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

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 (ccw12 Delta) indicated that CCW12 plays major roles in cell wall organization. Cluster II (ccw14 Delta, flo11 Delta, srl1 Delta, and tir3 Delta) mutants exhibited altered mother cell size and shape. Mutants of cluster III and IV exhibited no or very small morphological defects. Cluster V (dse2 Delta, egt2 Delta, and sun4 Delta) consisted of endoglucanase mutants with cell separation defects due to incomplete septum digestion. The cluster VI mutant cells (ecm33 Delta) exhibited perturbation of apical bud growth. Cluster VII mutant cells (sag1 Delta) 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