Structural rearrangement of the actin cytoskeleton in regenerating protoplasts of budding yeasts

Gabriel, Miroslav; Kopecká, Marie; Svoboda, Augustín

doi:10.1099/00221287-138-10-2229

Cited by 15 publications

(27 citation statements)

References 15 publications

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“…Protoplast cell-wall regeneration and electron microscopy of these cell walls Protoplasts were regenerated in 30% gelatinecontaining N1 medium. Their cell walls were isolated, washed and studied under transmission electron microscopy after metal shadowing [30,31].…”

Section: Formation Of Protoplasts and Isolation Of Their Fibrillar Nementioning

confidence: 99%

“…In contrast to what is observed in yeasts, the unicellular green alga Chlamydomonas reinhardii has cell walls that selfassemble in vitro from dissociated wall glycoprotein subunits in the presence of wall 'nucleation centres' [25,26]. Previously, we observed that yeast protoplasts that are devoid of their original cell walls can partially regenerate a new cell wall when grown on agar [27] or can completely regenerate a new cell wall when grown inside gelatine [28][29][30][31]. Our analyses of naked yeast protoplasts led us to hypothesize that the wall polymers synthesized and secreted to the external surface of naked protoplasts self-assemble de novo into cell walls around the regenerating protoplasts.…”

Section: Introductionmentioning

confidence: 99%

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Yeast and fungal cell-wall polysaccharides can self-assemblein vitrointo an ultrastructure resemblingin vivoyeast cell walls

Kopecká

2012

Microscopy (Tokyo)

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Polysaccharides account for more than 90% of the content of fungal cell walls, but the mechanism underlying the formation of the architecture of the cell walls, which consist of microfibrils embedded in an amorphous wall matrix, remains unknown. We used electron microscopy to investigate ten different fungal cell-wall polysaccharides to determine whether they could self-assemble into the fibrillar or amorphous component of fungal cell walls in a test tube without enzymes. The ultrastructures formed by precipitating β-1,3-glucan and β-1,6-glucan are different depending on the existence of branching in the molecule. Linear β-1,3-glucan and linear β-1,6-glucan precipitate into a fibrillar ultrastructure. Branched β-1,6-glucan, mannan and glycogen precipitates are amorphous. Branched β-1,3-glucan forms a fibrillar plus amorphous ultrastructure. Self-assembly among combinations of different linear and branched cell-wall polysaccharides results in an ultrastructure that resembles that of a yeast cell wall, which suggests that self-assembly of polysaccharides may participate in the development of the three-dimensional architecture of the yeast cell wall.

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Section: Formation Of Protoplasts and Isolation Of Their Fibrillar Nementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Yeast and fungal cell-wall polysaccharides can self-assemblein vitrointo an ultrastructure resemblingin vivoyeast cell walls

Kopecká

2012

Microscopy (Tokyo)

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“…Actin was found at sites where the cell wall synthesis is active like the bud, growing apices, and sites of septum formation (19). In regenerating protoplasts of the Saccharomyces cerevisiae, actin patches were found over the surface on which a new cell wall was being synthesized (20). During encystation, E. invadens undergo morphological changes from highly motile trophozoites to an immobile spherical cyst, which shows the involvement of cytoskeleton in the stage conversion.…”

Section: Introductionmentioning

confidence: 99%

Novel insights intoEntamoebacyst wall formation and the importance of actin cytoskeleton

Ghosh

Krishnan

Nayak

2020

Preprint

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21The cyst wall of Entamoeba histolytica, the causative agent of the amoebiasis, is a 22 potential target for new drugs. The "Wattle and Daub" model of cyst wall formation of 23 Entamoeba invadens had already been reported. In this study, we demonstrate in more detail the 24 morphological stages of chitin wall formation in E. invadens using fluorescent chitin-binding 25 dyes and immunolocalization of the cyst wall proteins. Here, the expression and localization of 26 chitin synthase and the importance of actin cytoskeleton dynamics at cellular level, during 27 encystations have been demonstrated for the first time. Chitin deposition was found to be 28 initiated on the cell surface mostly from one distinct point, though multipoint initiation was also 29 observed sometimes. From these points, the wall grew outwards and gradually covered the entire 30 cyst surface with time. The initiation of chitin deposition was guided by the localization of chitin 31 synthase 1 on the plasma membrane. The gradual formation of the cyst wall follows the Wattle 32 and daub model. The chitin deposition occurred on the foundation of Jacob lectin at the cell 33 membrane, and the other cyst wall components, like chitinase, and Jessie were also found to be 34 present in the growing incomplete walls. In contrary to the Wattle and daub model, Jessie was 35 found to be expressed and localized in the growing wall at the early hours of encystations. 36During encystation, F-actin was reorganized into the cortical region within an hour encystation 37 initiation and remained intact until the completion of the chitin wall. Disruption of cortical actin 38 polymerization with 2, 3-Butanedione monoxime inhibited proper wall formation but produced 39 wall-less cysts or cysts with defective chitin wall. Malformations of cyst-walls were mainly due 40 to improper localization and activity of chitin synthases, which indicates the indispensability of 41 cortical actin cytoskeleton for the proper cyst wall formation.42 3 43 Author Summary 44 Entamoeba parasites reach new hosts using the resistant cyst form, so preventing its 45 formation can stop the spread of amoebiasis. The resistant nature of the cyst is due to the chitin 46 wall, and thus identifying the critical steps of wall formation could provide targets for designing 47 new drugs. Here we studied the morphological stages of the cyst wall formation by observing 48 how the chitin and other cell wall components were deposited on the cell surface using 49 fluorescent chitin-binding dyes and antibodies against cyst wall proteins. In most cases, the 50 chitin wall was found to start from one distinct point from which it spread all over the cell 51 surface, guided by chitin synthase. The composition of these incomplete walls was the same as a 52 mature cyst wall indicating that the wall may be a result of extracellular self-assembly of its 53 constituents from one starting point. We have also observed that F-actin polymerized in the 54 cortex of encysting cells and its disruption resulted in wall-less...

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“…Actin Staining with Rhodamine Phalloidin Gabriel et al 24) have reported that actin, which maintains cellular architecture, is distributed evenly in spheroplasts and is locally concentrated during the regeneration of spheroplasts into competent cells. We therefore examined the profile of actin distribution in control and HM-1-treated spheroplasts (Fig.…”

Section: Dna Staining With Dapimentioning

confidence: 99%

Round Shape Enlargement of the Yeast Spheroplast of Saccharomyces cerevisiae by HM-1 Toxin.

Komiyama

Kimura

Furuichi

2002

Biological & Pharmaceutical Bulletin

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HM-1 killer toxin (HM-1) is a strong anti-yeast protein produced by the yeast Williopsis saturnus var. mrakii IFO 0895, which was previously named Hansenura mrakii, and belongs to the K9-type killer toxin group.1-6) HM-1 consists of 88 amino acids and is stable in response to heat treatment and various pH conditions. The three-dimensional structure of this protein has been clarified using nuclear magnetic resonance.7) The interactions of HM-1 with sensitive yeast follows two steps: an initial binding to cell-wall polysaccharides and a secondary binding to its receptor localized in the cell membrane. [8][9][10][11] We have previously shown that two specific arginine residues (positions 82 and 86) are required for the cytocidal effect on yeast Saccharomyces cerevisiae cells. 12)HM-1 cytocidicity is effected by disturbing the cell-wall synthesis of sensitive cells by inhibiting b-1,3-glucan synthase and the formation of pores at the budding tips of dividing cells. 13,14) Cellular materials spouting from the pores made at the budding tip leads to cell death. 6) Yamamoto et al. have reported that the cytocidal effects of HM-1 are specific to b-1,3-glucan synthesis of sensitive cells, and that HM-1 does not inhibit other cellular events, including the synthesis of proteins, mannans, and chitin. 13) In our previous studies, we have noted that HM-1 does not kill sensitive yeast cells if the cell walls are removed and exist as spheroplasts (unpublished data). Curiously, although spheroplasts survive against HM-1, their morphology is affected by incubation with HM-1, resulting in a perfectly round shape with an increase in the volume of spheroplasts and cellular vacuoles. In the present study, we examined the basis for this unique phenomenon caused by HM-1. Aculeacin A and papulacandin B, lipophilic antifungal antibiotics that inhibit yeast glucan synthase, 5,15) were included as controls to compare the effects. MATERIALS AND METHODSCells and Reagents HM-1 was prepared as described previously.14) S. cerevisiae A451 (MATa, ura3, leu2, trp1, can1, aro7) and S. cerevisiae BJ1824 (MATa , ura3, leu2, trp1, pep4) cells are sensitive to HM-1. S. cerevisiae BJ1824rhk1D::URA3 (rhk1D::URA3 in BJ1824) cells are resistant to HM-1. 6,9,14) The YPD medium used to grow these cells consisted of 1% yeast extract and 2% each of peptone and glucose. The Zymolyase 100T and the DNA quantitation kits were products of Seikagaku Corporation (Tokyo, Japan) and Nippon Bio-Rad Laboratories (Tokyo), respectively. 46-Diamidino-2-phenylindol (DAPI) and rhodamine phalloidin were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and Molecular Probes, Inc. (Eugene, OR, U.S.A.), respectively. Phase-contrast light and fluorescence microscopes, model BHS, were the products of the Olympus Optical Co., Ltd. (Tokyo), and Kodak TMAX 400 and Fujichrome PROVIA 400 film was used.Preparation of Yeast Spheroplasts Spheroplasts were prepared using Zymolyase 100T, a cell-wall hydrolyzing enzyme, as described previously.16) In brief, logarithmically ...

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Structural rearrangement of the actin cytoskeleton in regenerating protoplasts of budding yeasts

Cited by 15 publications

References 15 publications

Yeast and fungal cell-wall polysaccharides can self-assemblein vitrointo an ultrastructure resemblingin vivoyeast cell walls

Yeast and fungal cell-wall polysaccharides can self-assemblein vitrointo an ultrastructure resemblingin vivoyeast cell walls

Novel insights intoEntamoebacyst wall formation and the importance of actin cytoskeleton

Round Shape Enlargement of the Yeast Spheroplast of Saccharomyces cerevisiae by HM-1 Toxin.

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