PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-25 (221979)

Clipboard (0)
None

Related Articles

1.  The SH3/PH Domain Protein AgBoi1/2 Collaborates with the Rho-Type GTPase AgRho3 To Prevent Nonpolar Growth at Hyphal Tips of Ashbya gossypii▿  
Eukaryotic Cell  2006;5(10):1635-1647.
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.
doi:10.1128/EC.00210-06
PMCID: PMC1595331  PMID: 16950929
2.  A Ras-like GTPase Is Involved in Hyphal Growth Guidance in the Filamentous Fungus Ashbya gossypiiV⃞ 
Molecular Biology of the Cell  2004;15(10):4622-4632.
Characteristic features of morphogenesis in filamentous fungi are sustained polar growth at tips of hyphae and frequent initiation of novel growth sites (branches) along the extending hyphae. We have begun to study regulation of this process on the molecular level by using the model fungus Ashbya gossypii. We found that the A. gossypii Ras-like GTPase Rsr1p/Bud1p localizes to the tip region and that it is involved in apical polarization of the actin cytoskeleton, a determinant of growth direction. In the absence of RSR1/BUD1, hyphal growth was severely slowed down due to frequent phases of pausing of growth at the hyphal tip. During pausing events a hyphal tip marker, encoded by the polarisome component AgSPA2, disappeared from the tip as was shown by in vivo time-lapse fluorescence microscopy of green fluorescent protein-labeled AgSpa2p. Reoccurrence of AgSpa2p was required for the resumption of hyphal growth. In the Agrsr1/bud1Δ deletion mutant, resumption of growth occurred at the hyphal tip in a frequently uncoordinated manner to the previous axis of polarity. Additionally, hyphal filaments in the mutant developed aberrant branching sites by mislocalizing AgSpa2p thus distorting hyphal morphology. These results define AgRsr1p/Bud1p as a key regulator of hyphal growth guidance.
doi:10.1091/mbc.E04-02-0104
PMCID: PMC519154  PMID: 15282338
3.  Maximal Polar Growth Potential Depends on the Polarisome Component AgSpa2 in the Filamentous Fungus Ashbya gossypiiV⃞ 
Molecular Biology of the Cell  2003;14(10):4140-4154.
We used actin staining and videomicroscopy to analyze the development from a spore to a young mycelium in the filamentous ascomycete Ashbya gossypii. The development starts with an initial isotropic growth phase followed by the emergence of germ tubes. The initial tip growth speed of 6–10 μm/h increases during early stages of development. This increase is transiently interrupted in response to the establishment of lateral branches or septa. The hyphal tip growth speed finally reaches a maximum of up to 200 μm/h, and the tips of these mature hyphae have the ability to split into two equally fast-growing hyphae. A search for A. gossypii homologs of polarisome components of the yeast Saccharomyces cerevisiae revealed a remarkable size difference between Spa2p of both organisms, with AgSpa2p being double as long as ScSpa2p due to an extended internal domain. AgSpa2 colocalizes with sites of polarized actin. Using time-lapse videomicroscopy, we show that AgSpa2p-GFP polarization is established at sites of branch initiation and then permanently maintained at hyphal tips. Polarization at sites of septation is transient. During apical branching the existing AgSpa2p-GFP polarization is symmetrically divided. To investigate the function of AgSpa2p, we generated two AgSPA2 mutants, a partial deletion of the internal domain alone, and a complete deletion. The mutations had an impact on the maximal hyphal tip growth speed, on the hyphal diameter, and on the branching pattern. We suggest that AgSpa2p is required for the determination of the area of growth at the hyphal tip and that the extended internal domain plays an important role in this process.
doi:10.1091/mbc.E03-03-0167
PMCID: PMC207006  PMID: 12937275
4.  Cdc42p regulation of the yeast formin Bni1p mediated by the effector Gic2p 
Molecular Biology of the Cell  2012;23(19):3814-3826.
Regulation of the formin Bni1p by Cdc42p in yeast does not require direct interaction between Bni1p and Cdc42p. The Cdc42p effector Gic2p can bind both Bni1p and GTP-Cdc42p, providing a novel regulatory input.
Actin filaments are dynamically reorganized to accommodate ever-changing cellular needs for intracellular transport, morphogenesis, and migration. Formins, a major family of actin nucleators, are believed to function as direct effectors of Rho GTPases, such as the polarity regulator Cdc42p. However, the presence of extensive redundancy has made it difficult to assess the in vivo significance of the low-affinity Rho GTPase–formin interaction and specifically whether Cdc42p polarizes the actin cytoskeleton via direct formin binding. Here we exploit a synthetically rewired budding yeast strain to eliminate the redundancy, making regulation of the formin Bni1p by Cdc42p essential for viability. Surprisingly, we find that direct Cdc42p–Bni1p interaction is dispensable for Bni1p regulation. Alternative paths linking Cdc42p and Bni1p via “polarisome” components Spa2p and Bud6p are also collectively dispensable. We identify a novel regulatory input to Bni1p acting through the Cdc42p effector, Gic2p. This pathway is sufficient to localize Bni1p to the sites of Cdc42p action and promotes a polarized actin organization in both rewired and wild-type contexts. We suggest that an indirect mechanism linking Rho GTPases and formins via Rho effectors may provide finer spatiotemporal control for the formin-nucleated actin cytoskeleton.
doi:10.1091/mbc.E12-05-0400
PMCID: PMC3459858  PMID: 22918946
5.  Ras1-Induced Hyphal Development in Candida albicans Requires the Formin Bni1 
Eukaryotic Cell  2005;4(10):1712-1724.
Formins are downstream effector proteins of Rho-type GTPases and are involved in the organization of the actin cytoskeleton and actin cable assembly at sites of polarized cell growth. Here we show using in vivo time-lapse microscopy that deletion of the Candida albicans formin homolog BNI1 results in polarity defects during yeast growth and hyphal stages. Deletion of the second C. albicans formin, BNR1, resulted in elongated yeast cells with cell separation defects but did not interfere with the ability of bnr1 cells to initiate and maintain polarized hyphal growth. Yeast bni1 cells were swollen, showed an increased random budding pattern, and had a severe defect in cytokinesis, with enlarged bud necks. Induction of hyphal development in bni1 cells resulted in germ tube formation but was halted at the step of polarity maintenance. Bni1-green fluorescent protein is found persistently at the hyphal tip and colocalizes with a structure resembling the Spitzenkörper of true filamentous fungi. Introduction of constitutively active ras1G13V in the bni1 strain or addition of cyclic AMP to the growth medium did not bypass bni1 hyphal growth defects. Similarly, these agents were not able to suppress hyphal growth defects in the wal1 mutant which is lacking the Wiskott-Aldrich syndrome protein (WASP) homolog. These results suggest that the maintenance of polarized hyphal growth in C. albicans requires coordinated regulation of two actin cytoskeletal pathways, including formin-mediated secretion and WASP-dependent endocytosis.
doi:10.1128/EC.4.10.1712-1724.2005
PMCID: PMC1265897  PMID: 16215178
6.  Axl2 Integrates Polarity Establishment, Maintenance, and Environmental Stress Response in the Filamentous Fungus Ashbya gossypii ▿ † 
Eukaryotic Cell  2011;10(12):1679-1693.
In budding yeast, new sites of polarity are chosen with each cell cycle and polarization is transient. In filamentous fungi, sites of polarity persist for extended periods of growth and new polarity sites can be established while existing sites are maintained. How the polarity establishment machinery functions in these distinct growth forms found in fungi is still not well understood. We have examined the function of Axl2, a transmembrane bud site selection protein discovered in Saccharomyces cerevisiae, in the filamentous fungus Ashbya gossypii. A. gossypii does not divide by budding and instead exhibits persistent highly polarized growth, and multiple axes of polarity coexist in one cell. A. gossypii axl2Δ (Agaxl2Δ) cells have wavy hyphae, bulbous tips, and a high frequency of branch initiations that fail to elongate, indicative of a polarity maintenance defect. Mutant colonies also have significantly lower radial growth and hyphal tip elongation speeds than wild-type colonies, and Agaxl2Δ hyphae have depolarized actin patches. Consistent with a function in polarity, AgAxl2 localizes to hyphal tips, branches, and septin rings. Unlike S. cerevisiae Axl2, AgAxl2 contains a Mid2 homology domain and may function to sense or respond to environmental stress. In support of this idea, hyphae lacking AgAxl2 also display hypersensitivity to heat, osmotic, and cell wall stresses. Axl2 serves to integrate polarity establishment, polarity maintenance, and environmental stress response for optimal polarized growth in A. gossypii.
doi:10.1128/EC.05183-11
PMCID: PMC3232731  PMID: 21984708
7.  Formin-dependent actin assembly is regulated by distinct modes of Rho signaling in yeast 
The Journal of Cell Biology  2003;161(6):1081-1092.
Formins are actin filament nucleators regulated by Rho-GTPases. In budding yeast, the formins Bni1p and Bnr1p direct the assembly of actin cables, which guide polarized secretion and growth. From the six yeast Rho proteins (Cdc42p and Rho1–5p), we have determined that four participate in the regulation of formin activity. We show that the essential function of Rho3p and Rho4p is to activate the formins Bni1p and Bnr1p, and that activated alleles of either formin are able to bypass the requirement for these Rho proteins. Through a separate signaling pathway, Rho1p is necessary for formin activation at elevated temperatures, acting through protein kinase C (Pkc1p), the major effector for Rho1p signaling to the actin cytoskeleton. Although Pkc1p also activates a MAPK pathway, this pathway does not function in formin activation. Formin-dependent cable assembly does not require Cdc42p, but in the absence of Cdc42p function, cable assembly is not properly organized during initiation of bud growth. These results show that formin function is under the control of three distinct, essential Rho signaling pathways.
doi:10.1083/jcb.200212040
PMCID: PMC2173006  PMID: 12810699
actin; polarity; Cdc42; PKC; MAPK
8.  Rsr1 Focuses Cdc42 Activity at Hyphal Tips and Promotes Maintenance of Hyphal Development in Candida albicans 
Eukaryotic Cell  2013;12(4):482-495.
The extremely elongated morphology of fungal hyphae is dependent on the cell's ability to assemble and maintain polarized growth machinery over multiple cell cycles. The different morphologies of the fungus Candida albicans make it an excellent model organism in which to study the spatiotemporal requirements for constitutive polarized growth and the generation of different cell shapes. In C. albicans, deletion of the landmark protein Rsr1 causes defects in morphogenesis that are not predicted from study of the orthologous protein in the related yeast Saccharomyces cerevisiae, thus suggesting that Rsr1 has expanded functions during polarized growth in C. albicans. Here, we show that Rsr1 activity localizes to hyphal tips by the differential localization of the Rsr1 GTPase-activating protein (GAP), Bud2, and guanine nucleotide exchange factor (GEF), Bud5. In addition, we find that Rsr1 is needed to maintain the focused localization of hyphal polarity structures and proteins, including Bem1, a marker of the active GTP-bound form of the Rho GTPase, Cdc42. Further, our results indicate that tip-localized Cdc42 clusters are associated with the cell's ability to express a hyphal transcriptional program and that the ability to generate a focused Cdc42 cluster in early hyphae (germ tubes) is needed to maintain hyphal morphogenesis over time. We propose that in C. albicans, Rsr1 “fine-tunes” the distribution of Cdc42 activity and that self-organizing (Rsr1-independent) mechanisms of polarized growth are not sufficient to generate narrow cell shapes or to provide feedback to the transcriptional program during hyphal morphogenesis.
doi:10.1128/EC.00294-12
PMCID: PMC3623438  PMID: 23223038
9.  Dynamic Localization and Function of Bni1p at the Sites of Directed Growth in Saccharomyces cerevisiae 
Molecular and Cellular Biology  2001;21(3):827-839.
Formin homology (FH) proteins are implicated in cell polarization and cytokinesis through actin organization. There are two FH proteins in the yeast Saccharomyces cerevisiae, Bni1p and Bnr1p. Bni1p physically interacts with Rho family small G proteins (Rho1p and Cdc42p), actin, two actin-binding proteins (profilin and Bud6p), and a polarity protein (Spa2p). Here we analyzed the in vivo localization of Bni1p by using a time-lapse imaging system and investigated the regulatory mechanisms of Bni1p localization and function in relation to these interacting proteins. Bni1p fused with green fluorescent protein localized to the sites of cell growth throughout the cell cycle. In a small-budded cell, Bni1p moved along the bud cortex. This dynamic localization of Bni1p coincided with the apparent site of bud growth. A bni1-disrupted cell showed a defect in directed growth to the pre-bud site and to the bud tip (apical growth), causing its abnormally spherical cell shape and thick bud neck. Bni1p localization at the bud tips was absolutely dependent on Cdc42p, largely dependent on Spa2p and actin filaments, and partly dependent on Bud6p, but scarcely dependent on polarized cortical actin patches or Rho1p. These results indicate that Bni1p regulates polarized growth within the bud through its unique and dynamic pattern of localization, dependent on multiple factors, including Cdc42p, Spa2p, Bud6p, and the actin cytoskeleton.
doi:10.1128/MCB.21.3.827-839.2001
PMCID: PMC86674  PMID: 11154270
10.  The Anaphase-Promoting Complex/Cyclosome Is Required for Anaphase Progression in Multinucleated Ashbya gossypii Cells▿ †  
Eukaryotic Cell  2006;6(2):182-197.
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.
doi:10.1128/EC.00364-06
PMCID: PMC1797942  PMID: 17158735
11.  Comparative Live-Cell Imaging Analyses of SPA-2, BUD-6 and BNI-1 in Neurospora crassa Reveal Novel Features of the Filamentous Fungal Polarisome 
PLoS ONE  2012;7(1):e30372.
A key multiprotein complex involved in regulating the actin cytoskeleton and secretory machinery required for polarized growth in fungi, is the polarisome. Recognized core constituents in budding yeast are the proteins Spa2, Pea2, Aip3/Bud6, and the key effector Bni1. Multicellular fungi display a more complex polarized morphogenesis than yeasts, suggesting that the filamentous fungal polarisome might fulfill additional functions. In this study, we compared the subcellular organization and dynamics of the putative polarisome components BUD-6 and BNI-1 with those of the bona fide polarisome marker SPA-2 at various developmental stages of Neurospora crassa. All three proteins exhibited a yeast-like polarisome configuration during polarized germ tube growth, cell fusion, septal pore plugging and tip repolarization. However, the localization patterns of all three proteins showed spatiotemporally distinct characteristics during the establishment of new polar axes, septum formation and cytokinesis, and maintained hyphal tip growth. Most notably, in vegetative hyphal tips BUD-6 accumulated as a subapical cloud excluded from the Spitzenkörper (Spk), whereas BNI-1 and SPA-2 partially colocalized with the Spk and the tip apex. Novel roles during septal plugging and cytokinesis, connected to the reinitiation of tip growth upon physical injury and conidial maturation, were identified for BUD-6 and BNI-1, respectively. Phenotypic analyses of gene deletion mutants revealed additional functions for BUD-6 and BNI-1 in cell fusion regulation, and the maintenance of Spk integrity. Considered together, our findings reveal novel polarisome-independent functions of BUD-6 and BNI-1 in Neurospora, but also suggest that all three proteins cooperate at plugged septal pores, and their complex arrangement within the apical dome of mature hypha might represent a novel aspect of filamentous fungal polarisome architecture.
doi:10.1371/journal.pone.0030372
PMCID: PMC3265482  PMID: 22291944
12.  Yeast Formins Bni1 and Bnr1 Utilize Different Modes of Cortical Interaction during the Assembly of Actin Cables 
Molecular Biology of the Cell  2007;18(5):1826-1838.
The budding yeast formins Bni1 and Bnr1 control the assembly of actin cables. These formins exhibit distinct patterns of localization and polymerize two different populations of cables: Bni1 in the bud and Bnr1 in the mother cell. We generated a functional Bni1-3GFP that improved the visualization of Bni1 in vivo at endogenous levels. Bni1 exists as speckles in the cytoplasm, some of which colocalize on actin cables. These Bni1 speckles display linear, retrograde-directed movements. Loss of polymerized actin or specifically actin cables abolished retrograde movement, and resulted in depletion of Bni1 speckles from the cytoplasm, with enhanced targeting of Bni1 to the bud tip. Mutations that impair the actin assembly activity of Bni1 abolished the movement of Bni1 speckles, even when actin cables were present. In contrast, Bnr1-GFP or 3GFP-Bnr1 did not detectably associate with actin cables and was not observed as cytoplasmic speckles. Finally, fluorescence recovery after photobleaching demonstrated that Bni1 was very dynamic, exchanging between polarized sites and the cytoplasm, whereas Bnr1 was confined to the bud neck and did not exchange with a cytoplasmic pool. In summary, our results indicate that formins can have distinct modes of cortical interaction during actin cable assembly.
doi:10.1091/mbc.E06-09-0820
PMCID: PMC1855024  PMID: 17344480
13.  Rho1p-Bni1p-Spa2p Interactions: Implication in Localization of Bni1p at the Bud Site and Regulation of the Actin Cytoskeleton in Saccharomyces cerevisiae 
Molecular Biology of the Cell  1998;9(5):1221-1233.
Rho1p is a yeast homolog of mammalian RhoA small GTP-binding protein. Rho1p is localized at the growth sites and required for bud formation. We have recently shown that Bni1p is a potential target of Rho1p and that Bni1p regulates reorganization of the actin cytoskeleton through interactions with profilin, an actin monomer-binding protein. Using the yeast two-hybrid screening system, we cloned a gene encoding a protein that interacted with Bni1p. This protein, Spa2p, was known to be localized at the bud tip and to be implicated in the establishment of cell polarity. The C-terminal 254 amino acid region of Spa2p, Spa2p(1213–1466), directly bound to a 162-amino acid region of Bni1p, Bni1p(826–987). Genetic analyses revealed that both the bni1 and spa2 mutations showed synthetic lethal interactions with mutations in the genes encoding components of the Pkc1p-mitogen-activated protein kinase pathway, in which Pkc1p is another target of Rho1p. Immunofluorescence microscopic analysis showed that Bni1p was localized at the bud tip in wild-type cells. However, in the spa2 mutant, Bni1p was not localized at the bud tip and instead localized diffusely in the cytoplasm. A mutant Bni1p, which lacked the Rho1p-binding region, also failed to be localized at the bud tip. These results indicate that both Rho1p and Spa2p are involved in the localization of Bni1p at the growth sites where Rho1p regulates reorganization of the actin cytoskeleton through Bni1p.
PMCID: PMC25343  PMID: 9571251
14.  Regulation of Cell Polarity by Interactions of Msb3 and Msb4 with Cdc42 and Polarisome Components 
Molecular and Cellular Biology  2005;25(19):8567-8580.
In Saccharomyces cerevisiae, polarized growth depends on interactions between the actin cytoskeleton and the secretory machinery. Here we show that the Rab GTPase-activating proteins (GAPs) Msb3 and Msb4 interact directly with Spa2, a scaffold protein of the “polarisome” that also interacts with the formin Bni1. Spa2 is required for the polarized localization of Msb3 and Msb4 at the bud tip. We also show that Msb3 and Msb4 bind specifically to Cdc42-GDP and Rho1-GDP in vitro and that Msb3 and Rho GDP dissociation inhibitor act independently but oppositely on Cdc42. Finally, we show that Msb3 and Msb4 are involved in Bni1-nucleated actin assembly in vivo. These results suggest that Msb3 and Msb4 regulate polarized growth by multiple mechanisms, directly regulating exocytosis through their GAP activity toward Sec4 and potentially coordinating the functions of Cdc42, Rho1, and Bni1 in the polarisome through their binding to these GTPases. A functional equivalent of the polarisome probably exists in other fungi and mammals.
doi:10.1128/MCB.25.19.8567-8580.2005
PMCID: PMC1265733  PMID: 16166638
15.  Septation of Infectious Hyphae Is Critical for Appressoria Formation and Virulence in the Smut Fungus Ustilago Maydis 
PLoS Pathogens  2011;7(5):e1002044.
Differentiation of hyphae into specialized infection structures, known as appressoria, is a common feature of plant pathogenic fungi that penetrate the plant cuticle. Appressorium formation in U. maydis is triggered by environmental signals but the molecular mechanism of this hyphal differentiation is largely unknown. Infectious hyphae grow on the leaf surface by inserting regularly spaced retraction septa at the distal end of the tip cell leaving empty sections of collapsed hyphae behind. Here we show that formation of retraction septa is critical for appressorium formation and virulence in U. maydis. We demonstrate that the diaphanous-related formin Drf1 is necessary for actomyosin ring formation during septation of infectious hyphae. Drf1 acts as an effector of a Cdc42 GTPase signaling module, which also consists of the Cdc42-specific guanine nucleotide exchange factor Don1 and the Ste20-like kinase Don3. Deletion of drf1, don1 or don3 abolished formation of retraction septa resulting in reduced virulence. Appressorium formation in these mutants was not completely blocked but infection structures were found only at the tip of short filaments indicating that retraction septa are necessary for appressorium formation in extended infectious hyphae. In addition, appressoria of drf1 mutants penetrated the plant tissue less frequently.
Author Summary
Pathogens exhibit various developmental stages during the process of infection and proliferation. The basidiomycete Ustilago maydis is a model organism for plant pathogenic fungi. On the plant surface U. maydis grows as a cell-cycle arrested filament. Growth of infectious hyphae involves regular formation of retraction septa leaving empty sections behind. The tip cell forms an appressorium and penetrates the cuticle. In this study we identified for the first time a signaling module regulating formation of retraction septa in fungal hyphae. The module consists of the highly conserved small GTPase Cdc42, its activator Don1 and the actin-organizing formin Drf1. After penetration of the plant, cell cycle arrest is released and hyphal septation is resumed in planta but was found to be independent of Cdc42 and Drf1. Thus, during infection Cdc42 signaling and Drf1 coordinate hyphal septation events specifically in infectious hyphae in U. maydis. The inability to form retraction septa affects filament elongation and appressorium formation resulting in significantly reduced virulence. We observed a threshold size of the cytoplasm filled tip compartment above which appressorium formation is blocked. These findings highlight that formation of retraction septa, a common feature of filamentous fungi, is an important virulence determinant of U. maydis.
doi:10.1371/journal.ppat.1002044
PMCID: PMC3098242  PMID: 21625538
16.  Regulation of exit from mitosis in multinucleate Ashbya gossypii cells relies on a minimal network of genes 
Molecular Biology of the Cell  2011;22(17):3081-3093.
Homologues of all components of the Saccharomyces cerevisiae FEAR and MEN pathways are expressed in the multinucleated hyphae of Ashbya gossypii despite the lack of controlled nuclear positioning and mitosis-dependent septum formation. In this system, MEN loses its dominant role, and nuclear divisions are controlled by FEAR homologues.
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.
doi:10.1091/mbc.E10-12-1006
PMCID: PMC3164456  PMID: 21737675
17.  Yeast-to-Hyphal Transition Triggers Formin-dependent Golgi Localization to the Growing Tip in Candida albicans 
Molecular Biology of the Cell  2006;17(10):4364-4378.
Rapid and long-distance secretion of membrane components is critical for hyphal formation in filamentous fungi, but the mechanisms responsible for polarized trafficking are not well understood. Here, we demonstrate that in Candida albicans, the majority of the Golgi complex is redistributed to the distal region during hyphal formation. Randomly distributed Golgi puncta in yeast cells cluster toward the growing tip during hyphal formation, remain associated with the distal portion of the filament during its extension, and are almost absent from the cell body. This restricted Golgi localization pattern is distinct from other organelles, including the endoplasmic reticulum, vacuole and mitochondria, which remain distributed throughout the cell body and hypha. Hyphal-induced positioning of the Golgi and the maintenance of its structural integrity requires actin cytoskeleton, but not microtubules. Absence of the formin Bni1 causes a hyphal-specific dispersal of the Golgi into a haze of finely dispersed vesicles with a sedimentation density no different from that of normal Golgi. These results demonstrate the existence of a hyphal-specific, Bni1-dependent cue for Golgi integrity and positioning at the distal portion of the hyphal tip, and suggest that filamentous fungi have evolved a novel strategy for polarized secretion, involving a redistribution of the Golgi to the growing tip.
doi:10.1091/mbc.E06-02-0143
PMCID: PMC1635370  PMID: 16855023
18.  Yeast formin Bni1p has multiple localization regions that function in polarized growth and spindle orientation 
Molecular Biology of the Cell  2012;23(3):412-422.
There are four distinct localization domains in formin Bni1p of budding yeast. Analysis of the functions of the domains in the actin cytoskeleton and in spindle orientation reveals unexpected complexity in the mechanism of formin localization and function.
Formins are conserved proteins that assemble unbranched actin filaments in a regulated, localized manner. Budding yeast's two formins, Bni1p and Bnr1p, assemble actin cables necessary for polarized cell growth and organelle segregation. Here we define four regions in Bni1p that contribute to its localization to the bud and at the bud neck. The first (residues 1–333) requires dimerization for its localization and encompasses the Rho-binding domain. The second (residues 334–821) covers the Diaphanous inhibitory–dimerization–coiled coil domains, and the third is the Spa2p-binding domain. The fourth region encompasses the formin homology 1–formin homology 2–COOH region of the protein. These four regions can each localize to the bud cortex and bud neck at the right stage of the cell cycle independent of both F-actin and endogenous Bni1p. The first three regions contribute cumulatively to the proper localization of Bni1p, as revealed by the effects of progressive loss of these regions on the actin cytoskeleton and fidelity of spindle orientation. The fourth region contributes to the localization of Bni1p in tiny budded cells. Expression of mislocalized Bni1p constructs has a dominant-negative effect on both growth and nuclear segregation due to mislocalized actin assembly. These results define an unexpected complexity in the mechanism of formin localization and function.
doi:10.1091/mbc.E11-07-0631
PMCID: PMC3268721  PMID: 22160598
19.  AgSwe1p Regulates Mitosis in Response to Morphogenesis and Nutrients in Multinucleated Ashbya gossypii Cells 
Molecular Biology of the Cell  2006;17(10):4494-4512.
Nuclei in the filamentous, multinucleated fungus Ashbya gossypii divide asynchronously. We have investigated what internal and external signals spatially direct mitosis within these hyphal cells. Mitoses are most common near cortical septin rings found at growing tips and branchpoints. In septin mutants, mitoses are no longer concentrated at branchpoints, suggesting that the septin rings function to locally promote mitosis near new branches. Similarly, cells lacking AgSwe1p kinase (a Wee1 homologue), AgHsl1p (a Nim1-related kinase), and AgMih1p phosphatase (the Cdc25 homologue that likely counteracts AgSwe1p activity) also have mitoses distributed randomly in the hyphae as opposed to at branchpoints. Surprisingly, however, no phosphorylation of the CDK tyrosine 18 residue, the conserved substrate of Swe1p kinases, was detected in normally growing cells. In contrast, abundant CDK tyrosine phosphorylation was apparent in starving cells, resulting in diminished nuclear density. This starvation-induced CDK phosphorylation is AgSwe1p dependent, and overexpressed AgSwe1p is sufficient to delay nuclei even in rich nutrient conditions. In starving cells lacking septins or AgSwe1p negative regulators, the nuclear density is further diminished compared with wild type. We have generated a model in which AgSwe1p may regulate mitosis in response to cell intrinsic morphogenesis cues and external nutrient availability in multinucleated cells.
doi:10.1091/mbc.E06-03-0215
PMCID: PMC1635347  PMID: 16899511
20.  Septin Ring Assembly Requires Concerted Action of Polarisome Components, a PAK Kinase Cla4p, and the Actin Cytoskeleton in Saccharomyces cerevisiae 
Molecular Biology of the Cell  2004;15(12):5329-5345.
Septins are filament-forming proteins that function in cytokinesis in a wide variety of organisms. In budding yeast, the small GTPase Cdc42p triggers the recruitment of septins to the incipient budding site and the assembly of septins into a ring. We herein report that Bni1p and Cla4p, effectors of Cdc42p, are required for the assembly of the septin ring during the initiation of budding but not for its maintenance after the ring converts to a septin collar. In bni1Δ cla4-75-td mutant, septins were recruited to the incipient budding site. However, the septin ring was not assembled, and septins remained at the polarized growing sites. Bni1p, a formin family protein, is a member of the polarisome complex with Spa2p, Bud6p, and Pea2p. All spa2Δ cla4-75-td, bud6Δ cla4-75-td, and pea2Δ cla4-75-td mutants showed defects in septin ring assembly. Bni1p stimulates actin polymerization for the formation of actin cables. Point mutants of BNI1 that are specifically defective in actin cable formation also exhibited septin ring assembly defects in the absence of Cla4p. Consistently, treatment of cla4Δ mutant with the actin inhibitor latrunculin A inhibited septin ring assembly. Our results suggest that polarisome components and Cla4p are required for the initial assembly of the septin ring and that the actin cytoskeleton is involved in this process.
doi:10.1091/mbc.E04-03-0254
PMCID: PMC532014  PMID: 15371547
21.  Mechanism and cellular function of Bud6 as an actin nucleation–promoting factor 
Molecular Biology of the Cell  2011;22(21):4016-4028.
Bud6 functions as an actin nucleation–promoting factor (NPF) for Bni1; thus formins can depend on NPFs like the Arp2/3 complex. Unexpected parallels exist between Bud6 and WASp. Bud6 is the first nonmetazoan example of formins pairing with actin monomer–binding proteins to stimulate nucleation, akin to Spire-Capu and APC-mDia1
Formins are a conserved family of actin assembly–promoting factors with diverse biological roles, but how their activities are regulated in vivo is not well understood. In Saccharomyces cerevisiae, the formins Bni1 and Bnr1 are required for the assembly of actin cables and polarized cell growth. Proper cable assembly further requires Bud6. Previously it was shown that Bud6 enhances Bni1-mediated actin assembly in vitro, but the biochemical mechanism and in vivo role of this activity were left unclear. Here we demonstrate that Bud6 specifically stimulates the nucleation rather than the elongation phase of Bni1-mediated actin assembly, defining Bud6 as a nucleation-promoting factor (NPF) and distinguishing its effects from those of profilin. We generated alleles of Bud6 that uncouple its interactions with Bni1 and G-actin and found that both interactions are critical for NPF activity. Our data indicate that Bud6 promotes filament nucleation by recruiting actin monomers to Bni1. Genetic analysis of the same alleles showed that Bud6 regulation of formin activity is critical for normal levels of actin cable assembly in vivo. Our results raise important mechanistic parallels between Bud6 and WASP, as well as between Bud6 and other NPFs that interact with formins such as Spire.
doi:10.1091/mbc.E11-05-0404
PMCID: PMC3204064  PMID: 21880892
22.  Regulation of Cdc42 GTPase Activity in the Formation of Hyphae in Candida albicans 
Molecular Biology of the Cell  2007;18(1):265-281.
The human fungal pathogen Candida albicans can switch between yeast, pseudohyphal, and hyphal morphologies. To investigate whether the distinctive characteristics of hyphae are due to increased activity of the Cdc42 GTPase, strains lacking negative regulators of Cdc42 were constructed. Unexpectedly, the deletion of the Cdc42 Rho guanine dissociation inhibitor RDI1 resulted in reduced rather than enhanced polarized growth. However, when cells lacking both Cdc42 GTPase-activating proteins, encoded by RGA2 and BEM3, were grown under pseudohyphal-promoting conditions the bud was highly elongated and lacked a constriction at its base, so that its shape resembled a hyphal germ tube. Moreover, a Spitzenkörper was present at the bud tip, a band of disorganized septin was present at bud base, true septin rings formed within the bud, and nuclei migrated out of the mother cell before the first mitosis. These are all characteristic features of a hyphal germ tube. Intriguingly, we observed hyphal-specific phosphorylation of Rga2, suggesting a possible mechanism for Cdc42 activation during normal hyphal development. In contrast, expression of Cdc42G12V, which is constitutively GTP bound because it lacks GTPase activity, resulted in swollen cells with prominent and stable septin bars. These results suggest the development of hyphal-specific characteristics is promoted by Cdc42-GTP in a process that also requires the intrinsic GTPase activity of Cdc42.
doi:10.1091/mbc.E06-05-0411
PMCID: PMC1751335  PMID: 17093060
23.  Functional Characterization and Cellular Dynamics of the CDC-42 – RAC – CDC-24 Module in Neurospora crassa 
PLoS ONE  2011;6(11):e27148.
Rho-type GTPases are key regulators that control eukaryotic cell polarity, but their role in fungal morphogenesis is only beginning to emerge. In this study, we investigate the role of the CDC-42 – RAC – CDC-24 module in Neurospora crassa. rac and cdc-42 deletion mutants are viable, but generate highly compact colonies with severe morphological defects. Double mutants carrying conditional and loss of function alleles of rac and cdc-42 are lethal, indicating that both GTPases share at least one common essential function. The defects of the GTPase mutants are phenocopied by deletion and conditional alleles of the guanine exchange factor (GEF) cdc-24, and in vitro GDP-GTP exchange assays identify CDC-24 as specific GEF for both CDC-42 and RAC. In vivo confocal microscopy shows that this module is organized as membrane-associated cap that covers the hyphal apex. However, the specific localization patterns of the three proteins are distinct, indicating different functions of RAC and CDC-42 within the hyphal tip. CDC-42 localized as confined apical membrane-associated crescent, while RAC labeled a membrane-associated ring excluding the region labeled by CDC42. The GEF CDC-24 occupied a strategic position, localizing as broad apical membrane-associated crescent and in the apical cytosol excluding the Spitzenkörper. RAC and CDC-42 also display distinct localization patterns during branch initiation and germ tube formation, with CDC-42 accumulating at the plasma membrane before RAC. Together with the distinct cellular defects of rac and cdc-42 mutants, these localizations suggest that CDC-42 is more important for polarity establishment, while the primary function of RAC may be maintaining polarity. In summary, this study identifies CDC-24 as essential regulator for RAC and CDC-42 that have common and distinct functions during polarity establishment and maintenance of cell polarity in N. crassa.
doi:10.1371/journal.pone.0027148
PMCID: PMC3210136  PMID: 22087253
24.  Regulation of the Formin for3p by cdc42p and bud6p 
Molecular Biology of the Cell  2007;18(10):4155-4167.
Formins are conserved actin nucleators responsible for the assembly of diverse actin structures. Many formins are controlled through an autoinhibitory mechanism involving the interaction of a C-terminal DAD sequence with an N-terminal DID sequence. Here, we show that the fission yeast formin for3p, which mediates actin cable assembly and polarized cell growth, is regulated by a similar autoinhibitory mechanism in vivo. Multiple sites govern for3p localization to cell tips. The localization and activity of for3p are inhibited by an intramolecular interaction of divergent DAD and DID-like sequences. A for3p DAD mutant expressed at endogenous levels produces more robust actin cables, which appear to have normal organization and dynamics. We identify cdc42p as the primary Rho GTPase involved in actin cable assembly and for3p regulation. Both cdc42p, which binds at the N terminus of for3p, and bud6p, which binds near the C-terminal DAD-like sequence, are needed for for3p localization and full activity, but a mutation in the for3p DAD restores for3p localization and other phenotypes of cdc42 and bud6 mutants. In particular, the for3p DAD mutation suppresses the bipolar growth (NETO) defect of bud6Δ cells. These findings suggest that cdc42p and bud6p activate for3p by relieving autoinhibition.
doi:10.1091/mbc.E07-02-0094
PMCID: PMC1995706  PMID: 17699595
25.  Cortical actin dynamics 
Bioarchitecture  2011;1(4):165-168.
The actin cytoskeleton plays essential roles in cell polarization and cell morphogenesis of the budding yeast Saccharomyces cerevisiae. Yeast cells utilize formin-generated actin cables as tracks for polarized transport, which forms the basis for a positive feedback loop driving Cdc42-dependent cell polarization. Previous studies on cable organization mostly focused on polarized actin cables in budded cells and their role as relatively static tracks for myosin-dependent organelle transport. Using quantitative live cell imaging, we have recently characterized the dynamics of cortical actin cables throughout the yeast cell cycle. Surprisingly, randomly oriented actin cables in G1 cells exhibited the highest level of dynamics, while cable dynamics was markedly slowed down upon cell polarization. We further demonstrated that the rapid dynamics of randomly oriented cables were driven by the formin Bni1 and Myosin V. Our data suggested a precise spatio-temporal regulation of the two yeast formins, as well as an unexpected mechanism of actin cable rearrangement through myosins. Here we discuss the immediate significance of these findings, which illustrates the importance of generating randomness for cellular organization.
doi:10.4161/bioa.1.4.17314
PMCID: PMC3210520  PMID: 22069508
actin; formin; myosin; polarity; self organization

Results 1-25 (221979)