scholarly journals Regulation of exit from mitosis in multinucleate Ashbya gossypii cells relies on a minimal network of genes

2011 ◽  
Vol 22 (17) ◽  
pp. 3081-3093 ◽  
Author(s):  
Mark R. Finlayson ◽  
A. Katrin Helfer-Hungerbühler ◽  
Peter Philippsen
Keyword(s):  
Regulatory Networks ◽  
Temporal Constraints ◽  
Ashbya Gossypii ◽  
Metaphase Arrest ◽  
Minimal Network ◽  
Filamentous Ascomycete ◽  
Low Degree ◽  
Mitotic Exit Network ◽  
Early Anaphase ◽  
Core Components

In Saccharomyces cerevisiae, mitosis is coupled to cell division by the action of the Cdc fourteen early anaphase release (FEAR) and mitotic exit network (MEN) regulatory networks, which mediate exit from mitosis by activation of the phosphatase Cdc14. The closely related filamentous ascomycete Ashbya gossypii provides a unique cellular setting to study the evolution of these networks. Within its multinucleate hyphae, nuclei are free to divide without the spatial and temporal constraints described for budding yeast. To investigate how this highly conserved system has adapted to these circumstances, we constructed a series of mutants lacking homologues of core components of MEN and FEAR and monitored phenomena such as progression through mitosis and Cdc14 activation. MEN homologues in A. gossypii were shown to have diverged from their anticipated role in Cdc14 release and exit from mitosis. We observed defects in septation, as well as a partial metaphase arrest, in Agtem1Δ, Agcdc15Δ, Agdbf2/dbf20Δ, and Agmob1Δ. A. gossypii homologues of the FEAR network, on the other hand, have a conserved and more pronounced role in regulation of the M/G1 transition. Agcdc55Δ mutants are unable to sequester AgCdc14 throughout interphase. We propose a reduced model of the networks described in yeast, with a low degree of functional redundancy, convenient for further investigations into these networks.

scholarly journals The Role of the Polo Kinase Cdc5 in Controlling Cdc14 Localization

2003 ◽  
Vol 14 (11) ◽  
pp. 4486-4498 ◽  
Author(s):  
Rosella Visintin ◽  
Frank Stegmeier ◽  
Angelika Amon
Keyword(s):  
Protein Phosphatase ◽  
Regulatory Networks ◽  
Competitive Inhibitor ◽  
S Phase ◽  
Mitotic Exit ◽  
Late Anaphase ◽  
Mitotic Exit Network ◽  
Early Anaphase ◽  
The Relationship

In budding yeast, the protein phosphatase Cdc14 controls exit from mitosis. Its activity is regulated by a competitive inhibitor Cfi1/Net1, which binds to and sequesters Cdc14 in the nucleolus. During anaphase, Cdc14 is released from its inhibitor by the action of two regulatory networks. The Cdc Fourteen Early Anaphase Release (FEAR) network initiates Cdc14 release from Cfi1/Net1 during early anaphase, and the Mitotic Exit Network (MEN) promotes Cdc14 release during late anaphase. Here, we investigate the relationship among FEAR network components and propose an order in which they function to promote Cdc14 release from the nucleolus. Furthermore, we examine the role of the protein kinase Cdc5, which is a component of both the FEAR network and the MEN, in Cdc14 release from the nucleolus. We find that overexpression of CDC5 led to Cdc14 release from the nucleolus in S phase-arrested cells, which correlated with the appearance of phosphorylated forms of Cdc14 and Cfi1/Net1. Cdc5 promotes Cdc14 phosphorylation and, by stimulating the MEN, Cfi1/Net1 phosphorylation. Furthermore, we suggest that Cdc14 release from the nucleolus only occurs when Cdc14 and Cfi1/Net1 are both phosphorylated.

scholarly journals Putting the Brake on FEAR: Tof2 Promotes the Biphasic Release of Cdc14 Phosphatase during Mitotic Exit

2009 ◽  
Vol 20 (1) ◽  
pp. 245-255 ◽  
Author(s):  
William G. Waples ◽  
Charly Chahwan ◽  
Marta Ciechonska ◽  
Brigitte D. Lavoie
Keyword(s):  
Chromosome Segregation ◽  
Genetic Data ◽  
Mitotic Exit ◽  
Genetic Interactions ◽  
Genetic Screens ◽  
Rdna Array ◽  
Mitotic Exit Network ◽  
M Phase ◽  
Early Anaphase ◽  
Biphasic Release

The completion of chromosome segregation during anaphase requires the hypercondensation of the ∼1-Mb rDNA array, a reaction dependent on condensin and Cdc14 phosphatase. Using systematic genetic screens, we identified 29 novel genetic interactions with budding yeast condensin. Of these, FOB1, CSM1, LRS4, and TOF2 were required for the mitotic condensation of the tandem rDNA array localized on chromosome XII. Interestingly, whereas Fob1 and the monopolin subunits Csm1 and Lrs4 function in rDNA condensation throughout M phase, Tof2 was only required during anaphase. We show that Tof2, which shares homology with the Cdc14 inhibitor Net1/Cfi1, interacts with Cdc14 phosphatase and its deletion suppresses defects in mitotic exit network (MEN) components. Consistent with these genetic data, the onset of Cdc14 release from the nucleolus was similar in TOF2 and tof2Δ cells; however, the magnitude of the release was dramatically increased in the absence of Tof2, even when the MEN pathway was compromised. These data support a model whereby Tof2 coordinates the biphasic release of Cdc14 during anaphase by restraining a population of Cdc14 in the nucleolus after activation of the Cdc14 early anaphase release (FEAR) network, for subsequent release by the MEN.

scholarly journals Homologous recombination as the main mechanism for DNA integration and cause of rearrangements in the filamentous ascomycete Ashbya gossypii.

Genetics ◽  
1995 ◽  
Vol 140 (3) ◽  
pp. 973-987 ◽  
Author(s):  
S Steiner ◽  
J Wendland ◽  
M C Wright ◽  
P Philippsen
Keyword(s):  
Resistance Gene ◽  
Gene Replacement ◽  
Phytopathogenic Fungus ◽  
Ashbya Gossypii ◽  
Aminoglycoside Resistance ◽  
Genetic Changes ◽  
Apparent Lack ◽  
Dna Integration ◽  
Filamentous Ascomycete ◽  
One Step

Abstract A slow and a fast growth phenotype were observed after transformation of the phytopathogenic fungus Ashbya gossypii using a plasmid carrying homologous DNA and as selectable marker the Tn903 aminoglycoside resistance gene expressed from a strong A. gossypii promoter. Transformations with circular plasmids yielded slowly and irregularly growing geneticin-resistant mycelia in which 1% of nuclei contained plasmid sequences. Occasionally, fast growing sectors appeared which were shown to be initiated by homologous integration of the transforming DNA. Transformants obtained with plasmids linearized within the homology region immediately exhibited fast radial growth. In all 28 transformants analyzed plasmid DNA was integrated homologously. Such apparent lack of nonhomologous recombination has so far not been observed in filamentous ascomycetes. In 14 transformants two to four tandemly integrated plasmid copies were found. They underwent several types of genetic changes, mainly in the older mycelium: excision of whole plasmid copies and rearrangements within the integrated DNA (inversions and deletions). These internal rearrangements involved 360-bp inverted repeats, remnants of IS-elements flanking the resistance gene, and 156-bp direct repeats, originating from the strong A. gossypii promoter. Improved vectors lacking sequence repetitions were constructed and used for stable one-step gene replacement in A. gossypii.

scholarly journals The SH3/PH Domain Protein AgBoi1/2 Collaborates with the Rho-Type GTPase AgRho3 To Prevent Nonpolar Growth at Hyphal Tips of Ashbya gossypii

2006 ◽  
Vol 5 (10) ◽  
pp. 1635-1647 ◽  
Author(s):  
Philipp Knechtle ◽  
Jürgen Wendland ◽  
Peter Philippsen
Keyword(s):  
Fluorescent Protein ◽  
Ashbya Gossypii ◽  
Ph Domain ◽  
Filamentous Ascomycete ◽  
Actin Cable ◽  
Single Deletion ◽  
Tip Extension ◽  
Green Fluorescent ◽  
Actin Cables ◽  
Polar Tip Growth

ABSTRACT Unlike most other cells, hyphae of filamentous fungi permanently elongate and lack nonpolar growth phases. We identified AgBoi1/2p in the filamentous ascomycete Ashbya gossypii as a component required to prevent nonpolar growth at hyphal tips. Strains lacking AgBoi1/2p frequently show spherical enlargement at hyphal tips with concomitant depolarization of actin patches and loss of tip-located actin cables. These enlarged tips can repolarize and resume hyphal tip extension in the previous polarity axis. AgBoi1/2p permanently localizes to hyphal tips and transiently to sites of septation. Only the tip localization is important for sustained elongation of hyphae. In a yeast two-hybrid experiment, we identified the Rho-type GTPase AgRho3p as an interactor of AgBoi1/2p. AgRho3p is also required to prevent nonpolar growth at hyphal tips, and strains deleted for both AgBOI1/2 and AgRHO3 phenocopied the respective single-deletion strains, demonstrating that AgBoi1/2p and AgRho3p function in a common pathway. Monitoring the polarisome of growing hyphae using AgSpa2p fused to the green fluorescent protein as a marker, we found that polarisome disassembly precedes the onset of nonpolar growth in strains lacking AgBoi1/2p or AgRho3p. AgRho3p locked in its GTP-bound form interacts with the Rho-binding domain of the polarisome-associated formin AgBni1p, implying that AgRho3p has the capacity to directly activate formin-driven actin cable nucleation. We conclude that AgBoi1/2p and AgRho3p support polarisome-mediated actin cable formation at hyphal tips, thereby ensuring permanent polar tip growth.

Determination of cell polarity in germinated spores and hyphal tips of the filamentous ascomycete Ashbya gossypii requires a rhoGAP homolog

2000 ◽  
Vol 113 (9) ◽  
pp. 1611-1621 ◽  
Author(s):  
J. Wendland ◽  
P. Philippsen
Keyword(s):  
Filamentous Fungi ◽  
Cell Polarity ◽  
Germ Cells ◽  
Rho Gtpases ◽  
Protein Domain ◽  
Ashbya Gossypii ◽  
Cortical Actin ◽  
Filamentous Ascomycete ◽  
Bud Emergence ◽  
Hyphal Tip

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 tip. The initial steps of hyphal tip formation in filamentous fungi, therefore, resemble processes taking place prior to and during bud emergence of unicellular yeast-like fungi. We investigated whether phenotypic similarities between these distinct events extended to the molecular level. To this end we isolated and characterized the A. gossypii homolog of the Saccharomyces cerevisiae BEM2 gene which is part of a network of rho-GTPases and their regulators required for bud emergence and bud growth in yeast. Here we show 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. Surprisingly, the complete loss of cell polarity which lead to spherical hyphal tips was overcome by the establishment of new cell polarities and the formation of multiple new hyphal tips. In conclusion these results and other findings demonstrate that establishment of cell polarity, maintenance of cell polarity, and polarized hyphal growth in filamentous fungi require members of Ρ-GTPase modules.

scholarly journals Of Bars and Rings: Hof1-Dependent Cytokinesis in Multiseptated Hyphae of Ashbya gossypii

2008 ◽  
Vol 29 (3) ◽  
pp. 771-783 ◽  
Author(s):  
Andreas Kaufmann ◽  
Peter Philippsen
Keyword(s):  
Ashbya Gossypii ◽  
Actin Ring ◽  
Src Homology 3 ◽  
Filamentous Ascomycete ◽  
Multinucleated Cells ◽  
Ring Assembly ◽  
Src Homology ◽  
Type Ii Myosin ◽  
First Time

ABSTRACT We analyzed the development of multiple septa in elongated multinucleated cells (hyphae) of the filamentous ascomycete Ashbya gossypii in which septation is apparently uncoupled from nuclear cycles. A key player for this compartmentalization is the PCH protein Hof1. Hyphae that are lacking this protein form neither actin rings nor septa but still elongate at wild-type speed. Using in vivo fluorescence microscopy, we present for the first time the coordination of cytokinesis and septation in multiseptated and multinucleated cells. Hof1, the type II myosin Myo1, the landmark protein Bud3, and the IQGAP Cyk1 form collars of cortical bars already adjacent to hyphal tips, thereby marking the sites of septation. While hyphae continue to elongate, these proteins gradually form cortical rings. This bar-to-ring transition depends on Hof1 and Cyk1 but not Myo1 and is required for actin ring assembly. The Fes/CIP4 homology (FCH) domain of Hof1 ensures efficient localization of Hof1, whereas ring integrity is conferred by the Src homology 3 (SH3) domain. Up to several hours after site selection, actin ring contraction leads to membrane invagination and subsequent cytokinesis. Simultaneously, a septum forms between the adjacent hyphal compartments, which do not separate. During evolution, A. gossypii lost the homologs of two enzymes essential for cell separation in Saccharomyces cerevisiae.

scholarly journals The Anaphase-Promoting Complex/Cyclosome Is Required for Anaphase Progression in Multinucleated Ashbya gossypii Cells

2006 ◽  
Vol 6 (2) ◽  
pp. 182-197 ◽  
Author(s):  
Amy S. Gladfelter ◽  
Nicoleta Sustreanu ◽  
A. Katrin Hungerbuehler ◽  
Sylvia Voegeli ◽  
Virginie Galati ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Nuclear Division ◽  
Eukaryotic Cell ◽  
Ashbya Gossypii ◽  
Anaphase Promoting Complex ◽  
Cycle Progression ◽  
Filamentous Ascomycete ◽  
Multinucleated Cells ◽  
Mitotic Cyclin

ABSTRACT Regulated protein degradation is essential for eukaryotic cell cycle progression. The anaphase-promoting complex/cyclosome (APC/C) is responsible for the protein destruction required for the initiation of anaphase and the exit from mitosis, including the degradation of securin and B-type cyclins. We initiated a study of the APC/C in the multinucleated, filamentous ascomycete Ashbya gossypii to understand the mechanisms underlying the asynchronous mitosis observed in these cells. These experiments were motivated by previous work which demonstrated that the mitotic cyclin AgClb1/2p persists through anaphase, suggesting that the APC/C may not be required for the division cycle in A. gossypii. We have now found that the predicted APC/C components AgCdc23p and AgDoc1p and the targeting factors AgCdc20p and AgCdh1p are essential for growth and nuclear division. Mutants lacking any of these factors arrest as germlings with nuclei blocked in mitosis. A likely substrate of the APC/C is the securin homologue AgPds1p, which is present in all nuclei in hyphae except those in anaphase. The destruction box sequence of AgPds1p is required for this timed disappearance. To investigate how the APC/C may function to degrade AgPds1p in only the subset of anaphase nuclei, we localized components and targeting subunits of the APC/C. Remarkably, AgCdc23p, AgDoc1p, and AgCdc16p were found in all nuclei in all cell cycle stages, as were the APC/C targeting factors AgCdc20p and AgCdh1p. These data suggest that the AgAPC/C may be constitutively active across the cell cycle and that proteolysis in these multinucleated cells may be regulated at the level of substrates rather than by the APC/C itself.

scholarly journals Cell Polarity and Hyphal Morphogenesis Are Controlled by Multiple Rho-Protein Modules in the Filamentous Ascomycete Ashbya gossypii

Genetics ◽  
2001 ◽  
Vol 157 (2) ◽  
pp. 601-610
Author(s):  
J Wendland ◽  
P Philippsen
Keyword(s):  
Cell Growth ◽  
Filamentous Fungi ◽  
Cell Polarity ◽  
Hyphal Growth ◽  
Ashbya Gossypii ◽  
Rho Proteins ◽  
Filamentous Ascomycete ◽  
Polarized Cell Growth ◽  
Rho Protein ◽  
Protein Modules

Abstract 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 as well as other processes. Hyphal growth in filamentous fungi represents an ideal model to investigate mechanisms involved in generating cell polarity and establishing polarized cell growth. 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.

scholarly journals Cytoplasmic dynein is required to oppose the force that moves nuclei towards the hyphal tip in the filamentous ascomycete Ashbya gossypii

2001 ◽  
Vol 114 (5) ◽  
pp. 975-986 ◽  
Author(s):  
C. Alberti-Segui ◽  
F. Dietrich ◽  
R. Altmann-Johl ◽  
D. Hoepfner ◽  
P. Philippsen
Keyword(s):  
Long Range ◽  
Nuclear Migration ◽  
Cytoplasmic Dynein ◽  
Mitotic Division ◽  
Chain Gene ◽  
Ashbya Gossypii ◽  
Yeast Saccharomyces Cerevisiae ◽  
Filamentous Ascomycete ◽  
Nuclear Clusters ◽  
Actin Cables

We have followed the migration of GFP-labelled nuclei in multinucleate hyphae of Ashbya gossypii. For the first time we could demonstrate that the mode of long range nuclear migration consists of oscillatory movements of nuclei with, on average, higher amplitudes in the direction of the growing tip. We could also show that mitotic division proceeds at a constant rate of 0. 64 microm/minute which differs from the biphasic kinetics described for the yeast Saccharomyces cerevisiae. Furthermore we were able to identify the microtubule-based motor dynein as a key element in the control of long range nuclear migration. For other filamentous fungi it had already been demonstrated that inactivating mutations in dynein led to severe problems in nuclear migration, i.e. generation of long nuclei-free hyphal tips and clusters of nuclei throughout the hyphae. This phenotype supported the view that dynein is important for the movement of nuclei towards the tip. In A. gossypii the opposite seems to be the case. A complete deletion of the dynein heavy chain gene leads to nuclear clusters exclusively at the hyphal tips and to an essentially nucleus-free network of hyphal tubes and branches. Anucleate hyphae and branches in the vicinity of nuclear clusters show actin cables and polarized actin patches, as well as microtubules. The slow growth of this dynein null mutant could be completely reverted to wild-type-like growth in the presence of benomyl, which can be explained by the observed redistribution of nuclei in the hyphal network.

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