scholarly journals From Function to Shape: A Novel Role of a Formin in Morphogenesis of the Fungus Ashbya gossypii

2006 ◽  
Vol 17 (1) ◽  
pp. 130-145 ◽  
Author(s):  
Hans-Peter Schmitz ◽  
Andreas Kaufmann ◽  
Michael Köhli ◽  
Pierre Philippe Laissue ◽  
Peter Philippsen
Keyword(s):  
Giant Cells ◽  
Polar Growth ◽  
Ashbya Gossypii ◽  
Essential Factor ◽  
Wild Type ◽  
Actin Cable ◽  
Lateral Branches ◽  
Actin Cables ◽  
Two Hybrid

Morphogenesis of filamentous ascomycetes includes continuously elongating hyphae, frequently emerging lateral branches, and, under certain circumstances, symmetrically dividing hyphal tips. We identified the formin AgBni1p of the model fungus Ashbya gossypii as an essential factor in these processes. AgBni1p is an essential protein apparently lacking functional overlaps with the two additional A. gossypii formins that are nonessential. Agbni1 null mutants fail to develop hyphae and instead expand to potato-shaped giant cells, which lack actin cables and thus tip-directed transport of secretory vesicles. Consistent with the essential role in hyphal development, AgBni1p locates to tips, but not to septa. The presence of a diaphanous autoregulatory domain (DAD) indicates that the activation of AgBni1p depends on Rho-type GTPases. Deletion of this domain, which should render AgBni1p constitutively active, completely changes the branching pattern of young hyphae. New axes of polarity are no longer established subapically (lateral branching) but by symmetric divisions of hyphal tips (tip splitting). In wild-type hyphae, tip splitting is induced much later and only at much higher elongation speed. When GTP-locked Rho-type GTPases were tested, only the young hyphae with mutated AgCdc42p split at their tips, similar to the DAD deletion mutant. Two-hybrid experiments confirmed that AgBni1p interacts with GTP-bound AgCdc42p. These data suggest a pathway for transforming one axis into two new axes of polar growth, in which an increased activation of AgBni1p by a pulse of activated AgCdc42p stimulates additional actin cable formation and tip-directed vesicle transport, thus enlarging and ultimately splitting the polarity site.

scholarly journals Mechanism and cellular function of Bud6 as an actin nucleation–promoting factor

2011 ◽  
Vol 22 (21) ◽  
pp. 4016-4028 ◽  
Author(s):  
Brian R. Graziano ◽  
Amy Grace DuPage ◽  
Alphee Michelot ◽  
Dennis Breitsprecher ◽  
James B. Moseley ◽  
...  
Keyword(s):  
Actin Assembly ◽  
Actin Cable ◽  
Polarized Cell Growth ◽  
Cable Assembly ◽  
Nucleation Promoting Factor ◽  
Actin Cables ◽  
Elongation Phase

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.

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.

scholarly journals Identification of Rhodospirillum rubrum GlnB Variants That Are Altered in Their Ability To Interact with Different Targets in Response to Nitrogen Status Signals

2006 ◽  
Vol 188 (5) ◽  
pp. 1866-1874 ◽  
Author(s):  
Yu Zhu ◽  
Mary C. Conrad ◽  
Yaoping Zhang ◽  
Gary P. Roberts
Keyword(s):  
Hybrid System ◽  
Structural Changes ◽  
Rhodospirillum Rubrum ◽  
Single Copy ◽  
Wild Type ◽  
Yeast Two Hybrid System ◽  
Dinitrogenase Reductase ◽  
Nitrogen Excess ◽  
Two Hybrid

ABSTRACT In Rhodospirillum rubrum, NifA, the transcriptional activator for the nif genes, is posttranslationally activated only by the uridylylated form of GlnB, one of three PII homologs in the organism. We have used the yeast two-hybrid system to detect variants of GlnB that interact better with NifA than does wild-type GlnB. When examined for physiological effects in R. rubrum, these GlnB* variants activated NifA in the presence of NH4 +, which normally blocks NifA activation completely, and in the absence of GlnD, whose uridylylation of GlnB is also normally essential for NifA activation. When these variants were tested in the two-hybrid system for their interaction with NtrB, a receptor that should interact with the nonuridylylated form of GlnB, they were uniformly weaker than wild-type GlnB in that interaction. When expressed in R. rubrum either as single-copy integrants or on multiple-copy plasmids, these variants were also dramatically altered in terms of their ability to regulate several other receptors involved in nitrogen metabolism, including GlnE, NtrB/NtrC, and DRAT (dinitrogenase reductase ADP-ribosyl transferase)-DRAG (dinitrogenase reductase-activating glycohydrolase). The consistent pattern throughout is that these GlnB variants partially mimic the uridylylated form of wild-type GlnB, even under nitrogen-excess conditions and in strains lacking GlnD. The results suggest that the role of uridylylation of GlnB is primarily to shift the equilibrium of GlnB from a “nitrogen-sufficient” form to a “nitrogen-deficient” form, each of which interacts with different but overlapping receptor proteins in the cell. These GlnB variants apparently shift that equilibrium through direct structural changes.

scholarly journals Role of the Carboxy Terminus of Escherichia coli FtsA in Self-Interaction and Cell Division

2000 ◽  
Vol 182 (22) ◽  
pp. 6366-6373 ◽  
Author(s):  
Lucía Yim ◽  
Guy Vandenbussche ◽  
Jesús Mingorance ◽  
Sonsoles Rueda ◽  
Mercedes Casanova ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Biological Function ◽  
Wild Type ◽  
Carboxy Terminus ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Carboxy Terminal ◽  
Two Hybrid

ABSTRACT The role of the carboxy terminus of the Escherichia coli cell division protein FtsA in bacterial division has been studied by making a series of short sequential deletions spanning from residue 394 to 420. Deletions as short as 5 residues destroy the biological function of the protein. Residue W415 is essential for the localization of the protein into septal rings. Overexpression of theftsA alleles harboring these deletions caused a coiled cell phenotype previously described for another carboxy-terminal mutation (Gayda et al., J. Bacteriol. 174:5362–5370, 1992), suggesting that an interaction of FtsA with itself might play a role in its function. The existence of such an interaction was demonstrated using the yeast two-hybrid system and a protein overlay assay. Even these short deletions are sufficient for impairing the interaction of the truncated FtsA forms with the wild-type protein in the yeast two-hybrid system. The existence of additional interactions between FtsA molecules, involving other domains, can be postulated from the interaction properties shown by the FtsA deletion mutant forms, because although unable to interact with the wild-type and with FtsAΔ1, they can interact with themselves and cross-interact with each other. The secondary structures of an extensive deletion, FtsAΔ27, and the wild-type protein are indistinguishable when analyzed by Fourier transform infrared spectroscopy, and moreover, FtsAΔ27 retains the ability to bind ATP. These results indicate that deletion of the carboxy-terminal 27 residues does not alter substantially the structure of the protein and suggest that the loss of biological function of the carboxy-terminal deletion mutants might be related to the modification of their interacting properties.

scholarly journals Loose Plant Architecture 1-Interacting Kinesin-like Protein KLP Promotes Rice Resistance to Sheath Blight Disease

2021 ◽  
Author(s):  
Jin Chu ◽  
Han Xu ◽  
Hai Dong ◽  
Yuanhu Xuan
Keyword(s):  
Transcriptional Activation ◽  
Chromatin Immunoprecipitation ◽  
Plant Architecture ◽  
Wild Type ◽  
Yeast Two Hybrid ◽  
Sheath Blight Disease ◽  
Blight Disease ◽  
Two Hybrid ◽  
Wild Type Control

Abstract BackgroundSheath blight disease (ShB) is a destructive disease affecting rice production. Previously, we have reported that Loose Plant Architecture 1 (LPA1) promotes resistance to ShB. However, the mechanisms by which LPA1 confers resistance against this disease have not been extensively investigated. Notably, interactors that regulate LPA-1 activity remain elusive.FindingsIn this study, we identified the interaction of kinesin-like protein (KLP) with LPA1 in the nucleus of rice cells by yeast two-hybrid, bimolecular fluorescent complimentary (BiFC), and co-immunoprecipitation (co-IP) assays. To investigate the role of KLP in promoting resistance to ShB, wild-type, klp mutant, and KLP overexpressor (KLP OX) rice plants were inoculated with Rhizoctonia solani AG1-IA. The results indicated that, compared with the wild-type control, klp mutants were more susceptible while KLP OX plants were less susceptible to ShB. Since LPA1 transcriptionally activates PIN-FORMED 1a (PIN1a), we examined the expression of 8 related PIN genes. The results showed that only the expression of PIN1a and PIN3b coincided with KLP expression levels. In addition, a chromatin immunoprecipitation (ChIP) assay showed that KLP bound directly to the promoter region of PIN1a but not of PIN3b. Transient expression assays confirmed that LPA1 and KLP transcriptionally activate PIN1a, and that coexpression of KLP and LPA1 had an additive effect on the activation of PIN1a, suggesting that KLP enhances LPA1 transcriptional activation activity. ConclusionsTaken together, our results show that KLP is a novel LPA1 interactor that promotes resistance of rice to ShB.

The Role of Integrins aMb2 (CD11b/CD18) and aDb2 (CD11d/CD18) in Macrophage Fusion.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2138-2138
Author(s):  
YiFei Wu ◽  
Evgeniya Kushchaeva ◽  
Tatiana Ugarova
Keyword(s):  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Dominant Role ◽  
Granulomatous Inflammation ◽  
Giant Cells ◽  
Peritoneal Macrophages ◽  
Minor Role ◽  
Wild Type ◽  
Cell Nuclei

Abstract Abstract 2138 Macrophage fusion leading to the formation of multinucleated giant cells (MGCs) is a hallmark of many chronic inflammatory reactions. MGCs are an invariable constituent of tuberculoid lesions and also found in a variety of conditions leading to granulomatous inflammation as well as the foreign body reaction. Despite the prominent phenotype, the molecular mechanisms underlying macrophage fusion are not well understood. MGCs originate from macrophages that are recruited to sites of chronic inflammation. The major myelo-monocytic integrin αMβ2 (CD11b/CD18, Mac-1), together with a related integrin αDβ2 (CD11d/CD18), mediate critical adhesive reactions of monocyte/macrophages. The function of β2 integrins in macrophage fusion remains controversial. Some studies using function blocking antibodies implicated αMβ2 in the cell/substrate adhesive interactions that are required for MGC formation, whereas one recent report indicated that this integrin plays a minor role. Moreover, the contribution of αDβ2, a receptor with recognition specificity overlapping that of αMβ2, to macrophage fusion is unknown. To evaluate the role of αMβ2 and αDβ2 in MGC formation, we examined fusion of inflammatory peritoneal macrophages isolated from wild-type mice and mice with deficiency of αM or αD integrin subunits. Macrophages were isolated at day 3 after thioglycollate injection and cultured for 24–72 hours in the presence of IL-4 to induce fusion. Percentage fusion was quantified as the number of giant cell nuclei (≥2 nuclei) to the number of total nuclei. The number of fused macrophages isolated from wild-type mice gradually increased and ∼40–50% macrophages formed MGCs after IL-4 stimulation by day 3. Analyses of fusion of αMβ2-deficient macrophages demonstrated that fusion was significantly reduced. By day 3, macrophage fusion of αMβ2-deficient macrophages was 23 ± 2% of wild-type macrophages. Fusion of αDβ2-deficient macrophages was also decreased and the change was statistically significant, albeit to a smaller degree (75 ± 4%). Using a mouse model of sterile peritonitis induced by thioglycollate injection, we also examined the formation of MGCs in vivo. In wild-type mice, the number of MGCs gradually increased from 2.5 ± 0.3% at day 0 (resident macrophages) to 17 ± 2% at day 18 (the resolution phase of inflammation). Moreover, expression of αMβ2 and αDβ2 on peritoneal macrophages increased by ∼2 and 1.6-fold, respectively, on 18th day after induction of inflammation. In αMβ2-deficient mice, the number of MGCs was reduced by 2.1-fold compared to wild-type mice. Furthermore, while the size of wild-type and αMβ2-deficient giant cells was the same, the number of cells with 3 and more nuclei in fused αMβ2-deficient MGCs was 4-fold less than in wild-type cells. The results indicate that both αMβ2 and αDβ2 integrins support macrophage fusion with αMβ2 playing a dominant role. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Role of RIM101 for Sporulation at Alkaline pH in Ashbya gossypii

2021 ◽  
Vol 7 (7) ◽  
pp. 527
Author(s):  
Lisa Wasserstrom ◽  
Jürgen Wendland
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fungal Species ◽  
Alkaline Media ◽  
Ashbya Gossypii ◽  
Alkaline Ph ◽  
Wild Type ◽  
Liquid Media ◽  
High Ph ◽  
The Family

Microorganisms need to sense and adapt to fluctuations in the environmental pH. In fungal species, this response is mediated by the conserved pacC/RIM101 pathway. In Aspergillus nidulans, PacC activates alkaline-expressed genes and represses acid-controlled genes in response to alkaline pH and has important functions in regulating growth and conidia formation. In Saccharomyces cerevisiae, the PacC homolog Rim101 is required for adaptation to extracellular pH and to regulate transcription of IME1, the Initiator of MEiosis. S. cerevisiae rim101 mutants are defective in sporulation. In Ashbya gossypii, a filamentous fungus belonging to the family of Saccharomycetaceae, little is known about the role of pH in regulating growth and sporulation. Here, we deleted the AgRIM101 homolog (AFR190C). Our analyses show that Rim101 is important for growth and essential for sporulation at alkaline pH in A. gossypii. Acidic liquid sporulation media were alkalinized by sporulating strains, while the high pH of alkaline media (starting pH = 8.6) was reduced to a pH ~ 7.5 by these strains. However, Agrim101 mutants were unable to sporulate in alkaline media and failed to reduce the initial high pH, while they were capable of sporulation in acidic liquid media in which they increased the pH like the wild type.

scholarly journals Loose Plant Architecture 1-Interacting Kinesin-like Protein KLP Promotes Rice Resistance to Sheath Blight Disease

Rice ◽  
2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Jin Chu ◽  
Han Xu ◽  
Hai Dong ◽  
Yuan Hu Xuan
Keyword(s):  
Transcriptional Activation ◽  
Chromatin Immunoprecipitation ◽  
Plant Architecture ◽  
Sheath Blight ◽  
Wild Type ◽  
Sheath Blight Disease ◽  
Blight Disease ◽  
Two Hybrid ◽  
Wild Type Control

Abstract Background Sheath blight disease (ShB) is a destructive disease affecting rice production. Previously, we have reported that Loose Plant Architecture 1 (LPA1) promotes resistance to ShB. However, the mechanisms by which LPA1 confers resistance against this disease have not been extensively investigated. Notably, interactors that regulate LPA-1 activity remain elusive. Findings In this study, we identified the interaction of kinesin-like protein (KLP) with LPA1 in the nucleus of rice cells by yeast two-hybrid, bimolecular fluorescent complimentary (BiFC), and co-immunoprecipitation (co-IP) assays. To investigate the role of KLP in promoting resistance to ShB, wild-type, klp mutant, and KLP overexpressor (KLP OX) rice plants were inoculated with Rhizoctonia solani AG1-IA. The results indicated that, compared with the wild-type control, klp mutants were more susceptible while KLP OX plants were less susceptible to ShB. Since LPA1 transcriptionally activates PIN-FORMED 1a (PIN1a), we examined the expression of 8 related PIN genes. The results showed that only the expression of PIN1a and PIN3b coincided with KLP expression levels. In addition, a chromatin immunoprecipitation (ChIP) assay showed that KLP bound directly to the promoter region of PIN1a but not of PIN3b. Transient expression assays confirmed that LPA1 and KLP transcriptionally activate PIN1a, and that coexpression of KLP and LPA1 had an additive effect on the activation of PIN1a, suggesting that KLP enhances LPA1 transcriptional activation activity. Conclusions Taken together, our results show that KLP is a novel LPA1 interactor that promotes resistance of rice to ShB.

scholarly journals 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

2010 ◽  
Vol 21 (21) ◽  
pp. 3680-3692 ◽  
Author(s):  
Sandrine Grava ◽  
Peter Philippsen
Keyword(s):  
Budding Yeast ◽  
Ashbya Gossypii ◽  
Wild Type ◽  
Major Function ◽  
Bud Neck ◽  
Pulling Forces ◽  
Cytoplasmic Microtubules ◽  
Hyphal Tip

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.

Differential Role of Components of the Fibrinolytic System in the Formation and Removal of Thrombus Induced by Endothelial Injury

1999 ◽  
Vol 81 (04) ◽  
pp. 601-604 ◽  
Author(s):  
Hiroyuki Matsuno ◽  
Osamu Kozawa ◽  
Masayuki Niwa ◽  
Shigeru Ueshima ◽  
Osamu Matsuo ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Thrombus Formation ◽  
Endothelial Injury ◽  
Fibrinolytic System ◽  
Tissue Type ◽  
Clot Lysis ◽  
Wild Type ◽  
Type Plasminogen Activator ◽  
Pai 1

SummaryThe role of fibrinolytic system components in thrombus formation and removal in vivo was investigated in groups of six mice deficient in urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA), or plasminogen activator inhibitor-1 (PAI-1) (u-PA-/-, t-PA-/- or PAI-1-/-, respectively) or of their wild type controls (u-PA+/+, t-PA+/+ or PAI-1+/+). Thrombus was induced in the murine carotid artery by endothelial injury using the photochemical reaction between rose bengal and green light (540 nm). Blood flow was continuously monitored for 90 min on day 0 and for 20 min on days 1, 2 and 3. The times to occlusion after the initiation of endothelial injury in u-PA+/+, t-PA+/+ or PAI-1+/+ mice were 9.4 ± 1.3, 9.8 ± 1.1 or 9.7 ± 1.6 min, respectively. u-PA-/- and t-PA-/- mice were indistinguishable from controls, whereas that of PAI-1-/- mice were significantly prolonged (18.4 ± 3.7 min). Occlusion persisted for the initial 90 min observation period in 10 of 18 wild type mice and was followed by cyclic reflow and reocclusion in the remaining 8 mice. At day 1, persistent occlusion was observed in 1 wild type mouse, 8 mice had cyclic reflow and reocclusion and 9 mice had persistent reflow. At day 2, all injured arteries had persistent reflow. Persistent occlusion for 90 min on day 0 was observed in 3 u-PA-/-, in all t-PA-/- mice at day 1 and in 2 of the t-PA-/-mice at day 2 (p <0.01 versus wild type mice). Persistent patency was observed in all PAI-1-/- mice at day 1 and in 5 of the 6 u-PA-/- mice at day 2 (both p <0.05 versus wild type mice). In conclusion, t-PA increases the rate of clot lysis after endothelial injury, PAI-1 reduces the time to occlusion and delays clot lysis, whereas u-PA has little effect on thrombus formation and spontaneous lysis.

Sign in / Sign up

Export Citation Format

Share Document