1 Max-Planck-Institute for Terrestrial Microbiology, Department of Biochemistry, Karl-von Frisch Str., 35043 Marburg, Germany
2 University of Karlsruhe, Applied Microbiology, Hertzstr. 16, 76187 Karlsruhe, Germany
3 Department of Pharmacology, 675 Hoes Lane, UMDNJ-R.W. Johnson Medical School, Piscataway, NJ 08854-5635, USA
* Author for correspondence (e-mail: reinhard.fischer{at}bio.uni-karlsruhe.de)
Accepted 13 May 2005
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Summary |
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Key words: Spindle pole body, Nuclear movement, Dynein, Kinesin, Microtubules, MTOC
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Introduction |
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Whereas S. cerevisiae mutants with defects in components of the nuclear distribution machinery do not display a severe growth defect, nuclear migration is essential in filamentous fungi to distribute nuclei within the extremely polarized cells (Morris, 1976; Suelmann et al., 1997
). Nuclear migration is best studied in Aspergillus nidulans where the molecular analysis had its basis in a mutant screening (Fischer, 1999
; Morris, 1976
; Morris et al., 1995
; Xiang and Morris, 1999
). R. Morris isolated several temperature sensitive mutants (nud=nuclear distribution), in which nuclei did not migrate out of the conidiospores at restrictive temperature although mitosis and polar hyphal extension were not affected early after germination (Morris, 1976
). Cloning of the corresponding genes as well as analysis of mutants isolated in later screenings revealed several subunits of the dynein protein complex as well as regulatory components (Efimov, 2003
; Efimov and Morris, 2000
; Osmani et al., 1990
; Xiang et al., 1994
; Xiang and Fischer, 2004
). The components are evolutionarily conserved and important in higher eukaryotes (Morris et al., 1998a
; Morris et al., 1998b
; Xiang et al., 1995a
). For instance, malfunction of the human homolog of nudF, Lis1, causes severe brain defects and patients have only a short life expectation. However, to understand the phenomenon of nuclear distribution in A. nidulans, the subcellular arrangement and the interaction of the two main players, the microtubule cytoskeleton and the dynein motor protein need to be considered. Recently, MTs were visualized with GFP and their dynamics was studied (Han et al., 2001
). It was found that in a growing hyphal tip MTs are generally oriented with the plus end towards the tip. Their inherent dynamic instability causes a change between elongation periods and shrinkage. However, it remains unclear how these dynamics could contribute to nuclear distribution. With regards to the motor protein dynein, it is also not clear yet how the motor can move nuclei. In early attempts, the motor was identified by immunolocalization at hyphal tips (Xiang et al., 1995b
). If it were fixed there, it could attach to the MTs, which reach the cortex and subsequently start moving along them. This would cause a pulling of the MTs and could lead to a movement of connected nuclei. This model resembles the proposed mechanism of nuclear translocation in S. cerevisiae (see above). However, nuclei in the tip compartment of A. nidulans move with different velocities and stop moving at different times (Suelmann et al., 1997
). It is difficult to imagine that the individual motor molecules at the tip are regulated differently while being close to each other. In addition, MT bending along the cortex of the tip or a shortening of the filaments is not detectable (our unpublished data) and (Han et al., 2001
). Recently, dynein and other components of the machinery were in addition found at the growing plus end of MTs (Morris, 2003
; Xiang et al., 2000
; Zhang et al., 2002
; Zhang et al., 2003
). Analyses of the dynein distribution pattern in a conventional kinesin mutant of A. nidulans suggested that this motor is responsible for plus end localization (Zhang et al., 2003
). The localization pattern of dynein is in agreement with the pattern in S. cerevisiae. In this organism, tip localization delivers the motor at the cortex and ensures the contact between the growing MT end and protein complexes in the membrane (Sheeman et al., 2003
). However, for the reasons discussed above it seems unlikely that in A. nidulans the dynein at MT plus ends is directly involved in nuclear migration, although it could effect nuclear migration indirectly by effecting MT dynamics.
In addition to `core'-nuclear migration components, two other A. nidulans genes, apsA and apsB were identified by mutagenesis and found to control nuclear positioning (Clutterbuck, 1994). The apsA gene encodes a 186 kDa coiled-coil protein with similarity to the cortical Num1 protein of S. cerevisiae, whereas ApsB is a 121 kDa coiled-coil protein and was originally localized as spots in the cytoplasm (Suelmann et al., 1998
). Both aps mutants were long known to have nuclear migration defects (see supplementary material, Movies 1-5), but the reason was unclear (Fischer and Timberlake, 1995
; Suelmann et al., 1997
; Suelmann et al., 1998
). In this paper, we show that the apsA and the apsB mutations have effects on the MT cytoskeleton and describe ApsB as a novel spindle-pole body associated protein. ApsB appears to be involved in MT production mainly from non-spindle-pole body centrosomes.
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Materials and Methods |
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Molecular techniques
Standard DNA transformation procedures were used for A. nidulans (Yelton et al., 1984) and E. coli (Sambrook and Russel, 1999
). For PCR experiments, standard protocols were applied using a capillary Rapid Cycler (Idaho Technology, Idaho Falls, ID, USA) for the reaction cycles. Genomic DNA was extracted from A. nidulans with the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Southern blotting was performed according to published protocols (Sambrook and Russel, 1999
). DNA sequencing was done commercially (MWG, Ebersberg, Germany).
N-terminal tagging of apsB
To facilitate making N-terminal GFP fusions, the terminator codon of the gfp2-5 gene in plasmid pMCB17 (Fernandez-Abalos et al., 1998) and the subsequent sequence up to the BamHI site were replaced with the sequence GGCGCGCCGGCTTAATTAA containing AscI and PacI recognition sites. The XbaI insert in the resulting plasmid (pMCB17ap) was removed to produce plasmid pMCB17apx. The first 1469 bp of the apsB gene were amplifed by PCR with genomic DNA using primers 5'-TTTGGCGCGCCCGGCATGACTCTAAAAGAGCAAAGTAGTACG-3' and 5'-GGGTTAATTAAGCTCTTCTCCAAAGATTCCATCTCTTC-3'. The PCR product was cut with AscI and PacI and cloned at the AscI-PacI sites of pMCB17apx to give p17apx-apsB. The PCR-derived regions of p17apx-apsB were confirmed by sequencing. GR5 strain was transformed with supercoiled p17apx-apsB. A single homologous integration of p17apx-apsB at the apsB locus was confirmed by PCRs and Southern blottings for five independent pyr4+ transformants. These strains express GFP2-5 protein sequence followed by Gly-Ala-Pro-Gly sequence and complete ApsB protein sequence. All five transformants displayed wild-type phenotypes on non-repressing medium (minimal glycerol), and apsB-like phenotypes (reduced conidiation and colony size) on repressing medium (minimal glucose). For co-localization experiments apsB was also tagged with mRFP1. mRFP1 was PCR-amplified from pDM2 using primers 5'-CGGTACCATGGCCTCCTCCGAGG-3' (including KpnI restriction side) and 5'-CGGCGCGCCGGCGCCGGTGGAG-3' (including AscI restriction side). The PCR fragment was cloned into pCR2.1-Topo to give pDM6. Subsequently mRFP1 was cut out of pDM6 with KpnI and AscI and ligated into p17apx-apsB, which was linearised with KpnI and AscI (GFP was cut out) to give the final plasmid pDM8.
C-terminal tagging of apsB
The apsB gene and GFP were fused as described in (Suelmann et al., 1997) to give pRS48. The gpd-promoter was released from pJH19 with BamHI and ligated into pRS48 after linearization with BamHI. This resulted in the final plasmid pDM5.
Light and fluorescence microscopy
For live-cell imaging, cells were grown in glass-bottom dishes (World Precision Instruments, Berlin, Germany) in 4 ml of minimal medium containing either 2% glycerol or 2% glucose as carbon source. Medium was supplemented with pyridoxine, p-aminobenzoic acid, biotin, arginine, uracil or uridine depending on auxotrophy of the strains. Cells were incubated at room temperature for 1-2 days and images were captured using an Axiophot microscope (Zeiss, Jena, Germany), a Plan-apochromatic 63x or 100x oil immersion objective lens, and a HBO50 Hg lamp. Fluorescence was observed using standard Zeiss filter combinations No. 09 (FITC, GFP), No. 15 (DsRed) and No. 01 (DAPI). Images were collected and analyzed with a Hamamatsu Orca ER II camera system and the Wasabi software (version 1.2). Time-lapse series were obtained with an automated Wasabi program that acquires series of images with 2- or 3-second intervals, 0.1- or 0.75-second exposure time, and about 100 exposures in a sequence. Image and video processing were done with the Wasabi software from Hamamatsu, Adobe Photoshop, ImageJ (NIH, Bethesda, Maryland, USA), and virtual dub (http://www.virtualdub.org). For benomyl studies, the drug was added 15 minutes before observation at a final concentration of 1.5 µg ml1 to germlings grown for 1-2 days at room temperature and observed for 3 hours.
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Results |
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ApsB is a novel spindle-pole body (SPB) associated protein
AspB was described as a cytoplasmic protein with a spot-like distribution when expressed at high levels under the control of the inducible alcA-promoter (Suelmann et al., 1998). We reinvestigated the distribution of ApsB at lower expression levels and compared the localization pattern of C-terminally tagged ApsB-GFP with a homologously integrated construct in which ApsB was tagged with GFP at the N-terminus (GFP-ApsB). With both constructs we observed a spot-like distribution. But in comparison to earlier studies, the number of spots was reduced and an even spacing of ApsB along hyphae became obvious. We assumed that the even distribution was due to co-localization with nuclei. We proved this co-localization through the analysis of GFP- or mRFP1-tagged ApsB in strains with GFP-, DsRedT4- or DAPI-stained nuclei. The result was further confirmed by immunostaining of ApsB-HA. Besides nuclear localized ApsB, extra ApsB spots (20-60%) were found within the cytoplasm, associated with MTs. These spots were highly mobile and moved along MTs with an average speed of 0.2-0.5 µm second1 up to maximum speeds of more than 6 µm second1. The movement along a given MT occurred in both directions. Occasionally, the spots rotated around the MT axis and were able to change between different adjacent filaments without a noticeable delay. C-terminally and N-terminally tagged ApsB behaved alike. The ApsB protein does not have any similarities to known motor proteins and thus the observed movement depends on other forces (see below).
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The apsB mutation affects MTOC activities
The MT array in A. nidulans is produced by the activity of MTOCs at the SPB, in the cytoplasm and at septa (Konzack et al., 2005). Because ApsB localized to the MTOC at the SPB, we anticipated that the staining pattern at the septa was also due to co-localization with the MTOC there. We used the MT plus-end localized kinesin-like motor protein KipA as plus-end marker [as described (Konzack et al., 2005
)] to determine the activity of MTOCs at SPBs and septa. Comparing wild type and apsB-mutant strains during a 5 minute time period, we observed a reduction of newly emanating GFP-KipA signals in the mutant. At SPBs, GFP-KipA signal counts were only slightly reduced by about 30% in apsB mutants, while the situation was much more dramatic at septa, where a reduction of counts of more than 60% in apsB mutants compared to wild type was measured (Fig. 4) (see supplementary material, Movie 11). To test whether the presence or absence of septa influences MTOC activities, we analyzed the MT organization in a sepAts mutant at permissive and at a restrictive temperature (Harris et al., 1994
). We could not detect any effect on the number of cytoplasmic MTs, as we did in the apsB mutant. This was not surprising, because cytoplasmic and SPB associated MTOC activites were unaffected in the sepA mutant (data not shown).
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Mutation of apsA and kinA are synthetically lethal whereas mutations in apsB suppress dynein mutations
Nuclear distribution depends on the function of dynein (nudA) and, to a certain extent, on conventional kinesin (kinA) (Requena et al., 2001; Xiang et al., 1994
). To analyze whether ApsA or ApsB functionally interact with one of these motor proteins, we created corresponding double mutants. The combination of
apsA and
kinA caused a drastic reduction of the growth rate, which is not observed in either single mutant (Fig. 6A). No obvious growth phenotype was found for the combination of
apsA with mutants of the two other A. nidulans kinesin motors kipA or kipB (data not shown). Similarly, the apsA5/
nudA double mutant (or the double mutant
apsA/nudA1) displayed no special phenotype and was identical to the nudA mutant (Fig. 6B). The apsA5 mutation also has no effect on the nudF deletion, which has also a nuclear distribution defect (Efimov, 2003
). In case of the apsB6 mutation, a synthetic lethality was not observed when combined with the
kinA mutation. Surprisingly, the apsB14 mutation, as well as apsB deletion, caused a suppression of the
nudA and
nudF growth phenotypes (Fig. 6B). DAPI staining of nuclei in germinating spores showed that nuclear migration was also slightly improved in the double mutants compared to dynein deletion strains.
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ApsB accumulates in the hyphal tip in the absence of dynein
As discussed above, ApsB moves rapidly along MTs. Such movements can be explained by an action of a processive motor protein associated with ApsB rather than by the dynamics of MTs. To address this question, we investigated ApsB movement in three different kinesin mutants (kinA,
kipA, and
kipB) and in a
nudA-mutant background. Whereas the deletion of either kinesin did not affect the movement of ApsB, the lack of dynein caused an accumulation of ApsB near the apex of growing hyphae. However, the speed and bidirectionality of the ApsB movement along MTs were not affected (Fig. 7) (see supplementary material Movies 17-18). To rule out the possibility that ApsB accumulated at the tip due to the increased number of MT ends at the tip in dynein mutant strains (see Fig. 1A,d), the ApsB localization pattern was observed in apsA1 mutant strains (Fig. 7F), which showed an increase in MT number, too (Fig. 1A,c). However, no accumulation of ApsB was detected. Therefore, ApsB accumulation appears to be the consequence of the defect in the dynein motor, but not of the MT organization itself. The localization of ApsB at the SPB was not altered in
nudA,
kinA or
kipA mutant background.
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Discussion |
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In apsA mutants MTs appear curved and thinner than in wild-type A. nidulans. This could be explained if we assume that ApsA serves a similar function as Num1 in S. cerevisiae, as sequence similarities suggest (Fischer and Timberlake, 1995). Lee et al. suggested that Num1 interacts with MT-plus end localized dynein at the cortex and may be involved in the offloading from the MT tip to the cortex (Lee et al., 2003
). After cortical dynein is activated, it tugs at MTs and pulls attached nuclei. The situation could be similar in A. nidulans where ApsA could serve as a docking place for MTs. If ApsA is absent, the contact cannot be established and MTs continue to grow after reaching the cortex. That astral MTs exert a force at the elongating spindle can be concluded from the observation that mitotic spindles do not oscillate in apsA mutant strains and mitotic progression is delayed. Previously, Clutterbuck observed the presence of some giant nuclei in aps mutant hyphae, although experiments with a mitotic inhibitor or diploid stability tests did not prove a specific mitotic function of the Aps-proteins (Clutterbuck, 1994
). A MT-cortex interaction appears not to be crucial for nuclear migration, because nuclei in apsA mutants move similarly to nuclei in A. nidulans wild type (Suelmann et al., 1997
). The only differences are that the number of nuclei in hyphal compartments of apsA mutants is increased and that they are not evenly distributed. Thus MT-cortex interactions appear to be necessary for nuclear positioning or anchoring once the nuclei are distributed. Conversely, the organization of MTs could be responsible for an even spacing of nuclei in the cell. Plamann et al. suggested that nuclei are interconnected through overlapping MTs and equal forces acting on each side of a nucleus cause their even spacing (Plamann et al., 1994
). If this model applies, any disturbance of the MT cytoskeleton will affect nuclear distribution (see below).
Interestingly, we found genetic interaction between apsA and conventional kinesin, kinA. KinA is not responsible for the localization of ApsA at the cortex (not shown). A link between the two proteins could be dynein, because the heavy chain of dynein, NudA, is possibly transported by KinA along MTs and accumulates at the MT-plus end (Zhang et al., 2003). However, a nudA nuclear distribution phenotype was not observed in the small colonies of the double mutant. Nuclei were even more clustered than in apsA mutants but still migrated out of the conidiospore (data not shown). This suggests that ApsA and KinA serve functions besides nuclear migration and positioning. It is also possible that synthetic inhibitory effect is caused by the stabilization of cytoplasmic MTs caused by kinA deletion (Requena et al., 2001
).
In apsB mutants the number of cytoplasmic MTs was reduced due to a reduction of the MT producing activity of MTOCs. During mitosis the number of astral MTs was reduced whereas the mitotic spindle did not look altered in comparison to wild type. Mitosis itself appeared also not to be affected. In interphase cells, long MTs are oriented longitudinal and span the entire compartment. The number of those MT filaments was reduced to one or two in apsB mutants. It was shown recently that MT nucleation occurs at three different types of MTOCs in A. nidulans (Konzack et al., 2005). One important MTOC is the SPB, the A. nidulans centrosome equivalent, but MT nucleation also occurs at MTOCs in the cytoplasm, close to nuclei and at septa. The cytoplasmic and septal MTOCs are very poorly understood. The lack of ApsB has a more drastic effect on the activity of septal MTOCs than on the activity of the SPB. This suggests that the nucleation centres are not identical. We found that the C-terminally GFP-tagged ApsB protein caused a dominant-negative effect with regards to MT formation. Interestingly, the protein localized still at the SPB but not at the septa anymore. This suggests that the C-terminus of ApsB is crucial for this localization and one can speculate that the C-terminus may be required for protein-protein interaction. These results are similar to results obtained recently for the S. pombe protein Mod20 (=Mbo1p=Mto1p). This protein was identified in a mutant screen designed to identify genes involved in cytoskeleton organization and polarity and was isolated at the same time as a component of the
-tubulin complex (Sawin et al., 2004
; Venkatram et al., 2005
; Venkatram et al., 2004
). Mod20 displays only a weak similarity to ApsB, but considering our findings, Mod20 probably represents a functional homologue of ApsB.
Given that the MTOCs at the septa are more drastically affected by the lack of ApsB and that the number of cytoplasmic MTs is largely reduced, we suggest that the normal MT array in A. nidulans is dependent on the activity of septal MTOCs. Whether ApsB is directly involved in MTOC function or is used to recruit proteins of the -tubulin complex to the MTOCs, as it was suggested recently in S. pombe (Sawin et al., 2004
), cannot be decided yet.
Another question is how the different ApsB pools in the cell are connected. We observed that ApsB-GFP aligns and moves rapidly along MTs into both directions. ApsB, when transported to the MT minus end, would arrive at the MTOC where it could assemble into the complex. The movement of ApsB along MTs does not appear to depend on conventional kinesin, KipA or KipB. Although dynein appears to have an effect, the bidirectional transport of ApsB still occurs, which can be explained if a second motor moves along anti-parallel MT filaments. However, the nature of the movement remains to be determined.
Why does the loss of apsB function suppress mutations in the dynein pathway? This could be due to the effect of mutations on the MT cytoskeleton. MTs are less dynamic in the absence of dynein (Han et al., 2001) and dynein mutants can be partially rescued by the MT destabilizing drug benomyl or destabilising mutations in alpha tubulin (Willins et al., 1995
). If we assume that there is a force that moves nuclei in the absence of dynein that is not MT-dependent (e.g., cytoplasmic streaming), then hyperstable MTs attached to nuclei would only hamper such movements. The reduced number of cytoplasmic MTs in the apsB mutants could facilitate residual nuclear movement in the absence of dynein by freeing nuclei from microtubules. Moreover, since ApsB localizes to septa, it could tether MTs and attached nuclei to septa. Indeed, previous studies showed that nuclei move more freely along the hyphae in apsB mutants (Suelmann et al., 1998
) (see supplementary material Movies 2 and 3).
In this study, we attempt to document how nuclei are pulled through attached MTs. The important question is where the pulling force is localized. Several scenarios can be envisaged (Fig. 8). (1) The mechanism could be similar to S. cerevisiae where dynein is transported to the MT-plus end and then transfers to the cortex. Once there, it could pull the attached MT and thus translocate the nucleus. Our result that cortical protein ApsA is required for mitotic spindle oscillation indicates that MTs are indeed pulled from the cortex, at least during mitosis. Our finding that nuclei still move in apsB strains, in which the number of MTs is largely reduced and interactions of MTs emanating from the nuclear SPB were not observed, speaks against this model as the only mechanism. However, it could be that only very few MT-cortex interactions are sufficient and that those were overlooked in our experiments. (2) Another possibility is that nuclei move along MTs driven by dynein or kinesin attached to the SPB. Although we observed what appears as movement of SPBs along MTs (Fig. 3) (see supplementary material Movies 8 and 9), at the moment we cannot distinguish such movement from pulling of SPBs by attached MTs. (3) We observed that nuclei are sometimes connected by a MT and move synchronously (Fig. 5C) (see supplementary material Movies 14-15). Therefore overlapping MTs, could play an important role for nuclear migration as suggested already by Plamann and coworkers (Plamann et al., 1994). Although the exact mechanism still remains to be elucidated, it is possible that several or even all three of these mechanisms contribute to nuclear distribution in filamentous fungi.
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Acknowledgments |
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Footnotes |
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References |
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Adams, N. R. and Cooper, J. A. (2000). Microtubule interactions with the cell cortex causing nuclear movements in Saccharomcyces cerevisiae. J. Cell Biol. 149, 863-874.
Ahringer, J. (2003). Control of cell polarity and mitotic spindle positioning in animal cells. Curr. Opin. Cell Biol. 15, 73-81.[CrossRef][Medline]
Aramayo, R., Adams, T. H. and Timberlake, W. E. (1989). A large cluster of highly expressed genes is dispensable for growth and development in Aspergillus nidulans. Genetics 122, 65-71.
Berns, M. W., Aist, J. R., Wright, W. H. and Liang, H. (1992). Optical trapping in animal and fungal cells using a tunable, near-infrared titanium-sapphire laser. Exp. Cell Res. 198, 375-378.[CrossRef][Medline]
Bloom, K. (2000). It's a kar9ochore to capture microtubules. Nat. Cell Biol. 2, E96-E98.[CrossRef][Medline]
Bloom, K. (2001). Nuclear migration: cortical anchors for cytoplasmic dynein. Curr. Biol. 11, R326-R329.[CrossRef][Medline]
Clutterbuck, A. J. (1969). A mutational analysis of conidial development in Aspergillus nidulans. Genetics 63, 317-327.
Clutterbuck, A. J. (1994). Mutants of Aspergillus nidulans deficient in nuclear migration during hyphal growth and conidiation. Microbiology 140, 1169-1174.[Medline]
Cottingham, F. R. and Hoyt, M. A. (1997). Mitotic spindle positioning in Saccharomyces cerevisiae is accomplished by antagonistically acting microtubule motor proteins. J. Cell Biol. 138, 1041-1053.
Cottingham, F. R., Gheber, L., Miller, D. L. and Hoyt, M. A. (1999). Novel roles for Saccharomyces cerevisiae mitotic spindle motors. J. Cell Biol. 147, 335-349.
Efimov, V. P. (2003). Roles of NUDE and NUDF protein of Aspergillus nidulans: Insights from intracellular localization and overexpression effects. Mol. Biol. Cell 14, 871-888.
Efimov, V. P. and Morris, N. R. (2000). The LIS1-related NUDF protein of Aspergillus nidulans interacts with the coiled-coil domain of the NUDE/RO11 protein. J. Cell Biol. 150, 681-688.
Farkasovsky, M. and Küntzel, H. (2001). Cortical Num1p interacts with the dynein intermediate chain Pac11p and cytoplasmic microtubules. J. Cell Biol. 152, 251-262.
Fernandez-Abalos, J. M., Fox, H., Pitt, C., Wells, B. and Doonan, J. H. (1998). Plant-adapted green fluorescent protein is a versatile vital reporter for gene expression, protein localization and mitosis in the filamentous fungus Aspergillus nidulans. Mol. Microbiol. 27, 121-130.[CrossRef][Medline]
Fischer, R. (1999). Nuclear movement in filamentous fungi. FEMS Microbiol. Rev. 23, 38-69.
Fischer, R. and Timberlake, W. E. (1995). Aspergillus nidulans apsA (anucleate primary sterigmata) encodes a coiled-coil protein necessary for nuclear positioning and completion of asexual development. J. Cell Biol. 128, 485-498.[Abstract]
Fox, H., Hickey, P. C., Fernández-Ábalos, J. M., Lunness, P., Read, N. D. and Doonan, J. H. (2002). Dynamic distribution of BIMGPP1 in living hyphae of Aspergillus indicates a novel role in septum formation. Mol. Microbiol. 45, 1219-1230.[CrossRef][Medline]
Gundersen, G. G. and Bretscher, A. (2003). Microtubule asymmetry. Science 300, 2040-2041.
Han, G., Liu, B., Zhang, J., Zuo, W., Morris, N. R. and Xiang, X. (2001). The Aspergillus cytoplasmic dynein heavy chain and NUDF localize to microtubule ends and affect microtubule dynamics. Curr. Biol. 11, 19-24.
Harris, S. D., Morrell, J. L. and Hamer, J. E. (1994). Identification and characterization of Aspergillus nidulans mutants defective in cytokinesis. Genetics 136, 517-532.
Hill, T. W. and Käfer, E. (2001). Improved protocols for Aspergillus minimal medium: trace element and minimal medium salt stock solutions. Fungal Genet. Newsl. 48, 20-21.
Hoepfner, D., Brachat, A. and Philippsen, P. (2000). Time-lapse video microscopy analysis reveals astral microtubule detachment in the yeast spindle pole mutant cnm67v. Mol. Biol. Cell 11, 1197-1211.
Inoue, S., Turgeon, B. G., Yoder, O. C. and Aist, J. R. (1998). Role of fungal dynein in hyphal growth, microtubule organization, spindle pole body motility and nuclear migration. J. Cell Sci. 111, 1555-1566.
Ketelaar, T., Faivre-Moskalenko, C., Esseling, J. J., de Ruijter, N. C., Grierson, C. S., Dogterom, M. and Emons, A. M. (2002). Positioning of nuclei in Arabidopsis root hairs: an actin-regulated process of tip growth. Plant Cell 14, 2941-2955.
Konzack, S., Rischitor, P., Enke, C. and Fischer, R. (2005). The role of the kinesin motor KipA in microtubule organization and polarized growth of Aspergillus nidulans. Mol. Biol. Cell 16, 497-506.
Lee, W. L., Oberle, J. R. and Cooper, J. A. (2003). The role of the lissencephaly protein Pac1 during nuclear migration in budding yeast. J. Cell Biol. 160, 355-364.
Maekawa, H., Usui, T., Knop, M. and Schiebel, E. (2003). Yeast Cdk1 translocates to the plus end of cytoplasmic microtubules to regulate but cortex interactions. EMBO J. 22, 438-449.
Miller, R. K. and Rose, M. D. (1998). Kar9p is a novel cortical protein required for cytoplasmic microtubule orientation in yeast. J. Cell Biol. 140, 377-390.
Miller, R. K., Heller, K. K., Frisèn, L., Wallack, D. L., Loayza, D., Gammie, A. E. and Rose, M. D. (1998). The kinesin-related proteins, Kip2p and Kip3p, function differently in nuclear migration in yeast. Mol. Biol. Cell 9, 2051-2068.
Miller, R. K., Matheos, D. and Rose, M. D. (1999). The cortical localization of the microtubule orientation protein, Kar9p, is dependent upon actin and proteins required for polarization. J. Cell Biol. 144, 963-975.
Minke, P. F., Lee, I. H. and Plamann, M. (1999). Microscopic analysis of Neurospora ropy mutants defective in nuclear distribution. Fungal Genet. Biol. 28, 55-67.[CrossRef][Medline]
Morris, N. R. (1976). Mitotic mutants of Aspergillus nidulans. Genet. Res. 26, 237-254.
Morris, N. R. (2003). Nuclear positioning: the means is at the ends. Curr. Opin. Cell Biol. 15, 54-59.[CrossRef][Medline]
Morris, N. R., Xiang, X. and Beckwith, S. M. (1995). Nuclear migration advances in fungi. Trends Cell Biol. 5, 278-282.[CrossRef][Medline]
Morris, N. R., Efimov, V. P. and Xiang, X. (1998a). Nuclear migration, nucleokinesis and lissencephaly. Trends Cell Biol. 8, 467-470.[CrossRef][Medline]
Morris, S. M., Albrecht, U., Reiner, O., Eichele, G. and Yu-Lee, L.-y. (1998b). The lissencephaly gene product Lis1, a protein involved in neuronal migration, interacts with a nuclear movement protein, NudC. Curr. Biol. 8, 603-609.[CrossRef][Medline]
Osmani, A. H., Osmani, S. A. and Morris, N. R. (1990). The molecular cloning and identification of a gene product specifically required for nuclear movement in Aspergillus nidulans. J. Cell Biol. 111, 543-551.[Abstract]
Osmani, A. H., Davies, J., Oakley, C. E., Oakley, B. R. and Osmani, S. A. (2003). TINA interacts with the NIMA kinase in Aspergillus nidulans and negatively regulates astral microtubules during metaphase arrest. Mol. Biol. Cell 14, 3169-3179.
Plamann, M., Minke, P. F., Tinsley, J. H. and Bruno, K. S. (1994). Cytoplasmic dynein and actin-related protein Arp1 are required for normal nuclear distribution in filamentous fungi. J. Cell Biol. 127, 139-149.[Abstract]
Requena, N., Alberti-Segui, C., Winzenburg, E., Horn, C., Schliwa, M., Philippsen, P., Liese, R. and Fischer, R. (2001). Genetic evidence for a microtubule-destabilizing effect of conventional kinesin and analysis of its consequences for the control of nuclear distribution in Aspergillus nidulans. Mol. Microbiol. 42, 121-132.[CrossRef][Medline]
Rischitor, P., Konzack, S. and Fischer, R. (2004). The Kip3-like kinesin KipB moves along microtubules and determines spindle position during synchronized mitoses in Aspergillus nidulans hyphae. Eukaryot. Cell 3, 632-645.
Sambrook, J. and Russel, D. W. (1999). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.
Sawin, K. E., Lourenco, P. C. C. and Snaith, H. A. (2004). Microtubule nucleation at non-spindle pole body microtubule-organizing centers requires fission yeast centrosomin-related protein mod20p. Curr. Biol. 14, 763-775.[CrossRef][Medline]
Segal, M., Bloom, K. and Reed, S. I. (2002). Kar9p-independent microtubule capture at Bud6p cortical sites primes spindle polarity before bud emergence in Saccharomyces cerevisiae. Mol. Biol. Cell 13, 4141-4155.
Shaw, S. L., Yeh, E., Maddox, P., Salmon, E. D. and Bloom, K. (1997). Astral microtubule dynamics in yeast: A microtubule-based searching mechanism for spindle orientation and nuclear migration into the bud. J. Cell Biol. 139, 985-994.
Shaw, S. L., Maddox, P., Skibbens, R. V., Yeh, E., Salmon, E. D. and Bloom, K. (1998). Nuclear and spindle dynamics in budding yeast. Mol. Biol. Cell 9, 1627-1631.
Sheeman, B., Carvalho, P., Sagot, I., Geiser, J., Kho, D., Hoyt, M. A. and Pellman, D. (2003). Determinants of S. cerevisiae dynein localization and activation: Implications for the mechanism of spindle positioning. Curr. Biol. 13, 364-372.[CrossRef][Medline]
Stringer, M. A., Dean, R. A., Sewall, T. C. and Timberlake, W. E. (1991). Rodletless, a new Aspergillus developmental mutant induced by directed gene inactivation. Genes Dev. 5, 1161-1171.[Abstract]
Su, W., Li, S., Oakley, B. R. and Xiang, X. (2004). Dual-color imaging of nucelar division and mitotic spindle elongation in live cells of Aspergillus nidulans. Eukaryot. Cell 3, 553-556.
Suelmann, R., Sievers, N. and Fischer, R. (1997). Nuclear traffic in fungal hyphae: In vivo study of nuclear migration and positioning in Aspergillus nidulans. Mol. Microbiol. 25, 757-769.[CrossRef][Medline]
Suelmann, R., Sievers, N., Galetzka, D., Robertson, L., Timberlake, W. E. and Fischer, R. (1998). Increased nuclear traffic chaos in hyphae of apsB mutants of Aspergillus nidulans: Molecular characterization of apsB and in vivo observation of nuclear behaviour. Mol. Microbiol. 30, 831-842.[CrossRef][Medline]
Toews, M. W., Warmbold, J., Konzack, S., Rischitor, P. E., Veith, D., Vienken, K., Vinuesa, C., Wei, H. and Fischer, R. (2004). Establishment of mRFP1 as fluorescent marker in Aspergillus nidulans and construction of expression vectors for high-throughput protein tagging using recombination in Escherichia coli (GATEWAY). Curr. Genet. 45, 383-389.[CrossRef][Medline]
Tran, P. T., Marsh, L., Doye, V., Inoue, S. and Chang, F. (2001). A mechanism for nuclear positioning in fission yeast based on microtubule pushing. J. Cell Biol. 153, 397-411.
Venkatram, S., Tasto, J. J., Feoktistova, A., Jennings, J. L., Link, A. J. and Gould, K. L. (2004). Identification and characterization of two novel proteins affecting fission yeast -tubulin complex function. Mol. Biol. Cell 15, 2287-2301.
Venkatram, S., Jennings, J. L., Link, A. and Gould, K. L. (2005). Mto2p, a novel fission yeast protein required for cytoplasmic microtubule organization and anchoring of the cytokinetic actin ring. Mol. Biol. Cell 16, 3052-3063.
Waring, R. B., May, G. S. and Morris, N. R. (1989). Characterization of an inducible expression system in Aspergillus nidulans using alcA and tubulin coding genes. Gene 79, 119-130.[CrossRef][Medline]
Willins, D. A., Xiang, X. and Morris, N. R. (1995). An alpha tubulin mutation suppresses nuclear migration mutations in Aspergillus nidulans. Genetics 141, 1287-1298.
Xiang, S., Han, G., Winkelmann, D. A., Zuo, W. and Morris, N. R. (2000). Dynamics of cytoplasmic dynein in living cells and the effect of a mutation in the dynactin complex actin-related protein Arp1. Curr. Biol. 10, 603-606.[CrossRef][Medline]
Xiang, X. and Morris, R. (1999). Hyphal tip growth and nuclear migration. Curr. Opin. Microbiol. 2, 636-640.[CrossRef][Medline]
Xiang, X. and Fischer, R. (2004). Nuclear migration and positioning in filamentous fungi. Fungal Genet. Biol. 41, 411-419.[CrossRef][Medline]
Xiang, X., Beckwith, S. M. and Morris, N. R. (1994). Cytoplasmic dynein is involved in nuclear migration in Aspergillus nidulans. Proc. Natl. Acad. Sci. USA 91, 2100-2104.
Xiang, X., Osmani, A. H., Osmani, S. A., Xin, M. and Morris, N. R. (1995a). NudF, a nuclear migration gene in Aspergillus nidulans, is similar to the human LIS-1 gene required for neuronal migration. Mol. Biol. Cell 6, 297-310.[Abstract]
Xiang, X., Roghi, C. and Morris, N. R. (1995b). Characterization and localization of the cytoplasmic dynein heavy chain in Aspergillus nidulans. Proc. Natl. Acad. Sci. USA 92, 9890-9894.
Yang, L., Ukil, L., Osmani, A., Nahm, F., Davies, J., De Souza, C. P. C., Dou, X., Perez-Balguer, A. and Osmani, S. A. (2004). Rapid production of gene replacement constructs and generation of a green fluorescent protein-tagged centromeric marker in Aspergillus nidulans. Eukaryot. Cell 3, 1359-1362.
Yeh, E., Yang, C., Chin, E., Maddox, P., Salmon, E. D., Lew, D. J. and Bloom, K. (2000). Dynamic positioning of mitotic spindles in yeast: Role of microtubule motors and cortical determinants. Mol. Biol. Cell 11, 3949-3961.
Yelton, M. M., Hamer, J. E. and Timberlake, W. E. (1984). Transformation of Aspergillus nidulans by using a trpC plasmid. Proc. Natl. Acad. Sci. USA 81, 1470-1474.
Zhang, J., Han, G. and Xiang, X. (2002). Cytoplasmic dynein intermediate chain and heavy chain are dependent upon each other for microtubule end localization in Aspergillus nidulans. Mol. Microbiol. 44, 381-392.[CrossRef][Medline]
Zhang, J., Li, S., Fischer, R. and Xiang, X. (2003). The accumulation of cytoplasmic dynein and dynactin at microtubule plus-ends is kinesin dependent in Aspergillus nidulans. Mol. Biol. Cell 14, 1479-1488.[CrossRef][Medline]