Protein interactors of Spindle Pole Body (SPB) components and septal proteins in fungus Neurospora crassa: A mass spectrometry-based dataset.

Microtubule Organizing Centers (MTOC) are subcellular structures in eukaryotic cells where nucleation of microtubules (MTs) takes place and represents the filament's minus end. Their localization depends on the species, cell type, and cell cycle stage. Along the fungal kingdom, the Spindle Pole Body (SPB) in the nucleus (an equivalent to Centrosomes in animal cells) is the principal MTOC. Other MTOCs have been identified in filamentous fungi, such as the Spitzenkörper in the hyphal tips of Schizosaccharomyces pombe or the septal pore of Aspergillus nidulans. However, in the fungal-model organism Neurospora crassa, these alternative MTOCs have not been recognized. Here, we present a Mass spectrometry-based dataset of proteins interacting with four MTOC components of N. crassa tagged with fluorescent proteins: γ-Tubulin-sGFP (main nucleator at the SPB), MZT-1-sGFP (structural SPB microprotein), APS-2-dRFP (septal protein and recognized SPB component), and SPA-10-sGFP (septal MTOC protein). A WT and a cytosolic GFP expressing strain were included as controls. The protein interactors were pulled down by Co-IP1, using GFP-Magnetic agarose that captures recombinant GFP proteins (including GFP-derivatives) in their native state. Bounded proteins were separated by SDS-PAGE and identified by nano LC-MS/MS2. The protein annotation was done using the N. crassa protein database.

The cytoplasmic microtubule array in Neurospora crassa depends on microtubule-organizing centers at spindle pole bodies and microtubule +end-depending pseudo-MTOCs at septa.

γ-Tubulin ring complexes (γ-TuRC) mediate nucleation and anchorage of microtubules (MTs) to microtubule organizing centers (MTOCs). In fungi, the spindle pole body (SPB) is the functional equivalent of the centrosome, which is the main MTOC. In addition, non-centrosomal MTOCs (ncMTOCs) contribute to MT formation in some fungi like Schizosaccharomyces pombe and Aspergillus nidulans. In A. nidulans, MTOCs are anchored at septa (sMTOC) and share components of the outer plaque of the SPB. Here we show that the Neurospora crassa SPB is embedded in the nuclear envelope, with the γ-TuRC targeting proteins PCP-1Pcp1/PcpA located at the inner plaque and APS-2Mto1/ApsB located at the outer plaque of the SPB. PCP-1 was a specific component of nuclear MTOCs, while APS-2 was also present at the septal pore. Although γ-tubulin was only detected at the nucleus, spontaneous MT nucleation occurred in the apical and subapical cytoplasm during recovery from benomyl-induced MT depolymerization experiments. There was no evidence for MT nucleation at septa. However, without benomyl treatment MT plus-ends were organized in the septal pore through MTB-3EB1. Those septal MT plus ends polymerized MTs from septa in interphase cells Thus we conclude that the SPB is the only MT nucleation site in N. crassa, but the septal pore aids the MT network arrangement through the anchorage of the MT plus-ends through a pseudo-MTOC.

F-actin dynamics following mechanical injury of Trichoderma atroviride and Neurospora crassa hyphae.

We investigated hyphae regeneration in Trichoderma atroviride and Neurospora crassa, with particular focus on determining the role of the actin cytoskeleton after mechanical injury. Filamentous actin (F-actin) dynamics was observed by live-cell confocal microscopy in both T. atroviride and N. crassa strains expressing Lifeact-GFP. In growing hyphae of both fungi, F-actin localized in three different structural forms: patches, cables and actomyosin rings. Most patches were conspicuously arranged in a collar in the hyphal subapex. A strong F-actin signal, likely actin filaments, colocalized with the core of the Spitzenkörper. Filaments and cables of F-actin were observed along the cortex throughout hyphae. Following mechanical damage at the margin of growing mycelia of T. atroviride and N. crassa, the severed hyphae lost their cytoplasmic contents, but plugging of the septal pore by a Woronin body occured, and the rest of the hyphal tube remained whole. In both fungi, patches of F-actin began accumulating next to the plugged septum. Regeneration was attained by the emergence of a new hyphal tube as an extension of the plugged septum wall. The septum wall was gradually remodeled into the apical wall of the emerging hypha. Whereas in T. atroviride the re-initiation of polarized growth took  âˆ¼ 1 h, in N. crassa, actin patch accumulation began almost immediately, and new growing hyphae were observed âˆ¼ 30 min after injury. By confocal microscopy, we found that chitin synthase 1 (CHS-1), a microvesicle (chitosome) component, accumulated next to the plugged septum in regenerating hyphae of N. crassa. We concluded that the actin cytoskeleton plays a key role in hyphal regeneration by supporting membrane remodeling, helping to facilitate transport of vesicles responsible for new wall growth and organization of the new tip-growth apparatus.

Transcriptomic analysis of polyaromatic hydrocarbon degradation by the halophilic fungus Aspergillus sydowii at hypersaline conditions.

Polycyclic aromatic hydrocarbons (PAHs) are among the most persistent xenobiotic compounds, with high toxicity effects. Mycoremediation with halophilic Aspergillus sydowii was used for their removal from a hypersaline medium (1 M NaCl). A. sydowii metabolized PAHs as sole carbon sources, resulting in the removal of up to 90% for both PAHs [benzo [a] pyrene (BaP) and phenanthrene (Phe)] after 10 days. Elimination of Phe and BaP was almost exclusively due to biotransformation and not adsorption by dead mycelium and did not correlate with the activity of lignin modifying enzymes (LME). Transcriptomes of A. sydowii grown on PAHs, or on glucose as control, both at hypersaline conditions, revealed 170 upregulated and 76 downregulated genes. Upregulated genes were related to starvation, cell wall remodelling, degradation and metabolism of xenobiotics, DNA/RNA metabolism, energy generation, signalling and general stress responses. Changes of LME expression levels were not detected, while the chloroperoxidase gene, possibly related to detoxification processes in fungi, was strongly upregulated. We propose that two parallel metabolic pathways (mitochondrial and cytosolic) are involved in degradation and detoxification of PAHs in A. sydowii resulting in intracellular oxidation of PAHs. To the best of our knowledge, this is the most comprehensive transcriptomic analysis on fungal degradation of PAHs.

The GUL-1 Protein Binds Multiple RNAs Involved in Cell Wall Remodeling and Affects the MAK-1 Pathway in Neurospora crassa.

The Neurospora crassa GUL-1 is part of the COT-1 pathway, which plays key roles in regulating polar hyphal growth and cell wall remodeling. We show that GUL-1 is a bona fide RNA-binding protein (RBP) that can associate with 828 "core" mRNA species. When cell wall integrity (CWI) is challenged, expression of over 25% of genomic RNA species are modulated (2,628 mRNAs, including the GUL-1 mRNA). GUL-1 binds mRNAs of genes related to translation, cell wall remodeling, circadian clock, endoplasmic reticulum (ER), as well as CWI and MAPK pathway components. GUL-1 interacts with over 100 different proteins, including stress-granule and P-body proteins, ER components and components of the MAPK, COT-1, and STRIPAK complexes. Several additional RBPs were also shown to physically interact with GUL-1. Under stress conditions, GUL-1 can localize to the ER and affect the CWI pathway-evident via altered phosphorylation levels of MAK-1, interaction with mak-1 transcript, and involvement in the expression level of the transcription factor adv-1. We conclude that GUL-1 functions in multiple cellular processes, including the regulation of cell wall remodeling, via a mechanism associated with the MAK-1 pathway and stress-response.

Haloadaptative Responses of Aspergillus sydowii to Extreme Water Deprivation: Morphology, Compatible Solutes, and Oxidative Stress at NaCl Saturation.

Water activity (aw) is critical for microbial growth, as it is severely restricted at aw < 0.90. Saturating NaCl concentrations (~5.0 M) induce extreme water deprivation (aw ≅ 0.75) and cellular stress responses. Halophilic fungi have cellular adaptations that enable osmotic balance and ionic/oxidative stress prevention to grow at high salinity. Here we studied the morphology, osmolyte synthesis, and oxidative stress defenses of the halophile Aspergillus sydowii EXF-12860 at 1.0 M and 5.13 M NaCl. Colony growth, pigmentation, exudate, and spore production were inhibited at NaCl-saturated media. Additionally, hyphae showed unpolarized growth, lower diameter, and increased septation, multicellularity and branching compared to optimal NaCl concentration. Trehalose, mannitol, arabitol, erythritol, and glycerol were produced in the presence of both 1.0 M and 5.13 M NaCl. Exposing A. sydowii cells to 5.13 M NaCl resulted in oxidative stress evidenced by an increase in antioxidant enzymes and lipid peroxidation biomarkers. Also, genes involved in cellular antioxidant defense systems were upregulated. This is the most comprehensive study that investigates the micromorphology and the adaptative cellular response of different non-enzymatic and enzymatic oxidative stress biomarkers in halophilic filamentous fungi.

Transcriptional profiling and localization of GUL-1, a COT-1 pathway component, in Neurospora crassa.

Impairment of theNeurospora crassaCOT-1 kinase results in defects in hyphal polarity. Some of these effects are partially suppressed by inactivation of gul-1 (encoding an mRNA-binding protein involved in translational regulation). Here, we report on the transcriptional profiling of cot-1 inactivation and demonstrate that gul-1 affects transcript abundance of multiple genes in the COT-1 pathway, including processes such as cell wall remodeling, nitrogen and amino acid metabolism. The GUL-1 protein itself was found to be distributed within the entire hyphal cell, along with a clear presence of aggregates that traffic within the cytoplasm. Live imaging of GUL-1-GFP demonstrated that GUL-1 transport is microtubule-dependent. Cellular stress, as imposed by the presence of the cell wall biosynthesis inhibitor Nikkomycin Z or by nitrogen limitation, resulted in a 2-3-fold increase of GUL-1 aggregate association with nuclei. Taken together, this study demonstrates that GUL-1 affects multiple processes, its function is stress-related and linked with cellular traffic and nuclear association.

The actin motor MYO-5 effect in the intracellular organization of Neurospora crassa.

In filamentous fungi, polarized growth is the result of vesicle secretion at the hyphal apex. Motor proteins mediate vesicle transport to target destinations on the plasma membrane via actin and microtubule cytoskeletons. Myosins are motor proteins associated with actin filaments. Specifically, class V myosins are responsible for cargo transport in eukaryotes. We studied the dynamics and localization of myosin V in wild type hyphae of Neurospora crassa and in hyphae that lacked MYO-5. In wild type hyphae, MYO-5-GFP was localized concentrated in the hyphal apex and colocalized with Spitzenkörper. Photobleaching studies showed that MYO-5-GFP was transported to the apex from subapical hyphal regions. The deletion of the class V myosin resulted in a reduced rate of hyphal growth, apical hyperbranching, and intermittent loss of hyphal polarity. MYO-5 did not participate in breaking the symmetrical growth during germination but contributed in the apical organization upon establishment of polarized growth. In the Δmyo-5 mutant, actin was organized into thick cables in the apical and subapical hyphal regions, and the number of endocytic patches was reduced. The microvesicles-chitosomes observed with CHS-1-GFP were distributed as a cloud occupying the apical dome and not in the Spitzenkörper as the WT strain. The mitochondrial movement was not associated with MYO-5, but tubular vacuole position is MYO-5-dependent. These results suggest that MYO-5 plays a role in maintaining apical organization and the integrity of the Spitzenkörper and is required for normal hyphal growth, polarity, septation, conidiation, and proper conidial germination.

[Factors related to oral candidiasis in HIV children and adolescents, species characterization and antifungal susceptibility]. / Factores relacionados a candidiasis oral en niños y adolescentes con VIH, caracterización de especies y susceptibilidad antifúngica.

BACKGROUND: Factors associated with candidiasis and colonization in HIV-positive children and adolescents in developing countries are not well understood. AIM: To identify the factors associated with oral Candida colonization and candidiasis in institutionalized HIV-positive children and adolescents in Tijuana, México, as well as the response of the isolates to antifungals. MATERIALS AND METHODS: Sample of the oral mucosa of 30 HIV positive children and adolescents were obtained to isolate and identify Candida species by culture and metabolic profile. Antifungal drugs susceptibility was determined according to CLSI. Indicators of immunological and virologic failure were classified in accordance to WHO criteria. RESULTS: Six Candida species were identified from oral mucosa, 53% colonizers and 47% in candidiasis. Factors associated with candidiasis and oral colonization were viral load (p = 0,001), CD4+ counts (p = 0,002) and HAART regimen (p ≤ 0,014). The most prevalent species was C. glabrata (33%), but C. albicans (27%) was more resistant to fluconazole (p = 0,001). Itraconazol resistant species were identified in regimens that include an NNRTI (p = 0,041). CONCLUSION: HIV-positive children and adolescents living in an orphanage showed high prevalence of colonizing Candida spp. and resistance to antifungals, related to NNRTI.

Factores relacionados a candidiasis oral en niños y adolescentes con VIH, caracterización de especies y susceptibilidad antifúngica / Factors related to oral candidiasis in HIV children and adolescents, species characterization and antifungal susceptibility

Resumen Introducción: Se desconocen los factores asociados a la candidiasis oral en población pediátrica con infección por VIH de los países en desarrollo. Objetivo: Identificar los factores asociados a la colonización por Candida, candidiasis oral y la susceptibilidad in vitro a antifúngicos, en niños y adolescentes con infección por VIH institucionalizados en la ciudad de Tijuana, México. Materiales y Métodos: Se examinó la cavidad oral de 30 niños y adolescentes con infección por VIH, se obtuvo una muestra de la mucosa oral para identificar las especies de Candida mediante cultivo y auxonograma. La susceptibilidad a los antifúngicos se determinó de acuerdo al CLSI. Los indicadores del estado inmunológico y falla virológica se clasificaron conforme a la OMS. Resultados: Se identificaron seis especies de Candida, 53% colonizantes y 47% causantes de candidiasis. Los factores asociados a candidiasis fueron alta carga viral (p = 0,001), menor recuento de LTCD4+ (p = 0,002) y esquema TARAA (p ≤ 0,014). La especie prevalente fue C. glabrata (33%); sin embargo, C. albicans (27%) fue más resistente a fluconazol (p = 0,001). Las especies resistentes a itraconazol se identificaron en esquemas que incluyen un INNTR (p = 0,041). Conclusiones: Los niños y adolescentes con infección por VIH institucionalizados mostraron una prevalencia elevada de Candida spp. colonizante y resistencia a los antifúngicos relacionada con los INNTR .

Background: Factors associated with candidiasis and colonization in HIV-positive children and adolescents in developing countries are not well understood. Aim: To identify the factors associated with oral Candida colonization and candidiasis in institutionalized HIV-positive children and adolescents in Tijuana, México, as well as the response of the isolates to antifungals. Materials and Methods: Sample of the oral mucosa of 30 HIV positive children and adolescents were obtained to isolate and identify Candida species by culture and metabolic profile. Antifungal drugs susceptibility was determined according to CLSI. Indicators of immunological and virologic failure were classified in accordance to WHO criteria. Results: Six Candida species were identified from oral mucosa, 53% colonizers and 47% in candidiasis. Factors associated with candidiasis and oral colonization were viral load (p = 0,001), CD4+ counts (p = 0,002) and HAART regimen (p ≤ 0,014). The most prevalent species was C. glabrata (33%), but C. albicans (27%) was more resistant to fluconazole (p = 0,001). Itraconazol resistant species were identified in regimens that include an NNRTI (p = 0,041). Conclusion: HIV-positive children and adolescents living in an orphanage showed high prevalence of colonizing Candida spp. and resistance to antifungals, related to NNRTI.

Fungal Morphogenesis, from the Polarized Growth of Hyphae to Complex Reproduction and Infection Structures.

Filamentous fungi constitute a large group of eukaryotic microorganisms that grow by forming simple tube-like hyphae that are capable of differentiating into more-complex morphological structures and distinct cell types. Hyphae form filamentous networks by extending at their tips while branching in subapical regions. Rapid tip elongation requires massive membrane insertion and extension of the rigid chitin-containing cell wall. This process is sustained by a continuous flow of secretory vesicles that depends on the coordinated action of the microtubule and actin cytoskeletons and the corresponding motors and associated proteins. Vesicles transport cell wall-synthesizing enzymes and accumulate in a special structure, the Spitzenkörper, before traveling further and fusing with the tip membrane. The place of vesicle fusion and growth direction are enabled and defined by the position of the Spitzenkörper, the so-called cell end markers, and other proteins involved in the exocytic process. Also important for tip extension is membrane recycling by endocytosis via early endosomes, which function as multipurpose transport vehicles for mRNA, septins, ribosomes, and peroxisomes. Cell integrity, hyphal branching, and morphogenesis are all processes that are largely dependent on vesicle and cytoskeleton dynamics. When hyphae differentiate structures for asexual or sexual reproduction or to mediate interspecies interactions, the hyphal basic cellular machinery may be reprogrammed through the synthesis of new proteins and/or the modification of protein activity. Although some transcriptional networks involved in such reprogramming of hyphae are well studied in several model filamentous fungi, clear connections between these networks and known determinants of hyphal morphogenesis are yet to be established.

Candida species diversity and antifungal susceptibility patterns in oral samples of HIV/AIDS patients in Baja California, Mexico.

Candidiasis is the most common opportunistic fungal infection in HIV patients. The aims of this study were to identify the prevalence of carriers of Candida, Candida species diversity, and in vitro susceptibility to antifungal drugs. In 297 HIV/AIDS patients in Baja California, Mexico, Candida strains were identified by molecular methods (PCR-RFLP) from isolates of oral rinses of patients in Tijuana, Mexicali, and Ensenada. 56.3% of patients were colonized or infected with Candida. In Tijuana, there was a significantly higher percentage of carriers (75.5%). Out of the 181 strains that were isolated, 71.8% were Candida albicans and 28.2% were non-albicans species. The most common non-albicans species was Candida tropicalis (12.2%), followed by Candida glabrata (8.3%), Candida parapsilosis (2.2%), Candida krusei (1.7%), and Candida guilliermondii (1.1%). Candida dubliniensis was not isolated. Two associated species were found in 11 patients. In Mexicali and Ensenada, there was a lower proportion of Candida carriers compared to other regions in Mexico and worldwide, however, in Tijuana, a border town with many peculiarities, a higher carrier rate was found. In this population, only a high viral load was associated with oral Candida carriers. Other factors such as gender, use of antiretroviral therapy, CD4+ T-lymphocyte levels, time since diagnosis, and alcohol/ tobacco consumption, were not associated with Candida carriers.

Localization and role of MYO-1, an endocytic protein in hyphae of Neurospora crassa.

The subapical endocytic collar is a prominent feature of hyphae of Neurospora crassa. It comprises a dynamic collection of actin patches associated with a number of proteins required for endocytosis, namely, ARP-2/3 complex, fimbrin, coronin, etc. We presently show that MYO-1 is another key component of this endocytic collar. A myo-1 sequence was identified in the genome of N. crassa and used it to generate a strain with a myo-1-sgfp allele under the ccg1 promoter. Examination of living hyphae by confocal microscopy, revealed MYO-1-GFP located mainly as a dynamic collection of small patches arranged in collar-like fashion in the hyphal subapex. Dual tagging showed MYO-1-GFP partially colocalized with two other endocytic proteins, fimbrin and coronin. MYO-1 was also present during septum formation. By recovering a viable strain, albeit severely inhibited, after deletion of myo-1, it was possible to investigate the phenotypic consequences of the elimination of MYO-1. Deletion of myo-1 caused a severe reduction in growth rate (95%), near absence of aerial mycelium and no conidiation. A reduced uptake of the lipophilic dye FM4-64 indicated a deficiency in endocytosis in the Δmyo-1 mutant. Hyphae were produced by the Δmyo-1 mutant but their morphogenesis was severely affected; hyphal morphology was distorted displaying irregular periods of isotropic and polarized growth. The morphological alterations were accompanied, and presumably caused, by a disruption in the organization and dynamics of a myosin-deprived actin cytoskeleton that, ultimately, compromised the stability and function of the Spitzenkörper as a vesicle supply center.

Two microtubule-plus-end binding proteins LIS1-1 and LIS1-2, homologues of human LIS1 in Neurospora crassa.

LIS1 is a microtubule (Mt) plus-end binding protein that interacts with the dynein/dynactin complex. In humans, LIS1 is required for proper nuclear and organelle migration during cell growth. Although gene duplication is absent from Neurospora crassa, we found two paralogues of human LIS1. We named them LIS1-1 and LIS1-2 and studied their dynamics and function by fluorescent tagging. At the protein level, LIS1-1 and LIS1-2 were very similar. Although, the characteristic coiled-coil motif was not present in LIS1-2. LIS1-1-GFP and LIS1-2-GFP showed the same cellular distribution and dynamics, but LIS1-2-GFP was less abundant. Both LIS1 proteins were found in the subapical region as single fluorescent particles traveling toward the cell apex, they accumulated in the apical dome forming prominent short filament-like structures, some of which traversed the Spitzenkörper (Spk). The fluorescent structures moved exclusively in anterograde fashion along straight paths suggesting they traveled on Mts. There was no effect in the filament behavior of LIS1-1-GFP in the Δlis1-2 mutant but the dynamics of LIS1-2-GFP was affected in the Δlis1-1 mutant. Microtubular integrity and the dynein-dynactin complex were necessary for the formation of filament-like structures of LIS1-1-GFP in the subapical and apical regions; however, conventional kinesin (KIN-1) was not. Deletion mutants showed that the lack of lis1-1 decreased cell growth by ∼75%; however, the lack of lis1-2 had no effect on growth. A Δlis1-1;Δlis1-2 double mutant showed slower growth than either single mutant. Conidia production was reduced but branching rate increased in Δlis1-1 and the Δlis1-1;Δlis1-2 double mutants. The absence of LIS1-1 had a strong effect on Mt organization and dynamics and indirectly affected nuclear and mitochondrial distribution. The absence of LIS1-1 filaments in dynein mutants (ropy mutants) or in benomyl treated hyphae indicates the strong association between this protein and the regulation of the dynein-dynactin complex and Mt organization. LIS1-1 and LIS1-2 had a high amino acid homology, nevertheless, the absence of the coiled-coil motif in LIS1-2 suggests that its function or regulation may be distinct from that of LIS1-1.

Proper actin ring formation and septum constriction requires coordinated regulation of SIN and MOR pathways through the germinal centre kinase MST-1.

Nuclear DBF2p-related (NDR) kinases constitute a functionally conserved protein family of eukaryotic regulators that control cell division and polarity. In fungi, they function as effector kinases of the morphogenesis (MOR) and septation initiation (SIN) networks and are activated by pathway-specific germinal centre (GC) kinases. We characterized a third GC kinase, MST-1, that connects both kinase cascades. Genetic and biochemical interactions with SIN components and life cell imaging identify MST-1 as SIN-associated kinase that functions in parallel with the GC kinase SID-1 to activate the SIN-effector kinase DBF-2. SID-1 and MST-1 are both regulated by the upstream SIN kinase CDC-7, yet in an opposite manner. Aberrant cortical actomyosin rings are formed in Δmst-1, which resulted in mis-positioned septa and irregular spirals, indicating that MST-1-dependent regulation of the SIN is required for proper formation and constriction of the septal actomyosin ring. However, MST-1 also interacts with several components of the MOR network and modulates MOR activity at multiple levels. MST-1 functions as promiscuous enzyme and also activates the MOR effector kinase COT-1 through hydrophobic motif phosphorylation. In addition, MST-1 physically interacts with the MOR kinase POD-6, and dimerization of both proteins inactivates the GC kinase hetero-complex. These data specify an antagonistic relationship between the SIN and MOR during septum formation in the filamentous ascomycete model Neurospora crassa that is, at least in part, coordinated through the GC kinase MST-1. The similarity of the SIN and MOR pathways to the animal Hippo and Ndr pathways, respectively, suggests that intensive cross-communication between distinct NDR kinase modules may also be relevant for the homologous NDR kinases of higher eukaryotes.

MTB-3, a microtubule plus-end tracking protein (+TIP) of Neurospora crassa.

The microtubule (MT) "plus end" constitutes the platform for the accumulation of a structurally and functionally diverse group of proteins, collectively called "MT plus-end tracking proteins" (+TIPs). +TIPs control MT dynamics and link MTs to diverse sub-cellular structures. Neurospora crassaMicroTubule Binding protein-3 (MTB-3) is the homolog of yeast EB1, a highly conserved +TIP. To address the function of MTB-3, we examined strains with mtb-3 deletions, and we tagged MTB-3 with GFP to assess its dynamic behavior. MTB-3-GFP was present as comet-like structures distributed more or less homogeneously within the hyphal cytoplasm, and moving mainly towards the apex at speeds up to 4× faster than the normal hyphal elongation rates. MTB-3-GFP comets were present in all developmental stages, but were most abundant in mature hyphae. MTB-3-GFP comets were observed moving in anterograde and retrograde direction along the hypha. Retrograde movement was also observed as originating from the apical dome. The integrity of the microtubular cytoskeleton affects the presence and dynamics of MTB-3-GFP comets, while actin does not seem to play a role. The size of MTB-3-GFP comets is affected by the absence of dynactin and conventional kinesin. We detected no obvious morphological phenotypes in Δmtb-3 mutants but there were fewer MTs in Δmtb-3, MTs were less bundled and less organized. Compared to WT, both MT polymerization and depolymerization rates were significantly decreased in Δmtb-3. In summary, the lack of MTB-3 affects overall growth and morphological phenotypes of N. crassa only slightly, but deletion of mtb-3 has strong effect on MT dynamics.

Coronin is a component of the endocytic collar of hyphae of Neurospora crassa and is necessary for normal growth and morphogenesis.

Coronin plays a major role in the organization and dynamics of actin in yeast. To investigate the role of coronin in a filamentous fungus (Neurospora crassa), we examined its subcellular localization using fluorescent proteins and the phenotypic consequences of coronin gene (crn-1) deletion in hyphal morphogenesis, Spitzenkörper behavior and endocytosis. Coronin-GFP was localized in patches, forming a subapical collar near the hyphal apex; significantly, it was absent from the apex. The subapical patches of coronin colocalized with fimbrin, Arp2/3 complex, and actin, altogether comprising the endocytic collar. Deletion of crn-1 resulted in reduced hyphal growth rates, distorted hyphal morphology, uneven wall thickness, and delayed establishment of polarity during germination; it also affected growth directionality and increased branching. The Spitzenkörper of Δcrn-1 mutant was unstable; it appeared and disappeared intermittently giving rise to periods of hyphoid-like and isotropic growth respectively. Uptake of FM4-64 in Δcrn-1 mutant indicated a partial disruption in endocytosis. These observations underscore coronin as an important component of F-actin remodeling in N. crassa. Although coronin is not essential in this fungus, its deletion influenced negatively the operation of the actin cytoskeleton involved in the orderly deployment of the apical growth apparatus, thus preventing normal hyphal growth and morphogenesis.

Visualization of F-actin localization and dynamics with live cell markers in Neurospora crassa.

Filamentous actin (F-actin) plays essential roles in filamentous fungi, as in all other eukaryotes, in a wide variety of cellular processes including cell growth, intracellular motility, and cytokinesis. We visualized F-actin organization and dynamics in living Neurospora crassa cells via confocal microscopy of growing hyphae expressing GFP fusions with homologues of the actin-binding proteins fimbrin (FIM) and tropomyosin (TPM-1), a subunit of the Arp2/3 complex (ARP-3) and a recently developed live cell F-actin marker, Lifeact (ABP140 of Saccharomyces cerevisiae). FIM-GFP, ARP-3-GFP, and Lifeact-GFP associated with small patches in the cortical cytoplasm that were concentrated in a subapical ring, which appeared similar for all three markers but was broadest in hyphae expressing Lifeact-GFP. These cortical patches were short-lived, and a subset was mobile throughout the hypha, exhibiting both anterograde and retrograde motility. TPM-1-GFP and Lifeact-GFP co-localized within the Spitzenkörper (Spk) core at the hyphal apex, and were also observed in actin cables throughout the hypha. All GFP fusion proteins studied were also transiently localized at septa: Lifeact-GFP first appeared as a broad ring during early stages of contractile ring formation and later coalesced into a sharper ring, TPM-1-GFP was observed in maturing septa, and FIM-GFP/ARP3-GFP-labeled cortical patches formed a double ring flanking the septa. Our observations suggest that each of the N. crassa F-actin-binding proteins analyzed associates with a different subset of F-actin structures, presumably reflecting distinct roles in F-actin organization and dynamics. Moreover, Lifeact-GFP marked the broadest spectrum of F-actin structures; it may serve as a global live cell marker for F-actin in filamentous fungi.

Live-cell imaging of microtubule dynamics in hyphae of Neurospora crassa.

Due to the large number of microtubules in the wild-type strain, total internal reflection fluorescence (TIRF) microscopy was used to study cortical microtubule dynamics in leading hyphae of Neurospora crassa expressing beta-tubulin-GFP. Detection of plus-end dynamics of individual microtubule was much improved with this approach compared to the other commonly used methods such as confocal and widefield fluorescence microscopy. In order to address the roles of motor proteins in microtubule dynamics, microtubule-motor mutant strains, deltankin and ro-1 were examined. Unlike the wild-type strain, there were fewer microtubules in these hyphal cells; therefore, imaging was done using widefield fluorescence microscopy. We have shown that polymerization and depolymerization rates as well as hyphal extension rates were reduced by one half relative to those of wild type. Therefore, we believe that the hyphal extension rates are dependent upon the dynamic characteristics of microtubules, which are then regulated by microtubule motors in N. crassa.