scholarly journals Cdc28–Cln3 phosphorylation of Sla1 regulates actin patch dynamics in different modes of fungal growth

2012 ◽  
Vol 23 (17) ◽  
pp. 3485-3497 ◽  
Author(s):  
Guisheng Zeng ◽  
Yan-Ming Wang ◽  
Yue Wang
Keyword(s):  
Dynamic Balance ◽  
Fungal Growth ◽  
Patch Dynamics ◽  
Hyphal Growth ◽  
Cortical Actin ◽  
Growth Modes ◽  
Targeted Transport ◽  
Endocytic Machinery ◽  
Cdk Phosphorylation ◽  
Actin Patch

A dynamic balance between targeted transport and endocytosis is critical for polarized cell growth. However, how actin-mediated endocytosis is regulated in different growth modes remains unclear. Here we report differential regulation of cortical actin patch dynamics between the yeast and hyphal growth in Candida albicans. The mechanism involves phosphoregulation of the endocytic protein Sla1 by the cyclin-dependent kinase (CDK) Cdc28–Cln3 and the actin-regulating kinase Prk1. Mutational studies of the CDK phosphorylation sites of Sla1 revealed that Cdc28–Cln3 phosphorylation of Sla1 enhances its further phosphorylation by Prk1, weakening Sla1 association with Pan1, an activator of the actin-nucleating Arp2/3 complex. Sla1 is rapidly dephosphorylated upon hyphal induction and remains so throughout hyphal growth. Consistently, cells expressing a phosphomimetic version of Sla1 exhibited markedly reduced actin patch dynamics, impaired endocytosis, and defective hyphal development, whereas a nonphosphorylatable Sla1 had the opposite effect. Taken together, our findings establish a molecular link between CDK and a key component of the endocytic machinery, revealing a novel mechanism by which endocytosis contributes to cell morphogenesis.

Sla1p couples the yeast endocytic machinery to proteins regulating actin dynamics

2002 ◽  
Vol 115 (8) ◽  
pp. 1703-1715 ◽  
Author(s):  
Derek T. Warren ◽  
Paul D. Andrews ◽  
Campbell W. Gourlay ◽  
Kathryn R. Ayscough
Keyword(s):  
Immunofluorescence Microscopy ◽  
Fluid Phase ◽  
Actin Dynamics ◽  
Cell Cortex ◽  
Cortical Actin ◽  
Binding Studies ◽  
Endocytic Machinery ◽  
Single Complex ◽  
Actin Patch ◽  
First Time

Sla1p is a protein required for cortical actin patch structure and organisation in budding yeast. Here we use a combination of immunofluorescence microscopy and biochemical approaches to demonstrate interactions of Sla1p both with proteins regulating actin dynamics and with proteins required for endocytosis. Using Sla1p-binding studies we reveal association of Sla1p with two proteins known to be important for activation of the Arp2/3 complex in yeast, Abp1p and the yeast WASP homologue Las17p/Bee1p. A recent report of Sla1p association with Pan1p puts Sla1p in the currently unique position of being the only yeast protein known to interact with all three known Arp2/3-activating proteins in yeast. Localisation of Sla1p at the cell cortex is, however, dependent on the EH-domain-containing protein End3p, which is part of the yeast endocytic machinery. Using spectral variants of GFP on Sla1p(YFP) and on Abp1p (CFP) we show for the first time that these proteins can exist in discrete complexes at the cell cortex. However, the detection of a significant FRET signal means that these proteins also come close together in a single complex, and it is in this larger complex that we propose that Sla1p binding to Abp1p and Las17p/Bee1p is able to link actin dynamics to the endocytic machinery. Finally, we demonstrate marked defects in both fluid-phase and receptor-mediated endocytosis in cells that do not express SLA1, indicating that Sla1p is central to the requirement in yeast to couple endocytosis with the actin cytoskeleton.

scholarly journals Dynamic phosphoregulation of the cortical actin cytoskeleton and endocytic machinery revealed by real-time chemical genetic analysis

2003 ◽  
Vol 162 (5) ◽  
pp. 765-772 ◽  
Author(s):  
Mariko Sekiya-Kawasaki ◽  
Aaron Chris Groen ◽  
M. Jamie T.V. Cope ◽  
Marko Kaksonen ◽  
Hadiya A. Watson ◽  
...  
Keyword(s):  
Actin Assembly ◽  
Cortical Actin ◽  
Chemical Genetic ◽  
Endocytic Machinery ◽  
Microscopy Analysis ◽  
Actin Regulatory Proteins ◽  
Actin Patch ◽  
Related Process ◽  
Endocytic Proteins

We used chemical genetics to control the activity of budding yeast Prk1p, which is a protein kinase that is related to mammalian GAK and AAK1, and which targets several actin regulatory proteins implicated in endocytosis. In vivo Prk1p inhibition blocked pheromone receptor endocytosis, and caused cortical actin patches to rapidly aggregate into large clumps that contained Abp1p, Sla2p, Pan1p, Sla1p, and Ent1p. Clump formation depended on Arp2p, suggesting that this phenotype might result from unregulated Arp2/3-stimulated actin assembly. Electron microscopy/immunoelectron microscopy analysis and tracking of the endocytic membrane marker FM4-64 revealed vesicles of likely endocytic origin within the actin clumps. Upon inhibitor washout, the actin clumps rapidly disassembled, and properly polarized actin patches reappeared. Our results suggest that actin clumps result from blockage at a normally transient step during which actin assembly is stimulated by endocytic proteins. Thus, we revealed tight phosphoregulation of an intrinsically dynamic, actin patch–related process, and propose that Prk1p negatively regulates the actin assembly–stimulating activity of endocytic proteins.

scholarly journals The Role of SreA-Mediated Iron Regulation in Maintaining Epichloë festucae–Lolium perenne Symbioses

2019 ◽  
Vol 32 (10) ◽  
pp. 1324-1335 ◽  
Author(s):  
Natasha T. Forester ◽  
Geoffrey A. Lane ◽  
Catherine M. McKenzie ◽  
Iain L. Lamont ◽  
Linda J. Johnson
Keyword(s):  
Iron Uptake ◽  
Iron Homeostasis ◽  
Iron Acquisition ◽  
Functional Characterization ◽  
Fungal Growth ◽  
Hyphal Growth ◽  
Epichloë Festucae ◽  
Gradual Loss ◽  
Growth Assay ◽  
Iron Management

In ascomycetes and basidiomycetes, iron-responsive GATA-type transcriptional repressors are involved in regulating iron homeostasis, notably to prevent iron toxicity through control of iron uptake. To date, it has been unknown whether this iron regulator contributes toward mutualistic endosymbiosis of microbes with plants, a system where the endophyte must function within the constraints of an in-host existence, including a dependency on the host for nutrient acquisition. Functional characterization of one such protein, SreA from Epichloë festucae, a fungal endosymbiont of cool-season grasses, indicates that regulation of iron homeostasis processes is important for symbiotic maintenance. The deletion of the sreA gene (ΔsreA) led to iron-dependent aberrant hyphal growth and the gradual loss of endophyte hyphae from perennial ryegrass. SreA negatively regulates the siderophore biosynthesis and high-affinity iron uptake systems of E. festucae, similar to other fungi, resulting in iron accumulation in mutants. Our evidence suggests that SreA is involved in the processes that moderate Epichloë iron acquisition from the plant apoplast, because overharvesting of iron in ΔsreA mutants was detected as premature chlorosis of the host using a hydroponic plant growth assay. E. festucae appears to have a tightly regulated iron management system, involving SreA that balances endophyte growth with its survival and prevents overcompetition with the host for iron in the intercellular niche, thus promoting mutualistic associations. Mutations that interfere with Epichloë iron management negatively affect iron-dependent fungal growth and destabilize mutualistic Epichloë –ryegrass associations.

scholarly journals G protein–coupled receptor/arrestin3 modulation of the endocytic machinery

2002 ◽  
Vol 156 (4) ◽  
pp. 665-676 ◽  
Author(s):  
Francesca Santini ◽  
Ibragim Gaidarov ◽  
James H. Keen
Keyword(s):  
G Protein ◽  
Fluorescent Protein ◽  
De Novo ◽  
Membrane Skeleton ◽  
Live Cells ◽  
G Protein Coupled Receptor ◽  
Cortical Actin ◽  
Endocytic Machinery ◽  
Green Fluorescent ◽  
G Protein Coupled

Nonvisual arrestins (arr) modulate G protein–coupled receptor (GPCR) desensitization and internalization and bind to both clathrin (CL) and AP-2 components of the endocytic coated pit (CP). This raises the possibility that endocytosis of some GPCRs may be a consequence of arr-induced de novo CP formation. To directly test this hypothesis, we examined the behavior of green fluorescent protein (GFP)-arr3 in live cells expressing β2-adrenergic receptors and fluorescent CL. After agonist stimulation, the diffuse GFP-arr3 signal rapidly became punctate and colocalized virtually completely with preexisting CP spots, demonstrating that activated complexes accumulate in previously formed CPs rather than nucleating new CP formation. After arr3 recruitment, CP appeared larger: electron microscopy analysis revealed an increase in both CP number and in the occurrence of clustered CPs. Mutant arr3 proteins with impaired binding to CL or AP-2 displayed reduced recruitment to CPs, but were still capable of inducing CP clustering. In contrast, though constitutively present in CPs, the COOH-terminal moiety of arr3, which contains CP binding sites but lacks receptor binding, did not induce CP clustering. Together, these results indicate that recruitment of functional arr3–GPCR complexes to CP is necessary to induce clustering. Latrunculin B or 16°C blocked CP rearrangements without affecting arr3 recruitment to CP. These results and earlier studies suggest that discrete CP zones exist on cell surfaces, each capable of supporting adjacent CPs, and that the cortical actin membrane skeleton is intimately involved with both the maintenance of existing CPs and the generation of new structures.

Interactions of the nonhost French bean plant (Phaseolus vulgaris) with parasitic and saprophytic fungi. IV. Effect of preinoculation with the bean rust fungus on growth of parasitic fungi nonpathogenic on beans

1991 ◽  
Vol 69 (8) ◽  
pp. 1642-1646 ◽  
Author(s):  
Myriam R. Fernandez ◽  
Michèle C. Heath
Keyword(s):  
Rust Fungus ◽  
Fungal Growth ◽  
Hyphal Growth ◽  
French Bean ◽  
Bean Rust ◽  
Protein Synthesis Inhibitors ◽  
Rust Infection ◽  
Parasitic Fungi ◽  
Wall Appositions ◽  
Bean Leaves

Bean leaves inoculated 24 h previously with the bean rust fungus were inoculated with spores of Cochliobolus heterostrophus, Stemphylium sarcinaeforme, Stemphylium botryosum, or Cladosporium fulvum. For all species except C. fulvum, hyphal growth resulting from stomatal penetrations was greater than that in leaves that were not rust-infected but did not continue for more than about 24 h. The incidence of direct penetrations for these three fungi also was increased by prior rust infection, and the incidence of epidermal wall appositions was reduced. Growth of C. fulvum in rust-infected leaves only exceeded that in control leaves when spores were injected into the intercellular spaces of the mesophyll tissue. Rust infection either had little effect on the incidence of cell death, normally induced by all of the tested fungi except C. fulvum, or it enhanced this response in association with greater fungal growth. From this and previous studies, it seems that successful rust infection increases the growth of a wider array of fungi nonparasitic to beans than treatments with growth regulators or intercellular washing fluids from rusted tissue. Its effect is most closely mimicked by preinoculation treatments with heat or protein synthesis inhibitors, but it does not induce indiscriminate susceptibility. Its effect may, in part, be due to the suppression of defenses involving wall modifications. Key words: Uromyces appendiculatus, induced susceptibility, nonhost resistance.

Quantifying the growth of arbuscular mycorrhizal fungi: usefulness of the fractal dimension

Botany ◽  
2009 ◽  
Vol 87 (4) ◽  
pp. 387-400 ◽  
Author(s):  
Christine Juge ◽  
Annie Champagne ◽  
Andrew P. Coughlan ◽  
Nicolas Juge ◽  
Lael Parrott ◽  
...  
Keyword(s):  
Fractal Dimension ◽  
Mycorrhizal Fungi ◽  
Growth And Development ◽  
Time Course ◽  
Glomus Intraradices ◽  
Fungal Growth ◽  
Arbuscular Mycorrhizal ◽  
Hyphal Growth ◽  
Growth Cycle

The present study is, to the best of our knowledge, the first to investigate the use of the fractal dimension (FD) to quantify the growth and development of undisturbed, fully functional arbuscular mycorrhizal (AM) hyphae developing in vitro. The majority of the work focused on the model AM fungus Glomus intraradices DAOM 181602. The time course study and final measurements of an intact mature extraradical mycelium allowed us to compare the development of the mycelium and the FD value. The final FD value of 1.62 for the mature mycelium is similar to that obtained for highly branched root systems and tree crowns. The FD method was used to characterize the morphology of germinative and presymbiotic hyphae in the presence of stimulatory (strigolactone GR-24, 0.1 µmol·L–1 and bisphenol A, 10 µmol·L–1) and inhibitory (NaCl, 80 mmol·L–1) molecules, and the extraradical phase in the presence of an inhibitory molecule (NaCl, 80 mmol·L–1). Where possible, results were compared with those obtained using the traditional grid-line (GL) technique. The FD approach allowed treatment effects to be accurately quantified, both in germinative and extraradical phases. In the second case, this technique provided a single quantitative value of extraradical hyphal growth that included runner hyphae (RH) networks, and fine-branching (FB) ramifications. This is in contrast to the GL technique, which provides a value for the estimation of RH, but which is not suitable for accurately measuring FB hyphae. Given the ease with which the FD values can be calculated, and the fact that this method can provide a single value for the quantification of extraradical hyphal growth and development, we suggest that this method is useful for in vitro studies. Furthermore under certain situations of germinative or presymbiotic growth, it may be used in concert with the GL method to provide a greater degree of information about hyphal morphology. The usefulness and limits of the FD method at different stages of the AM fungal growth cycle are discussed.

scholarly journals Functional Analysis of Sterol Transporter Orthologues in the Filamentous Fungus Aspergillus nidulans

2015 ◽  
Vol 14 (9) ◽  
pp. 908-921 ◽  
Author(s):  
Nicole Bühler ◽  
Daisuke Hagiwara ◽  
Norio Takeshita
Keyword(s):  
Plasma Membrane ◽  
Aspergillus Nidulans ◽  
Filamentous Fungi ◽  
Gene Deletion ◽  
Fluorescent Protein ◽  
Fungal Growth ◽  
Hyphal Growth ◽  
Apical Plasma Membrane ◽  
Important Species ◽  
Content Type

ABSTRACT Polarized growth in filamentous fungi needs a continuous supply of proteins and lipids to the growing hyphal tip. One of the important membrane compounds in fungi is ergosterol. At the apical plasma membrane ergosterol accumulations, which are called sterol-rich plasma membrane domains (SRDs). The exact roles and formation mechanism of the SRDs remained unclear, although the importance has been recognized for hyphal growth. Transport of ergosterol to hyphal tips is thought to be important for the organization of the SRDs. Oxysterol binding proteins, which are conserved from yeast to human, are involved in nonvesicular sterol transport. In Saccharomyces cerevisiae seven oxysterol-binding protein homologues (OSH1 to -7) play a role in ergosterol distribution between closely located membranes independent of vesicle transport. We found five homologous genes ( oshA to oshE ) in the filamentous fungi Aspergillus nidulans . The functions of OshA-E were characterized by gene deletion and subcellular localization. Each gene-deletion strain showed characteristic phenotypes and different sensitivities to ergosterol-associated drugs. Green fluorescent protein-tagged Osh proteins showed specific localization in the late Golgi compartments, puncta associated with the endoplasmic reticulum, or diffusely in the cytoplasm. The genes expression and regulation were investigated in a medically important species Aspergillus fumigatus , as well as A. nidulans . Our results suggest that each Osh protein plays a role in ergosterol distribution at distinct sites and contributes to proper fungal growth.

scholarly journals Candida albicans Vrp1 is required for polarized morphogenesis and interacts with Wal1 and Myo5

Microbiology ◽  
2010 ◽  
Vol 156 (10) ◽  
pp. 2962-2969 ◽  
Author(s):  
Nicole Borth ◽  
Andrea Walther ◽  
Patrick Reijnst ◽  
Sigyn Jorde ◽  
Yvonne Schaub ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Actin Cytoskeleton ◽  
Hyphal Growth ◽  
Potential Interaction ◽  
Wild Type ◽  
Terminal Part ◽  
Cortical Actin ◽  
Interacting Protein ◽  
Growth Defects ◽  
Interaction Sites

Recently, a link between endocytosis and hyphal morphogenesis has been identified in Candida albicans via the Wiskott–Aldrich syndrome gene homologue WAL1. To get a more detailed mechanistic understanding of this link we have investigated a potentially conserved interaction between Wal1 and the C. albicans WASP-interacting protein (WIP) homologue encoded by VRP1. Deletion of both alleles of VRP1 results in strong hyphal growth defects under serum inducing conditions but filamentation can be observed on Spider medium. Mutant vrp1 cells show a delay in endocytosis – measured as the uptake and delivery of the lipophilic dye FM4-64 into small endocytic vesicles – compared to the wild-type. Vacuolar morphology was found to be fragmented in a subset of cells and the cortical actin cytoskeleton was depolarized in vrp1 daughter cells. The morphology of the vrp1 null mutant could be complemented by reintegration of the wild-type VRP1 gene at the BUD3 locus. Using the yeast two-hybrid system we could demonstrate an interaction between the C-terminal part of Vrp1 and the N-terminal part of Wal1, which contains the WH1 domain. Furthermore, we found that Myo5 has several potential interaction sites on Vrp1. This suggests that a Wal1–Vrp1–Myo5 complex plays an important role in endocytosis and the polarized localization of the cortical actin cytoskeleton to promote polarized hyphal growth in C. albicans.

scholarly journals Sla1p Is a Functionally Modular Component of the Yeast Cortical Actin Cytoskeleton Required for Correct Localization of Both Rho1p-GTPase and Sla2p, a Protein with Talin Homology

1999 ◽  
Vol 10 (4) ◽  
pp. 1061-1075 ◽  
Author(s):  
Kathryn R. Ayscough ◽  
Jennifer J. Eby ◽  
Thomas Lila ◽  
Hilary Dewar ◽  
Keith G. Kozminski ◽  
...  
Keyword(s):  
Cell Walls ◽  
Binding Protein ◽  
Actin Binding ◽  
Actin Assembly ◽  
Cortical Actin ◽  
Actin Binding Protein ◽  
Actin Patch ◽  
Mutant Forms ◽  
Modular Component ◽  
Terminal Repeat Region

SLA1 was identified previously in budding yeast in a genetic screen for mutations that caused a requirement for the actin-binding protein Abp1p and was shown to be required for normal cortical actin patch structure and organization. Here, we show that Sla1p, like Abp1p, localizes to cortical actin patches. Furthermore, Sla1p is required for the correct localization of Sla2p, an actin-binding protein with homology to talin implicated in endocytosis, and the Rho1p-GTPase, which is associated with the cell wall biosynthesis enzyme β-1,3-glucan synthase. Mislocalization of Rho1p in sla1 null cells is consistent with our observation that these cells possess aberrantly thick cell walls.  Expression of mutant forms of Sla1p in which specific domains were deleted showed that the phenotypes associated with the full deletion are functionally separable. In particular, a region of Sla1p encompassing the third SH3 domain is important for growth at high temperatures, for the organization of cortical actin patches, and for nucleated actin assembly in a permeabilized yeast cell assay. The apparent redundancy between Sla1p and Abp1p resides in the C-terminal repeat region of Sla1p. A homologue of SLA1 was identified inSchizosaccharomyces pombe. Despite relatively low overall sequence homology, this gene was able to rescue the temperature sensitivity associated with a deletion of SLA1 inSaccharomyces cerevisiae.

scholarly journals In silico screening leads to novel scaffolds with both antifungal and anti-NLRP3 inflammasome activity

2021 ◽  
Vol 3 (12) ◽  
Author(s):  
David Lowes ◽  
Rand Al-waqfi ◽  
Kirk Hevener ◽  
Brian Peters
Keyword(s):  
Nlrp3 Inflammasome ◽  
In Silico ◽  
Fungal Growth ◽  
Hyphal Growth ◽  
Acetohydroxyacid Synthase ◽  
In Silico Screening ◽  
Chemical Structures ◽  
Ic50 Values

Due to structural similarities that exist between established inhibitors of the NLRP3-inflammasome, sulfonylureas Glyburide and MCC-950, and herbicidal-sulfonylureas, that specifically target fungal acetohydroxyacid synthase (AHAS), we sought to determine the potential for compounds to block both inflammation and inhibit fungal growth. In silico screening of ∼250,000 compounds was used to identify a prioritized list of chemical structures capable of inhibiting both targets. Prioritization of the top 1% of scores identified ∼70 compounds with a diverse set of scaffolds for testing in vitro. Selected hits were used to assess anti-inflammatory function in a THP-1 challenge model with LPS+ATP and resulting IC50 values were obtained. MIC and hyphal-growth assays were conducted to determine potential antifungal activity using media depleted of branched chain amino acids isoleucine and valine, to confirm on target AHAS inhibition. Identification of hits that exhibited low micromolar activity for NLRP3 and AHAS inhibition were selected for SAR study. In vitro testing of the analogs along with molecular docking led to increased knowledge for lead optimization of the potential hits. In silico screening has resulted in IC50 (IL-1β release) and MIC50 (fungal growth) values with low μM potency against several Candida species. In vivo validation will further confirm the potential of the scaffolds for further synthetic-modification for the rationale design of novel dual-purpose drugs

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