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1.  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.
PMCID: PMC519154  PMID: 15282338
2.  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.
PMCID: PMC207006  PMID: 12937275
3.  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.
PMCID: PMC1595331  PMID: 16950929
4.  Of Bars and Rings: Hof1-Dependent Cytokinesis in Multiseptated Hyphae of Ashbya gossypii▿ §  
Molecular and Cellular Biology  2008;29(3):771-783.
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.
PMCID: PMC2630676  PMID: 19029253
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.
PMCID: PMC1265897  PMID: 16215178
6.  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.
PMCID: PMC1855024  PMID: 17344480
7.  FH3, A Domain Found in Formins, Targets the Fission Yeast Formin Fus1 to the Projection Tip During Conjugation  
The Journal of Cell Biology  1998;141(5):1217-1228.
Formins are involved in diverse aspects of morphogenesis, and share two regions of homology: FH1 and FH2. We describe a new formin homology region, FH3. FH3 is an amino-terminal domain that differs from the Rho binding site identified in Bni1p and p140mDia. The Schizosaccharomyces pombe formin Fus1 is required for conjugation, and is localized to the projection tip in cells of mating pairs. We replaced genomic fus1+ with green fluorescent protein (GFP)- tagged versions that lacked either the FH1, FH2, or FH3 domain. Deletion of any FH domain essentially abolished mating. FH3, but neither FH1 nor FH2, was required for Fus1 localization. An FH3 domain–GFP fusion protein localized to the projection tips of mating pairs. Thus, the FH3 domain alone can direct protein localization. The FH3 domains of both Fus1 and the S. pombe cytokinesis formin Cdc12 were able to localize GFP to the spindle pole body in half of the late G2 cells in a vegetatively growing population. Expression of both FH3-GFP fusions also affected cytokinesis. Overexpression of the spindle pole body component Sad1 altered the distribution of both Sad1 and the FH3-GFP domain. Together these data suggest that proteins at multiple sites can interact with FH3 domains.
PMCID: PMC2137179  PMID: 9606213
8.  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.
PMCID: PMC3268721  PMID: 22160598
9.  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.
PMCID: PMC1635347  PMID: 16899511
10.  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.
PMCID: PMC3164456  PMID: 21737675
11.  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.
PMCID: PMC2173006  PMID: 12810699
actin; polarity; Cdc42; PKC; MAPK
12.  Dynamics of Multiple Nuclei in Ashbya gossypii Hyphae Depend on the Control of Cytoplasmic Microtubules Length by Bik1, Kip2, Kip3, and Not on a Capture/Shrinkage Mechanism 
Molecular Biology of the Cell  2010;21(21):3680-3692.
The multinucleate growth mode of A. gossypii has resulted in a unique control of cytoplasmic MT dynamics. Our analyses of MT +tips behavior and cMT–cell cortex interactions show the necessity of A. gossypii to produce very long cMTs for nuclear migration to compensate the lack of MT capture/shrinkage mechanisms important in budding yeast.
Ashbya gossypii has a budding yeast-like genome but grows exclusively as multinucleated hyphae. In contrast to budding yeast where positioning of nuclei at the bud neck is a major function of cytoplasmic microtubules (cMTs), A. gossypii nuclei are constantly in motion and positioning is not an issue. To investigate the role of cMTs in nuclear oscillation and bypassing, we constructed mutants potentially affecting cMT lengths. Hyphae lacking the plus (+)end marker Bik1 or the kinesin Kip2 cannot polymerize long cMTs and lose wild-type nuclear movements. Interestingly, hyphae lacking the kinesin Kip3 display longer cMTs concomitant with increased nuclear oscillation and bypassing. Polymerization and depolymerization rates of cMTs are 3 times higher in A. gossypii than in budding yeast and cMT catastrophes are rare. Growing cMTs slide along the hyphal cortex and exert pulling forces on nuclei. Surprisingly, a capture/shrinkage mechanism seems to be absent in A. gossypii. cMTs reaching a hyphal tip do not shrink, and cMT +ends accumulate in hyphal tips. Thus, differences in cMT dynamics and length control between budding yeast and A. gossypii are key elements in the adaptation of the cMT cytoskeleton to much longer cells and much higher degrees of nuclear mobilities.
PMCID: PMC2965685  PMID: 20844079
13.  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.
PMCID: PMC3232731  PMID: 21984708
14.  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.
PMCID: PMC1635370  PMID: 16855023
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.
PMCID: PMC3098242  PMID: 21625538
16.  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.
PMCID: PMC3459858  PMID: 22918946
17.  Stable and Dynamic Axes of Polarity Use Distinct Formin Isoforms in Budding YeastD⃞V⃞ 
Molecular Biology of the Cell  2004;15(11):4971-4989.
Bud growth in yeast is guided by myosin-driven delivery of secretory vesicles from the mother cell to the bud. We find transport occurs along two sets of actin cables assembled by two formin isoforms. The Bnr1p formin assembles cables that radiate from the bud neck into the mother, providing a stable mother-bud axis. These cables also depend on septins at the neck and are required for efficient transport from the mother to the bud. The Bni1p formin assembles cables that line the bud cortex and target vesicles to varying locations in the bud. Loss of these cables results in morphological defects as vesicles accumulate at the neck. Assembly of these cables depends on continued polarized secretion, suggesting vesicular transport provides a positive feedback signal for Bni1p activation, possibly by rho-proteins. By coupling different formin isoforms to unique cortical landmarks, yeast uses common cytoskeletal elements to maintain stable and dynamic axes in the same cell.
PMCID: PMC524755  PMID: 15371545
18.  Structure of the FH2 domain of Daam1: implications for formin regulation of actin assembly 
Journal of molecular biology  2007;369(5):1258-1269.
Daam1 (Dishevelled-associated activator of morphogenesis-1) is a diaphanous-related formin first studied as a novel dishevelled-binding protein and shown to be crucial for the planar cell polarity (PCP) pathway in Xenopus. Daam1, like other formins, directs nucleation and elongation of new actin filaments using its conserved formin-homology-2 (FH2) domain. Here we report the crystal structure of a large C-terminal fragment of human Daam1 containing the FH2 domain. The structure, determined at 2.25 Å resolution using the SAD phasing method, reveals a “tethered dimer” architecture that is similar to that previously described for the FH2 domain of the yeast formin Bni1, which shares ~21% sequence identity with Daam1. Despite the overall similarity with the dimeric FH2 domain of Bni1 and with a truncated monomeric structure of mDia1, the Daam1 FH2 structure reveals a number of differences in secondary structure elements and in the “lasso/post” dimerization interface that may be functionally important. Most strikingly, the two halves of the crystallographic dimer pack together in a manner that occludes their actin binding surfaces. This “locked” conformation is stabilized by two novel, interacting β-strands formed by the ends of the linkers that connect the two sides of the dimer. The Daam1 FH2 domain has weak actin assembly activity as compared with other mammalian formins, but mutations that disrupt the β-strand lock increase activity ~10-fold to a level comparable to other formins, suggesting that this occluded conformation may represent an autoinhibited conformation of the Daam1 FH2 domain.
PMCID: PMC1939941  PMID: 17482208
protein structure; formin; FH2 domain; actin assembly; dishevelled
19.  Structural Mutants of the Spindle Pole Body Cause Distinct Alteration of Cytoplasmic Microtubules and Nuclear Dynamics in Multinucleated Hyphae 
Molecular Biology of the Cell  2010;21(5):753-766.
To determine how microtubule (MT) nucleation and nuclear migration are controlled in multinucleated hyphae we deleted genes encoding MTOC subunits and AgStu2. The novel phenotypes we observed in these mutants compared with analogous deletions in budding yeast allowed us to assign functions to the two types of cMTs that we observe in A. gossypii.
In the multinucleate fungus Ashbya gossypii, cytoplasmic microtubules (cMTs) emerge from the spindle pole body outer plaque (OP) in perpendicular and tangential directions. To elucidate the role of cMTs in forward/backward movements (oscillations) and bypassing of nuclei, we constructed mutants potentially affecting cMT nucleation or stability. Hyphae lacking the OP components AgSpc72, AgNud1, AgCnm67, or the microtubule-stabilizing factor AgStu2 grew like wild- type but showed substantial alterations in the number, length, and/or nucleation sites of cMTs. These mutants differently influenced nuclear oscillation and bypassing. In Agspc72Δ, only long cMTs were observed, which emanate tangentially from reduced OPs; nuclei mainly moved with the cytoplasmic stream but some performed rapid bypassing. Agnud1Δ and Agcnm67Δ lack OPs; short and long cMTs emerged from the spindle pole body bridge/half-bridge structures, explaining nuclear oscillation and bypassing in these mutants. In Agstu2Δ only very short cMTs emanated from structurally intact OPs; all nuclei moved with the cytoplasmic stream. Therefore, long tangential cMTs promote nuclear bypassing and short cMTs are important for nuclear oscillation. Our electron microscopy ultrastructural analysis also indicated that assembly of the OP occurs in a stepwise manner, starting with AgCnm67, followed by AgNud1 and lastly AgSpc72.
PMCID: PMC2828962  PMID: 20053682
20.  Control of Mitotic Spindle Position by the Saccharomyces cerevisiae Formin Bni1p  
The Journal of Cell Biology  1999;144(5):947-961.
Alignment of the mitotic spindle with the axis of cell division is an essential process in Saccharomyces cerevisiae that is mediated by interactions between cytoplasmic microtubules and the cell cortex. We found that a cortical protein, the yeast formin Bni1p, was required for spindle orientation. Two striking abnormalities were observed in bni1Δ cells. First, the initial movement of the spindle pole body (SPB) toward the emerging bud was defective. This phenotype is similar to that previously observed in cells lacking the kinesin Kip3p and, in fact, BNI1 and KIP3 were found to be in the same genetic pathway. Second, abnormal pulling interactions between microtubules and the cortex appeared to cause preanaphase spindles in bni1Δ cells to transit back and forth between the mother and the bud. We therefore propose that Bni1p may localize or alter the function of cortical microtubule-binding sites in the bud. Additionally, we present evidence that other bipolar bud site determinants together with cortical actin are also required for spindle orientation.
PMCID: PMC2148193  PMID: 10085293
mitotic spindle apparatus; cell division; microtubule; actin; Saccharomyces cerevisiae
21.  A comparative sequence analysis reveals a common GBD/FH3-FH1-FH2-DAD architecture in formins from Dictyostelium, fungi and metazoa 
BMC Genomics  2005;6:28.
Formins are multidomain proteins defined by a conserved FH2 (formin homology 2) domain with actin nucleation activity preceded by a proline-rich FH1 (formin homology 1) domain. Formins act as profilin-modulated processive actin nucleators conserved throughout a wide range of eukaryotes.
We present a detailed sequence analysis of the 10 formins (ForA to J) identified in the genome of the social amoeba Dictyostelium discoideum. With the exception of ForI and ForC all other formins conform to the domain structure GBD/FH3-FH1-FH2-DAD, where DAD is the Diaphanous autoinhibition domain and GBD/FH3 is the Rho GTPase-binding domain/formin homology 3 domain that we propose to represent a single domain. ForC lacks a FH1 domain, ForI lacks recognizable GBD/FH3 and DAD domains and ForA, E and J have additional unique domains. To establish the relationship between formins of Dictyostelium and other organisms we constructed a phylogenetic tree based on the alignment of FH2 domains. Real-time PCR was used to study the expression pattern of formin genes. Expression of forC, D, I and J increased during transition to multi-cellular stages, while the rest of genes displayed less marked developmental variations. During sexual development, expression of forH and forI displayed a significant increase in fusion competent cells.
Our analysis allows some preliminary insight into the functionality of Dictyostelium formins: all isoforms might display actin nucleation activity and, with the exception of ForI, might also be susceptible to autoinhibition and to regulation by Rho GTPases. The architecture GBD/FH3-FH1-FH2-DAD appears common to almost all Dictyostelium, fungal and metazoan formins, for which we propose the denomination of conventional formins, and implies a common regulatory mechanism.
PMCID: PMC555941  PMID: 15740615
22.  Initial Polarized Bud Growth by Endocytic Recycling in the Absence of Actin Cable–dependent Vesicle Transport in Yeast 
Molecular Biology of the Cell  2010;21(7):1237-1252.
Budding yeast mutants in assembly of actin cables, which are thought to be the only actin structures essential for budding, still could form a small bud. Mutations in actin patch endocytic machineries/endocytic recycling factors inhibited this budding, suggesting a mechanism that promotes polarized growth by local recycling of endocytic vesicles.
The assembly of filamentous actin is essential for polarized bud growth in budding yeast. Actin cables, which are assembled by the formins Bni1p and Bnr1p, are thought to be the only actin structures that are essential for budding. However, we found that formin or tropomyosin mutants, which lack actin cables, are still able to form a small bud. Additional mutations in components for cortical actin patches, which are assembled by the Arp2/3 complex to play a pivotal role in endocytic vesicle formation, inhibited this budding. Genes involved in endocytic recycling were also required for small-bud formation in actin cable-less mutants. These results suggest that budding yeast possesses a mechanism that promotes polarized growth by local recycling of endocytic vesicles. Interestingly, the type V myosin Myo2p, which was thought to use only actin cables to track, also contributed to budding in the absence of actin cables. These results suggest that some actin network may serve as the track for Myo2p-driven vesicle transport in the absence of actin cables or that Myo2p can function independent of actin filaments. Our results also show that polarity regulators including Cdc42p were still polarized in mutants defective in both actin cables and cortical actin patches, suggesting that the actin cytoskeleton does not play a major role in cortical assembly of polarity regulators in budding yeast.
PMCID: PMC2847527  PMID: 20147449
23.  Functional Characterization of Aspergillus nidulans Homologues of Saccharomyces cerevisiae Spa2 and Bud6 
Eukaryotic Cell  2006;5(6):881-895.
The importance of polarized growth for fungi has elicited significant effort directed at better understanding underlying mechanisms of polarization, with a focus on yeast systems. At sites of tip growth, multiple protein complexes assemble and coordinate to ensure that incoming building material reaches the appropriate destination sites, and polarized growth is maintained. One of these complexes is the polarisome that consists of Spa2, Bud6, Pea2, and Bni1 in Saccharomyces cerevisiae. Filamentous hyphae differ in their development and life style from yeasts and likely regulate polarized growth in a different way. This is expected to reflect on the composition and presence of protein complexes that assemble at the hyphal tip. In this study we searched for polarisome homologues in the model filamentous fungus Aspergillus nidulans and characterized the S. cerevisiae Spa2 and Bud6 homologues, SpaA and BudA. Compared to the S. cerevisiae Spa2, SpaA lacks domain II but has three additional domains that are conserved within filamentous fungi. Gene replacement strains and localization studies show that SpaA functions exclusively at the hyphal tip, while BudA functions at sites of septum formation and possibly at hyphal tips. We show that SpaA is not required for the assembly or maintenance of the Spitzenkörper. We propose that the core function of the polarisome in polarized growth is maintained but with different contributions of polarisome components to the process.
PMCID: PMC1489272  PMID: 16757736
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.
PMCID: PMC1995706  PMID: 17699595
25.  Septin ring size scaling and dynamics require the coiled-coil region of Shs1p 
Molecular Biology of the Cell  2012;23(17):3391-3406.
How the size and dynamics of higher-order septin structures is determined is not well understood in any system. In this paper, we show that the coiled-coil domain of the septin Shs1p limits septin ring size and dynamics in the filamentous fungus Ashbya gossypii, providing a link between protein exchange and the scaling of septin assemblies.
Septins are conserved GTP-binding proteins that assemble into heteromeric complexes that form filaments and higher-order structures in cells. What directs filament assembly, determines the size of higher-order septin structures, and governs septin dynamics is still not well understood. We previously identified two kinases essential for septin ring assembly in the filamentous fungus Ashbya gossypii and demonstrate here that the septin Shs1p is multiphosphorylated at the C-terminus of the protein near the predicted coiled-coil domain. Expression of the nonphosphorylatable allele shs1-9A does not mimic the loss of the kinase nor does complete truncation of the Shs1p C-terminus. Surprisingly, however, loss of the C-terminus or the predicted coiled-coil domain of Shs1p generates expanded zones of septin assemblies and ectopic septin fibers, as well as aberrant cell morphology. The expanded structures form coincident with ring assembly and are heteromeric. Interestingly, while septin recruitment to convex membranes is increased, septin localization is diminished at concave membranes in these mutants. Additionally, the loss of the coiled-coil leads to increased mobility of Shs1p. These data indicate the coiled-coil of Shs1p is an important negative regulator of septin ring size and mobility, and its absence may make septin assembly sensitive to local membrane curvature.
PMCID: PMC3431940  PMID: 22767579

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