RESUMEN
SUMMARYThe endoplasmic reticulum (ER) is one of the most extensive organelles in eukaryotic cells. It performs crucial roles in protein and lipid synthesis and Ca2+ homeostasis. Most information on ER types, functions, organization, and domains comes from studies in uninucleate animal, plant, and yeast cells. In contrast, there is limited information on the multinucleate cells of filamentous fungi, i.e., hyphae. We provide an analytical review of existing literature to categorize different types of ER described in filamentous fungi while emphasizing the research techniques and markers used. Additionally, we identify the knowledge gaps that need to be resolved better to understand the structure-function correlation of ER in filamentous fungi. Finally, advanced technologies that can provide breakthroughs in understanding the ER in filamentous fungi are discussed.
Asunto(s)
Proteínas Fúngicas , Hongos , Animales , Proteínas Fúngicas/metabolismo , Hongos/metabolismo , Retículo Endoplásmico/metabolismo , Saccharomyces cerevisiae/metabolismo , HifaRESUMEN
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.
Asunto(s)
Proteínas Portadoras , Proteínas Fúngicas , Proteínas Asociadas a Microtúbulos , Neurospora crassa , Benomilo/metabolismo , Proteínas Portadoras/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Centro Organizador de los Microtúbulos/metabolismo , Microtúbulos/metabolismo , Neurospora crassa/genética , Neurospora crassa/metabolismo , Cuerpos Polares del Huso/metabolismo , Tubulina (Proteína)/genéticaRESUMEN
Extracellular vesicles (EVs) are membranous compartments produced by yeast and mycelial forms of several fungal species. One of the difficulties in perceiving the role of EVs during the fungal life, and particularly in cell wall biogenesis, is caused by the presence of a thick cell wall. One alternative to have better access to these vesicles is to use protoplasts. This approach has been investigated here with Aspergillus fumigatus, one of the most common opportunistic fungal pathogens worldwide. Analysis of regenerating protoplasts by scanning electron microscopy and fluorescence microscopy indicated the occurrence of outer membrane projections in association with surface components and the release of particles with properties resembling those of fungal EVs. EVs in culture supernatants were characterized by transmission electron microscopy and nanoparticle tracking analysis. Proteomic and glycome analysis of EVs revealed the presence of a complex array of enzymes related to lipid/sugar metabolism, pathogenic processes, and cell wall biosynthesis. Our data indicate that (i) EV production is a common feature of different morphological stages of this major fungal pathogen and (ii) protoplastic EVs are promising tools for undertaking studies of vesicle functions in fungal cells.IMPORTANCE Fungal cells use extracellular vesicles (EVs) to export biologically active molecules to the extracellular space. In this study, we used protoplasts of Aspergillus fumigatus, a major fungal pathogen, as a model to evaluate the role of EV production in cell wall biogenesis. Our results demonstrated that wall-less A. fumigatus exports plasma membrane-derived EVs containing a complex combination of proteins and glycans. Our report is the first to characterize fungal EVs in the absence of a cell wall. Our results suggest that protoplasts represent a promising model for functional studies of fungal vesicles.
Asunto(s)
Aspergillus fumigatus/fisiología , Vesículas Extracelulares/fisiología , Proteómica , Protoplastos/fisiología , Pared Celular/metabolismo , Vesículas Extracelulares/ultraestructura , Proteínas Fúngicas/metabolismo , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Biogénesis de Organelos , Protoplastos/ultraestructuraRESUMEN
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.
Asunto(s)
Citoesqueleto de Actina/genética , Hifa/genética , Miosina Tipo V/genética , Neurospora crassa/genética , Membrana Celular/genética , Polaridad Celular/genética , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica/genética , Proteínas Fluorescentes Verdes/genética , Hifa/crecimiento & desarrollo , Neurospora crassa/crecimiento & desarrolloRESUMEN
In Neurospora crassa hyphae the localization of all seven chitin synthases (CHSs) at the Spitzenkörper (SPK) and at developing septa has been well analyzed. Hitherto, the mechanisms of CHSs traffic and sorting from synthesis to delivery sites remain largely unexplored. In Saccharomyces cerevisiae exit of Chs3p from the endoplasmic reticulum (ER) requires chaperone Chs7p. Here, we analyzed the role of CSE-7, N. crassa Chs7p orthologue, in the biogenesis of CHS-4 (orthologue of Chs3p). In a N. crassa Δcse-7 mutant, CHS-4-GFP no longer accumulated at the SPK and septa. Instead, fluorescence was retained in hyphal subapical regions in an extensive network of elongated cisternae (NEC) referred to previously as tubular vacuoles. In a complemented strain expressing a copy of cse-7 the localization of CHS-4-GFP at the SPK and septa was restored, providing evidence that CSE-7 is necessary for the localization of CHS-4 at hyphal tips and septa. CSE-7 was revealed at delimited regions of the ER at the immediacies of nuclei, at the NEC, and remarkably also at septa and the SPK. The organization of the NEC was dependent on the cytoskeleton. SEC-63, an extensively used ER marker, and NCA-1, a SERCA-type ATPase previously localized at the nuclear envelope, were used as markers to discern the nature of the membranes containing CSE-7. Both SEC-63 and NCA-1 were found at the nuclear envelope, but also at regions of the NEC. However, at the NEC only NCA-1 co-localized extensively with CSE-7. Observations by transmission electron microscopy revealed abundant rough ER sheets and distinct electron translucent smooth flattened cisternae, which could correspond collectively to the NEC, thorough the subapical cytoplasm. This study identifies CSE-7 as the putative ER receptor for its cognate cargo, the polytopic membrane protein CHS-4, and elucidates the complexity of the ER system in filamentous fungi.
Asunto(s)
Quitina Sintasa/genética , Hifa/genética , Proteínas de la Membrana/genética , Chaperonas Moleculares/genética , Neurospora crassa/genética , Proteínas de Saccharomyces cerevisiae/genética , Núcleo Celular/genética , Citoplasma/genética , Retículo Endoplásmico/genética , Proteínas Fúngicas/genética , Proteínas Fluorescentes Verdes/genética , Hifa/crecimiento & desarrollo , Microtúbulos/genética , Neurospora crassa/crecimiento & desarrollo , Transporte de Proteínas/genética , Saccharomyces cerevisiae/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genéticaRESUMEN
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 â¼75%; 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.
Asunto(s)
1-Alquil-2-acetilglicerofosfocolina Esterasa/genética , Proteínas Fúngicas/genética , Proteínas Asociadas a Microtúbulos/genética , Neurospora crassa/genética , 1-Alquil-2-acetilglicerofosfocolina Esterasa/química , Secuencia de Aminoácidos , Núcleo Celular/metabolismo , Complejo Dinactina , Dineínas/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Expresión Génica , Humanos , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Mitocondrias/metabolismo , Datos de Secuencia Molecular , Mutación , Neurospora crassa/metabolismo , Unión Proteica , Transporte de Proteínas , Proteínas Recombinantes de Fusión , Alineación de SecuenciaRESUMEN
A field survey of ballistosporic yeasts in a Neotropical forest yielded a new species isolated from a fern leaf. The isolate is a cream-colored butyrous yeast that reproduces by budding. Budding occurs at both the apical and basal cell poles; occasionally multiple budding events co-occur, giving rise to rosette-like clusters of cells at both poles of the yeast mother cell. DNA sequences of large and small subunit and the internal transcribed spacer regions of the nuclear ribosomal DNA cistron indicated an affinity to Microbotryomycetes, Pucciniomycotina. A new genus, Meredithblackwellia, is proposed to accommodate the new species, M. eburnea (type strain MCA4105). Based on phylogenetic analyses, Meredithblackwellia is related to Kriegeria eriophori, a sedge parasite, to an aquatic fungus Camptobasidium hydrophilum and to several recently described anamorphic yeasts that have been isolated from plant material or psychrophilic environments. Morphological and ultrastructural studies confirm the relatedness of M. eburnea to these taxa and prompted the re-evaluation of higher-level classification within Microbotryomycetes. We propose here a new order, Kriegeriales, and place two families, Kriegeriaceae fam. nov. and Camptobasidiaceae R.T. Moore, within it. Our study re-emphasizes the need for systematic revision of species described in Rhodotorula.
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Basidiomycota/clasificación , Levaduras/clasificación , Secuencia de Bases , Basidiomycota/aislamiento & purificación , Basidiomycota/fisiología , Basidiomycota/ultraestructura , ADN de Hongos/química , ADN de Hongos/genética , ADN Ribosómico/química , ADN Ribosómico/genética , ADN Espaciador Ribosómico/química , ADN Espaciador Ribosómico/genética , Helechos/microbiología , Guyana , Datos de Secuencia Molecular , Tipificación de Secuencias Multilocus , Técnicas de Tipificación Micológica , Filogenia , Análisis de Secuencia de ADN , Esporas Fúngicas/ultraestructura , Levaduras/aislamiento & purificación , Levaduras/fisiología , Levaduras/ultraestructuraRESUMEN
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.
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Polaridad Celular , Hifa/citología , Neurospora crassa/crecimiento & desarrollo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hifa/genética , Hifa/crecimiento & desarrollo , Hifa/metabolismo , Modelos Biológicos , Neurospora crassa/citología , Neurospora crassa/genética , Neurospora crassa/metabolismoRESUMEN
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.
Asunto(s)
Citoesqueleto de Actina/ultraestructura , Actinas/análisis , Neurospora crassa/ultraestructura , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Biomarcadores/análisis , Proteínas Portadoras/análisis , Citocinesis , Citoplasma/metabolismo , Proteínas Fluorescentes Verdes/análisis , Hifa/química , Hifa/crecimiento & desarrollo , Hifa/metabolismo , Glicoproteínas de Membrana/análisis , Proteínas de Microfilamentos/análisis , Proteínas de Microfilamentos/metabolismo , Microscopía Confocal , Neurospora crassa/crecimiento & desarrollo , Neurospora crassa/metabolismo , Tropomiosina/análisis , Tropomiosina/metabolismoRESUMEN
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.
Asunto(s)
Núcleo Celular/metabolismo , Hifa/citología , Hifa/metabolismo , Movimiento , Neurospora crassa/citología , Neurospora crassa/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo , Proteínas Fluorescentes Verdes/metabolismo , Hifa/crecimiento & desarrollo , Interfase , Centro Organizador de los Microtúbulos/metabolismo , Microtúbulos/metabolismo , Modelos Biológicos , Neurospora crassa/crecimiento & desarrollo , Análisis de RegresiónRESUMEN
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.