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Research Group Prof. Peter Philippsen
Phone: +41 61 267 1480
Fax: +41 61 267 1481
E-mail: Peter.Philippsen@unibas.ch
http://www.biozentrum.unibas.ch/philippsen
Genomics as information basis for investigating dynamics of growth and nuclear migration in fungi
Group members:
Ch. Alberti-Segui, N.Alijoski, Y. Bauer, M. Bloch, F. Dietrich, Amy S. Gladfelter, H. Helfer, D. Hoepfner, K. Hungerbühler, A. Kaufmann, Ph. Knechtle, Ph. Laissue, A. Lerch, S. Lemire-Brachat, Ph. Lüdi, R. Rischatsch, F. Schärer, H.P. Schmitz, S. Voegeli, K. Wojnowska
In collaboration with B. André (Univ. Libre de Bruxelles, Belgium), J. Boeke (Johns Hopkins Univ., Baltimore, USA), H. Bussey (McGill Univ., Montreal, Canada), R. Davis (Stanford Univ., USA), R. Fischer (University of Marburg, Germany), F. Foury (Univ. de Louvain, Belgium), S. Friend (Rosetta Inpharmatics, Seattle, USA), T. Gaffney (Syngenta, Research Triangle Park, USA), J. Hegemann (Univ. Düsseldorf, Germany), E. Hurt (University of Heidelberg, Germany), M. Johnston (Washington Univ., St. Louis, USA), S. Kelly (Univ. of Wales, Aberystwyth, UK), P. Koetter (Univ. Frankfurt, Germany), B. Scherens (CERIA, Inst. de Recherches, Bruxelles, Belgium), J. Revuelta (Univ. de Salamanca, Spain), M. Snyder (Yale Univ., New Haven, USA), H. Tabak (University of Amsterdam, NL), G. Valle (Univ. di Padova, Italy), G. Volckaert (Katholieke Univ., Leuven, Belgium), R. Wing (Clemson Univ., USA)
Introduction
Over the previous years our research was significantly influenced by the genomic sequencing projects of two eukaryotic microorganisms, the unicellular fungus Saccharomyces cerevisiae and the filamentous fungus Ashbya gossypii. We use this novel knowledge for diverse types of gene targeting and follow-up experiments with the aim to study two cellular processes: Dynamics of nuclei in S. cerevisiae and in A. gossypii and, as a new project, the control of hyphal growth in A. gossypii. One final aim is to understand coordination between growth and nuclear dynamics in this fungus.
Our interest in Ashbya gossypii originates from several interesting features of this organism. It belongs to the important group of filamentous Ascomycetes and grows as multinucleated mycelium. It has been described as a plant pathogen and it is able to overproduce riboflavin. Our analysis of the A. gossypii genome reveals that it consists of only 9x106 base pairs, the smallest genome of a free-living eukaryote, coding for 4700 proteins (25% less than S. cerevisiae). Gene manipulations in this fungus by homologous recombination are straight forward allowing for example efficient PCR-based gene targeting including fusions to fluorescence markers like GFP.
Genomics as information basis for investigating growth and proliferation in fungi
With the completion of the DNA sequence of the S. cerevisiae genome in 1996 it became apparent that 40% of the 6‘200 predicted genes code for proteins of unknown function. In 1996 several European labs which had participated in the yeast genome sequence project initiated a functional analysis network (EUROFAN) with the aim to find the cellular roles of novel proteins. We participated in both phases of this EU program in order to find novel components involved in nuclear migration and division (see also previous reports). In addition, we coordinated during the second phase of EUROFAN the European activities of a transatlantic consortium which generated bar-coded deletions for all annotated S. cerevisiae open reading frames. This unique deletion collection is now used in academia and industry for functional profiling of the S. cerevisiae genome. It allows to apply an almost unlimited number of growth, stress, or selection conditions to liquid cultures each carrying the mixture of all gene deletions. The best growing and the non-growing deletions are detected with the help of the bar codes (two unique 20-mer sequences associated with each deletion). Fluorescently labeled copies of all bar codes present in the culture at the end of the experiment are prepared by PCR and hybridized to commercially available DNA chips carrying complementary sequences to all bar codes.
Our group initiated in 1995 a genome project with the filamentous fungus A. gossypii. In 1997 we gained support from Novartis Agro (now Syngenta) which allowed us to complete over 90% of the genome analysis by the end of 2001. Key results are the almost complete map of close to 4700 ORFs which surprisingly shows extensive synteny to the gene order in S. cerevisiae and the apparent lack of gene duplications (with very few exceptions). The data are a rich source for novel approaches to analyze the dynamics of fungal growth and its molecular control.
Genes controlling nuclear migration in S. cerevisiae
S. cerevisiae is a budding yeast and nuclear migration comprises two major steps during the mitotic cycle. During the first step the nucleus moves from an apparently random position in the mother cell to a site close to the bud neck. The insertion of the dividing nucleus into the daughter cell during anaphase marks the second step. Nuclear migration and positioning of the mitotic spindle relative to the mother-daughter axis are dependent on the dynamic action of cytoplasmic microtubules. Other proteins required for correct migration and segregation of nuclei include microtubule-based motor proteins as well as putative components of the dynactin complex and the actin cytoskeleton.
The spindle pole body (SPB) of S. cerevisiae is the functional homolog of the mammalian centrosome, responsible for the organization of the tubulin cytoskeleton. Cytoplasmic (astral) microtubules essential for the proper segregation of the nucleus into the daughter cell are attached at the outer plaque on the SPB cytoplasmic face. Previously, we had shown that Cnm67p is an integral component of this structure; cells deleted for CNM67 are lacking the SPB outer plaque and thus experience severe nuclear migration defects. With the use of partial deletion mutants of CNM67, we show that the N-and C-terminal domains of the protein are important for nuclear migration. The C terminus, not the N terminus, is essential for Cnm67p localization to the SPB. On the other hand, only the N terminus is subject to protein phosphorylation of yet unknown function. Electron microscopy of SPB serial thin sections reveals that deletion of the N- or C-terminal domains disturbs outer plaque formation, whereas mutations in the central coiled-coil domain of Cnm67p change the distance between the SPB core and the outer plaque. We conclude that Cnm67p is the protein that connects the outer plaque to the central plaque embedded in the nuclear envelope, adjusting the space between them by the length of its coiled-coil.
We also investigated the role of Spc72, the receptor of the cytoplasmic g-tubulin complex. By using in-vivo fluorescence microscopy, we showed that cells lacking Spc72 can only generate very short (<1 mm) and unstable astral microtubules. Consequently, nuclear migration to the bud neck and orientation of the anaphase spindle along the mother-bud axis are absent in these cells. However, SPC72 deletion is not lethal because elongated but misaligned spindles can frequently reorient in mother cells permitting delayed but otherwise correct nuclear segregation. High-resolution time-lapse sequences revealed that this spindle reorientation was most likely accomplished by cortex interactions of the very short astral microtubules. In addition, a set of double mutants suggested that reorientation was dependent on the SPB outer plaque and the astral microtubule motor function of Kar3 but not Kip2/Kip3/Dhc1, or the cortex components Kar9/Num1. Our observations suggest that Spc72 is required for astral microtubule formation at the SPB half-bridge and for stabilization of astral microtubules at the SPB outer plaque. In addition, our data excludes involvement of Spc72 in spindle formation and elongation functions.
Motor proteins involved in nuclear dynamics of A. gossypii
In order to follow nuclear movement within the hyphae during fungal growths an in-frame GFP fusion to the histone H4 has been carried out which resulted in a strong fluorescent labeling of nuclei. Video fluorescence microscopy and time-lapse studies revealed an active traffic of nuclei within the hyphae, including continuous oscillations, frequent mitotic events, bypassing of nuclei and movement through septa. This dynamic behavior results in a uniform distribution of nuclei along the hyphae. To better characterize nuclear movement, we also measured some dynamic parameters. Under time-lapse conditions (minimal medium, 24°C), we estimated the nuclear velocity to be 0.3 mm per minute which was similar to the hyphal tip extension, whereas oscillation velocity could reach 4 mm per minute.
We identified seven homologs to S. cerevisiae genes coding for molecular motors which interact with microtubules: Six kinesin-related proteins, KRP1-6, and one dynein heavy chain, DHC1. One-step gene targeting has been used to delete the corresponding genes in A. gossypii. Deletion of three of the kinesin-related proteins (KRP1, KRP2 and KRP4) did not affect to an observable degree nuclear distribution or migration. Deletion of KRP3 lead to a lower density and more random distribution of nuclei in the hyphae and to a prolonged time for mitosis. Analysis of deletions of KRP5 revealed a role in nuclear oscillation. Analysis of KRP6 is still in progress. We observed the most severe defect in nuclear migration when the complete dynein heavy chain gene had been removed. In contrast to dynein mutants of Aspergillus nidulans where nuclei fail to move into the germ tube, we observed in the DHC1 deletion of A. gossypii clumping of nuclei at the tip of hyphae. For the future we plan to study the deletion mutants of kinesin motors in hyphae with GFP-labeled spindle pole bodies and GFP-labeled organelles.
Functional similarities and differences between homologous growth control genes of A. gossypii and S. cerevisiae
In the filamentous ascomycete Ashbya gossypii, like in other filamentous fungi, onset of growth in dormant spores occurs as an isotropic growth phase generating spherical germ cells. Thereafter, a switch to polarized growth results in the formation of the first hyphal tube (monopolar germling). The initial steps of hyphal tube formation in filamentous fungi, therefore, resemble processes taking place prior to and during bud emergence of unicellular yeast-like fungi. In contrast to yeast species, where growth of the tip of an emerging bud is temporally limited, filamentous fungi exhibit sustained polarized growth of the hyphal tip. This, together with frequent lateral branching, generates the typical network of hyphae (mycelium).
The genome sequence of A. gossypii revealed homologs to all S. cerevisiae genes involved in polar growth despite substantial differences in the growth modes of these two organisms. Therefore it was very intriguing to analyze the function of the Ashbya homologs with respect to filamentous growth control.
Polarized cell growth requires a polarized organization of the actin cytoskeleton. Small GTP-binding proteins of the Rho-family have been shown to be involved in the regulation of actin polarization. Since a potential role of Rho-proteins has not been studied so far in filamentous fungi we isolated and characterized the Ashbya gossypii homologs of the Saccharomyces cerevisiae CDC42, CDC24, RHO1, and RHO3 genes. The AgCDC42 and AgCDC24 genes can both complement conditional mutations in the S. cerevisiae CDC42 and CDC24 genes and both proteins are required for the establishment of actin polarization in A. gossypii germ cells. Agrho1 mutants show a cell lysis phenotype. Null mutant strains of Agrho3 show periodic swelling of hyphal tips that is overcome by repolarization and polar hyphal growth in a manner resembling the germination pattern of spores. Thus different Rho-protein modules are required for distinct steps during polarized hyphal growth of A. gossypii.
Next we characterized the A. gossypii homolog of the S. cerevisiae BEM2 gene which is part of a network of Rho-GTPases and their regulators. ScBem2 is required for bud emergence and bud growth in yeast. We showed that the AgBem2 protein contains a GAP-(GTPase activating protein) domain for Rho-like GTPases at its carboxy terminus, and that this part of AgBem2p is required for complementation of an Agbem2 null strain. Germination of spores resulted in enlarged Agbem2 germ cells that were unable to generate the bipolar branching pattern found in wild-type germ cells. In addition, mutant hyphae were swollen due to defects in polarized cell growth indicated by the delocalized distribution of chitin and cortical actin patches in swollen hyphal tips. Surprisingly, the complete loss of cell polarity which leads to spherical hyphal tips was often overcome by the establishment of new cell polarities and the formation of multiple new hyphal tips. These results demonstrate that establishment of cell polarity, maintenance of cell polarity, and polarized hyphal growth in filamentous fungi require similar proteins as in S. cerevisiae.
We were also able to identify for the first time a fungal gene important for hyphal maturation. This novel A. gossypii gene encodes a presumptive PAK (p21-activated kinase)-like kinase. Its closest homolog is the S. cerevisiae Cla4 protein kinase; the A. gossypii protein is therefore called AgCla4p. Agcla4 deletion strains are no longer able to perform the developmental switch from young to mature hyphae, and GFP (green fluorescent protein)-tagged AgCla4p localizes with much higher frequency in mature hyphal tips than in young hyphal tips. Both results support the importance of AgCla4p in hyphal maturation. AgCla4p is also required for septation, indicated by the inability of Agcla4 deletion strains to properly form actin rings and chitin rings. Despite the requirement of AgCla4p for the development of fast-growing hyphae, AgCla4p is not necessary for actin polarization per se, because tips enriched in cortical patches and hyphae with a fully developed network of actin cables can be seen in Agcla4 deletion strains.
The function of the Ashbya BUD1/RSR1 and BUD2 homolog were investigated by using time-lapse video microscopy. These movies revealed that AgBUD2 is important for a stable growth axis of the elongating hyphae; Ashbya hyphae lacking this gene frequently change direction of growth. AgRSR1/BUD1 seems to play a role in the maintenance of hyphal tip growth and in the elongation of initiated hyphal branches. Ashbya hyphae lacking this gene frequently paused growth at the tip and showed many unsuccessful branching events. Pausing of tip growth and frequent failures in lateral branch growth coincided with the loss of green fluorescence at tips and at the basis of branches in strains carrying a polarisome-GFP marker in addition to the rsr1/bud1 deletion.
These first data on hyphal growth control allow the conclusion that homologous proteins of S. cerevisiae and A. gossypii like Cla4, Rsr1/Bud1 and Bud2 serve different functions in the cellular environments of both organisms.
Effects of myosin gene deletions on growth and development of A. gossypii
Three myosin genes, AgMYO1, AgMYO2 and AgMYO3 were identified in the genome of the filamentous fungus Ashbya gossypii. These encode type II, type V, type I myosins, respectively. Thus, Ashbya is the eukaryotic organism with the smallest number of myosins found so far. In order to learn whether these myosins fulfill similar functions in A. gossypii as their homologs in S. cerevisiae we performed a phenotypic analysis of deletion strains.
Agmyo1 null mutants grew at rates close to those of wild type. Hyphal morphology was unaltered. However, actin rings were absent, but septa still formed. Sporulation in this mutant was severely decreased. Agmyo2 deletion mutants were non-viable. Although the knock out is lethal, spores still geminated and often grew to a stage of deformed germlings before growth arrested or lysis occurred. Enlarged forms of actin rings, thick actin ribbons as well as single cables, could be observed. Agmyo3 deletion mutants showed severely reduced growth rates. This is most likely due to an impaired organization of the actin cytoskeleton, manifested in faint or entirely missing actin caps and heterogeneous patch size. In addition, this deletion did not sporulate. These data show that homologous myosins perform similar roles in A. gossypii and S. cerevisiae.
Publications
Ayad-Durieux, Y., Knechtle, P., Goff, S., Dietrich, F. & Philippsen, P. (2000). A PAK-like protein kinase is required for maturation of young hyphae and septation in the filamentous ascomycete Ashbya gossypii. J Cell Sci 113 Pt 24, 4563-75.
Brachat, A., Liebundguth, N., Rebischung, C., Lemire, S., Schärer, F., Hoepfner, D., Demchyshyn, V., Howald, I., Düsterhöft, A., Möstl, D., Pöhlmann, R., Kotter, P., Hall, M. N., Wach, A. & Philippsen, P. (2000). Analysis of deletion phenotypes and GFP fusions of 21 novel Saccharomyces cerevisiae open reading frames. Yeast 16, 241-53.
Hoepfner, D., Brachat, A. & Philippsen, P. (2000). Time-lapse video microscopy analysis reveals astral microtubule detachment in the yeast spindle pole mutant cnm67. Mol Biol Cell 11, 1197-211.
Wendland, J., Ayad-Durieux, Y., Knechtle, P., Rebischung, C. & Philippsen, P. (2000). PCR-based gene targeting in the filamentous fungus Ashbya gossypii. Gene 242, 381-91.
Wendland, J. & Philippsen, P. (2000). Determination of cell polarity in germinated spores and hyphal tips of the filamentous ascomycete Ashbya gossypii requires a rhoGAP homolog. J Cell Sci 113 ( Pt 9), 1611-21.
Alberti-Segui, C., Dietrich, F., Altmann-Jöhl, R., Hoepfner, D. & Philippsen, P. (2001). Cytoplasmic dynein is required to oppose the force that moves nuclei towards the hyphal tip in the filamentous ascomycete Ashbya gossypii. J Cell Sci 114, 975-86.
Gadal, O., Strauss, D., Braspenning, J., Hoepfner, D., Petfalski, E., Philippsen, P., Tollervey, D. & Hurt, E. (2001). A nuclear AAA-type ATPase (Rix7p) is required for biogenesis and nuclear export of 60S ribosomal subunits. EMBO J 20, 3695-704.
Hoepfner, D., Schaerer, F., Brachat, A., Wach, A., and Philippsen, P. (2001). Reorientation of mispositioned spindles in the short astral microtubule mutant spc72 is dependent on the spindle pole body outer plaque and the Kar3 motor protein. Mol Biol Cell 13, 1366-1380.
Hoepfner, D., van den Berg, M., Philippsen, P., Tabak, H. F. & Hettema, E. H. (2001). A role for Vps1p, actin, and the Myo2p motor in peroxisome abundance and inheritance in Saccharomyces cerevisiae. J Cell Biol 155, 979-90.
Requena, N., Alberti-Segui, C., Winzenburg, E., Horn, C., Schliwa, M., Philippsen, P., Liese, R. & 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, 121-32.
Schaerer, F., Morgan, G., Winey, M. & Philippsen, P. (2001). Cnm67p is a spacer protein of the Saccharomyces cerevisiae spindle pole body outer plaque. Mol Biol Cell 12, 2519-33.
Wendland, J. & Philippsen, P. (2001). Cell polarity and hyphal morphogenesis are controlled by multiple rho-protein modules in the filamentous ascomycete Ashbya gossypii. Genetics 157, 601-10.
Wendland, J. & Philippsen, P. (in press). An IQGAP-related protein, encoded by AgCYK1, is required for septation in the filamentous fungus Ashbya gossypii. Fungal Genetics and Biology.
Giaever, G., Chu, A., Ni, L., Connelly, C., Riles, L., Bussey, H., Boeke, J., Snyder, M., Philippsen, P., Davis, R.W & Johnston, M. (in press). Functional Profiling of the S. cerevisiae Genome.