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Nuclear migration in fungi – different motors at work
Res. Microbiol. 151 (2000) 247–254 © 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S0923250800001510/REV
Mini-review Nuclear migration in fungi – different motors at work Rüdiger Suelmann*, Reinhard Fischer** Laboratorium für Mikrobiologie, Philipps-Universität Marburg and Max-Planck-Insitut für terrestrische Mikrobiologie, Karl-vonFrisch-Str., 35043 Marburg, Germany (Submitted 11 January 2000; accepted 22 January 2000)
Abstract — Lower fungi such as Saccharomyces cerevisiae and Aspergillus nidulans are ideal organisms for studying the molecular biology underlying nuclear migration in eukaryotic cells. In this review, the role of different motor proteins such as dynein, kinesin and myosin will be discussed. © 2000 Éditions scientifiques et médicales Elsevier SAS fungi, lower / organelle movement / motor protein / microtubule / Saccharomyces cerevisiae / Aspergillus nidulans
1. Introduction Nuclear migration has been observed in eukaryotic cells for some time. However, a detailed molecular analysis of the process has mainly been performed during the past 10 years. The progress in the field has been reviewed with emphasis on nuclear migration in fungi [9, 20], nuclear migration during yeast mating [23], nuclear positioning [22], the role of motor proteins in organelle movement [27] and the correlation between nuclear migration in fungi and brain development in mammals [19]. Several recent publications merit a review on the involvement of different motor proteins in the process in yeast and in filamentous fungi.
2. The phenomenon In Saccharomyces cerevisiae cells, which duplicate by budding, the nucleus migrates from a random position in the mother cell to the bud-
* Present address: Bayer AG, Zentrale Forschung, 51368 Leverkusen, Germany ** Correspondence and reprints Tel.: +49 6421 178 330; fax: 49 6421 178 309; [email protected]
ding neck and undergoes localized mitosis to provide each cell with a nucleus (figure 1). Therefore, the distance over which the nucleus has to migrate from the center of the cell is rather short and the mitotic machinery contributes significantly to this distribution process [34]. In filamentous fungi, nuclei migrate long distances to follow the growing hyphal tip. During the migration process nuclei divide and individual nuclei are left behind to populate the entire mycelium (figure 1). This movement has been followed in living hyphae in a number of different fungi using phase contrast or fluorescence microscopy for GFP-labeled nuclei [14, 29]. Nuclei travel with velocities of 0.1–1.2 µm per min through the mycelium of Aspergillus nidulans and can reach speeds of up to 40 µm per min. Nuclei mostly follow the direction of hyphal tip elongation although other movements have also been observed [29]. One important tool to study the molecular components required for nuclear migration is the use of mutants with defects in nuclear migration (figure 2).
3. The tracks To drive dynamic processes such as organelle movement, two components are required, a
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Figure 1. Nuclear distribution in the yeast S. cerevisiae (A–C) and the filamentous fungus A. nidulans (D, E). Yeast cells were fixed and nuclei were stained with DAPI. In (A) the nucleus migrated close to the budding neck where it divided (B) to provide both daughter cells with a nucleus (C). Cytokinesis occurred afterwards. Nuclei in A. nidulans were stained with GFP and nuclear migration observed in live cells (D, E). A spore germinated and nuclei moved into the germ tube (D). As the germ tube elongated, nuclei followed the tip and duplicated through mitosis (E). The arrow indicates the place of septum formation. Video sequences showing nuclear migration in the filamentous fungus are available in the Internet: http://www.uni-marburg.de/mpi/movies/movies.htm. Modified after [9].
motor and a stator. The interaction between these is highly specific and thus different motor proteins require specific static structures. Two main cytoskeletal elements that serve the stator function are microtubules and actin. Both consist of polar strands which are maintained in a dynamic equilibrium. The subunits, actin and a/b-tubulin, respectively, assemble at one end with a higher rate than they disassemble and thus this growing end is called the plus end. In contrast, at the minus end, the disassembling rate exceeds the assembling rate. Microtubules and actin can serve as tracks along which motor proteins can carry their cargo. The orientation of the tracks within the cell determines the direction of movement of the motor proteins. Whereas dynein migrates along microtubules
Figure 2. Scheme of nuclear migration mutants (dynein) in S. cerevisiae and A. nidulans. Nuclei divide but do not migrate to appropriate positions.
toward the minus end, most kinesins move toward the plus end. Thus, the orientation of the tracks is very important to allow organelle transport in one or the other direction. Microtubules emanate from microtubule organizing centers such as the spindle pole body, which is embedded in the nuclear envelope. There is evidence in some fungi for additional cytoplasmic microtubule organizing centers at the apex of a hypha, which suggests a mixed polarization of microtubules in these cells. Whether this is true for all fungi currently remains an open question. It is largely accepted that microtubules perform an important function in nuclear migration in fungi. A study of microtubule dynamics in living S. cerevisiae cells was performed with GFP-labeled a-tubulin [3] and GFP-labeled dynein [26]. Astral microtubules emanated from the spindle pole body and transiently made contact with the cortex of the cell. Continuous growth of those microtubules caused pushing of the nucleus, whereas shrinkage of a filament led to pulling of the nucleus toward the attachment site. The dynamic interaction of microtubules with the cortex was cell-cycle-dependentregulated and more focused in budding cells,
Nuclear migration in fungi
where microtubules swept the cortex, made contact and displayed a shrinking behavior. If the attachment of microtubules to the cortex is crucial for nuclear movement, the question is raised: through which proteins could this contact be mediated? Two candidates were recently suggested for this function. The first is Num1 protein (Num1p), which is localized in a cellcycle-dependent manner with the cortex of the mother cell and which causes a nuclear distribution phenotype when absent. The Num1p could thus confer contact of microtubules only in one of the two cells, namely the mother cell. A protein with the capacity to mediate the contact of microtubules with the cortex of the daughter cell is Kar9p, localized as GFP-Kar9p at the tip of the emerging bud [8, 18]. Localization of Kar9p was independent of microtubules but dependent on an intact actin cytoskeleton. In addition, its distribution was altered by mutations in genes involved in the polarization of the yeast cell. This is an apt example of how microtubule cytoskeletal functions are interwoven with those of the actin cytoskeleton. In filamentous fungi, the cytological function of microtubules is less clear than in the singlecelled yeast. However, GFP-tagged tubulin derivatives are now available in A. nidulans and should help to unravel their function in nuclear migration (Xiang and Morris, pers. comm.). The situation in A. nidulans might be far more complicated than in yeast, because cell compartments contain several nuclei, which move independently toward the growing tip but are also able to move backwards. A ‘pushing’ or ‘pulling’ mechanism of interphase nuclei through growing or shrinking microtubules was not observed [30]. Moreover, in Nectria haematococca, it was shown with laser optical trap experiments that interphase nuclei are rather fixed at their position [14]. However, a homologue of the yeast Num1p, which could be important for the attachment of microtubules to the cortex, also exists in filamentous fungi such as A. nidulans and Podospora anserina [10] (Picard, pers. comm.). In A. nidulans the protein, ApsA, was localized along the cortex of the cell, although there is no evidence yet that the pro-
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tein is involved in the regulation of microtubule stability as in yeast, or that it might serve as an attachment site for microtubules. Nevertheless, mutagenesis of apsA leads to nuclear misdistribution in hyphae and, because of a misdistribution during conidiophore production, to a strong developmental block. This indicates the importance of exact nuclear migration and positioning in developmental processes [10]. A second gene, apsB, was described in A. nidulans, which had the same phenotype when mutated as apsA mutants and thus might be involved in the same process. GFP-tagged nuclei in an apsB-mutated strain revealed a saltatory movement of individual nuclei within one hyphal compartment, which led to transient clustering of nuclei. In contrast, nuclei are evenly distributed in wildtype cells [30]. A homologue of the ApsB protein can be found in Schizosaccharomyces pombe (sequencing project) but not in S. cerevisiae. Besides actin and microtubules, intermediate filaments are structural components in higher eukaryotic cells. This class of proteins was not described in fungi until recently. Yaffe et al. described a protein, Mdm1, with features of intermediate filaments, and showed that this protein serves roles in nuclear migration and in mitochondrial movement [11] (and previous work of that group). Recently, a similar gene was found in S. pombe (sequencing project). This suggests that this type of protein is likely to be found in other fungi besides yeast and S. pombe as well, and it should be very exciting to unravel the cellular functions of Mdm1p-like proteins in other fungal species.
4. The motors Motor proteins convert chemical energy into conformational changes in the protein, which result in their movement along the cytoskeletal tracks. The exact mechanisms of energy transformation are still not understood for all motor proteins and intensive research is being carried out to solve this fundamental question. For proper functioning of a cell, many different dynamic processes are required. Since the number of cytoskeletal elements is rather limited,
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specificity mainly can be achieved through motor proteins or their associated regulatory components. Indeed, the yeast cell, as a simple eukaryote, contains six kinesins, one dynein and five myosins. The number of motor proteins increases dramatically in higher eukaryotes [12] (see also: http://tubulin.cb.m.utokyo.ac.jp/KIF/index.html). To understand the function of these motors and to dissect their specific roles in different cellular processes is one of the major goals in the field. Lower eukaryotes such as S. cerevisiae and A. nidulans should serve as excellent models to unravel the basic principles. There is good evidence that the microtubuledependent motor protein dynein is one major player for nuclear migration (table I). Dynein is a large protein complex that consists of two heavy chains and several smaller subunits. Using conserved regions of different dynein heavy chains, the corresponding regions were PCR-amplified from S. cerevisiae and functionally analyzed. Disruption of the gene (DYN1 = DHC1) caused an increased percentage of mother cells harboring two nuclei, indicating that the correct positioning and orientation of the mitotic spindle at the budding neck was impaired. Initial movement of the nucleus toward the neck region was not affected but the nucleus did not migrate as close to the neck as in the wild type. It was concluded that dynein is required for proper spindle orientation during mitosis. Nevertheless, dynein-deficient yeast cells are still able to form colonies. The protein was localized as a b-galactosidase fusion protein along cytoplasmic microtubules, at the spindle pole body and at the cortex [34]. Recently, dynein protein distribution was analyzed in live budding yeast cells using a GFP–dynein hybrid protein, which efficiently decorated cytoplasmic microtubules but which was not observed at the cortex (see above) [26]. On the basis of these results Carminati and Stearns [3] suggested a model of how dynein could be involved in nuclear movement. They proposed that microtubules sweep the cortex until they make contact with a protein or protein complex, which serves as a dynein attachment site. Once attached, microtubules
start shrinking and thus pulling in the nucleus. Dynein could directly pull the microtubules or it could catalyze their depolymerization. According to this model, dynein should be transiently localized at the cortex. Recently, deZwaan et al. suggested an additional role for dynein in an oscillatory movement, which the nucleus undergoes at the budding neck in the absence of Kip3 [6]. In A. nidulans the dynein heavy chain gene was discovered in a screen for temperaturesensitive mutants with defects in nuclear migration and subsequently cloned by complementation [32, 33]. In germinating spores, nuclei divided but remained in the spore and did not move out into the germ tube (figure 2). This defect leads to a severe reduction in hyphal growth. The protein was localized through secondary immunofluorescence at the tip of the elongating hyphae, which could point to a pulling force on the microtubules. However, in recent investigations using a dynein–GFP hybrid protein, the motor was also found at the tips of cytoplasmic microtubules (Xiang and Morris, pers. comm.). As dynein is a minus-end-directed motor, this suggests that it is either transported to the tip by another motor, perhaps as part of a kinesin-driven vesicle, or has an affinity for some other tip structure at the tip. Whether this cytoplasmic microtubule dynein mediates nuclear migration remains to be determined. Similar phenotypes of dynein heavy chain mutants as in A. nidulans were described for Neurospora crassa and Nectria haematococca. In N. crassa and in N. haematococca, additional characteristics of the mutants were observed. In part, a thorough analysis in N. haematococca suggested the involvement of dynein in spindle pole body motility, nuclear positioning and vesicle transport toward the hyphal tip [14]. However, whereas in dynein mutants of the mentioned fungal species nuclei remained in the rear of the hyphae, in Ashbya gossypii the situation appeared to be the contrary. In the absence of the dynein motor protein, nuclei accumulated at the hyphal tip (Philippsen, pers. comm.). The fact that deletion of the dynein gene in several fungi does not completely prevent
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Table I. Motor proteins involved in nuclear migration in A. nidulans, N. crassa and S. cerevisiae and some of their interactions in yeast. Gene Dynein nudA nudG ro-1 DYN1 = DHC1 Kinesins Nkin
Species
Function
Reference
A. nidulans A. nidulans
nuclear movement towards the hyphal tip dynein light chain, required for localization of dynein heavy chain nuclear movement towards the hyphal tip orientation of mitotic spindle, movement through the budding neck, oscillatory movement of nucleus in budding neck
[32, 33] [2]
N. crassa S. cerevisiae
N. crassa
KAR3
S. cerevisiae
KIP2 KIP3
S. cerevisiae S. cerevisiae
vesicle movement, nuclear distribution in hyphae meitosis, meiosis, spindle positioning in the absence of dynein nuclear migration, counteracting Kip3 and Dyn1 nuclear migration towards the budding neck, spindle positioning in the absence of dynein
Double and triple mutants in yeast dyn1/kip3 dyn1/kip3/kip2 dyn1/kip2 kip2/kar3 kip2/kar9 kip3/kar9 kip3/kar3 kip3/kar3/kip2 kip3/kip2 dyn1/kar3 dyn1/kar3/kip2
nuclear migration indicates that other motor proteins might be involved in nuclear movement and can at least partially substitute for the dynein function. Candidates for this role are the microtubule-dependent kinesins. In S. cerevisiae six kinesins are found in the genome, three of which, KIP2, KIP3 and KAR3, play a role in the nuclear migration process [4– 6, 17]. In addition, KIP3 and KAR3 have functions in other cellular processes such as chromosome segregation and karyogamy. Whereas Kip2p localized only to cytoplasmic microtubules, Kip3p also associated with spindle microtubules. Deletion of any of the three kinesin genes only slightly compromised nuclear distribution, with the result that an increased number of binucleate mother and anucleate daughter cells were produced, similar to the dynein mutants. New insights into the process of nuclear migration were gained from phenotypic analyses of deletion mutants of single motor genes compared to double and
[21] [6, 7, 16]
[25] [4] [17] [4, 6, 17] [4, 6, 17]
synthetically lethal viable viable viable synthetically lethal viable synthetically lethal synthetically lethal viable synthetically lethal viable
triple mutants. It was suggested that nuclear migration occurs in two distinct steps. First the nucleus moves close to the budding neck, which requires Kip3p. In a second phase the nucleus migrates through the neck, which depends on dynein function (figure 3) [6]. The Kar3p motor also plays a role in nuclear movement, because double mutants of kip3 and kar3 as well as kar3 and dyn1 were lethal. Similar analyses of double and triple deletion mutants led Miller et al. [17] to propose a dynein-dependent and a Kar9dependent pathway of nuclear migration. They found that the kip2 dyn1 double mutant was viable but a kip2 kar9 double mutant was lethal. In contrast kip3 dyn1 strains were not viable whereas the combination of kip3 and kar9 was viable. Interestingly, the lethality of kip3 dyn1 could be suppressed by the deletion of kip2, suggesting a counteracting effect of this motor. Kip2 deletion also suppressed the lethal phenotype of dyn1 kar3 double mutants. In contrast,
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Figure 3. Scheme of possible roles for motors in nuclear migration in S. cerevisiae. Different kinesins and dynein have overlapping or counteracting functions. Kip3p and Kar3p together with Dyn1 are involved in the migration of the nucleus toward and through the bud, whereas Kip2p antagonizes the forces of the other three motors. The oscillations observed at the budding neck are dependent on dynein. Modified after [4, 6].
the kip3 kar3 double deletion could not be suppressed by additional deletion of kip2. According to these results, nuclear migration would require three motors, Dyn1p, Kip3p and Kar3p, which move the nucleus toward the bud and one motor, Kip2p, with a counteracting force (figure 3). It is interesting to note that if the three motors exert force in the same direction, the mechanisms have to be different because dynein and kinesins move along microtubules in opposite directions, the minus and the plus end, respectively. One possible explanation could be that one important feature of motor proteins is their capacity to modify the stability of microtubules (see below). Further deletion of additional motor proteins revealed that yeast cells are able to grow only with two functional kinesin motor proteins, the BimC-type motor Cin8p and either Kar3p or Kip3p, even in the absence of the dynein heavy chain [5]. This demonstrates that kinesin functions are highly redundant. Whereas in S. cerevisiae, the understanding of the role of kinesins in nuclear migration is well under way, in filamentous fungi several kinesins are described, one of which, the conventional kinesin, has been implicated in nuclear
positioning. This kinesin has no homologue in S. cerevisiae and seems to have diverse functions in different fungi. It appears to be involved in tipward vesicle movement and nuclear positioning in N. crassa and N. haematococca, in mitochondrial positioning in N. haematococca and in vacuole movement in Ustilago maydis [15, 24, 28]. An interesting question is whether one can identify homologues of the yeast kinesins Kip2p and Kip3p, which serve distinct roles in the nuclear migration process in the unicellular fungus S. cerevisiae. If they exist in filamentous fungi it will be interesting to analyze their roles in nuclear movement in filaments and during developmental processes. It should be much easier to observe overlapping or counteracting forces of the kinesins to dynein in filamentous fungi, because of the long distance movement of the nucleus. In addition, the complexity of their life cycles probably requires different motor proteins during specific stages. Whereas the active orientation of the mitotic spindle does not seem to be of great importance in filaments of fungi it could become crucial during developmental processes, such as conidiation. Here a switch from the syncytial to the cellular stage takes place [1]. The recent results clearly show the involvement of different motor proteins in nuclear migration. One open question is how the motors perform their forces. One possibility is the migration of motor proteins along cytoplasmic tracks, carrying the cargo connected to the tail-domain of the motor protein. This model is well accepted for the transport of vesicles along microtubules. However, dynein and kinesins were not detected directly attached to the nuclear envelope. Only dynein was found at the spindle pole body and could theoretically serve such a function. This is unlikely because dynein is a minus-end-directed motor and would migrate toward the spindle pole body. More likely, dynein exerts its function along microtubules or at the cortex. One additional feature of dynein and kinesin motor proteins is that they influence the stability of microtubules. Whereas Dyn1p, Kar3p and Kip3p destabilize microtubules, Kip2p stabi-
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lizes them. This leads to longer cytoplasmic microtubules in the one class of mutant cells but shorter microtubules in kip2 deletion cells. The localization of kinesin proteins along microtubules would be in agreement with an effect on stability. The precise regulation of the integrity of the microtubule cytoskeleton could thus contribute significantly to the movement of the nucleus. This would nicely explain the migration of interphase nuclei in yeast, which are moved within the cell with only a transient contact of astral microtubules to the cortex (see above). Future research should attempt to dissect the different possibilities for motor action and their contribution to the different stages of nuclear migration. Besides the involvement of microtubules and microtubule-dependent motor proteins, there is good recent evidence that the actin cytoskeleton also contributes significantly to the nuclear migration process [18, 31]. This suggests that actin-dependent motor proteins might be required for certain steps in nuclear movement. It will be very interesting to investigate the exact interaction of the two cytoskeletal systems and the different motor proteins [13]. However, direct evidence for a role of myosin-like motors is not yet available.
4. Conclusions Whereas mitosis has been analyzed for many years, the importance of nuclear migration during interphase was acknowledged only much later. There is good evidence now that nuclei are driven through different motor proteins with redundant, overlapping or counteracting functions. A unified model for the interaction of motors and tracks has not yet emerged but it can well be that different mechanisms may be operating at different stages of the fungal life cycle. Although nuclear migration seemed to be a simple cellular function, the continuous progress of the studies over the last few years demonstrates how complicated the process indeed is. The studies of nuclear migration in different fungi revealed common functions of the motor
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proteins, although some have as yet been described only in certain species. It also reflects the differing suitability of some fungi for investigations of certain phenomena and it clearly demonstrates that we have to retain this variety of different organisms for molecular analyses in order to develop a unified model and to discover species-specific features. The yeast S. cerevisiae is undoubtedly a fantastic model organism for unravelling basic features of a eukaryotic cell. However, filamentous fungi add several properties, such as filamentous growth, developmental processes and symbiotic or pathogenic interactions with higher eukaryotes, which allow us to study the interdependence of these cellular functions at a molecular level. The tools developed for filamentous fungi in the past promise a fruitful and fascinating area of future research.
Acknowledgments We wish to thank X. Xiang, N.R. Morris (Piscataway, NJ, USA), M. Picard (Paris, France) and P. Philippsen (Basel, Switzerland) for sharing unpublished results and M.D. Rose (Princeton, USA) for providing figure 1 (A–C). The work of the authors was supported by the Deutsche Forschungsgemeinschaft, the Boehringer Ingelheim Fonds, the Max-PlanckInstitute for terrestrial Microbiology and the Philipps-University of Marburg.
References [1] Adams T.H., Wieser J.K., Yu J.H., Asexual sporulation in Aspergillus nidulans, Microbiol. Mol. Biol. Rev. 62 (1998) 35–54. [2] Beckwith S.M., Roghi C.H., Liu B., Morris N.R., The ‘8-kD’ cytoplasmic dynein light chain is required for nuclear migration and for dynein heavy chain localization in Aspergillus nidulans, J. Cell Biol. 143 (1998) 1239–1247. [3] Carminati J.L., Stearns T., Microbutules orient the mitotic spindle in yeast through dynein-dependent interactions with the cell cortex, J. Cell Biol. 138 (1997) 629–641. [4] Cottingham F.R., Hoyt M.A., Mitotic spindle positioning in Saccharomyces cerevisiae is accomplished by antagonistically acting microtubule motor proteins, J. Cell Biol. 138 (1997) 1041–1053. [5] Cottingham F.R., Gheber L., Miller D.L., Hoyt M.A., Novel roles for Saccharomyces cerevisiae mitotic spindle motors, J. Cell Biol. 147 (1999) 335–349. [6] DeZwaan T.M., Ellingson E., Pellman D., Roof D.M., Kinesin-related KIP3 of Saccharomyces cerevisiae is required for a distinct step in nuclear migration, J. Cell Biol. 138 (1997) 1023–1040.
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[7] Eshel D., Urrestarazu L.A., Vissers S., Jauniaux J.C., Van VlietReedijk J.C., Planta R.J., Gibbons I.R., Cytoplasmic dynein is required for normal nuclear segregation in yeast, Proc. Natl. Acad. Sci. USA 90 (1993) 11172–11176. [8] Farkasovsky M., Küntzel H., Yeast Num1p associates with the mother cell cortex during S/G2 phase and affects microtubular functions, J. Cell Biol. 131 (1995) 1003–1014. [9] Fischer R., Nuclear movement in filamentous fungi, FEMS Microbiol. Rev. 23 (1999) 38–69. [10] Fischer R., Timberlake W.E., Aspergillus nidulans apsA (anucleate primary sterigmata) encodes a coiled-coil protein necessary for nuclear positioning and completion of asexual development, J. Cell Biol. 128 (1995) 485–498. [11] Fisk H.A., Yaffe M.P., Mutational analysis of Mdm1p function in nuclear and mitochondrial inheritance, J. Cell Biol. 138 (1997) 485–494. [12] Hirokawa N., Kinesin and dynein superfamily proteins and the mechanism of organelle transport, Science 279 (1998) 519–526. [13] Huang J.D., Brady S.T., Richards B.W., Stenoien D., Resua J.H., Copeland N.G., Jenkins N.A., Direct interaction of microtubuleand actin-based transport motors, Nature 397 (1999) 267–270. [14] Inoue S., Turgeon B.G., Yoder O.C., Aist J.R., Role of fungal dynein in hyphal growth, microtubule organization, spindle pole body motility and nuclear migration, J. Cell Sci. 111 (1998) 1555–1566. [15] Lehmler C., Steinberg G., Snetselaar K.M., Schliwa M., Kahmann R., Bölker M., Identification of a motor protein required for filamentous growth in Ustilago maydis, EMBO J. 16 (1997) 3464–3473. [16] Li Y.Y., Yeh E., Hays T., Bloom K., Disruption of mitotic spindle orientation in a yeast dynein mutant, Proc. Natl. Acad. Sci. USA 90 (1993) 10096–10100. [17] Miller R.K., Heller K.K., Frisèn L., Wallck D.L., Loayza D., Gammie A.E., Rose M.D., The kinesin-related proteins, Kip2p and Kip3p, function differently in nuclear migration in yeast, Mol. Biol. Chem. 9 (1998) 2051–2068. [18] Miller R.K., Matheos D., Rose M.D., The cortical localization of the microtubule orientation protein, Kar9p, is dependent upon actin and proteins required for polarization, J. Cell Biol. 144 (1999) 963–975. [19] Morris N.R., Efimov V.P., Xiang X., Nuclear migration, nucleokinesis and lissencephaly, Trends Cell. Biol. 8 (1998) 467–470. [20] Morris N.R., Xiang X., Beckwith S.M., Nuclear migration advances in fungi, Trends Cell. Biol. 5 (1995) 278–282. [21] Plamann M., Minke P.F., Tinsley J.H., Bruno K.S., Cytoplasmic dynein and actin-related protein Arp1 are required for normal nuclear distribution in filamentous fungi, J. Cell Biol. 127 (1994) 139–149.
[22] Reinsch S., Gönczy P., Mechanisms of nuclear positioning, J. Cell Sci. 111 (1998) 2283–2295. [23] Rose M.D., Nuclear fusion in the yeast Saccharomyces cerevisiae, Annu. Rev. Cell Dev. Biol. 12 (1996) 663–695. [24] Seiler S., Nargang F.E., Steinberg G., Schliwa M., Kinesin is essential for cell morphogenesis and polarized secretion in Neurospora crassa, EMBO J. 16 (1997) 3025–3034. [25] Seiler S., Plamann M., Schliwa M., Kinesin and dynein mutants provide novel insights into the roles of vesicle traffic during cell morphogenesis in Neurospora, Curr. Biol. 9 (1999) 779–785. [26] Shaw S.L., Yeh E., Maddox P., Salmon E.D., Bloom K., Astral microtubule dynamics in yeast: A microtubule-based searching mechanism for spindle orientation and nuclear migration into the bud, J. Cell Biol. 139 (1997) 985–994. [27] Steinberg G., Organelle transport and molecular motors in fungi, Fungal Genet. Biol. 24 (1998) 161–177. [28] Steinberg G., Schliwa M., Lehmler C., Bölker M., Kahmann R., McIntosh J.R., Kinesin from the plant pathogenic fungus Ustilago maydis is involved in vacuole formation and cytoplasmic migration, J. Cell Sci. 111 (1998) 2235–2246. [29] Suelmann R., Sievers N., Fischer R., Nuclear traffic in fungal hyphae: In vivo study of nuclear migration and positioning in Aspergillus nidulans, Mol. Microbiol. 25 (1997) 757–769 (http://www. blacksci. co. uk/products/elecpro/anims/mole254.gif). [30] Suelmann R., Sievers N., Galetzka D., Robertson L., Timberlake W.E., Fischer R., 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 (1998) 831–842 (http://www. uni-marburg. de/mpi/movies/apsbnew. mov). [31] Theesfeld C.L., Irazoqui J.E., Bloom K., Lew D.J., The role of actin in spindle orientation changes during the Saccharomyces cerevisiae cell cycle, J. Cell Biol. 146 (1999) 1019–1031. [32] Xiang X., Beckwith S.M., Morris N.R., Cytoplasmic dynein is involved in nuclear migration in Aspergillus nidulans, Proc. Natl. Acad. Sci. USA 91 (1994) 2100–2104. [33] Xiang X., Roghi C., Morris N.R., Characterization and localization of the cytoplasmic dynein heavy chain in Aspergillus nidulans, Proc. Natl. Acad. Sci. USA 92 (1995) 9890–9894. [34] Yeh E., Skibbens R.V., Cheng J.W., Salmon E.D., Bloom K., Spindle dynamics and cell cycle regulation of dynein in the budding yeast, Saccharomyces cerevisiae, J. Cell Biol. 130 (1995) 687–700.
Mini-review Nuclear migration in fungi – different motors at work Rüdiger Suelmann*, Reinhard Fischer** Laboratorium für Mikrobiologie, Philipps-Universität Marburg and Max-Planck-Insitut für terrestrische Mikrobiologie, Karl-vonFrisch-Str., 35043 Marburg, Germany (Submitted 11 January 2000; accepted 22 January 2000)
Abstract — Lower fungi such as Saccharomyces cerevisiae and Aspergillus nidulans are ideal organisms for studying the molecular biology underlying nuclear migration in eukaryotic cells. In this review, the role of different motor proteins such as dynein, kinesin and myosin will be discussed. © 2000 Éditions scientifiques et médicales Elsevier SAS fungi, lower / organelle movement / motor protein / microtubule / Saccharomyces cerevisiae / Aspergillus nidulans
1. Introduction Nuclear migration has been observed in eukaryotic cells for some time. However, a detailed molecular analysis of the process has mainly been performed during the past 10 years. The progress in the field has been reviewed with emphasis on nuclear migration in fungi [9, 20], nuclear migration during yeast mating [23], nuclear positioning [22], the role of motor proteins in organelle movement [27] and the correlation between nuclear migration in fungi and brain development in mammals [19]. Several recent publications merit a review on the involvement of different motor proteins in the process in yeast and in filamentous fungi.
2. The phenomenon In Saccharomyces cerevisiae cells, which duplicate by budding, the nucleus migrates from a random position in the mother cell to the bud-
* Present address: Bayer AG, Zentrale Forschung, 51368 Leverkusen, Germany ** Correspondence and reprints Tel.: +49 6421 178 330; fax: 49 6421 178 309; [email protected]
ding neck and undergoes localized mitosis to provide each cell with a nucleus (figure 1). Therefore, the distance over which the nucleus has to migrate from the center of the cell is rather short and the mitotic machinery contributes significantly to this distribution process [34]. In filamentous fungi, nuclei migrate long distances to follow the growing hyphal tip. During the migration process nuclei divide and individual nuclei are left behind to populate the entire mycelium (figure 1). This movement has been followed in living hyphae in a number of different fungi using phase contrast or fluorescence microscopy for GFP-labeled nuclei [14, 29]. Nuclei travel with velocities of 0.1–1.2 µm per min through the mycelium of Aspergillus nidulans and can reach speeds of up to 40 µm per min. Nuclei mostly follow the direction of hyphal tip elongation although other movements have also been observed [29]. One important tool to study the molecular components required for nuclear migration is the use of mutants with defects in nuclear migration (figure 2).
3. The tracks To drive dynamic processes such as organelle movement, two components are required, a
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Figure 1. Nuclear distribution in the yeast S. cerevisiae (A–C) and the filamentous fungus A. nidulans (D, E). Yeast cells were fixed and nuclei were stained with DAPI. In (A) the nucleus migrated close to the budding neck where it divided (B) to provide both daughter cells with a nucleus (C). Cytokinesis occurred afterwards. Nuclei in A. nidulans were stained with GFP and nuclear migration observed in live cells (D, E). A spore germinated and nuclei moved into the germ tube (D). As the germ tube elongated, nuclei followed the tip and duplicated through mitosis (E). The arrow indicates the place of septum formation. Video sequences showing nuclear migration in the filamentous fungus are available in the Internet: http://www.uni-marburg.de/mpi/movies/movies.htm. Modified after [9].
motor and a stator. The interaction between these is highly specific and thus different motor proteins require specific static structures. Two main cytoskeletal elements that serve the stator function are microtubules and actin. Both consist of polar strands which are maintained in a dynamic equilibrium. The subunits, actin and a/b-tubulin, respectively, assemble at one end with a higher rate than they disassemble and thus this growing end is called the plus end. In contrast, at the minus end, the disassembling rate exceeds the assembling rate. Microtubules and actin can serve as tracks along which motor proteins can carry their cargo. The orientation of the tracks within the cell determines the direction of movement of the motor proteins. Whereas dynein migrates along microtubules
Figure 2. Scheme of nuclear migration mutants (dynein) in S. cerevisiae and A. nidulans. Nuclei divide but do not migrate to appropriate positions.
toward the minus end, most kinesins move toward the plus end. Thus, the orientation of the tracks is very important to allow organelle transport in one or the other direction. Microtubules emanate from microtubule organizing centers such as the spindle pole body, which is embedded in the nuclear envelope. There is evidence in some fungi for additional cytoplasmic microtubule organizing centers at the apex of a hypha, which suggests a mixed polarization of microtubules in these cells. Whether this is true for all fungi currently remains an open question. It is largely accepted that microtubules perform an important function in nuclear migration in fungi. A study of microtubule dynamics in living S. cerevisiae cells was performed with GFP-labeled a-tubulin [3] and GFP-labeled dynein [26]. Astral microtubules emanated from the spindle pole body and transiently made contact with the cortex of the cell. Continuous growth of those microtubules caused pushing of the nucleus, whereas shrinkage of a filament led to pulling of the nucleus toward the attachment site. The dynamic interaction of microtubules with the cortex was cell-cycle-dependentregulated and more focused in budding cells,
Nuclear migration in fungi
where microtubules swept the cortex, made contact and displayed a shrinking behavior. If the attachment of microtubules to the cortex is crucial for nuclear movement, the question is raised: through which proteins could this contact be mediated? Two candidates were recently suggested for this function. The first is Num1 protein (Num1p), which is localized in a cellcycle-dependent manner with the cortex of the mother cell and which causes a nuclear distribution phenotype when absent. The Num1p could thus confer contact of microtubules only in one of the two cells, namely the mother cell. A protein with the capacity to mediate the contact of microtubules with the cortex of the daughter cell is Kar9p, localized as GFP-Kar9p at the tip of the emerging bud [8, 18]. Localization of Kar9p was independent of microtubules but dependent on an intact actin cytoskeleton. In addition, its distribution was altered by mutations in genes involved in the polarization of the yeast cell. This is an apt example of how microtubule cytoskeletal functions are interwoven with those of the actin cytoskeleton. In filamentous fungi, the cytological function of microtubules is less clear than in the singlecelled yeast. However, GFP-tagged tubulin derivatives are now available in A. nidulans and should help to unravel their function in nuclear migration (Xiang and Morris, pers. comm.). The situation in A. nidulans might be far more complicated than in yeast, because cell compartments contain several nuclei, which move independently toward the growing tip but are also able to move backwards. A ‘pushing’ or ‘pulling’ mechanism of interphase nuclei through growing or shrinking microtubules was not observed [30]. Moreover, in Nectria haematococca, it was shown with laser optical trap experiments that interphase nuclei are rather fixed at their position [14]. However, a homologue of the yeast Num1p, which could be important for the attachment of microtubules to the cortex, also exists in filamentous fungi such as A. nidulans and Podospora anserina [10] (Picard, pers. comm.). In A. nidulans the protein, ApsA, was localized along the cortex of the cell, although there is no evidence yet that the pro-
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tein is involved in the regulation of microtubule stability as in yeast, or that it might serve as an attachment site for microtubules. Nevertheless, mutagenesis of apsA leads to nuclear misdistribution in hyphae and, because of a misdistribution during conidiophore production, to a strong developmental block. This indicates the importance of exact nuclear migration and positioning in developmental processes [10]. A second gene, apsB, was described in A. nidulans, which had the same phenotype when mutated as apsA mutants and thus might be involved in the same process. GFP-tagged nuclei in an apsB-mutated strain revealed a saltatory movement of individual nuclei within one hyphal compartment, which led to transient clustering of nuclei. In contrast, nuclei are evenly distributed in wildtype cells [30]. A homologue of the ApsB protein can be found in Schizosaccharomyces pombe (sequencing project) but not in S. cerevisiae. Besides actin and microtubules, intermediate filaments are structural components in higher eukaryotic cells. This class of proteins was not described in fungi until recently. Yaffe et al. described a protein, Mdm1, with features of intermediate filaments, and showed that this protein serves roles in nuclear migration and in mitochondrial movement [11] (and previous work of that group). Recently, a similar gene was found in S. pombe (sequencing project). This suggests that this type of protein is likely to be found in other fungi besides yeast and S. pombe as well, and it should be very exciting to unravel the cellular functions of Mdm1p-like proteins in other fungal species.
4. The motors Motor proteins convert chemical energy into conformational changes in the protein, which result in their movement along the cytoskeletal tracks. The exact mechanisms of energy transformation are still not understood for all motor proteins and intensive research is being carried out to solve this fundamental question. For proper functioning of a cell, many different dynamic processes are required. Since the number of cytoskeletal elements is rather limited,
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specificity mainly can be achieved through motor proteins or their associated regulatory components. Indeed, the yeast cell, as a simple eukaryote, contains six kinesins, one dynein and five myosins. The number of motor proteins increases dramatically in higher eukaryotes [12] (see also: http://tubulin.cb.m.utokyo.ac.jp/KIF/index.html). To understand the function of these motors and to dissect their specific roles in different cellular processes is one of the major goals in the field. Lower eukaryotes such as S. cerevisiae and A. nidulans should serve as excellent models to unravel the basic principles. There is good evidence that the microtubuledependent motor protein dynein is one major player for nuclear migration (table I). Dynein is a large protein complex that consists of two heavy chains and several smaller subunits. Using conserved regions of different dynein heavy chains, the corresponding regions were PCR-amplified from S. cerevisiae and functionally analyzed. Disruption of the gene (DYN1 = DHC1) caused an increased percentage of mother cells harboring two nuclei, indicating that the correct positioning and orientation of the mitotic spindle at the budding neck was impaired. Initial movement of the nucleus toward the neck region was not affected but the nucleus did not migrate as close to the neck as in the wild type. It was concluded that dynein is required for proper spindle orientation during mitosis. Nevertheless, dynein-deficient yeast cells are still able to form colonies. The protein was localized as a b-galactosidase fusion protein along cytoplasmic microtubules, at the spindle pole body and at the cortex [34]. Recently, dynein protein distribution was analyzed in live budding yeast cells using a GFP–dynein hybrid protein, which efficiently decorated cytoplasmic microtubules but which was not observed at the cortex (see above) [26]. On the basis of these results Carminati and Stearns [3] suggested a model of how dynein could be involved in nuclear movement. They proposed that microtubules sweep the cortex until they make contact with a protein or protein complex, which serves as a dynein attachment site. Once attached, microtubules
start shrinking and thus pulling in the nucleus. Dynein could directly pull the microtubules or it could catalyze their depolymerization. According to this model, dynein should be transiently localized at the cortex. Recently, deZwaan et al. suggested an additional role for dynein in an oscillatory movement, which the nucleus undergoes at the budding neck in the absence of Kip3 [6]. In A. nidulans the dynein heavy chain gene was discovered in a screen for temperaturesensitive mutants with defects in nuclear migration and subsequently cloned by complementation [32, 33]. In germinating spores, nuclei divided but remained in the spore and did not move out into the germ tube (figure 2). This defect leads to a severe reduction in hyphal growth. The protein was localized through secondary immunofluorescence at the tip of the elongating hyphae, which could point to a pulling force on the microtubules. However, in recent investigations using a dynein–GFP hybrid protein, the motor was also found at the tips of cytoplasmic microtubules (Xiang and Morris, pers. comm.). As dynein is a minus-end-directed motor, this suggests that it is either transported to the tip by another motor, perhaps as part of a kinesin-driven vesicle, or has an affinity for some other tip structure at the tip. Whether this cytoplasmic microtubule dynein mediates nuclear migration remains to be determined. Similar phenotypes of dynein heavy chain mutants as in A. nidulans were described for Neurospora crassa and Nectria haematococca. In N. crassa and in N. haematococca, additional characteristics of the mutants were observed. In part, a thorough analysis in N. haematococca suggested the involvement of dynein in spindle pole body motility, nuclear positioning and vesicle transport toward the hyphal tip [14]. However, whereas in dynein mutants of the mentioned fungal species nuclei remained in the rear of the hyphae, in Ashbya gossypii the situation appeared to be the contrary. In the absence of the dynein motor protein, nuclei accumulated at the hyphal tip (Philippsen, pers. comm.). The fact that deletion of the dynein gene in several fungi does not completely prevent
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Table I. Motor proteins involved in nuclear migration in A. nidulans, N. crassa and S. cerevisiae and some of their interactions in yeast. Gene Dynein nudA nudG ro-1 DYN1 = DHC1 Kinesins Nkin
Species
Function
Reference
A. nidulans A. nidulans
nuclear movement towards the hyphal tip dynein light chain, required for localization of dynein heavy chain nuclear movement towards the hyphal tip orientation of mitotic spindle, movement through the budding neck, oscillatory movement of nucleus in budding neck
[32, 33] [2]
N. crassa S. cerevisiae
N. crassa
KAR3
S. cerevisiae
KIP2 KIP3
S. cerevisiae S. cerevisiae
vesicle movement, nuclear distribution in hyphae meitosis, meiosis, spindle positioning in the absence of dynein nuclear migration, counteracting Kip3 and Dyn1 nuclear migration towards the budding neck, spindle positioning in the absence of dynein
Double and triple mutants in yeast dyn1/kip3 dyn1/kip3/kip2 dyn1/kip2 kip2/kar3 kip2/kar9 kip3/kar9 kip3/kar3 kip3/kar3/kip2 kip3/kip2 dyn1/kar3 dyn1/kar3/kip2
nuclear migration indicates that other motor proteins might be involved in nuclear movement and can at least partially substitute for the dynein function. Candidates for this role are the microtubule-dependent kinesins. In S. cerevisiae six kinesins are found in the genome, three of which, KIP2, KIP3 and KAR3, play a role in the nuclear migration process [4– 6, 17]. In addition, KIP3 and KAR3 have functions in other cellular processes such as chromosome segregation and karyogamy. Whereas Kip2p localized only to cytoplasmic microtubules, Kip3p also associated with spindle microtubules. Deletion of any of the three kinesin genes only slightly compromised nuclear distribution, with the result that an increased number of binucleate mother and anucleate daughter cells were produced, similar to the dynein mutants. New insights into the process of nuclear migration were gained from phenotypic analyses of deletion mutants of single motor genes compared to double and
[21] [6, 7, 16]
[25] [4] [17] [4, 6, 17] [4, 6, 17]
synthetically lethal viable viable viable synthetically lethal viable synthetically lethal synthetically lethal viable synthetically lethal viable
triple mutants. It was suggested that nuclear migration occurs in two distinct steps. First the nucleus moves close to the budding neck, which requires Kip3p. In a second phase the nucleus migrates through the neck, which depends on dynein function (figure 3) [6]. The Kar3p motor also plays a role in nuclear movement, because double mutants of kip3 and kar3 as well as kar3 and dyn1 were lethal. Similar analyses of double and triple deletion mutants led Miller et al. [17] to propose a dynein-dependent and a Kar9dependent pathway of nuclear migration. They found that the kip2 dyn1 double mutant was viable but a kip2 kar9 double mutant was lethal. In contrast kip3 dyn1 strains were not viable whereas the combination of kip3 and kar9 was viable. Interestingly, the lethality of kip3 dyn1 could be suppressed by the deletion of kip2, suggesting a counteracting effect of this motor. Kip2 deletion also suppressed the lethal phenotype of dyn1 kar3 double mutants. In contrast,
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Figure 3. Scheme of possible roles for motors in nuclear migration in S. cerevisiae. Different kinesins and dynein have overlapping or counteracting functions. Kip3p and Kar3p together with Dyn1 are involved in the migration of the nucleus toward and through the bud, whereas Kip2p antagonizes the forces of the other three motors. The oscillations observed at the budding neck are dependent on dynein. Modified after [4, 6].
the kip3 kar3 double deletion could not be suppressed by additional deletion of kip2. According to these results, nuclear migration would require three motors, Dyn1p, Kip3p and Kar3p, which move the nucleus toward the bud and one motor, Kip2p, with a counteracting force (figure 3). It is interesting to note that if the three motors exert force in the same direction, the mechanisms have to be different because dynein and kinesins move along microtubules in opposite directions, the minus and the plus end, respectively. One possible explanation could be that one important feature of motor proteins is their capacity to modify the stability of microtubules (see below). Further deletion of additional motor proteins revealed that yeast cells are able to grow only with two functional kinesin motor proteins, the BimC-type motor Cin8p and either Kar3p or Kip3p, even in the absence of the dynein heavy chain [5]. This demonstrates that kinesin functions are highly redundant. Whereas in S. cerevisiae, the understanding of the role of kinesins in nuclear migration is well under way, in filamentous fungi several kinesins are described, one of which, the conventional kinesin, has been implicated in nuclear
positioning. This kinesin has no homologue in S. cerevisiae and seems to have diverse functions in different fungi. It appears to be involved in tipward vesicle movement and nuclear positioning in N. crassa and N. haematococca, in mitochondrial positioning in N. haematococca and in vacuole movement in Ustilago maydis [15, 24, 28]. An interesting question is whether one can identify homologues of the yeast kinesins Kip2p and Kip3p, which serve distinct roles in the nuclear migration process in the unicellular fungus S. cerevisiae. If they exist in filamentous fungi it will be interesting to analyze their roles in nuclear movement in filaments and during developmental processes. It should be much easier to observe overlapping or counteracting forces of the kinesins to dynein in filamentous fungi, because of the long distance movement of the nucleus. In addition, the complexity of their life cycles probably requires different motor proteins during specific stages. Whereas the active orientation of the mitotic spindle does not seem to be of great importance in filaments of fungi it could become crucial during developmental processes, such as conidiation. Here a switch from the syncytial to the cellular stage takes place [1]. The recent results clearly show the involvement of different motor proteins in nuclear migration. One open question is how the motors perform their forces. One possibility is the migration of motor proteins along cytoplasmic tracks, carrying the cargo connected to the tail-domain of the motor protein. This model is well accepted for the transport of vesicles along microtubules. However, dynein and kinesins were not detected directly attached to the nuclear envelope. Only dynein was found at the spindle pole body and could theoretically serve such a function. This is unlikely because dynein is a minus-end-directed motor and would migrate toward the spindle pole body. More likely, dynein exerts its function along microtubules or at the cortex. One additional feature of dynein and kinesin motor proteins is that they influence the stability of microtubules. Whereas Dyn1p, Kar3p and Kip3p destabilize microtubules, Kip2p stabi-
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lizes them. This leads to longer cytoplasmic microtubules in the one class of mutant cells but shorter microtubules in kip2 deletion cells. The localization of kinesin proteins along microtubules would be in agreement with an effect on stability. The precise regulation of the integrity of the microtubule cytoskeleton could thus contribute significantly to the movement of the nucleus. This would nicely explain the migration of interphase nuclei in yeast, which are moved within the cell with only a transient contact of astral microtubules to the cortex (see above). Future research should attempt to dissect the different possibilities for motor action and their contribution to the different stages of nuclear migration. Besides the involvement of microtubules and microtubule-dependent motor proteins, there is good recent evidence that the actin cytoskeleton also contributes significantly to the nuclear migration process [18, 31]. This suggests that actin-dependent motor proteins might be required for certain steps in nuclear movement. It will be very interesting to investigate the exact interaction of the two cytoskeletal systems and the different motor proteins [13]. However, direct evidence for a role of myosin-like motors is not yet available.
4. Conclusions Whereas mitosis has been analyzed for many years, the importance of nuclear migration during interphase was acknowledged only much later. There is good evidence now that nuclei are driven through different motor proteins with redundant, overlapping or counteracting functions. A unified model for the interaction of motors and tracks has not yet emerged but it can well be that different mechanisms may be operating at different stages of the fungal life cycle. Although nuclear migration seemed to be a simple cellular function, the continuous progress of the studies over the last few years demonstrates how complicated the process indeed is. The studies of nuclear migration in different fungi revealed common functions of the motor
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proteins, although some have as yet been described only in certain species. It also reflects the differing suitability of some fungi for investigations of certain phenomena and it clearly demonstrates that we have to retain this variety of different organisms for molecular analyses in order to develop a unified model and to discover species-specific features. The yeast S. cerevisiae is undoubtedly a fantastic model organism for unravelling basic features of a eukaryotic cell. However, filamentous fungi add several properties, such as filamentous growth, developmental processes and symbiotic or pathogenic interactions with higher eukaryotes, which allow us to study the interdependence of these cellular functions at a molecular level. The tools developed for filamentous fungi in the past promise a fruitful and fascinating area of future research.
Acknowledgments We wish to thank X. Xiang, N.R. Morris (Piscataway, NJ, USA), M. Picard (Paris, France) and P. Philippsen (Basel, Switzerland) for sharing unpublished results and M.D. Rose (Princeton, USA) for providing figure 1 (A–C). The work of the authors was supported by the Deutsche Forschungsgemeinschaft, the Boehringer Ingelheim Fonds, the Max-PlanckInstitute for terrestrial Microbiology and the Philipps-University of Marburg.
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