scholarly journals Defining Functions of Mannoproteins in Saccharomyces cerevisiae by High-Dimensional Morphological Phenotyping

2021 ◽  
Vol 7 (9) ◽  
pp. 769
Author(s):  
Farzan Ghanegolmohammadi ◽  
Hiroki Okada ◽  
Yaxuan Liu ◽  
Kaori Itto-Nakama ◽  
Shinsuke Ohnuki ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Cell Size ◽  
Gaussian Mixture ◽  
High Dimensional ◽  
Actin Organization ◽  
Apical Bud ◽  
Biochemical Assays ◽  
Morphological Defects ◽  
Mutant Cells

Mannoproteins are non-filamentous glycoproteins localized to the outermost layer of the yeast cell wall. The physiological roles of these structural components have not been completely elucidated due to the limited availability of appropriate tools. As the perturbation of mannoproteins may affect cell morphology, we investigated mannoprotein mutants in Saccharomyces cerevisiae via high-dimensional morphological phenotyping. The mannoprotein mutants were morphologically classified into seven groups using clustering analysis with Gaussian mixture modeling. The pleiotropic phenotypes of cluster I mutant cells (ccw12Δ) indicated that CCW12 plays major roles in cell wall organization. Cluster II (ccw14Δ, flo11Δ, srl1Δ, and tir3Δ) mutants exhibited altered mother cell size and shape. Mutants of cluster III and IV exhibited no or very small morphological defects. Cluster V (dse2Δ, egt2Δ, and sun4Δ) consisted of endoglucanase mutants with cell separation defects due to incomplete septum digestion. The cluster VI mutant cells (ecm33Δ) exhibited perturbation of apical bud growth. Cluster VII mutant cells (sag1Δ) exhibited differences in cell size and actin organization. Biochemical assays further confirmed the observed morphological defects. Further investigations based on various omics data indicated that morphological phenotyping is a complementary tool that can help with gaining a deeper understanding of the functions of mannoproteins.

scholarly journals Cell Wall-Related Bionumbers and Bioestimates of Saccharomyces cerevisiae and Candida albicans

2013 ◽  
Vol 13 (1) ◽  
pp. 2-9 ◽  
Author(s):  
Frans M. Klis ◽  
Chris G. de Koster ◽  
Stanley Brul
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Candida Albicans ◽  
Cell Size ◽  
Pathogenic Fungus ◽  
Turgor Pressure ◽  
Hyphal Growth ◽  
Biological Research ◽  
Content Type ◽  
Starting Point

ABSTRACTBionumbers and bioestimates are valuable tools in biological research. Here we focus on cell wall-related bionumbers and bioestimates of the budding yeastSaccharomyces cerevisiaeand the polymorphic, pathogenic fungusCandida albicans. We discuss the linear relationship between cell size and cell ploidy, the correlation between cell size and specific growth rate, the effect of turgor pressure on cell size, and the reason why using fixed cells for measuring cellular dimensions can result in serious underestimation ofin vivovalues. We further consider the evidence that individual buds and hyphae grow linearly and that exponential growth of the population results from regular formation of new daughter cells and regular hyphal branching. Our calculations show that hyphal growth allowsC. albicansto cover much larger distances per unit of time than the yeast mode of growth and that this is accompanied by strongly increased surface expansion rates. We therefore predict that the transcript levels of genes involved in wall formation increase during hyphal growth. Interestingly, wall proteins and polysaccharides seem barely, if at all, subject to turnover and replacement. A general lesson is how strongly most bionumbers and bioestimates depend on environmental conditions and genetic background, thus reemphasizing the importance of well-defined and carefully chosen culture conditions and experimental approaches. Finally, we propose that the numbers and estimates described here offer a solid starting point for similar studies of other cell compartments and other yeast species.

Low temperature highlights the functional role of the cell wall integrity pathway in the regulation of growth in Saccharomyces cerevisiae

2012 ◽  
Vol 446 (3) ◽  
pp. 477-488 ◽  
Author(s):  
Isaac Córcoles-Sáez ◽  
Lídia Ballester-Tomas ◽  
Maria A. de la Torre-Ruiz ◽  
Jose A. Prieto ◽  
Francisca Randez-Gil
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Low Temperatures ◽  
Cell Wall Integrity ◽  
Target Of Rapamycin ◽  
Cold Sensitivity ◽  
Cold Response ◽  
Response Component ◽  
Cell Wall Integrity Pathway ◽  
Mutant Cells

Unlike other stresses, the physiological significance and molecular mechanisms involved in the yeast cold response are largely unknown. In the present study, we show that the CWI (cell wall integrity) pathway plays an important role in the growth of Saccharomyces cerevisiae at low temperatures. Cells lacking the Wsc1p (wall integrity and stress response component 1) membrane sensor or the MAPKs (mitogen-activated protein kinases) Bck1p (bypass of C kinase 1), Mkk (Mapk kinase) 1p/Mkk2p or Slt2p (suppressor of lyt2) exhibited cold sensitivity. However, there was no evidence of either a cold-provoked perturbation of the cell wall or a differential cold expression program mediated by Slt2p. The results of the present study suggest that Slt2p is activated by different inputs in response to nutrient signals and mediates growth control through TORC1 (target of rapamycin 1 complex)–Sch9p (suppressor of cdc25) and PKA (protein kinase A) at low temperatures. We found that absence of TOR1 (target of rapamycin 1) causes cold sensitivity, whereas a ras2Δ mutant shows increased cold growth. Lack of Sch9p alleviates the phenotype of slt2Δ and bck1Δ mutant cells, as well as attenuation of PKA activity by overexpression of BCY1 (bypass of cyclase mutations 1). Interestingly, swi4Δ mutant cells display cold sensitivity, but the phenotype is neither mediated by the Slt2p-regulated induction of Swi4p (switching deficient 4)-responsive promoters nor influenced by osmotic stabilization. Hence, cold signalling through the CWI pathway has distinct features and might mediate still unknown effectors and targets.

scholarly journals Immunofluorescence localization of the Saccharomyces cerevisiae CDC12 gene product to the vicinity of the 10-nm filaments in the mother-bud neck.

1987 ◽  
Vol 7 (10) ◽  
pp. 3678-3687 ◽  
Author(s):  
B K Haarer ◽  
J R Pringle
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Gene Product ◽  
Polyclonal Antibodies ◽  
Affinity Purification ◽  
Neck Region ◽  
Yeast Saccharomyces Cerevisiae ◽  
Bud Growth ◽  
10 Nm Filaments ◽  
Mutant Cells

Budding cells of the yeast Saccharomyces cerevisiae possess a ring of 10-nm-diameter filaments, of unknown biochemical nature, that lies just inside the plasma membrane in the neck connecting the mother cell to its bud (B. Byers and L. Goetsch, J. Cell Biol. 69:717-721, 1976). Mutants defective in any of four genes (CDC3, CDC10, CDC11, and CDC12) lack these filaments and display a pleiotropic phenotype that involves abnormal bud growth and cell-wall deposition and an inability to complete cytokinesis. We fused the cloned CDC12 gene to the Escherichia coli lacZ and trpE genes and used the resulting fusion proteins to raise polyclonal antibodies specific for the CDC12 gene product. In immunofluorescence experiments with affinity-purified antibodies, the neck region of wild-type and mutant cells stained in patterns consistent with the hypothesis that the CDC12 gene product is a constituent of the ring of 10-nm filaments. Without careful affinity purification of the CDC12-specific antibodies, these staining patterns were completely obscured by the staining of residual cell wall components in the neck by antibodies present even in the "preimmune" sera of all rabbits tested.

scholarly journals Piecemeal Microautophagy of the Nucleus Requires the Core Macroautophagy Genes

2008 ◽  
Vol 19 (10) ◽  
pp. 4492-4505 ◽  
Author(s):  
R. Krick ◽  
Y. Muehe ◽  
T. Prick ◽  
S. Bremer ◽  
P. Schlotterhose ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Saccharomyces Cerevisiae ◽  
Fusion Genes ◽  
Vacuole Membrane ◽  
The Core ◽  
Atg Proteins ◽  
Biochemical Assays ◽  
Atg Genes ◽  
Mutant Cells ◽  
Specific Membrane

Autophagy is a diverse family of processes that transport cytoplasm and organelles into the lysosome/vacuole lumen for degradation. During macroautophagy cargo is packaged in autophagosomes that fuse with the lysosome/vacuole. During microautophagy cargo is directly engulfed by the lysosome/vacuole membrane. Piecemeal microautophagy of the nucleus (PMN) occurs in Saccharomyces cerevisiae at nucleus-vacuole (NV) junctions and results in the pinching-off and release into the vacuole of nonessential portions of the nucleus. Previous studies concluded macroautophagy ATG genes are not absolutely required for PMN. Here we report using two biochemical assays that PMN is efficiently inhibited in atg mutant cells: PMN blebs are produced, but vesicles are rarely released into the vacuole lumen. Electron microscopy of arrested PMN structures in atg7, atg8, and atg9 mutant cells suggests that NV-junction–associated micronuclei may normally be released from the nucleus before their complete enclosure by the vacuole membrane. In this regard PMN is similar to the microautophagy of peroxisomes (micropexophagy), where the side of the peroxisome opposite the engulfing vacuole is capped by a structure called the “micropexophagy-specific membrane apparatus” (MIPA). The MIPA contains Atg proteins and facilitates terminal enclosure and fusion steps. PMN does not require the complete vacuole homotypic fusion genes. We conclude that a spectrum of ATG genes is required for the terminal vacuole enclosure and fusion stages of PMN.

Note. Morphological Changes in Saccharomyces cerevisiae during the Second Fermentation of Sparkling Wines

2008 ◽  
Vol 14 (4) ◽  
pp. 393-398 ◽  
Author(s):  
R. Gonzalez ◽  
A. Vian ◽  
A.V. Carrascosa
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Molecular Markers ◽  
Stationary Phase ◽  
Cell Size ◽  
Morphological Changes ◽  
Size Reduction ◽  
Death Phase ◽  
The Moment

This study shows the morphological changes of Saccharomyces cerevisiae EC1118 during the second fermentation of Spanish cava wines, in relation with progression of fermentation and aging. In the first stages of active fermentation, and associated with the increase in viable counts, budding cells and a relative homogeneity in cell size were observed. Close to the moment of sugar exhaustion cells acquired the morphology of stationary phase, to finally enter in a death phase with cell size reduction, and cytoplasm alterations including inhomogeneity, refringency, and detachment of the cell wall. At the beginning of this step structures reminiscent to autophagosomes are observed. This is in accordance with the appearance of molecular markers of autophagy described elsewhere in similar winemaking conditions.

scholarly journals Suppression of the Profilin-Deficient Phenotype by the RHO2 Signaling Pathway in Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 156 (2) ◽  
pp. 579-592 ◽  
Author(s):  
Nathaly Marcoux ◽  
Simon Cloutier ◽  
Ewa Zakrzewska ◽  
Pierre-Mathieu Charest ◽  
Yves Bourbonnais ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Signaling Pathway ◽  
Transmembrane Protein ◽  
Cortical Actin ◽  
Uncharacterized Protein ◽  
Actin Organization ◽  
Multicopy Suppressor ◽  
Multicopy Suppressors ◽  
Actin Cables

Abstract Profilin plays an important role in actin organization in all eukaryotic cells through mechanisms that are still poorly understood. We had previously shown that Mid2p, a transmembrane protein and a potential cell wall sensor, is an effective multicopy suppressor of the profilin-deficient phenotype in Saccharomyces cerevisiae. To better understand the role of Mid2p in the organization of the actin cytoskeleton, we isolated five additional multicopy suppressors of pfy1Δ cells that are Rom1p, Rom2p, Rho2p, Smy1p, and the previously uncharacterized protein Syp1p. The problems of caffeine and NaCl sensitivity, growth defects at 30° and 37°, the accumulation of intracellular vesicular structures, and a random budding pattern in pfy1Δ cells are corrected by all the suppressors tested. This is accompanied by a partial repolarization of the cortical actin patches without the formation of visible actin cables. The overexpression of Mid2p, Rom2p, and Syp1p, but not the overexpression of Rho2p and Smy1p, results in an abnormally thick cell wall in wild-type and pfy1Δ cells. Since none of the suppressors, except Rho2p, can correct the phenotype of the pfy1-111/rho2Δ strain, we propose a model in which the suppressors act through the Rho2p signaling pathway to repolarize cortical actin patches.

Immunofluorescence localization of the Saccharomyces cerevisiae CDC12 gene product to the vicinity of the 10-nm filaments in the mother-bud neck

1987 ◽  
Vol 7 (10) ◽  
pp. 3678-3687
Author(s):  
B K Haarer ◽  
J R Pringle
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Gene Product ◽  
Polyclonal Antibodies ◽  
Affinity Purification ◽  
Neck Region ◽  
Yeast Saccharomyces Cerevisiae ◽  
Bud Growth ◽  
10 Nm Filaments ◽  
Mutant Cells

Budding cells of the yeast Saccharomyces cerevisiae possess a ring of 10-nm-diameter filaments, of unknown biochemical nature, that lies just inside the plasma membrane in the neck connecting the mother cell to its bud (B. Byers and L. Goetsch, J. Cell Biol. 69:717-721, 1976). Mutants defective in any of four genes (CDC3, CDC10, CDC11, and CDC12) lack these filaments and display a pleiotropic phenotype that involves abnormal bud growth and cell-wall deposition and an inability to complete cytokinesis. We fused the cloned CDC12 gene to the Escherichia coli lacZ and trpE genes and used the resulting fusion proteins to raise polyclonal antibodies specific for the CDC12 gene product. In immunofluorescence experiments with affinity-purified antibodies, the neck region of wild-type and mutant cells stained in patterns consistent with the hypothesis that the CDC12 gene product is a constituent of the ring of 10-nm filaments. Without careful affinity purification of the CDC12-specific antibodies, these staining patterns were completely obscured by the staining of residual cell wall components in the neck by antibodies present even in the "preimmune" sera of all rabbits tested.

scholarly journals Novel Rho GTPase Involved in Cytokinesis and Cell Wall Integrity in the Fission Yeast Schizosaccharomyces pombe

2003 ◽  
Vol 2 (3) ◽  
pp. 521-533 ◽  
Author(s):  
Beatriz Santos ◽  
Javier Gutiérrez ◽  
Teresa M. Calonge ◽  
Pilar Pérez
Keyword(s):  
Cell Wall ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Rho Gtpases ◽  
Rho Gtpase ◽  
Cell Wall Integrity ◽  
Glucan Synthase ◽  
Actin Organization ◽  
Morphological Defects

ABSTRACT The Rho family of GTPases is present in all eukaryotic cells from yeast to mammals; they are regulators in signaling pathways that control actin organization and morphogenetic processes. In yeast, Rho GTPases are implicated in cell polarity processes and cell wall biosynthesis. It is known that Rho1 and Rho2 are key proteins in the construction of the cell wall, an essential structure that in Schizosaccharomyces pombe is composed of β-glucan, α-glucan, and mannoproteins. Rho1 regulates the synthesis of 1,3-β-d-glucan by activation of the 1,3-β-d-glucan synthase, and Rho2 regulates the synthesis of α-glucan by the 1,3-α-d-glucan synthase Mok1. Here we describe the characterization of another Rho GTPase in fission yeast, Rho4. rho4Δ cells are viable but display cell separation defects at high temperature. In agreement with this observation, Rho4 localizes to the septum. Overexpression of rho4 + causes lysis and morphological defects. Several lines of evidence indicate that both rho4 + deletion or rho4 + overexpression result in a defective cell wall, suggesting an additional role for Rho4 in cell wall integrity. rho4Δ cells also accumulate secretory vesicles around the septum and are defective in actin polarization. We propose that Rho4 could be involved in the regulation of the septum degradation during cytokinesis.

Ultrastructure and Morphology of the Neurospora Crassa Cell Wall Mutants, Slime And Slime X, Using Tem and Sem

1976 ◽  
Vol 34 ◽  
pp. 54-55
Author(s):  
Karen S. Howard ◽  
H. D. Braymer ◽  
M. D. Socolofsky ◽  
S. A. Milligan
Keyword(s):  
Cell Wall ◽  
Neurospora Crassa ◽  
Wild Type ◽  
Morphological Comparison ◽  
Small Areas ◽  
Solid Media ◽  
Thin Sectioning ◽  
X Cells ◽  
Mutant Cells ◽  
Isolated Cell

The recently isolated cell wall mutant slime X of Neurospora crassa was prepared for ultrastructural and morphological comparison with the cell wall mutant slime. The purpose of this article is to discuss the methods of preparation for TEM and SEM observations, as well as to make a preliminary comparison of the two mutants.TEM: Cells of the slime mutant were prepared for thin sectioning by the method of Bigger, et al. Slime X cells were prepared in the same manner with the following two exceptions: the cells were embedded in 3% agar prior to fixation and the buffered solutions contained 5% sucrose throughout the procedure.SEM: Two methods were used to prepare mutant and wild type Neurospora for the SEM. First, single colonies of mutant cells and small areas of wild type hyphae were cut from solid media and fixed with OSO4 vapors similar to the procedure used by Harris, et al. with one alteration. The cell-containing agar blocks were dehydrated by immersion in 2,2-dimethoxypropane (DMP).

Cell‐Wall Beta‐Glucans of Saccharomyces cerevisiae

2002 ◽  
Author(s):  
Gerrit J. P. Dijkgraaf ◽  
Huijuan Li ◽  
Howard Bussey
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Beta Glucans
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