
For live microscopic observation of plants, single-well chamber slides (Sigma, St Louis, MO) were supplemented with a 3-mm layer of growth medium (3 mM KNO3, 2 mM Ca(NO3)2.4H2O, 0.5 mM MgSO4.7H2O, 300 mg L-1 myo-inositol, 0.5 g L-1 MES, 1 mg ml-1 thiamine, 0.7% sucrose, 0.4% phytagel (Sigma, St Louis, MO), and micronutrients: 25 µM KCl, 17.5 µM H3BO3, 1 µM MnSO4.7H2O, 0.25 µM CuSO4.5H2O, 0.25 µM (NH4)6MoO24.4H2O, 25 µM (ethylene-dinitrilo tetraacetic acid (Fe–Na EDTA), pH 5.7). Seeds were pushed through the medium with a sterile toothpick until they reached the coverslip at the bottom of the chamber. Chambers were then placed at an angle, allowing growth of roots along the coverslip to facilitate viewing. Seedlings were observed 3–7 d after germination.
Fluorescence Microscopy
Images were collected using a Plan-Neofluar 40x (N.A. 1.3) oil-immersion objective (Zeiss, Thornwood, NY, USA). Wide-field microscopy was conducted using a shutter-equipped Zeiss Axiovert TV-100, and images were captured with a Coolsnap HQ CCD camera (Roper Industries, Duluth, GA) controlled by ESEE software (Inovision Corp., Durham, NC). GFP fluorescence was observed with a 460–500-nm excitation filter and a 510–560-nm emission filter. The light source was a variable intensity 100-watt mercury-vapor short-arc lamp (FluoArc, Zeiss). Typical lamp intensity was 25%, and exposure times were 0.5–1 s.
Confocal Microscopy
Confocal imaging was performed with a 40x plan-apochromatic water immersion objective mounted on a Zeiss LSM 510, using the 488-nm line from an argon laser. GFP fluorescence was observed using a 488-nm dichroic filter and a 505–545-nm emission filter. Typical scan times were 4 s, using a line averaging of 2 s. Slice thickness was 1.5 µm. Scan intervals were variable, and are indicated in figure legends.
Drug Treatments
Where indicated, 20 µM Latrunculin B (Calbiochem; San Diego, CA) was added from a 20-µM ethanol stock solution. A stock solution of 10 µM taxol (Sigma, St Louis, MO) was prepared in DMSO, and diluted to 20 µM in water immediately prior to experiments. Taxol treatments were at 5 µM.
Image Processing and Analysis
Image analysis was performed using ImageJ software (http://rsb.info.nih.gov/ij/).
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SUPPLEMENTARY DATA |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Supplementary Data are available at Molecular Plant Online.
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FUNDING |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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This work was funded by grants from the National Science Foundation, the United States Department of Agriculture and Department of Energy. J.C.A. was supported by a National Science Foundation training grant.
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Acknowledgements |
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We thank D. Fisher for critical reading of the manuscript, T. Hashimoto for the generous gift of GFP:TUB6, and the Salk Institute Genomic Analysis Laboratory for providing the sequence-indexed Arabidopsis T-DNA insertion mutants. No conflict of interest declared.
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