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Vol. 17, Issue 7, 3242-3253, July 2006
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Max-Planck-Institut für terrestrische Mikrobiologie, D-35043 Marburg, Germany
Submitted December 8, 2005;
Revised April 21, 2006;
Accepted April 24, 2006
Monitoring Editor: Yixian Zheng
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-tubulin–containing MTOCs at the boundary of mother and daughter cell, which results in a polarized MT array. In addition, free MTs and MTOCs move rapidly throughout the cytoplasm. Disruption of MTs with benomyl and subsequent washout led to an equal distribution of the MTOC and random formation of highly motile and randomly oriented MTs throughout the cytoplasm. Within 3 min after washout, MTOCs returned to the neck region and the polarized MT array was reestablished. MT motility and polarity of the MT array was lost in dynein mutants, indicating that dynein-based transport of MTs and MTOCs polarizes the MT cytoskeleton. Observation of green fluorescent protein-tagged dynein indicated that this is achieved by off-loading dynein from the plus-ends of motile MTs. We propose that MT organization in U. maydis involves dynein-mediated motility of MTs and nucleation sites. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Lane and Allan, 1998). MTs are tubulin polymers that possess an intrinsic polarity (Desai and Mitchison, 1997). Their minus-end is usually embedded in a microtubule-organizing center (MTOC), whereas most tubulin exchange occurs at the dynamic plus-end. The polarity of MTs is used by two opposing types of motor complexes, plus-end–directed kinesins and minus-end–directed dyneins (Hirokawa, 1998; Karki and Holzbaur, 1999; Vale, 2003) that move organelles and other "cargo" along MTs. The specific activity of these opposing motors participate in numerous essential processes, such as spindle formation and chromosome segregation (Sharp et al., 2000; Karsenti and Vernos, 2001; Gadde and Heald, 2004), organization of the endomembrane system (Lippincott-Schwartz et al., 1995; Burkhardt, 1998; Lane and Allan, 1998; Lippincott-Schwartz, 1998; Lane and Allan, 1999), and dynamic rearrangement of granules in melanophores (Gross et al., 2002; Barral and Seabra, 2004) or endosomes in fungal cells (Wedlich-Soldner et al., 2002b). Motors use the given polarity of the MT array that is usually determined by the cellular localization of the MTOC. Thus, the localization of the MTOC is a key factor in MT-based cellular organization. In addition, for animal cells it is known that other mechanisms, such as treadmilling of MTs (Rodionov and Borisy, 1997a; Maly and Borisy, 2002) or directed transport of assembled MTs (Keating et al., 1997; Rodionov and Borisy, 1997b; Mogensen, 1999) participates in the organization of the MT array. In particular in neurons MTs are highly motile (Tanaka and Kirschner, 1991; Dent et al., 1999; Yu et al., 2001). These MTs are released from the MTOC within the cell body (Yu et al., 1993) and actively transported into the axon (Baas and Ahmad, 1993). This process is known as slow axonal transport (Baas and Buster, 2004), and it is mediated by cytoplasmic dynein (Ahmad et al., 1998; He et al., 2005) that most likely is anchored to the cortex by interacting with F-actin (Garces et al., 1999; Hasaka et al., 2004). In contrast, the combined activity of kinesins and dynein leads to an anti-polar MT orientation in dendrites (Sharp et al., 1997a; Yu et al., 1997; Baas and Buster, 2004). Thus, in neurons MT orientation mainly results from the activity of motor complexes, whereas the site of MT nucleation is of minor importance for the organization of the MT arrays in these cells.
In yeast-like cells of the fungus Ustilago maydis, MT organization is thought to be determined by cell cycle-specific activation of cytoplasmic and nuclear MTOCs (Steinberg et al., 2001; Straube et al., 2003). In unbudded cells in G1 or S phase, MTs are nucleated at numerous cytoplasmic sites and organize themselves into antipolar bundles (Straube et al., 2003). In contrast, budding cells in G2 phase contain an almost unipolar MT cytoskeleton, with plus-ends reaching to the distal cell pole and into the growing bud. The polarization of MTs is thought to be based on the activity of MTOCs that are located in the neck region between the mother and the daughter cell (Straube et al., 2003). Interestingly, MTs within the interphase array are highly dynamic and undergo numerous types of motility, including bending and sliding (Steinberg et al., 2001). In addition, free MTs are transported along the cortex at rates that are reminiscent of motor activity (Steinberg et al., 2001). However, neither the motor for this MT motility nor the cellular importance for MT transport in U. maydis is known. Here, we demonstrate that cytoplasmic dynein mediates transport of individual MTs and nucleation sites. This motility is essential for MT polarization, suggesting that motor activity organizes MTs in growing U. maydis cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). To visualize MT plus-ends in the green fluorescent protein (GFP)-
-tubulin–expressing strains FB2_GT (Steinberg et al., 2001) and FB1Dyn2ts_GT (Adamikova et al., 2004), the endogenous copy of peb1, an EB1-homologue in U. maydis (Straube et al., 2003), was replaced by a construct containing monomeric red fluorescent protein (RFP) (Campbell et al., 2002) C-terminally fused to peb1 that was followed by a phleomycine resistance cassette. In addition, a Peb1-yellow fluorescent protein (YFP) fusion construct (Straube et al., 2003) was homologously integrated into the temperature-sensitive dynein mutant strain FB1Dyn2ts (Wedlich-Soldner et al., 2002a). To analyze the relationship between the polar MTOC and the nucleus, the Peb1-YFP construct and GFP fused to a nuclear localization signal (Straube et al., 2001) were integrated into strain the wild-type strain FB2 (Banuett and Herskowitz, 1989; Table 1). All homologous integration events were confirmed by Southern blotting. All strains were grown at 28°C in complete medium supplemented with 1% glucose (CM-G; Holliday, 1974) and solid media contained 2% (wt/vol) bacto-agar. Temperature-sensitive (ts) dyn2ts strains and corresponding control strains were grown overnight at 22°C and incubated in a 31–32°C water bath for restrictive growth.
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-values was done in Image-Pro Plus, MetaMorph, or Photoshop (Adobe Systems, Mountain View, CA).
Speckle Analysis and Monitoring Microtubule Orientation
Speckle analysis was done using strain FB2rGFPTub1 (Steinberg et al., 2001). Grown in complete medium supplemented with 1% arabinose (CM-A), this strain expresses an additional copy of -tubulin that is fused to GFP. After shift to complete medium supplemented with 1% glucose, the crg-promoter is repressed (Bottin et al., 1996), and the amount of GFP--tubulin gradually decreases with time, which results in GFP speckles in the MTs after 
4 h in glucose. Quantitative linescan analysis was done using MetaMorph. MT orientation was investigated by following Peb1-RFP or Peb1-YFP fusion proteins in control or temperature-sensitive dynein strains (Table 1). Image series were taken at 1- to 1.5-s time interval, and the motility of Peb1 signals was monitored. Because Peb1 only binds to growing MT plus-ends (Straube et al., 2003), moving signals indicated MT elongation and thus orientation. For experiments using a temperature-sensitive dynein allele (dyn2ts; Wedlich-Soldner et al., 2002a), control cells and dynein mutants were cultivated overnight at 22°C and than shifted to a 31–32°C water bath for 70–90 min. Budding cells were microscopically observed using a temperature-adjustable 100x Apochromat objective that was prewarmed at 32°C.
Benomyl Experiments
Benomyl-induced depolymerization was monitored by placing 1 µl of logarithmically growing FB1GT cells on a 2% agar/20 or 30 µM benomyl cushion that was generated by flattening 100 µl of hot agar solution between two microscope slides. Depolymerization of MTs was immediately observed in the microscope, and z-axis stacks were taken at various time points. For benomyl recovery experiments, 5 ml of logarithmically growing cells was incubated with 20 µM benomyl (Sigma Chemie, Taufkirchen, Germany) for 30–90 min at 28°C, and disruption of GFP-MTs was microscopically checked. Three hundred microliters of the benomyl-containing suspension was sedimented at 3500 rpm for 10 s. The pellet was resuspended in 300 µl of fresh medium, and cells were rapidly sedimented at 3500 rpm for 5–10 s. After removal of the supernatant, cells were resuspended in 100 µl of fresh medium, embedded in low-melt agarose, and immediately observed under the microscope. For recovery experiments on temperature-sensitive dynein mutants, all media, the centrifuge, and the microscope objective were prewarmed to 32°C. Image stacks of budded cells were acquired for 200 ms at 300-nm z-axis steps before benomyl treatment and within 6 min after drug removal.
Quantitative Analysis of -Tubulin-GFP Distribution
Strain FB1rTub2_T2G_R2T1 contained 1) the endogenous copy of the -tubulin gene tub2 under the control of the repressible crg promoter, 2) an ectopic copy of tub2 fused to GFP, and 3) a double RFP fused to -tubulin (tub1). Cells were grown in CM-A, and tub2 expression was repressed after shift to CM-G for 4 h. Tub2-GFP that were visible for at least 1.5 s (3 frames) were counted in the neck region that was defined as the area ± 3.5 µm around the neck constriction and compared with the number of Tub2-GFP dots within the rest of the cytoplasm. All values were normalized to an area of 1 µm2.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-tubulin of U. maydis (Steinberg et al., 2001; for strains, see Table 1), z-axis projections of six to eight optical planes demonstrated that long MTs are focused at the neck region (Figure 1A, arrowheads) and extend into the mother and the daughter cell. In addition, many cells contained short MTs that had no contact with the neck region (Figure 1A, arrow). To monitor the orientation of MTs in this array, we labeled MTs with GFP--tubulin and the plus-ends with a fusion protein of RFP and Peb1, an EB1-homologue that binds to MT plus-ends in U. maydis (Straube et al., 2003; strain FB2Peb1R_GT; also see Table 1). Consistent with previous results, we found that Peb1-RFP localized to elongating MT plus-ends that grew toward the distal cell pole, but it was absent from rapidly shortening MTs (Figure 1B, arrowhead in series). Occasionally, we observed that two Peb1-labeled plus-ends occurred at the same site in the neck and left the site in opposite directions (Figure 1C, arrows in image series; enlarged area indicated by box). This supports the notion that the neck region contains MTOCs (Straube et al., 2003). This nucleation activity was not associated with the nuclear spindle pole body, because the nucleus, marked with GFP fused to a nuclear localization signal (Straube et al., 2001), was located in the mother cell (Figure 1C, strain FB2Peb1R_nGFP). A detailed quantitative analysis of the orientation of MTs revealed that yeast-like cells (strain FB2Peb1R_GT) contain three classes of MTs: 1) long MTs that reach into neck region (Figure 1D, 1), 2) short MTs that were in contact with these long MTs (Figure 1, 2), and short "free" MTs (Figure 1D, 3). Whereas MTs of class 1 extend 96.5% of their plus-ends to the cell poles, MTs of the other classes had a more random orientation, with 57.4 (class 2) and 42.2% (class 3) of all plus-ends directed to the cell poles.
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-tubulin–expressing cells on top of an agar cushion supplemented with benomyl, a fungicide that reversibly destroys the tubulin cytoskeleton in U. maydis (Straube et al., 2003; Fuchs et al., 2005). This experimental setting allowed the microscopic observation of MT depolymerization in real time. Under the assumption that most MTs are nucleated at polar MTOCs, we predicted that minus-ends of shrinking MTs would keep contact with the neck region while they depolymerize at their plus-ends. Indeed, MTs disappeared within 6 min under benomyl treatment (Figure 2A, 6:00 min). Consistent with our model, most long MTs were still in contact with the neck region when incubated for 1–3 min (Figure 2A, 1:15 and 2:15 min; arrowhead marks neck region). However, additional free MTs that were not in contact with the polar MTOC were frequently found (Figure 2A, arrow at 2:15 min). A quantitative analysis revealed that about one-half of all cells contained such short MTs, suggesting that free MTs are a regular part of the MT array of U. maydis. To investigate this further, we performed benomyl recovery experiments, in which MTs were depolymerized in the presence of 20 µM benomyl (Figure 2B, +ben), and their reappearance was monitored after washout of the drug. Indeed, 1–2 min after rinsing the cells with fresh medium, MTs occurred randomly in the mother cell (Figure 2B, arrow), suggesting that MTs were nucleated at cytoplasmic nucleation sites. To determine the orientation of these free MTs, we performed benomyl recovery experiments using strain FB2Peb1R_GT. These experiments surprisingly revealed that reappearing MTs initially had a random orientation, with almost 50% of all MT plus-ends growing to the distal cell pole (Figure 2C, 1.5 min after washout of the drug). However, with time the MT array rapidly repolarized and the normal degree of polarization was achieved 3–3.5 min after removal of the drug (Figure 2C, compare 3.5 min and untreated).
Microtubules Undergo Unidirectional Motility
Our results indicated that a considerable portion of MTs is nucleated at free sites in the cytoplasm of the mother cell. Interestingly, repolarization of the MT array was accompanied by a significant increase of MT motility (Figure 3A), and even very short MTs were moving in cells that recovered from benomyl (Figure 3B), suggesting that MT motility participates in MT organization. Rapid motility of MTs at rates up to 41 µm/min was previously described in interphase of U. maydis (Steinberg et al., 2001). This phenomenon was found in 5–8% of untreated cells of strain FB1GT, and MTs were often moving along the periphery of the cell (Figure 3C, arrows). To confirm that assembled MTs are transported, we performed a speckle analysis of this motility phenomenon. We made use of strain FB2rGFPTub1 that contains GFP--tubulin under the control of the inducible/repressible crg-promoter in addition to its endogenous -tubulin (Steinberg et al., 2001). Growing this strain under inductive conditions (see Materials and Methods), cells express GFP-Tub1, which is incorporated into the MTs. After shift to repressive conditions for 4 h, the level of GFP-Tub1 decreases, and speckles of the fusion protein occur in interphase MTs. Formation of speckles is expected to be a random process; therefore, speckled MTs are most likely individually polymers. These speckles were used as structural landmarks on moving MTs. In all cases of MT motility that were observed, these speckles remained constant while the MT was translocated (Figure 3D, arrowheads; compare with stationary signals marked by asterisk; and E, arrows). This strongly indicates that assembled MTs are transported by unknown motor activity. This transport is a frequent process, because >50% of all free or bundled MTs showed either directed motility or bending within 30-s observation time (Table 2, motility), and both populations undergo either polymerization or depolymerization (Table 2, dynamic instability).
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2.4 s). Based on the bipolarity of these MT structures and the unusual flexibility of the hinge, we speculate that the nonfluorescent structures are nucleation sites. To confirm this, we observed a fusion protein of -tubulin and GFP (Tub2-GFP), which is located at MTOCs in U. maydis (Straube et al., 2003). In strain FB1rT2_T2G_R2T1, grown for 4 h in CM-G, the endogenous copy of tub2 is repressed and Tub2 is replaced by Tub2-GFP fusion protein (Straube et al., 2003). Under these conditions, faint Tub2-GFP dots were seen at the nonfluorescent hinge of the bend MT structures (Figures 4C, arrow, and 5A). In addition, -tubulin-GFP was found at the end of moving MTs (our unpublished data), suggesting that both MTs and nucleation sites are transported. Simultaneous observation of Tub2-GFP and Peb1-RFP in strain FB1rT2_T2G_P1R confirmed that one or two plus-ends leave these -tubulin dots, suggesting that they are indeed sites of MT nucleation (Figure 4D, arrow). Three to four of these Tub2-GFP dots were usually found in the neck region of budding cells (Figure 5A, arrowheads, strain FB1rT2_T2G_R2T1). Interestingly, the -tubulin signals, together with short MTs (Figure 5A, details), were motile and showed frequent short-distance movements toward the bud (Figure 5B, arrow; series shows motility of TUB2-GFP dot shown at the bottom of Figure 5A). In addition, they suddenly occurred at the neck, which could be due to new formation of MTOCs or their appearance from regions not in the focal plane (Figure 5B, compare first frame with 2 signals and last frame with 4 dots; see also inset in Figure 5C). Concentration of Tub2-GFP was only seen in the neck of budding cells (Figure 5C, asterisk), indicating that the polar MTOC consists of numerous small and motile nucleation sites. Interestingly, the polar MTOC accumulation at the neck was lost after disruption of MTs with benomyl, and Tub2-GFP dots were randomly distributed (Figure 5D, arrows; spindle pole body indicated by arrowhead; neck indicated by asterisk). However, 3–4 min after washout of the MT inhibitor, Tub2-GFP dots reappeared at the neck (Figure 5E), which nicely corresponds with the reappearance of the polarized MT array (see above; Figure 2C).
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-tubulin–containing MTOCs move within the cell. Apparently, this motility concentrates MTOCs at the neck region and is therefore most likely responsible for the establishment of the polar MT array in budding cells. As a first step toward an understanding of the putative motor activity for MT transport, we set out to determine the orientation of moving MTs in strain FB2Peb1R_GT. Most of these free MTs carried a single Peb1-RFP signal and almost all MTs moved with their Peb1-RFP signal leading (Figure 6, A and C). Because Peb1-RFP marks growing plus-ends this result indicated that MTs slid as a consequence of the activity of a minus-directed motor that pushed the MT plus-end forward. In addition, MTs occasionally moved with the plus-end trailing behind (Figure 6C) or even showed a bidirectional to-and-fro motion (Figure 6B). This raises the possibility that unknown plus-directed motors also participate in MT motility. Studies in neurons and fibroblasts suggest that minus-end–directed cytoplasmic dynein mediates MT motility in animal systems. Thus, we speculated that dynein could be responsible for MT transport in U. maydis. To check this possibility, we investigated the motility of GFP--tubulin–labeled MTs after benomyl recovery in conditional dynein mutants (strain FB1Dyn2ts_GT). This mutant contains a temperature-sensitive allele of dyn2, which encodes the C-terminal half of the dynein heavy chain in U. maydis (Straube et al., 2001). At the restrictive temperature for Dyn2ts cells (Wedlich-Soldner et al., 2002a), benomyl-treated control cells showed prominent MT motility (Figure 7A, control; strain FB1GT). In contrast, in dynein mutants motility of free MTs was almost abolished, indicating that cytoplasmic dynein is the motor for MT motility in U. maydis (Figure 7A, Dyn2ts). Interestingly, MT motility was significantly impaired after disruption of F-actin by 20 µM latrunculin A (Figure 7B, +LatA), suggesting that some motility is mediated by an interaction of dynein with F-actin at the cortex. However, about one-half of all MT motility was still found in the absence of F-actin.
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-tubulin–labeled MTs (strain FB2G2Dyn1_RT; Table 1). The dynein motor complex localized to the tips of astral MTs in anaphase (Figure 7C, only half of an anaphase spindle is shown) and to MT plus-ends at the cell poles of interphase cells (Figure 7D, small bud shown), where it remains attached while MTs were shrinking (Figure 7D, arrow in series). Plus-ends of free MTs also accumulated dynein (Figure 7E1). On MT translocation, a minor fraction of dynein left the tip and dispersed along the length of all observed moving MTs (Figure 7E1, arrows), but it returned to the plus-end when the MT stopped moving (Figure 7E2, previous movement of MT is not shown; arrow marks dynein). Occasionally, distinct dynein signals were observed that remained stationary while the MT moved along the cell periphery (Figure 7E3, arrows), suggesting that MTs are sliding by dynein anchored to the cell cortex.
Dynein Is Required for Polarization of the Microtubule Array in Budding Cells
The described results suggested that MT translocation is based on dynein and that this motility participates in MT polarization. To gain further support for this notion, we next tested whether dynein is required for the rapid polarization of the MT array. We shifted the Peb1-YFP–expressing control strain FB2Peb1Y and the dynein mutant strain FB1Dyn2ts_P1Y (Table 1) to 31–32°C for 1 h, treated them with the solvent dimethyl sulfoxide (DMSO) or with benomyl, and monitored the polarization of the MT cytoskeleton after washout of the reagents (Figure 8A). Consistent with the previously described recovery experiments, control cells reestablished a polarized MT array within 4 min after removal of the drug (Figure 8A, black bars). In contrast, dynein mutants were already impaired in MT polarization and were almost unable to recover polarity of the MT array after benomyl treatment (Figure 8A, gray bars). We next asked whether inactivation of dynein affects the polarity of already established MT arrays. Quantitative analysis of Peb1-RFP motility in control cells (FB2Peb1R_GT) and dynein mutants (FB1Dyn2ts_P1R_GT) revealed no difference at permissive temperature (Figure 8B, 22°C; number of signals/cell number is given in parentheses). After 70–90 min at restrictive temperature, MT arrays in control cells were still polarized (31°C, control). In contrast, MT polarity was almost lost in dynein mutants (Figure 8B, 31°C, dyn2ts). These results added further support to the notion that dynein activity is essential to maintain a polarized MT array in budding cells, and this could be a consequence of reduced MT motility. To get further support for this idea, we disrupted the F-actin, a treatment that was shown to reduce MT motility (Figure 7B). After treatment with latrunculin A for 1 h at 22°C, MT polarization was significantly affected, which was most obvious in mother cells (Figure 8B, compare 64.9% polarized in mother cell in +LatA to 82.7% polarized in mother cells of control at 22°C). Together, these data are most consistent with a role of dynein-dependent MT motility in polarization of the MT array.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-Tubulin–containing Nucleation Sites). On bud formation, the tubulin cytoskeleton is thought to polarize due to the activity of MTOCs in the neck region that send MTs out into the mother and the daughter cells (Steinberg et al., 2001; Straube et al., 2003). Here, we show that these polar MTOCs consist of numerous smaller -tubulin–containing sites that show directional motility within the cell. It was previously reported that 85% of all MT plus-ends, labeled with an EB1-homologue, grow toward the distal cell pole, whereas 15% of the MTs had an opposite orientation (Straube et al., 2003). A more detailed analysis revealed that the polarity of the array is based on the long MTs that are bound to the neck, whereas the free and motile MTs have a random orientation (Figure 1D), suggesting that these MTs are not yet focused at the neck region. In U. maydis, MTs rapidly move through the cytoplasm (Steinberg et al., 2001; this study), and most of them were associated with -tubulin-GFP dots, suggesting that nucleation sites and associated MTs are transported. This idea is supported by the benomyl recovery experiments, in which the nucleation sites lost their polar localization and were randomly distributed within the cytoplasm, where they most likely nucleate the reappearing MTs. Moreover, both polar localization of the MTOCs and polarity of the array are rapidly restored. Thus, bipolar cytoplasmic nucleation sites organize the MT array not only in unbudded cells (Straube et al., 2003) but also during budding, where the MTOCs are concentrated in the neck region (Figure 9). Cytoplasmic nucleation has also been described for animal systems (Yvon and Wadsworth, 1997; Vorobjev et al., 1997), and we therefore consider it likely that this mechanism of MT organization might be more common than anticipated.
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). Motility of free MTs could result from elongation at one end, while the other end is shrinking at similar rates, a mechanism known as treadmilling (Margolis and Wilson, 1981; Rodionov and Borisy, 1997a). Most interestingly, this mechanism has the potential to organize MT arrays in a polar manner (Maly and Borisy, 2002) and might therefore participate in MT organization in U. maydis. However, MT motility happens at average rates of 30–42 µm/min (Steinberg et al., 2001), which is in the range of motor activity but much faster than the observed MT elongation rate of 10 µm/min (Steinberg et al., 2001; Adamikova et al., 2004). Moreover, our speckle analysis strongly argues against MT treadmilling but supports the notion that assembled MTs are transported by molecular motors. MT motility has been described in animal systems (Seitz-Tutter et al., 1988; Tanaka and Kirschner, 1991; Keating et al., 1997; Dent et al., 1999; Yu et al., 2001) and in the ameba Dictyostelium discoideum (Koonce et al., 1999; Brito et al., 2005), but it is not known for other fungi. According to the "sliding filament model," cytoplasmic dynein in the axon is anchored at the cortex and pushes MTs with their plus-end leading toward the synapsis (Ahmad et al., 1998; Hasaka et al., 2004; He et al., 2005), thereby orienting MTs within the axon (Baas and Buster, 2004). A similar situation might exist in U. maydis. In vivo observation of MT plus-ends, labeled with an EB1-homologue, indicated that most motility is driven by a minus-motor. Indeed, MT motility was almost abolished in dynein mutants, suggesting that this minus-motor is responsible for MT motility in the fungus U. maydis. In neurons, it is thought that dynein-based MT motility requires the anchorage of the motor to the cell cortex, which is most likely mediated by cortical F-actin (Hasaka et al., 2004). Interestingly, treatment with the antiactin reagent latrunculin A inhibited only 50% of the dynein-driven MT transport in U. maydis, suggesting that a significant portion of the observed motility occurs independently of cortical F-actin. Latrunculin-insensitive motility of individual MTs was also reported in D. discoideum (Brito et al., 2005), which argues that cortex-independent motility of assembled tubulin is a general phenomenon.
Dynein Translocates along the Moving Microtubule
In fungi, it was shown that dynein localizes to the plus-ends of MTs (Sheeman et al., 2003; Zhang et al., 2003), where it is thought to be stored in an inactive form until it becomes activated for minus-end–directed motility. The best-known example for this is spindle motility in Saccharomyces cerevisiae. Here, growing MTs take dynein to the cell cortex, where it becomes "off loaded" and activated, which results in sliding of the astral MT along the stationary dynein (Heil-Chapdelaine et al., 2000; Lee et al., 2003, 2005; Sheeman et al., 2003). Our results on dynein localization in U. maydis argue for a similar mechanism in motility of interphase MTs. In this model, inactive dynein is stored at MT plus-ends. On activation, it is anchored to a cellular matrix, such as the cortical actin and moves to the minus-end of the free MT, which results in sliding of the filament along the stationary motor (Figure 9A). The inhibition of MT motility by LatA adds support to such a model. Motility is followed by plus-end targeting of dynein, which delivers the minus-motor back to the MT tip for another round of motility.
A Model of Dynein Function in Organizing the MT Array in U. maydis
The results summarized here demonstrate that MT organization in U. maydis is a complex process that involves cytoplasmic nucleation sites, MTs, and dynein. MTs and associated -tubulin–containing nucleation sites move throughout the cytoplasm by the activity of dynein, which is required to maintain the MT array polarized. Thus, it is most likely that dynein activity takes nucleation sites toward the neck, thereby arranging the MT cytoskeleton (Figure 9B). However, motility of MTs and nucleation sites was also directed toward the cell poles (Figures 4A and 9B, 2), suggesting that dynein also exerts outward forces on the MT array. In U. maydis, MTs are often bundled and extend straight from the neck to the cell poles (Steinberg et al., 2001). Plus- and minus-motors exert forces on these MTs (Straube et al., 2006; this study), indicating that the regular organization of the array is a consequence of counteracting motor activity. Thus, poleward dynein activity might primarily stretch MTs into the cell body (Figure 9B, 1), while counteracting MT-based forces focus bipolar nucleation sites at the neck region, where MT bundling forces stabilize them. This idea is supported by the observation that polar accumulation of nucleation sites is abolished in the absence of MTs, suggesting that interaction between MTs is required for keeping the MTOCs at the neck.
In conclusion, our work further supports a concept in which motor proteins organize the MT array, and this process is conserved from neurons (Sharp et al., 1997b; Ahmad et al., 1998; Yu et al., 2000) to fungi (Carazo-Salas et al., 2005; Straube et al., 2006; this study). Our data show that MT organization in U. maydis is based on a complex interplay of MT nucleation at cytoplasmic sites and molecular motors. It is presently unknown whether dynein directly transports nucleation sites or supports their assembly. However, our results make it likely that MT motility participates in polarizing of the MT array. Although further studies are needed to elucidate the mechanistic details, our data indicate that the capacity of motor-based self-organization of MTs is conserved from animals to fungi.
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ACKNOWLEDGMENTS |
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Footnotes |
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The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
Address correspondence to: Gero Steinberg ( gero.steinberg{at}staff.uni-marburg.de)
Abbreviations used: GFP, green fluorescent protein; LatA, latrunculin A; MT, microtubule; MTOC, microtubule organizing center; RFP, red fluorescent protein; YFP, yellow fluorescent protein.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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