Searching for gold beyond mitosis: Mining intracellular membrane traffic in Aspergillus nidulans.
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ABSTRACT:
SUBMITTER: Penalva MA
PROVIDER: S-EPMC3355971 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Mitosis in Aspergillus nidulans is very rapid, requiring less than 5 min at 37 °C in germlings (Bergen and Morris, 1983). In this time the cytoplasmic microtubules (MTs) must disassemble, the mitotic spindle assemble, function and disassemble, and cytoplasmic MTs reassemble. It follows that cytoplasmic MTs must be extremely dynamic in this period and we were interested, in particular, in examining the processes of MT disassembly in prophase and reassembly in anaphase and telophase. We observed a diploid strain that expressed GFP-?-tubulin. We used a spinning disk confocal microscope that allowed rapid image capture, which proved necessary because microtubule dynamics were extremely rapid. We found, for the first time, that microtubule severing occurs in prophase in a filamentous fungus and that catastrophe rather than nucleation limits astral microtubule growth.
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Project description:Glutathione (GSH) is an abundant and widely distributed antioxidant in fungi. Hence, understanding cellular GSH metabolism is of vital importance to deciphering redox regulation in these microorganisms. In this study, we generated dugB (AN1879), dugC (AN1092), and dugB dugC double deletion mutants which display disruption of the GSH degradation pathway in Aspergillus nidulans. Deletion of dugB, dugC or both resulted in a moderate increase in GSH content under growing conditions and substantially slowed down the depletion of GSH pools under carbon starvation. Inactivation of dug genes caused reduced accumulation of reactive oxygen species, decreased autolytic cell wall degradation and extracellular enzyme production, increased sterigmatocystin formation but decreased viability in starving cultures. Changes in the transcriptomes suggested that enzyme secretions were controlled at post transcriptional level. In contrast, secondary metabolite production was also regulated at the level of mRNA abundance. Based on these findings, we suggest that GSH connects starvation and redox regulation to each other: A. nidulans cells utilize GSH as stored carbon source during starvation. The reduction of GSH contents of cells alters the redox state activating regulatory pathways responsible for carbon starvation stress responses. Under glucose rich conditions, inactivation of dug genes reduced conidia production of surface cultures, disturbed sexual development and down-regulated the transcription of genes encoding MAP kinase pathway proteins (e.g. steC, sskB, pbsA, hogA, mkkA) or proteins involved in the regulation of conidiogenesis or sexual differentiation (e.g. flbA,C,E, nosA, rosA, nsdC,D). These finding indicates that the authority of redox regulation goes far beyond the protection against redox stress; it affects development, stress responses (other than redox stress) and secondary metabolism as well.
2021-03-02 | GSE151461 | GEO
Project description:Membrane traffic between organelles is essential for a multitude of processes that maintain cell homeostasis. Many steps in these tightly regulated trafficking pathways take place in microdomains on the membranes of organelles, which require analysis at nanometer resolution. Electron microscopy (EM) can visualize these processes in detail and is mainly responsible for our current view of morphology on the subcellular level. This review discusses how EM can be applied to solve many questions of intracellular membrane traffic, with a focus on the endosomal system. We describe the expansion of the technique from purely morphological analysis to cryo-immuno-EM, correlative light electron microscopy (CLEM), and 3D electron tomography. In this review we go into some technical details of these various techniques. Furthermore, we provide a full protocol for immunolabeling on Lowicryl sections of high-pressure frozen cells as well as a detailed description of a simple CLEM method that can be applied to answer many membrane trafficking questions. We believe that these EM-based techniques are important tools to expand our understanding of the molecular details of endosomal sorting and intracellular membrane traffic in general.
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Project description:Aspergillus nidulans hyphae grow exclusively by apical extension. Golgi equivalents (GEs) labeled with mRFP-tagged PH(OSBP) domain form a markedly polarized, dynamic network of ring-shaped and fenestrated cisternae that remains intact during "closed" mitosis. mRFP-PH(OSBP) GEs advance associated with the growing apex where secretion predominates but do not undergo long-distance movement toward the tip that could account for their polarization. mRFP-PH(OSBP) GEs overlap with the trans-Golgi resident Sec7 but do not colocalize with also polarized accretions of the early Golgi marker GrhA(Grh1)-GFP, indicating that early and late Golgi membranes segregate spatially. AnSec23-GFP ER exit sites (ERES) are numerous, relatively static foci localizing across the entire cell. However, their density is greatest near the tip, correlating with predominance of early and trans-Golgi elements in this region. Whereas GrhA-GFP structures and ERES reach the apical dome, mRFP-PH(OSBP) GEs are excluded from this region, which contains the endosome dynein loading zone. After latrunculin-mediated F-actin disruption, mRFP-PH(OSBP) GEs fragment and, like AnSec23-GFP ERES, depolarize. Brefeldin A transiently collapses late and early GEs into distinct aggregates containing Sec7/mRFP-PH(OSBP) and GrhA-GFP, respectively, temporarily arresting apical extension. Rapid growth reinitiates after washout, correlating with reacquisition of the normal Golgi organization that, we conclude, is required for apical extension.
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Project description:The genetically amenable fungus Aspergillus nidulans is well suited for cell biology studies involving the secretory pathway and its relationship with hyphal tip growth by apical extension. We exploited live-cell epifluorescence microscopy of the ER labeled with the translocon component Sec63, endogenously tagged with GFP, to study the organization of 'secretory' ER domains. The Sec63 A. nidulans ER network includes brightly fluorescent peripheral strands and more faintly labeled nuclear envelopes. In hyphae, the most abundant peripheral ER structures correspond to plasma membrane-associated strands that are polarized, but do not invade the hyphal tip dome, at least in part because the subapical collar of endocytic actin patches constrict the cortical strands in this region. Thus the subapical endocytic ring might provide an attachment for ER strands, thereby ensuring that the growing tip remains 'loaded' with secretory ER. Acute disruption of secretory ER function by reductive stress-mediated induction of the unfolded protein response results in the reversible aggregation of ER strands, cessation of exocytosis and swelling of the hyphal tips. The secretory ER is insensitive to brefeldin A treatment and does not undergo changes during mitosis, in agreement with the reports that apical extension continues at normal rates during this period.
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Project description:How the nucleolus is segregated during mitosis is poorly understood and occurs by very different mechanisms during closed and open mitosis. Here we report a new mechanism of nucleolar segregation involving removal of the nucleolar-organizing regions (NORs) from nucleoli during Aspergillus nidulans mitosis. This involves a double nuclear envelope (NE) restriction which generates three NE-associated structures, two daughter nuclei (containing the NORs), and the nucleolus. Therefore, a remnant nucleolar structure can exist in the cytoplasm without NORs. In G1, this parental cytoplasmic nucleolus undergoes sequential disassembly releasing nucleolar proteins to the cytoplasm as nucleoli concomitantly reform in daughter nuclei. By depolymerizing microtubules and mutating spindle assembly checkpoint function, we demonstrate that a cycle of nucleolar "segregation" can occur without a spindle in a process termed spindle-independent mitosis (SIM). During SIM physical separation of the NOR from the nucleolus occurs, and NE modifications promote expulsion of the nucleolus to the cytoplasm. Subsequently, the cytoplasmic nucleolus is disassembled and rebuilt at a new site around the nuclear NOR. The data demonstrate the existence of a mitotic machinery for nucleolar segregation that is normally integrated with mitotic spindle formation but that can function without it.
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Project description:Glutathione (GSH) is an abundant tripeptide that plays a crucial role in shielding cellular macromolecules from various reactive oxygen and nitrogen species in fungi. Understanding GSH metabolism is of vital importance for deciphering redox regulation in these microorganisms. In the present study, to better understand the GSH metabolism in filamentous fungi, we investigated functions of the dugB and dugC genes in the model fungus Aspergillus nidulans These genes are orthologues of dug2 and dug3, which are involved in cytosolic GSH degradation in Saccharomyces cerevisiae The deletion of dugB, dugC, or both resulted in a moderate increase in the GSH content in mycelia grown on glucose, reduced conidium production, and disturbed sexual development. In agreement with these observations, transcriptome data showed that genes encoding mitogen-activated protein (MAP) kinase pathway elements (e.g., steC, sskB, hogA, and mkkA) or regulatory proteins of conidiogenesis and sexual differentiation (e.g., flbA, flbC, flbE, nosA, rosA, nsdC, and nsdD) were downregulated in the ΔdugB ΔdugC mutant. Deletion of dugB and/or dugC slowed the depletion of GSH pools during carbon starvation. It also reduced accumulation of reactive oxygen species and decreased autolytic cell wall degradation and enzyme secretion but increased sterigmatocystin formation. Transcriptome data demonstrated that enzyme secretions-in contrast to mycotoxin production-were controlled at the posttranscriptional level. We suggest that GSH connects starvation and redox regulation to each other: cells utilize GSH as a stored carbon source during starvation. The reduction of GSH content alters the redox state, activating regulatory pathways responsible for carbon starvation stress responses.IMPORTANCE Glutathione (GSH) is a widely distributed tripeptide in both eukaryotes and prokaryotes. Owing to its very low redox potential, antioxidative character, and high intracellular concentration, GSH profoundly shapes the redox status of cells. Our observations suggest that GSH metabolism and/or the redox status of cells plays a determinative role in several important aspects of fungal life, including oxidative stress defense, protein secretion, and secondary metabolite production (including mycotoxin formation), as well as sexual and asexual differentiations. We demonstrated that even a slightly elevated GSH level can substantially disturb the homeostasis of fungi. This information could be important for development of new GSH-producing strains or for any biotechnologically relevant processes where the GSH content, antioxidant capacity, or oxidative stress tolerance of a fungal strain is manipulated.
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Project description:The G2 DNA damage and slowing of S-phase checkpoints over mitosis function through tyrosine phosphorylation of NIMX(cdc2) in Aspergillus nidulans. We demonstrate that breaking these checkpoints leads to a defective premature mitosis followed by dramatic rereplication of genomic DNA. Two additional checkpoint functions, uvsB and uvsD, also cause the rereplication phenotype after their mutation allows premature mitosis in the presence of low concentrations of hydroxyurea. uvsB is shown to encode a rad3/ATR homologue, whereas uvsD displays homology to rad26, which has only previously been identified in Schizosaccharomyces pombe. uvsB(rad3) and uvsD(rad26) have G2 checkpoint functions over mitosis and another function essential for surviving DNA damage. The rereplication phenotype is accompanied by lack of NIME(cyclinB), but ectopic expression of active nondegradable NIME(cyclinB) does not arrest DNA rereplication. DNA rereplication can also be induced in cells that enter mitosis prematurely because of lack of tyrosine phosphorylation of NIMX(cdc2) and impaired anaphase-promoting complex function. The data demonstrate that lack of checkpoint control over mitosis can secondarily cause defects in the checkpoint system that prevents DNA rereplication in the absence of mitosis. This defines a new mechanism by which endoreplication of DNA can be triggered and maintained in eukaryotic cells.
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Project description:α-Glucan is a major cell wall component and a virulence and adhesion factor for fungal cells. However, the biosynthetic pathway of α-glucan was still unclear. α-Glucan was shown to be composed mainly of 1,3-glycosidically linked glucose, with trace amounts of 1,4-glycosidically linked glucose. Besides the α-glucan synthetases, amylase-like proteins were also important for α-glucan synthesis. In our previous work, we showed that Aspergillus nidulans AmyG was an intracellular protein and was crucial for the proper formation of α-glucan. In the present study, we expressed and purified AmyG in an Escherichia coli system. Enzymatic characterization found that AmyG mainly functioned as an α-amylase that degraded starch into maltose. AmyG also showed weak glucanotransferase activity. Most intriguingly, supplementation with maltose in shaken liquid medium could restore the α-glucan content and the phenotypic defect of a ΔamyG strain. These data suggested that AmyG functions mainly as an intracellular α-amylase to provide maltose during α-glucan synthesis in A. nidulans. IMPORTANCE Short α-1,4-glucan was suggested as the primer structure for α-glucan synthesis. However, the exact structure and its source remain elusive. AmyG was essential to promote α-glucan synthesis and had a major impact on the structure of α-glucan in the cell wall. Data presented here revealed that AmyG belongs to the GH13_5 family and showed strong amylase function, digesting starch into maltose. Supplementation with maltose efficiently rescued the phenotypic defect and α-glucan deficiency in an ΔamyG strain but not in an ΔagsB strain. These results provide the first piece of evidence for the primer structure of α-glucan in fungal cells, although it might be specific to A. nidulans.
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Project description:Cyanobacteria, the progenitors of plant and algal chloroplasts, enabled aerobic life on earth by introducing oxygenic photosynthesis. In most cyanobacteria, the photosynthetic membranes are arranged in multiple, seemingly disconnected, concentric shells. In such an arrangement, it is unclear how intracellular trafficking proceeds and how different layers of the photosynthetic membranes communicate with each other to maintain photosynthetic homeostasis. Using electron microscope tomography, we show that the photosynthetic membranes of two distantly related cyanobacterial species contain multiple perforations. These perforations, which are filled with particles of different sizes including ribosomes, glycogen granules and lipid bodies, allow for traffic throughout the cell. In addition, different layers of the photosynthetic membranes are joined together by internal bridges formed by branching and fusion of the membranes. The result is a highly connected network, similar to that of higher-plant chloroplasts, allowing water-soluble and lipid-soluble molecules to diffuse through the entire membrane network. Notably, we observed intracellular membrane-bounded vesicles, which were frequently fused to the photosynthetic membranes and may play a role in transport to these membranes.
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