Global study of holistic morphological effectors in the budding yeast Saccharomyces cerevisiae

Abstract

Background: The size of the phenotypic effect of a gene has been thoroughly investigated in terms of fitness and specific morphological traits in the budding yeast Saccharomyces cerevisiae, but little is known about gross morphological abnormalities.

Results: We identified 1126 holistic morphological effectors that cause severe gross morphological abnormality when deleted, and 2241 specific morphological effectors with weak holistic effects but distinctive effects on yeast morphology. Holistic effectors fell into many gene function categories and acted as network hubs, affecting a large number of morphological traits, interacting with a large number of genes, and facilitating high protein expression. Holistic morphological abnormality was useful for estimating the importance of a gene to morphology. The contribution of gene importance to fitness and morphology could be used to efficiently classify genes into functional groups.

Conclusion: Holistic morphological abnormality can be used as a reproducible and reliable gene feature for high-dimensional morphological phenotyping. It can be used in many functional genomic applications.

Keywords: CalMorph; Morphology; Phenotyping; Saccharomyces cerevisiae; Yeast.

Fig. 1

Degree of morphological abnormality. a Schematic representation of morphological abnormality (i.e., nucleus size) in budding yeast cells. Red and blue circles indicate the actin patch and nucleus, respectively. Inequality between mutants indicates a difference in the degree of morphological abnormality. b Schematic representation of holistic morphological abnormality in mutants. For each mutant, Euclidean distance from the mean of wild-type replicates is calculated in orthogonal phenotypic space to determine the degree of gross morphological abnormality. As an example, calculation of the Euclidean distance of mutant “a” in three-dimensional phenotypic space is shown. Red and orange spheres indicate mutant “a” and wild-type replicates, respectively. c Schematic representation of signature profiles and holistic morphological abnormalities in yeast morphological mutants. As an example, abnormalities of six morphological traits in a mutant from wild type are shown in spider charts. A center of a chart indicates no abnormality. In the left panel, red and blue lines indicate signature profiles of mutant A and B, respectively. In the right panel, red and blue areas indicate holistic morphological abnormalities of mutant A’ and B’, respectively. Sizes of the colored areas are proportional to degrees of the holistic morphological abnormalities

Fig. 2

Identification of genes with holistic effects on yeast morphology. a Distribution of Euclidian distances (Additional file 2: Table S1). Blue, gray, and yellow boxes indicate non-essential gene deletion mutants with significant holistic morphological abnormality (left axis), other deletion mutants (left axis), and 109 replicates of the wild type (right axis), respectively, in 57-dimensional orthogonal space. The vertical solid red line indicates false discovery rate (FDR) = 0.01, and the purple curved line indicates a gamma distribution fitted to the wild-type replicates. b Scatter plot of non-essential gene deletion mutants in terms of holistic morphological abnormality (x-axis) and specific morphological abnormality (y-axis). The specific effect (y-axis) was defined as the maximum negative value of log-transformed p values for each of the 501 traits (Additional file 2: Table S1). Horizontal and vertical solid red lines indicate FDR = 0.01. Blue, green, orange, and black circles indicate 1126 holistic morphological mutants, 2241 specific morphological mutants, 109 replicates of the wild type, and 1351 other mutants, respectively

Fig. 3

Comparison of morphological abnormality in holistic and specific morphological mutants. a Comparison of the number of abnormal phenotypes. Number of altered traits was counted for each mutant after detecting abnormal phenotypes at FDR = 0.01 (Additional file 2: Table S1). Asterisk indicates significant difference (p < 0.01 by Mann–Whitney U test). b Microscopic images of representative mutants of specific effectors and holistic effectors. The specific (bre2Δ) and holistic (dia2Δ) morphological mutants selected for Fig. 3a were extreme mutants. Scale bar indicates 5 μm. c Venn diagram showing overlap of altered traits between holistic and specific morphological mutants. The total number of altered traits in holistic and specific morphological mutants (FDR = 0.01) is shown

Fig. 4

Relationship between holistic morphological abnormality and fitness. a Scatter plot of non-essential gene deletion mutants in terms of holistic morphological abnormality (x-axis) and fitness (y-axis) (Additional file 2: Table S1). Blue, green, and black circles indicate holistic morphological mutants, specific morphological mutants, and other mutants, respectively. Horizontal and vertical solid red lines indicate FDR = 0.01. Each number indicates the number of deletion mutants classified based on fitness and morphology. b Fitness in holistic morphological mutants, specific morphological mutants, and other mutants. Horizontal solid red lines indicate median values. ** indicates a significant difference at p < 0.01 by the Mann–Whitney U test

Fig. 5

Comparison of holistic morphological effectors with other gene features. a The number of genetic interactions of holistic morphological effectors, specific morphological effectors, and others. b Protein expression levels of holistic morphological effectors, specific morphological effectors, and others. c Holistic morphological abnormality of singletons and duplicates. Horizontal solid red lines indicate median values. * and ** indicate significant differences at p < 0.05 and p < 0.01, respectively, as determined by the Mann–Whitney U test after Bonferroni correction

Fig. 6

Functional enrichment in gene groups specified by fitness and morphology. Functional enrichment in gene groups I–VI. The scatter plot, colored dots, and solid red lines are as shown in Fig. 4a. Bar graphs associated with each group indicate the fractions of genes annotated with each adjacent gene ontology (GO) term. Bar colors: dark blue, dark green, dark gray, blue, green, and gray indicate gene groups I, II, III, IV, V, and VI, respectively

Fig. 7

Distribution of deletion mutants for genes annotated to specific GOs. a Group I related to autophagy (GO:0016236). b Morphological similarity among autophagy-related gene deletion mutants. The subnetwork was described using Cytoscape (http://www.cytoscape.org/) and nodes were placed using the spring-embedded layout. Colors of nodes represent genes in group I and IV (blue), group II and V (green), group III and VI (gray) Red and blue edges indicate positive and negative correlations with morphological phenotype, respectively. Transparency of colors at edges is proportional to absolute R value. Wide, medium, and narrow edges indicate strong (0.6 < R value ≤0.8), moderate (0.4 < R value ≤0.6), and weak correlations (0.2 < R value ≤0.4), respectively. c Group IV related to negative regulation of transport (GO:0051051). d Groups II and III related to mitochondrial translation (GO:0032543). Blue, green, and dark gray circles indicate mutants of genes annotated with specific GOs. Gray circles indicate mutants of genes not annotated with the specified GOs. The number of annotated genes in each group is shown in parentheses. Red frames indicate gene groups related to a specific GO. The scatter plot, colored dots, and solid red lines are as shown in Fig. 4a

Fig. 8

Enrichment of specific gene groups in Group V and VI. a Enrichment of low-abundance and sporulation-specific genes in Group VI. b Enrichment of minor cell wall protein-modifying genes in Group V. The symbols and colors used are as defined in Fig. 7