Carboxypeptidase G and pterin deaminase metabolic pathways degrade folic acid in Variovorax sp. F1.

BACKGROUND: Folic acid (FA) is a synthetic vitamin (B9) and the oxidized form of a metabolic cofactor that is essential for life. Although the biosynthetic mechanisms of FA are established, its environmental degradation mechanism has not been fully elucidated. The present study aimed to identify bacteria in soil that degrade FA and the mechanisms involved. RESULTS: We isolated the soil bacterium Variovorax sp. F1 from sampled weed rhizospheres in a grassland and investigated its FA degradation mechanism. Cultured Variovorax sp. F1 rapidly degraded FA to pteroic acid (PA), indicating that FA hydrolysis to PA and glutamate. We cloned the carboxypeptidase G (CPG) gene and found widely distributed paralogs within the Variovorax genus. Recombinant CPG preferred FA and deaminofolic acid as substrates, indicating its involvement in FA degradation by Variovorax. Prolonged culture of Variovorax sp. F1 resulted in decreased rates of deaminofolic acid (DFA) and deaminopteroic acid (DPA) accumulation. This indicated that the deamination reaction also comprised a route of FA degradation. We also identified an F1 gene that was orthologous to the pterin deaminase gene (Arad3529) of Agrobacterium radiobacter. The encoded protein deaminated FA and PA to DFA and DPA, which was consistent with the deamination activity of FA and PA in bacterial cell-free extracts. CONCLUSION: We discovered that the two enzymes required for FA degradation pathways in isolates of Variovorax sp. F1 comprise CPG and pterin deaminase, and that DFA and PA are intermediates in the generation of DPA.

The spindle pole body of Aspergillus nidulans is asymmetrical and contains changing numbers of γ-tubulin complexes.

Centrosomes are important microtubule-organizing centers (MTOCs) in animal cells. In addition, non-centrosomal MTOCs (ncMTOCs) are found in many cell types. Their composition and structure are only poorly understood. Here, we analyzed nuclear MTOCs (spindle-pole bodies, SPBs) and septal MTOCs in Aspergillus nidulans They both contain γ-tubulin along with members of the family of γ-tubulin complex proteins (GCPs). Our data suggest that SPBs consist of γ-tubulin small complexes (γ-TuSCs) at the outer plaque, and larger γ-tubulin ring complexes (γ-TuRC) at the inner plaque. We show that the MztA protein, an ortholog of the human MOZART protein (also known as MZT1), interacted with the inner plaque receptor PcpA (the homolog of fission yeast Pcp1) at SPBs, while no interaction nor colocalization was detected between MztA and the outer plaque receptor ApsB (fission yeast Mto1). Septal MTOCs consist of γ-TuRCs including MztA but are anchored through AspB and Spa18 (fission yeast Mto2). MztA is not essential for viability, although abnormal spindles were observed frequently in cells lacking MztA. Quantitative PALM imaging revealed unexpected dynamics of the protein composition of SPBs, with changing numbers of γ-tubulin complexes over time during interphase and constant numbers during mitosis.This article has an associated First Person interview with the first author of the paper.

Control of Actin and Calcium for Chitin Synthase Delivery to the Hyphal Tip of Aspergillus.

Filamentous fungi are covered by a cell wall consisting mainly of chitin and glucan. The synthesis of chitin, a ß-1,4-linked homopolymer of N-acetylglucosamine, is essential for hyphal morphogenesis. Fungal chitin synthases are integral membrane proteins that have been classified into seven classes. ChsB, a class III chitin synthase, is known to play a key role in hyphal tip growth and has been used here as a model to understand the cell biology of cell wall biosynthesis in Aspergillus nidulans. Chitin synthases are transported on secretory vesicles to the plasma membrane for new cell wall synthesis. Super-resolution localization imaging as a powerful biophysical approach indicated dynamics of the Spitzenkörper where spatiotemporally regulated exocytosis and cell extension, whereas high-speed pulse-chase imaging has revealed ChsB transport mechanism mediated by kinesin-1 and myosin-5. In addition, live imaging analysis showed correlations among intracellular Ca2+ levels, actin assembly, and exocytosis in growing hyphal tips. This suggests that pulsed Ca2+ influxes coordinate the temporal control of actin assembly and exocytosis, which results in stepwise cell extension. It is getting clear that turgor pressure and cell wall pressure are involved in the activation of Ca2+ channels for Ca2+ oscillation and cell extension. Here the cell wall synthesis and tip growth meet again.

Pulses of Ca2+ coordinate actin assembly and exocytosis for stepwise cell extension.

Many eukaryotic cells grow by extending their cell periphery in pulses. The molecular mechanisms underlying this process are not yet fully understood. Here we present a comprehensive model of stepwise cell extension by using the unique tip growth system of filamentous fungi. Live-cell imaging analysis, including superresolution microscopy, revealed that the fungus Aspergillus nidulans extends the hyphal tip in an oscillatory manner. The amount of F-actin and secretory vesicles (SV) accumulating at the hyphal tip oscillated with a positive temporal correlation, whereas vesicle amounts were negatively correlated to the growth rate. The intracellular Ca2+ level also pulsed with a positive temporal correlation to the amount of F-actin and SV at the hyphal tip. Two Ca2+ channels, MidA and CchA, were needed for proper tip growth and the oscillations of actin polymerization, exocytosis, and the growth rate. The data indicate a model in which transient Ca2+ pluses cause depolymerization of F-actin at the cortex and promote SV fusion with the plasma membrane, thereby extending the cell tip. Over time, Ca2+ diffuses away and F-actin and SV accumulate again at the hyphal tip. Our data provide evidence that temporally controlled actin polymerization and exocytosis are coordinated by pulsed Ca2+ influx, resulting in stepwise cell extension.

Inhomogeneous Molecular Distributions and Cytochrome Types and Redox States in Fungal Cells Revealed by Raman Hyperspectral Imaging Using Multivariate Curve Resolution-Alternating Least Squares.

Hyphae of filamentous fungi consist of compartments that are distinct both spatially and functionally, thereby forming a unique multicellular system. Much work has been done mainly using fluorescence imaging to reveal what biomolecules are present in those different hyphal sections and what physiological roles they play. Nevertheless, a holistic understanding of hyphal functions including the polarized growth of hyphae is still lacking because of the difficulty in simultaneous acquisition of spatial and chemical information on various molecular components in living hyphae. Here, we used a multivariate curve resolution-alternating least-squares (MCR-ALS) analysis of Raman hyperspectral imaging data to study in vivo the spatial distributions and chemical properties of major cellular components in the tip, basal, and branching regions of the model fungus Aspergillus nidulans. The MCR-ALS Raman imaging method visualized, without any labeling, the characteristic distributions of cytochromes as well as other components including polysaccharides, noncytochrome proteins, nucleic acids, lipids, and ergosterol in the hyphal regions studied. Furthermore, the intrinsic Raman spectra derived for the first time from the MCR-ALS analysis enabled us to gain otherwise unobtainable chemical insights into those visualized components. We show variations in the relative abundance of cytochromes b and c and in their redox states (reduced vs oxidized form) among the three different representative compartments of A. nidulans hyphae, which could potentially be associated with specific physiological activities and functions of hyphae. The present results demonstrate that our MCR-ALS Raman imaging can serve as a useful tool complementary to the conventional approaches, for elucidating the diverse roles of filamentous fungi at the molecular level.

Super Resolution Fluorescence Microscopy and Tracking of Bacterial Flotillin (Reggie) Paralogs Provide Evidence for Defined-Sized Protein Microdomains within the Bacterial Membrane but Absence of Clusters Containing Detergent-Resistant Proteins.

Biological membranes have been proposed to contain microdomains of a specific lipid composition, in which distinct groups of proteins are clustered. Flotillin-like proteins are conserved between pro-and eukaryotes, play an important function in several eukaryotic and bacterial cells, and define in vertebrates a type of so-called detergent-resistant microdomains. Using STED microscopy, we show that two bacterial flotillins, FloA and FloT, form defined assemblies with an average diameter of 85 to 110 nm in the model bacterium Bacillus subtilis. Interestingly, flotillin microdomains are of similar size in eukaryotic cells. The soluble domains of FloA form higher order oligomers of up to several hundred kDa in vitro, showing that like eukaryotic flotillins, bacterial assemblies are based in part on their ability to self-oligomerize. However, B. subtilis paralogs show significantly different diffusion rates, and consequently do not colocalize into a common microdomain. Dual colour time lapse experiments of flotillins together with other detergent-resistant proteins in bacteria show that proteins colocalize for no longer than a few hundred milliseconds, and do not move together. Our data reveal that the bacterial membrane contains defined-sized protein domains rather than functional microdomains dependent on flotillins. Based on their distinct dynamics, FloA and FloT confer spatially distinguishable activities, but do not serve as molecular scaffolds.

Microtubule-organizing centers of Aspergillus nidulans are anchored at septa by a disordered protein.

Microtubule-organizing centers (MTOCs) are large, multi-subunit protein complexes. Schizosaccharomyces pombe harbors MTOCs at spindle pole bodies, transient MTOCs in the division plane (eMTOCs) and nuclear-envelope associated MTOCs in interphase cells (iMTOCs). In the filamentous fungus Aspergillus nidulans SPBs and septum-associated MTOCs were described. Although comparable to S. pombe eMTOCs, A. nidulans sMTOCS are permanent septum-associated structures. The composition of sMTOCs is poorly understood and how they are targeted to septa was unknown. Here, we show that in A. nidulans several SPB outer plaque proteins also locate to sMTOCs while other SPB proteins do not, including SfiA, a protein required for SPB duplication in Saccharomyces cerevisiae and S. pombe and PcpA, the anchor for γ-TuSCs at the SPB inner plaque. The A. nidulans disordered protein Spa18Mto2 and the centrosomin-domain containing protein ApsBMto1 were required for recruiting the γ-TuRC component GcpC to sMTOCs and for seeding MT formation from septa. Testing different septum-associated proteins for a role in sMTOC function, Spa10 was identified. It forms a septal pore disc structure, recruits Spa18 and ApsB to septa and is required for sMTOC activity. This is the first evidence for a septum-specific protein, Spa10, as anchor for a specific class of MTOCs.

Oscillatory fungal cell growth.

Cells are dynamic systems, the state of which undergoes constant alteration that results in morphological changes and movement. Many dynamic cellular processes that appear continuous are driven by underlying mechanisms that oscillate with distinct periods. For example eukaryotic cells do not grow continuously, but rather by pulsed extension of the periphery. Stepwise cell extension at the hyphal tips of several filamentous fungi was discovered 20 years ago, but only a few molecular details of the mechanism have been clarified since then. A recent study has provided evidence for correlations among intracellular Ca2+ levels, actin assembly, exocytosis and cell extension in growing hyphal tips. This suggests that pulsed Ca2+ influxes coordinate the temporal control of actin assembly and exocytosis, which results in stepwise cell extension. The coordinated oscillation of these machineries are likely to be ubiquitous among all eukaryotes. Indeed, intracellular Ca2+ levels and/or actin polymerization oscillate in mammalian and plant cells. This review summarizes the mechanisms of oscillation in several systems.

Spatial heterogeneity of glycogen and its metabolizing enzymes in Aspergillus nidulans hyphal tip cells.

Glycogen is a homopolymer of glucose and a ubiquitous cellular-storage carbon. This study investigated which Aspergillus nidulans genes are involved in glycogen metabolism. Gene disruptants of predicted glycogen synthase (gsyA) and glycogenin (glgA) genes accumulated less cellular glycogen than the wild type strain, indicating that GsyA and GlgA synthesize glycogen similarly to other eukaryotes. Meanwhile, gene disruption of gphA encoding glycogen phosphorylase increased the amount of glycogen to a higher degree than wild type during the stationary phase that accompanies carbon-source limitation. GFP-tagged GsyA and GphA were distributed in the cytosol and formed punctate and filamentous structures, respectively. Carbon starvation resulted in elongated GphA-GFP filaments and increased numbers of filaments. These structures were more frequently located in the basal regions of tip cells and adjacent cells than in the apical regions of tip cells. Cellular glycogen visualized by incorporation of a fluorescent glucose analog accumulated in cytoplasmic puncta that were more prevalent in the basal regions, a pattern similar to that seen for GsyA. The colocalization of glycogen and GsyA at punctate structures in tip and sub-apical cells likely represents the cellular machinery for synthesizing glycogen. More frequent colocalization in the basal, rather than tip cell apical regions indicated that tip cells have differentiated subcellular regions for glycogen synthesis. Our findings regarding glycogen, GsyA and GphA distribution evoke the spatial heterogeneity of glycogen metabolism in fungal hyphae.

Genetic evidence for a microtubule-capture mechanism during polarised growth of Aspergillus nidulans.

The cellular switch from symmetry to polarity in eukaryotes depends on the microtubule (MT) and actin cytoskeletons. In fungi such as Schizosaccharomyces pombe or Aspergillus nidulans, the MT cytoskeleton determines the sites of actin polymerization through cortical cell-end marker proteins. Here we describe A. nidulans MT guidance protein A (MigA) as the first ortholog of the karyogamy protein Kar9 from Saccharomyces cerevisiae in filamentous fungi. A. nidulans MigA interacts with the cortical ApsA protein and is involved in spindle positioning during mitosis. MigA is also associated with septal and nuclear MT organizing centers (MTOCs). Super-resolution photoactivated localization microscopy (PALM) analyses revealed that MigA is recruited to assembling and retracting MT plus ends in an EbA-dependent manner. MigA is required for MT convergence in hyphal tips and plays a role in correct localization of the cell-end markers TeaA and TeaR. In addition, MigA interacts with a class-V myosin, suggesting that an active mechanism exists to capture MTs and to pull the ends along actin filaments. Hence, the organization of MTs and actin depend on each other, and positive feedback loops ensure robust polar growth.

The Vip1 inositol polyphosphate kinase family regulates polarized growth and modulates the microtubule cytoskeleton in fungi.

Microtubules (MTs) are pivotal for numerous eukaryotic processes ranging from cellular morphogenesis, chromosome segregation to intracellular transport. Execution of these tasks requires intricate regulation of MT dynamics. Here, we identify a new regulator of the Schizosaccharomyces pombe MT cytoskeleton: Asp1, a member of the highly conserved Vip1 inositol polyphosphate kinase family. Inositol pyrophosphates generated by Asp1 modulate MT dynamic parameters independent of the central +TIP EB1 and in a dose-dependent and cellular-context-dependent manner. Importantly, our analysis of the in vitro kinase activities of various S. pombe Asp1 variants demonstrated that the C-terminal phosphatase-like domain of the dual domain Vip1 protein negatively affects the inositol pyrophosphate output of the N-terminal kinase domain. These data suggest that the former domain has phosphatase activity. Remarkably, Vip1 regulation of the MT cytoskeleton is a conserved feature, as Vip1-like proteins of the filamentous ascomycete Aspergillus nidulans and the distantly related pathogenic basidiomycete Ustilago maydis also affect the MT cytoskeleton in these organisms. Consistent with the role of interphase MTs in growth zone selection/maintenance, all 3 fungal systems show aspects of aberrant cell morphogenesis. Thus, for the first time we have identified a conserved biological process for inositol pyrophosphates.

Coordinated process of polarized growth in filamentous fungi.

Filamentous fungi are extremely polarized organisms, exhibiting continuous growth at their hyphal tips. The hyphal form is related to their pathogenicity in animals and plants, and their high secretion ability for biotechnology. Polarized growth requires a sequential supply of proteins and lipids to the hyphal tip. This transport is managed by vesicle trafficking via the actin and microtubule cytoskeleton. Therefore, the arrangement of the cytoskeleton is a crucial step to establish and maintain the cell polarity. This review summarizes recent findings unraveling the mechanism of polarized growth with special emphasis on the role of actin and microtubule cytoskeleton and polarity marker proteins. Rapid insertions of membranes via highly active exocytosis at hyphal tips could quickly dilute the accumulated polarity marker proteins. Recent findings by a super-resolution microscopy indicate that filamentous fungal cells maintain their polarity at the tips by repeating transient assembly and disassembly of polarity sites.

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

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.

The cell-end marker TeaA and the microtubule polymerase AlpA contribute to microtubule guidance at the hyphal tip cortex of Aspergillus nidulans to provide polarity maintenance.

In the absence of landmark proteins, hyphae of Aspergillus nidulans lose their direction of growth and show a zigzag growth pattern. Here, we show that the cell-end marker protein TeaA is important for localizing the growth machinery at hyphal tips. The central position of TeaA at the tip correlated with the convergence of the microtubule (MT) ends to a single point. Conversely, in the absence of TeaA, the MTs often failed to converge to a single point at the cortex. Further analysis suggested a functional connection between TeaA and AlpA (an ortholog of the MT polymerase Dis1/CKAP5/XMAP215) for proper regulation of MT growth at hyphal tips. AlpA localized at MT plus-ends, and bimolecular fluorescence complementation assays suggested that it interacted with TeaA after MT plus-ends reached the tip cortex. In vitro MT polymerization assays showed that AlpA promoted MT growth up to sevenfold. Addition of the C-terminal region of TeaA increased the catastrophe frequency of the MTs. Thus, the control of the AlpA activity through TeaA might be a novel principle for MT growth regulation after reaching the cortex. In addition, we present evidence that the curvature of hyphal tips also could be involved in the control of MT growth at hyphal tips.

F-box protein RcyA controls turnover of the kinesin-7 motor KipA in Aspergillus nidulans.

Fungal filamentous growth depends on continuous membrane insertion at the tip, the delivery of membrane-bound positional markers, and the secretion of enzymes for cell wall biosynthesis. This is achieved through exocytosis. At the same time, polarized growth requires membrane and protein recycling through endocytosis. Endocytic vesicles are thought to enter the protein degradation pathway or recycle their content to the cell surface. In Saccharomyces cerevisiae, the Rcy1 F-box protein is involved in the recycling process of a v-SNARE protein. We identified a Rcy1 orthologue, RcyA, in the filamentous fungus Aspergillus nidulans as a protein interacting with the KipA kinesin-7 motor protein in a yeast two-hybrid screen. The interaction was confirmed through bimolecular fluorescence complementation. RcyA possesses an F-box domain at the N terminus and a prenylation (CaaX) motif at the C terminus. RcyA shows also similarity to Sec10, a component of the exocyst complex. The RcyA protein localized to the hyphal tip and forming septa, likely through transportation on secretory vesicles and partially on early endosomes, but independently of KipA. Deletion of rcyA did not cause severe morphological changes but caused partial defects in the recycling of the SynA v-SNARE protein and the positioning of the cell end markers TeaA and TeaR. In addition, deletion of rcyA led to increased concentrations of the KipA protein, whereas the transcript concentration was unaffected. These results suggest that RcyA probably labels KipA for degradation and thereby controls the protein amount of KipA.

The Metabolic Regulation of Amino Acid Synthesis Counteracts Reactive Nitrogen Stress via Aspergillus nidulans Cross-Pathway Control.

Nitric oxide (NO) is a natural reactive nitrogen species (RNS) that alters proteins, DNA, and lipids and damages biological activities. Although microorganisms respond to and detoxify NO, the regulation of the cellular metabolic mechanisms that cause cells to tolerate RNS toxicity is not completely understood. We found that the proline and arginine auxotrophic proA5 and argB2 mutants of the fungus Aspergillus nidulans require more arginine and proline for normal growth under RNS stress that starves cells by accumulating fewer amino acids. Fungal transcriptomes indicated that RNS stress upregulates the expression of the biosynthetic genes required for global amino acids, including proline and arginine. A mutant of the gene disruptant, cpcA, which encodes the transcriptional regulation of the cross-pathway control of general amino acid synthesis, did not induce these genes, and cells accumulated fewer amino acids under RNS stress. These results indicated a novel function of CpcA in the cellular response to RNS stress, which is mediated through amino acid starvation and induces the transcription of genes for general amino acid synthesis. Since CpcA also controls organic acid biosynthesis, impaired intermediates of such biosynthesis might starve cells of amino acids. These findings revealed the importance of the mechanism regulating amino acid homeostasis for fungal responses to and survival under RNS stress.

The role of flotillin FloA and stomatin StoA in the maintenance of apical sterol-rich membrane domains and polarity in the filamentous fungus Aspergillus nidulans.

Apical sterol-rich plasma membrane domains (SRDs), which can be viewed using the sterol-binding fluorescent dye filipin, are gaining attention for their important roles in polarized growth of filamentous fungi. The microdomain scaffolding protein flotillin/reggie and related stomatin were thought to be good candidates involved in the formation of SRDs. Here, we show that the flotillin/reggie orthologue FloA tagged with GFP localized as stable dots along the plasma membrane except hyphal tips. Deletion of floA reduced the growth rate, often resulted in irregularly shaped hyphae and impaired SRDs. In contrast, the stomatin orthologue StoA, tagged with GFP, localized at the cortex of young branch tips and at the subapical cortex in long hyphal tips, and was transported bi-directionally along microtubules on endosomes. Deletion of stoA resulted in irregular hyphal morphology and increased branching especially in young hyphae, but did not obviously affect SRDs. Double deletion of floA and stoA enhanced the defects of growth and hyphal morphology. Our data suggest that the plasma membrane of hyphal tips and in subapical regions are distinct and that FloA is involved in membrane compartmentalization and probably indirectly in SRD maintenance.

Novel Insights into Microbial Behavior Gleaned Using Microfluidics.

Microorganisms develop into communities in nearly every environmental niche, which is typically replete with micrometer-scale gaps and features. In each of these habitats, microorganisms adapt to and are affected by their physical environment. Conventional culture methods use glass bottom dishes or millimeter-scale flow cells, which poorly mimic the complexity of natural micrometer-scale environments; therefore, the limitations associated with the creation of microbe-scale environments with granularity hinder the ability to examine their ecological behavior. Microfluidics is a tool that is increasingly being used to study microorganisms because it enables the manipulation of micrometer-scale flows while simultaneously facilitating real-time and live-cell imaging. In this review, we discuss several insights into the behavior of bacteria and fungi that were gained through the adoption of microfluidics to control complex micrometer-scale environments. We also discuss the potential of the increased adoption of this tool.

Local calcium signal transmission in mycelial network exhibits decentralized stress responses.

Many fungi live as mycelia, which are networks of hyphae. Mycelial networks are suited for the widespread distribution of nutrients and water. The logistical capabilities are critical for the extension of fungal survival areas, nutrient cycling in ecosystems, mycorrhizal symbioses, and virulence. In addition, signal transduction in mycelial networks is predicted to be vital for mycelial function and robustness. A lot of cell biological studies have elucidated protein and membrane trafficking and signal transduction in fungal hyphae; however, there are no reports visualizing signal transduction in mycelia. This paper, by using the fluorescent Ca2+ biosensor, visualized for the first time how calcium signaling is conducted inside the mycelial network in response to localized stimuli in the model fungus Aspergillus nidulans. The wavy propagation of the calcium signal inside the mycelium or the signal blinking in the hyphae varies depending on the type of stress and proximity to the stress. The signals, however, only extended around 1,500 µm, suggesting that the mycelium has a localized response. The mycelium showed growth delay only in the stressed areas. Local stress caused arrest and resumption of mycelial growth through reorganization of the actin cytoskeleton and membrane trafficking. To elucidate the downstream of calcium signaling, calmodulin, and calmodulin-dependent protein kinases, the principal intracellular Ca2+ receptors were immunoprecipitated and their downstream targets were identified by mass spectrometry analyses. Our data provide evidence that the mycelial network, which lacks a brain or nervous system, exhibits decentralized response through locally activated calcium signaling in response to local stress.

Live-cell imaging of septins and cell polarity proteins in the growing dikaryotic vegetative hypha of the model mushroom Coprinopsis cinerea.

The developmental biology underlying the morphogenesis of mushrooms remains poorly understood despite the essential role of fungi in the terrestrial environment and global carbon cycle. The mushroom Coprinopsis cinerea is a leading model system for the molecular and cellular basis of fungal morphogenesis. The dikaryotic vegetative hyphae of this fungus grow by tip growth with clamp cell formation, conjugate nuclear division, septation, subapical peg formation, and fusion of the clamp cell to the peg. Studying these processes provides many opportunities to gain insights into fungal cell morphogenesis. Here, we report the dynamics of five septins, as well as the regulators CcCla4, CcSpa2, and F-actin, visualized by tagging with fluorescent proteins, EGFP, PA-GFP or mCherry, in the growing dikaryotic vegetative hyphae. We also observed the nuclei using tagged Sumo proteins and histone H1. The five septins colocalized at the hyphal tip in the shape of a dome with a hole (DwH). CcSpa2-EGFP signals were observed in the hole, while CcCla4 signals were observed as the fluctuating dome at the hyphal tip. Before septation, CcCla4-EGFP was also occasionally recruited transiently around the future septum site. Fluorescent protein-tagged septins and F-actin together formed a contractile ring at the septum site. These distinct specialized growth machineries at different sites of dikaryotic vegetative hyphae provide a foundation to explore the differentiation program of various types of cells required for fruiting body formation.