Visualization of vacuoles in Aspergillus oryzae by expression of CPY–EGFP

Ohneda, Mamoru; Arioka, Manabu; Nakajima, Harushi; Kitamoto, Katsuhiko

doi:10.1016/s1087-1845(02)00033-6

Cited by 54 publications

(43 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In the region before the first septum the vacuolar markers were abundant in an interconnected network of thin (Ͻ1 m) tubules, often with small, attached vesicles. These results with N. crassa are generally consistent with observations made using GFPtagged VAM-3 in Aspergillus oryzae (45,54). N. crassa grows faster, and the diameter of hyphae can be fivefold larger.…”

Section: Downloaded Fromsupporting

confidence: 88%

Structure and Distribution of Organelles and Cellular Location of Calcium Transporters in Neurospora crassa

et al. 2009

View full text Add to dashboard Cite

We wanted to examine the cellular locations of four Neurospora crassa proteins that transport calcium. However, the structure and distribution of organelles in live hyphae of N. crassa have not been comprehensively described. Therefore, we made recombinant genes that generate translational fusions of putative organellar marker proteins with green or red fluorescent protein. We observed putative endoplasmic reticulum proteins, encoded by grp-78 and dpm, in the nuclear envelope and associated membranes. Proteins of the vacuolar membrane, encoded by vam-3 and vma-1, were in an interconnected network of small tubules and vesicles near the hyphal tip, while in more distal regions they were in large and small spherical vacuoles. Mitochondria, visualized with tagged ARG-4, were abundant in all regions of the hyphae. Similarly, we tagged the four N. crassa proteins that transport calcium with green or red fluorescent protein to examine their cellular locations. NCA-1 protein, a homolog of the SERCA-type Ca 2؉ -ATPase of animal cells, colocalized with the endoplasmic reticulum markers. The NCA-2 and NCA-3 proteins are homologs of Ca 2؉ -ATPases in the vacuolar membrane in yeast or in the plasma membrane in animal cells. They colocalized with markers in the vacuolar membrane, and they also occurred in the plasma membrane in regions of the hyphae more than 1 mm from the tip. The cax gene encodes a Ca 2؉ /H ؉ exchange protein found in vacuoles. As expected, the CAX protein localized to the vacuolar compartment. We observed, approximately 50 to 100 m from the tip, a few spherical organelles that had high amounts of tagged CAX protein and tagged subunits of the vacuolar ATPase (VMA-1 and VMA-5). We suggest that this organelle, not described previously in N. crassa, may have a role in sequestering calcium.

show abstract

Section: Downloaded Fromsupporting

confidence: 88%

Structure and Distribution of Organelles and Cellular Location of Calcium Transporters in Neurospora crassa

et al. 2009

View full text Add to dashboard Cite

show abstract

“…To observe the aerial hyphae and conidiophores, the conidia of CEDR1 were inoculated on minimal agar medium (CD) on glass slides and then coverslips were placed on top of the culture setup. After incubation for 3 days at 30°C, aerial hyphae and conidiophores elongating from the medium into the air were observed with a fluorescence microscope (23). The aerial hyphae and conidiophores of CEDR1 were observed by differential interference and fluorescence microscopy.…”

Section: Methodsmentioning

confidence: 99%

Functional Analysis of the ATG8 Homologue Ao atg8 and Role of Autophagy in Differentiation and Germination in Aspergillus oryzae

et al. 2006

Self Cite

View full text Add to dashboard Cite

Autophagy is a well-known degradation system, induced by nutrient starvation, in which cytoplasmic components and organelles are digested via vacuoles/lysosomes. Recently, it was reported that autophagy is involved in the turnover of cellular components, development, differentiation, immune responses, protection against pathogens, and cell death. In this study, we isolated the ATG8 gene homologue Aoatg8 from the filamentous fungus Aspergillus oryzae and visualized autophagy by the expression of DsRed2-AoAtg8 and enhanced green fluorescent protein-AoAtg8 fusion proteins in this fungus. While the fusion proteins were localized in dot structures which are preautophagosomal structure-like structures under normal growth conditions, starvation or rapamycin treatment caused their accumulation in vacuoles. DsRed2 expressed in the cytoplasm was also taken up into vacuoles under starvation conditions or during the differentiation of conidiophores and conidial germination. Deletion mutants of Aoatg8 did not form aerial hyphae and conidia, and DsRed2 was not localized in vacuoles under starvation conditions, indicating that Aoatg8 is essential for autophagy. Furthermore, Aoatg8 conditional mutants showed delayed conidial germination in the absence of nitrogen sources. These results suggest that autophagy functions in both the differentiation of aerial hyphae and in conidial germination in A. oryzae.Autophagy is a protein degradation system that is conserved in eukaryotic cells and used to recycle macromolecules and aid cell survival under nutritional starvation conditions (12, 13). When autophagy is induced, bulk cytoplasm and/or organelles are sequestered within double-membrane vesicles termed autophagosomes. The outer membranes of the autophagosomes then fuse to the vacuolar/lysosomal membrane and deliver single-membrane vesicles, called autophagic bodies, into the lumina of the vacuoles/lysosomes. The subsequent breakdown of the vesicle membranes allows the degradation of the contents of the autophagic bodies by vacuolar hydrolases. ATG8 is an autophagy-related gene found in Saccharomyces cerevisiae that plays an important role in the formation of autophagosomes (9). Atg8 is localized in the membranes of preautophagosomal structures (PAS), autophagosomes, and autophagic bodies and has therefore been used as a marker of these organelles (33).In addition to helping cells to survive starvation, autophagy is involved in stress-induced differentiation and development. In S. cerevisiae diploid cells, atg mutations block starvationinduced sporulation (34). In Dictyostelium discoideum, starvation, overcrowding, and high temperature induce the formation of fruiting bodies and atg mutations block these multicellular developmental processes (25). Additionally, in Caenorhabditis elegans, atg mutations result in abnormal dauer development, which is also induced by starvation, overcrowding, high temperature and so on (17). Recent studies have suggested that autophagy might participate in diseases such as cancer, liver disease, muscula...

show abstract

“…8) Here, we report thê rst in vivo observation of the intracellular distribution of a secretory protein in A. oryzae hyphae using an EGFP-tagged fusion system. We also observed the eŠects of cold or heat shock on the distribution of the fusion protein inside hyphae.…”

mentioning

confidence: 99%

In vivoVisualization of the Distribution of a Secretory Protein inAspergillus oryzaeHyphae Using the RntA-EGFP Fusion Protein

Masai

Maruyama

Nakajima

et al. 2003

Bioscience, Biotechnology, and Biochemistry

Self Cite

View full text Add to dashboard Cite

A fusion gene encoding ribonuclease T 1 -EGFP (rntAegfp) was constructed and expressed to use it as a tool for studies on the secretory pathway in Aspergillus oryzae. The successful secretion of the intact RntA-EGFP fusion protein was detected by ‰uorescence measurement and Western analysis. With use of the RntA-EGFP system, we were able to see high ‰uorescence at hyphal tips and observe concentrated ‰uorescence at septa in basal cells during growth at optimal conditions. Cold or heat shock during growth caused the accumulation of EGFP ‰uorescence in vacuoles.

show abstract

Visualization of vacuoles in Aspergillus oryzae by expression of CPY–EGFP

Cited by 54 publications

References 31 publications

Structure and Distribution of Organelles and Cellular Location of Calcium Transporters in Neurospora crassa

Structure and Distribution of Organelles and Cellular Location of Calcium Transporters in Neurospora crassa

Functional Analysis of the ATG8 Homologue Ao atg8 and Role of Autophagy in Differentiation and Germination in Aspergillus oryzae

In vivoVisualization of the Distribution of a Secretory Protein inAspergillus oryzaeHyphae Using the RntA-EGFP Fusion Protein

Contact Info

Product

Resources

About