A preparation method of specimens of the fungus <i>Penicillium chrysogenum</i> for ultrastructural and immuno‐electron microscopical studies

Müller, Wally H.; Krift, T.P. van der; Knoll, Gerd; Smaal, Erik B.; Verkleij, Arie J.

doi:10.1111/j.1365-2818.1991.tb03189.x

Cited by 16 publications

(6 citation statements)

References 25 publications

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“…Ultrathin HM20 sections of the yeast cells were mounted on nickel grids and incubated with affinity-purified anti-␤1,6-glucan polyclonal antibodies (1:300) (27). The antigen-antibody complex was visualized with secondary goat anti-rabbit antibodies (1:20) conjugated with 10-nm gold particles (Aurion, Wageningen, The Netherlands) (39). The labeled ultrathin sections were viewed in a Philips EM420 electron microscope, and micrographs were taken at an acceleration voltage of 80 kV.…”

Section: Methodsmentioning

confidence: 99%

Localization of Synthesis of β1,6-Glucan in Saccharomyces cerevisiae

et al. 1999

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β1,6-Glucan is a key component of the yeast cell wall, interconnecting cell wall proteins, β1,3-glucan, and chitin. It has been postulated that the synthesis of β1,6-glucan begins in the endoplasmic reticulum with the formation of protein-bound primer structures and that these primer structures are extended in the Golgi complex by two putative glucosyltransferases that are functionally redundant, Kre6 and Skn1. This is followed by maturation steps at the cell surface and by coupling to other cell wall macromolecules. We have reinvestigated the role of Kre6 and Skn1 in the biogenesis of β1,6-glucan. Using hydrophobic cluster analysis, we found that Kre6 and Skn1 show significant similarities to family 16 glycoside hydrolases but not to nucleotide diphospho-sugar glycosyltransferases, indicating that they are glucosyl hydrolases or transglucosylases instead of genuine glucosyltransferases. Next, using immunogold labeling, we tried to visualize intracellular β1,6-glucan in cryofixed sec1-1 cells which had accumulated secretory vesicles at the restrictive temperature. No intracellular labeling was observed, but the cell surface was heavily labeled. Consistent with this, we could detect substantial amounts of β1,6-glucan in isolated plasma membrane-derived microsomes but not in post-Golgi secretory vesicles. Taken together, our data indicate that the synthesis of β1,6-glucan takes place largely at the cell surface. An alternative function for Kre6 and Skn1 is discussed.

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Section: Methodsmentioning

confidence: 99%

Localization of Synthesis of β1,6-Glucan in Saccharomyces cerevisiae

et al. 1999

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“…It can be seen that there were no significant changes due to the treatment. with and without using the fixative of fixative of Mfiller et al(1991b). Miiller et al(1991b).…”

Section: Resultsmentioning

confidence: 99%

Fixation technique to preserve the vacuolation ofPenicillium chrysogenum hyphae

1994

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SummaryIt has been shown that a paraformaldehyde/glutaraldehyde/phosphate buffer saline fixative can be used to preserve vacuolation of Penicillium chrysogenum hyphae from submerged cultures for later image analysis characterisation. After fixing, the proportion of the hyphae as vacuoles and empty cells (in several samples from different fermentations with different initial percentage vacuolations ) did not change significantly .over about 70h, and there were only minor changes towards smaller vacuoles in the vacuolation size distributions over the same period. This fixation method will permit the storage of vacuolated hyphae for later analysis either for physiological studies or for dynamic studies of the links b~tween hyphal vacuolation and fragmentation.

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“…In basidiomycetes, Hoch and Howard (1981) reported that the SPC and the dolipore septa may be changed in their morphology because of the aldehyde fixation. Müller et al (1991) documented a distortion of the plasma membrane in chemically fixed and freeze-fractured hyphal cells compared with cryofixed and freeze-fractured hyphal cells. Although the combination of cryofixation and freeze substitution may result in better preservation of the subcellular ultrastructure in hyphal cells, many serial sections must be made for final viewing of the topology of their organelles.…”

Section: Discussionmentioning

confidence: 98%

“…However, chemical fixation of hyphae may cause ultrastructural changes of organelles and subcellular structures in hyphae from similar structures in living hyphae (Hoch and Howard 1981, Howard and O'Donnell 1987, Müller et al 1991. In basidiomycetes, Hoch and Howard (1981) reported that the SPC and the dolipore septa may be changed in their morphology because of the aldehyde fixation.…”

Section: Discussionmentioning

confidence: 99%

Field‐emission scanning electron microscopy of the internal cellular organization of fungi

et al. 2000

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Summary:Internal viewing of the cellular organization of hyphae by scanning electron microscopy is an alternative to observing sectioned fungal material with a transmission electron microscope. To study cytoplasmic organelles in the hyphal cells of fungi by SEM, colonies were chemically fixed with glutaraldehyde and osmium tetroxide and then immersed in dimethyl sulfoxide. Following this procedure, the colonies were frozen and fractured on a liquid nitrogen-precooled metal block. Next, the fractured samples were macerated in diluted osmium tetroxide to remove the cytoplasmic matrix and subsequently dehydrated by freeze substitution in methanol. After critical point drying, mounting, and sputter coating, fractured cells of several basidiomycetes were imaged with field-emission SEM. This procedure produced clear images of elongated and spherical mitochondria, the nucleus, intravacuolar structures, tubular-and plate-like endoplasmic reticulum, and different types of septal pore caps. This method is a powerful approach for studying the intracellular ultrastructure of fungi by SEM.

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A preparation method of specimens of the fungus Penicillium chrysogenum for ultrastructural and immuno‐electron microscopical studies

Cited by 16 publications

References 25 publications

Localization of Synthesis of β1,6-Glucan in Saccharomyces cerevisiae

Localization of Synthesis of β1,6-Glucan in Saccharomyces cerevisiae

Fixation technique to preserve the vacuolation ofPenicillium chrysogenum hyphae

Field‐emission scanning electron microscopy of the internal cellular organization of fungi

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