RESUMO

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.

RESUMO

For the first time, the International Symposium on Fungal Stress was joined by the XIII International Fungal Biology Conference. The International Symposium on Fungal Stress (ISFUS), always held in Brazil, is now in its fourth edition, as an event of recognized quality in the international community of mycological research. The event held in São José dos Campos, SP, Brazil, in September 2022, featured 33 renowned speakers from 12 countries, including: Austria, Brazil, France, Germany, Ghana, Hungary, México, Pakistan, Spain, Slovenia, USA, and UK. In addition to the scientific contribution of the event in bringing together national and international researchers and their work in a strategic area, it helps maintain and strengthen international cooperation for scientific development in Brazil.

Assuntos

Biologia , Brasil , França , Espanha , México

RESUMO

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.

Assuntos

Proteínas de Transporte , Proteínas Fúngicas , Proteínas Associadas aos Microtúbulos , Neurospora crassa , Benomilo/metabolismo , Proteínas de Transporte/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Centro Organizador dos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Neurospora crassa/genética , Neurospora crassa/metabolismo , Corpos Polares do Fuso/metabolismo , Tubulina (Proteína)/genética

RESUMO

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.

Assuntos

Hidrocarbonetos Policíclicos Aromáticos , Transcriptoma , Aspergillus/genética , Biodegradação Ambiental , Perfilação da Expressão Gênica , Transcriptoma/genética

RESUMO

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.

RESUMO

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.

Assuntos

Citoesqueleto de Actina/genética , Hifas/genética , Miosina Tipo V/genética , Neurospora crassa/genética , Membrana Celular/genética , Polaridade Celular/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/genética , Proteínas de Fluorescência Verde/genética , Hifas/crescimento & desenvolvimento , Neurospora crassa/crescimento & desenvolvimento

RESUMO

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.

Assuntos

Infecções Oportunistas Relacionadas com a AIDS/microbiologia , Candida albicans/isolamento & purificação , Candidíase Bucal/microbiologia , Infecções por HIV/complicações , Mucosa Bucal/microbiologia , Infecções Oportunistas Relacionadas com a AIDS/tratamento farmacológico , Adolescente , Adulto , Antifúngicos/uso terapêutico , Candida albicans/classificação , Candidíase Bucal/classificação , Candidíase Bucal/tratamento farmacológico , Criança , Pré-Escolar , Estudos Transversais , Farmacorresistência Fúngica , Feminino , Fluconazol/uso terapêutico , Infecções por HIV/tratamento farmacológico , Humanos , Lactente , Itraconazol/uso terapêutico , Masculino , México , Estudos Prospectivos , Fatores de Risco , Carga Viral , Adulto Jovem

RESUMO

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 .

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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.

Assuntos

Humanos , Masculino , Feminino , Lactente , Pré-Escolar , Criança , Adolescente , Adulto , Adulto Jovem , Candida albicans/isolamento & purificação , Candidíase Bucal/microbiologia , Infecções por HIV/complicações , Infecções Oportunistas Relacionadas com a AIDS/microbiologia , Mucosa Bucal/microbiologia , Candida albicans/classificação , Candidíase Bucal/classificação , Candidíase Bucal/tratamento farmacológico , Fluconazol/uso terapêutico , Infecções por HIV/tratamento farmacológico , Estudos Transversais , Estudos Prospectivos , Fatores de Risco , Infecções Oportunistas Relacionadas com a AIDS/tratamento farmacológico , Itraconazol/uso terapêutico , Carga Viral , Farmacorresistência Fúngica , México , Antifúngicos/uso terapêutico

RESUMO

The present study examines the notion that polarized exocytosis in the tips of growing hyphae creates an excess of plasma membrane and thus the need for its removal by endocytosis. To measure endocytosis experimentally, we developed a photobleaching (FRAP) procedure to count endocytic events in hyphae of Neurospora crassa carrying a fluorescent tag on the actin-binding protein fimbrin (FIM-1-GFP). Given 40 nm as the average diameter of endocytic vesicles, we calculated that about 12.5% of the plasma membrane discharged in the apex becomes endocytosed in the subapex. According to our calculations, the GFP-tagged hyphae of N. crassa, measured under the constrained conditions of confocal microscopic examination, needed about 8800 vesicles/min to extend their plasma membrane or about 9800/min, if we include predicted demands for cell wall growth and extracellular secretion. Our findings support the notion that exocytosis and endocytosis operate in tandem with the latter serving as a compensatory process to remove any excess of plasma membrane generated by the intense exocytosis in the hyphal tips. Presumably, this tandem arrangement evolved to support the hallmark features of fungi namely rapid cell extension and abundant secretion of hydrolytic enzymes.

Assuntos

Endocitose/genética , Exocitose/genética , Hifas/genética , Glicoproteínas de Membrana/genética , Proteínas dos Microfilamentos/genética , Citoesqueleto de Actina/genética , Membrana Celular/genética , Parede Celular/genética , Proteínas Fúngicas/genética , Proteínas de Fluorescência Verde/genética , Hifas/crescimento & desenvolvimento , Neurospora crassa/genética , Fotodegradação

RESUMO

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.

Assuntos

Antifúngicos/farmacologia , Candida/classificação , Candida/efeitos dos fármacos , Candidíase Bucal/epidemiologia , Portador Sadio/epidemiologia , Infecções por HIV/complicações , Adulto , Idoso , Idoso de 80 Anos ou mais , Candida/genética , Candida/isolamento & purificação , Candidíase Bucal/microbiologia , Portador Sadio/microbiologia , DNA Fúngico/genética , Feminino , Humanos , Masculino , México/epidemiologia , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Prevalência , Adulto Jovem

Assuntos

Fungos/crescimento & desenvolvimento , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fungos/genética , Fungos/metabolismo , Genoma Fúngico

RESUMO

Neosartorya fischeri, an Aspergillaceae fungus, was evaluated in its capacity to transform high molecular weight polycyclic aromatics hydrocarbons (HMW-PAHs) and the recalcitrant fraction of petroleum, the asphaltenes. N. fischeri was able to grow in these compounds as sole carbon source. Coronene, benzo(g,h,i)perylene, and indeno(1,2,3-c,d)pyrene, together with the asphaltenes, were assayed for fungal biotransformation. The transformation of the asphaltenes and HMW-PAHs was confirmed by reverse-phase high-performance liquid chromatography (HPLC), nano-LC mass spectrometry, and IR spectrometry. The formation of hydroxy and ketones groups on the PAH molecules suggest a biotransformation mediated by monooxygenases such as cytochrome P450 system (CYP). A comparative microarray with the complete genome from N. fischeri showed three CYP monooxygenases and one flavin monooxygenase genes upregulated. These findings, together with the internalization of aromatic substrates into fungal cells and the microsomal transformation of HMW-PAHs, strongly support the role of CYPs in the oxidation of these recalcitrant compounds.

Assuntos

Neosartorya/metabolismo , Petróleo , Hidrocarbonetos Policíclicos Aromáticos/metabolismo , Biotransformação , Cromatografia Líquida de Alta Pressão , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genoma Fúngico , Análise em Microsséries , Peso Molecular , Oxirredução , Hidrocarbonetos Policíclicos Aromáticos/química

RESUMO

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.

Assuntos

Citoesqueleto de Actina/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Miosinas/genética , Miosinas/metabolismo , Neurospora crassa/genética , Neurospora crassa/metabolismo , Endocitose , Genoma Fúngico , Proteínas de Fluorescência Verde , Hifas/crescimento & desenvolvimento , Hifas/metabolismo , Hifas/ultraestrutura , Morfogênese , Mutação , Neurospora crassa/crescimento & desenvolvimento , Fenótipo , Esporos Fúngicos/fisiologia

RESUMO

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.

Assuntos

1-Alquil-2-acetilglicerofosfocolina Esterase/genética , Proteínas Fúngicas/genética , Proteínas Associadas aos Microtúbulos/genética , Neurospora crassa/genética , 1-Alquil-2-acetilglicerofosfocolina Esterase/química , Sequência de Aminoácidos , Núcleo Celular/metabolismo , Complexo Dinactina , Dineínas/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Expressão Gênica , Humanos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Mutação , Neurospora crassa/metabolismo , Ligação Proteica , Transporte Proteico , Proteínas Recombinantes de Fusão , Alinhamento de Sequência

RESUMO

Hyphae of the Ascomycota are tubular cells compartmentalized by perforated septa, whose central pore allows the flow of organelles and cytoplasm. While in plants and yeast septation leads to cell separation, in filamentous fungi the formation of crosswalls appears to have an architectural role, limits the extent of mechanical damage thus maintaining hyphal integrity, and also is of fundamental importance as part of cell differentiation. The increasing number of available fungal genome sequences, knockout mutants, versatile tools for protein tagging, and the continuous improvement of fluorescence microscopes have allowed scientists to analyze living cells and reveal the molecular and cellular basis of septation with unprecedented detail. This review summarizes the recent advances in septum ontogenesis in Neurospora crassa. A "septal actomyosin tangle" is the first indication of impending septation. It assembles prior to any visible evidence of plasma membrane inward growth, which occurs concomitantly with the formation and constriction of a contractile actomyosin ring and synthesis of the septum wall. One of the key questions in septum biogenesis is how the septation machinery is assembled to construct a centripetally growing crosswall. Most of the machinery utilized in apical cell wall growth can be expected at septation sites to ensure an organized arrival and supply of vesicles leading to the formation of a septum. Yet, the intrinsically different architecture of the septum may require a different organization and regulation of the wall-synthesizing machinery.

Assuntos

Parede Celular/metabolismo , Hifas/metabolismo , Neurospora crassa/metabolismo , Citoesqueleto de Actina/ultraestrutura , Transporte Biológico , Parede Celular/fisiologia , Parede Celular/ultraestrutura , Proteínas Fúngicas/metabolismo , Hifas/crescimento & desenvolvimento , Neurospora crassa/crescimento & desenvolvimento , Neurospora crassa/fisiologia

RESUMO

Neurospora crassa has been at the forefront of biological research from the early days of biochemical genetics to current progress being made in understanding gene and genetic network function. Here, we discuss recent developments in analysis of the fundamental form of fungal growth, development and proliferation -- the hypha. Understanding the establishment and maintenance of polarity, hyphal elongation, septation, branching and differentiation are at the core of current research. The advances in the identification and functional dissection of regulatory as well as structural components of the hypha provide an expanding basis for elucidation of fundamental attributes of the fungal cell. The availability and continuous development of various molecular and microscopic tools, as utilized by an active and co-supportive research community, promises to yield additional important new discoveries on the biology of fungi.

Assuntos

Polaridade Celular , Hifas/citologia , Neurospora crassa/crescimento & desenvolvimento , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hifas/genética , Hifas/crescimento & desenvolvimento , Hifas/metabolismo , Modelos Biológicos , Neurospora crassa/citologia , Neurospora crassa/genética , Neurospora crassa/metabolismo

RESUMO

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.

Assuntos

Citoesqueleto de Actina/ultraestrutura , Actinas/análise , Neurospora crassa/ultraestrutura , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Biomarcadores/análise , Proteínas de Transporte/análise , Citocinese , Citoplasma/metabolismo , Proteínas de Fluorescência Verde/análise , Hifas/química , Hifas/crescimento & desenvolvimento , Hifas/metabolismo , Glicoproteínas de Membrana/análise , Proteínas dos Microfilamentos/análise , Proteínas dos Microfilamentos/metabolismo , Microscopia Confocal , Neurospora crassa/crescimento & desenvolvimento , Neurospora crassa/metabolismo , Tropomiosina/análise , Tropomiosina/metabolismo

RESUMO

We used confocal microscopy to evaluate nuclear dynamics in mature, growing hyphae of Neurospora crassa whose nuclei expressed histone H1-tagged green fluorescent protein (GFP). In addition to the H1-GFP wild-type (WT) strain, we examined nuclear displacement (passive transport) in four mutants deficient in microtubule-related motor proteins (ro-1, ro-3, kin-1, and a ro-1 kin-1 double mutant). We also treated the WT strain with benomyl and cytochalasin A to disrupt microtubules and actin microfilaments, respectively. We found that the degree of nuclear displacement in the subapical regions of all strains correlated with hyphal elongation rate. The WT strain and that the ro-1 kin-1 double mutant showed the highest correlation between nuclear movement and hyphal elongation. Although most nuclei seemed to move forward passively, presumably carried by the cytoplasmic bulk flow, a small proportion of the movement detected was either retrograde or accelerated anterograde. The absence of a specific microtubule motor in the mutants ro-1, ro-3, or kin-1 did not prevent the anterograde and retrograde migration of nuclei; however, in the ro-1 kin-1 double mutant retrograde migration was absent. In the WT strain, almost all nuclei were elongated, whereas in all other strains a majority of nuclei were nearly spherical. With only one exception, a sizable exclusion zone was maintained between the apex and the leading nucleus. The ro-1 mutant showed the largest nucleus exclusion zone; only the treatment with cytochalasin A abolished the exclusion zone. In conclusion, the movement and distribution of nuclei in mature hyphae appear to be determined by a combination of forces, with cytoplasmic bulk flow being a major determinant. Motor proteins probably play an active role in powering the retrograde or accelerated anterograde migrations of nuclei and may also contribute to passive anterograde displacement by binding nuclei to microtubules.

Assuntos

Núcleo Celular/metabolismo , Hifas/citologia , Hifas/metabolismo , Movimento , Neurospora crassa/citologia , Neurospora crassa/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Proteínas de Fluorescência Verde/metabolismo , Hifas/crescimento & desenvolvimento , Interfase , Centro Organizador dos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Modelos Biológicos , Neurospora crassa/crescimento & desenvolvimento , Análise de Regressão

RESUMO

By confocal microscopy, we analyzed microtubule (Mt) behavior during hyphal growth and branching in a Neurospora crassa strain whose Mts had been tagged with GFP. Images were assembled spatially and temporally to better understand the 3-D organization of the microtubular cytoskeleton and a clearer view of its dynamics. Cytoplasmic Mts were mainly arranged longitudinally along the hyphal tube. Straight segments were rare; most Mts showed a distinct helical curvature with a long pitch and a tendency to intertwine with one another to form a loosely braided network throughout the cytoplasm. This study revealed that the microtubular cytoskeleton of a hypha advances as a unit, i.e., as the cell elongates, it moves forward by bulk flow. Nuclei appeared trapped in the microtubular network and were carried forward in unison as the hypha elongated. During branching, one or more cortical Mts became associated with the incipient branch and were pulled into the emergence of the branch. As extension of the branch and distortion of the Mts continued, Mts soon were severed with both new Mt ends (+ and -) present in the new branch. Although the exact mechanisms for addition Mt recruitment into the branch remains an open question, the recorded evidence indicates both bulk insertion of established cortical parent-hypha Mts as well as in situ polymerization were involved. The latter conclusion was supported by FRAP studies showing evidence of Mt nucleation and polymerization assembly in the growing tip of the developing branch. Nuclei entered the branch entrapped in the advancing network of Mts.

Assuntos

Hifas/crescimento & desenvolvimento , Microtúbulos/fisiologia , Neurospora crassa/crescimento & desenvolvimento , Citoplasma/metabolismo , Recuperação de Fluorescência Após Fotodegradação/métodos , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Hifas/ultraestrutura , Microscopia Confocal/métodos , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Neurospora crassa/metabolismo , Neurospora crassa/ultraestrutura , Organelas/metabolismo

RESUMO

Objetivo. Se propone el cálculo de los años acumulados de vida productiva potencial perdidos (AAVPPP) para complementar la evaluación de las pérdidas por accidentes de trabajo. Material y métodos. A partir de los datos obtenidos del Departamento General de Medicina del Trabajo de la Gerencia Médica de Petróleos Mexicanos, se analizaron 8 638 accidentes de trabajo dictaminados entre el 1 de enero de 1990 y el 30 de junio de 1993. Las variables independientes consideradas fueron: sexo, edad, sitio y tipo de lesión, nivel de ingresos, y situación laboral; la variable dependiente fue el porcentaje de incapacidad permanente. A partir del indicador propuesto se calcularon los AAVPPP. Resultados. El 19.5 por ciento de los accidentados quedó con algún grado de secuela; el total de AAVPPP fue de 5 611, con un promedio por caso de 3.2 años; de las variables analizadas el sexo masculino, los niveles de ingreso más bajos, la situación contractual transitoria y las lesiones en mano y muñeca fueron las que más AAVPPP aportaron. Se observaron diferencias al comparar la jerarquización de algunas variables independientes en función de diversos criterios con el de AAVPPP. Conclusiones. Este indicador es aplicable en el área operativa de seguridad e higiene; complementa la información de indicadores similares y se considera útil para la planeación de programas y la evaluación de riesgos de trabajo

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There are different indicators to give priority to health problems within the area of occupational health; we propose the calculation of accummulated years of potential productive life lost (AYPPLL) to estimate the loss of material and human resources due to work accidents. Material and methods. A total of 8 638 work accidents were analized using this method. Independent variables were: sex, age, place and type of accident, subject's income and work conditions. The dependent variable was permanent disability. Results. Subjects who had some kind of permanent damage were 19.5%; the total number of AYPPLL was 5 611, with an average of 3.2 YPPLL per accident. Variables associated with the highest AYPPLL were the socio-demographic variables, male sex, lowest income levels, temporary work contracts and injuries of the hand and wrist. Conclusions. The AYPPLL method was found to be a valuable indicator of the safety and hygiene of the worksite and a useful tool for planning and evaluating work accident programs.

Assuntos

Humanos , Masculino , Feminino , Acidentes de Trabalho/estatística & dados numéricos , Estatísticas de Sequelas e Incapacidade , Avaliação da Deficiência , Eficiência