Abstract
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.
Highlights
The cell wall is a rigid structure that plays essential roles in establishing cell morphology and dictating the oval shape of budding yeast, Saccharomyces cerevisiae, and it confers robustness on the cell by stabilizing internal osmotic conditions and serving as a site for cell wall enzymes to exert their effects [1,2,3]
We found that cell morphology was remarkably altered: of 490 parameters, perturbations were detected in 136 parameters, consisting of 16, 77, and 43 parameters related to actin, cell, and nuclear DNA morphology, respectively (Wald test, false discovery rate (FDR) = 0.05; Table S4)
The results indicate that high-dimensional morphophological phenotyping of mannoprotein mutants is an effective approach for determinlogical phenotyping of mannoprotein mutants is an effective approach for determining ing the responsibility matrix of yeast mannoproteins, which is difficult to obtain with the responsibility matrix of yeast mannoproteins, which is difficult to obtain with other other omics technologies
Summary
The cell wall is a rigid structure that plays essential roles in establishing cell morphology and dictating the oval shape of budding yeast, Saccharomyces cerevisiae, and it confers robustness on the cell by stabilizing internal osmotic conditions and serving as a site for cell wall enzymes to exert their effects [1,2,3]. Previous studies have indicated that individual deletions of genes encoding mannoproteins may result in subtle growth defects [7,8,9,10]. This can be partly explained by gene duplication, as 26 of 36 mannoprotein genes are duplicated [11]. Mannoproteins play a collective role in maintaining the cell wall structure [13], but differences in the localization, structure, and probably the function of mannoproteins in the cell wall [3,8] suggest that the perturbation of individual genes may result in different morphological phenotypes [12]. Each mannoprotein is likely to have a distinct role in the cell wall, but the details have not been elucidated due to limited quantitative morphological analysis of mannoprotein mutants
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