Cell wall biosynthesis impairment affects the budding lifespan of the Saccharomyces cerevisiae yeast

Abstract

The Saccharomyces cerevisiae yeast is one of the most widely used model in studies of cellular and organismal biology, including as aging and proliferation. Although several constraints of aging and budding lifespan have been identified, these processes have not yet been fully understood. Previous studies of aging in yeast have focused mostly on the molecular basics of the underlying mechanisms, while physical aspects, particularly those related to the cell wall, were rather neglected. In this paper, we examine for the first time, to our knowledge, the impact of cell wall biosynthesis disturbances on the lifespan in the budding yeast. We have used a set of cell wall mutants, including knr4Δ, cts1Δ, chs3Δ, fks1Δ and mnn9Δ, which affect biosynthesis of all major cell wall compounds. Our results indicated that impairment of chitin biosynthesis and cell wall protein mannosylation reduced the budding lifespan, while disruption in the 1,3-β-glucan synthase activity had no adverse effect on that parameter. The impact varied in the severity and the most notable effect was observed for the mnn9Δ mutant. What was interesting, in the case of the dysfunction of the Knr4 protein playing the role of the transcriptional regulator of cell wall chitin and glucan synthesis, the lifespan increased significantly. We also report the phenotypic characteristics of cell wall-associated mutants as revealed by imaging of the cell wall using transmission electron microscopy, scanning electron microscopy and atomic force microscopy. In addition, our findings support the conviction that achievement of the state of hypertrophy may not be the only factor that determines the budding lifespan.

Keywords: AFM; Aging; Budding lifespan; Cell wall; SEM; TEM; Yeast.

Fig. 1

Comparison of the reproductive potential of the haploid wild type yeast strain BY4741 and isogenic mutant strains chs3Δ, cts1Δ, fks1Δ, knr4Δ, mnn9Δ. Mean values (for total 80 cells from two independent experiments) of the reproductive potential are shown in parentheses

Fig. 2

Dependence of cell volume on the number of daughters accomplished by mother yeast cells. The results represent values for all cells tested in two independent experiments (80 cells). The bars indicate SD

Fig. 3

Comparison of cell wall thickness between BY4741 (wild-type) and cell wall mutants (chs3Δ, cts1Δ, fks1Δ, knr4Δ and mnn9Δ) measured in the images from the transmission electron microscopy using the EM Menu4 software. The means were obtained for 15 cells. The bars indicate SD. Statistical significances were assessed using ANOVA and the Dunnett’s post hoc test (**p < 0.05, ***p < 0.001)

Fig. 4

Scanning electron microscopy images of Saccharomyces cerevisiae cells for the BY4741 wild-type strain and cell wall mutants: chs3Δ, cts1Δ, fks1Δ, knr4Δ, and mnn9Δ. The arrows in a indicate wrinkles on the cell wall surface observed in fks1Δ. The arrows in b indicate changes in bud scars structure occurring in the chs3Δ and cts1Δ mutants compared to wild-type

Fig. 5

Representative AFM images of Saccharomyces cerevisiae cell surface for the wild type strain BY4741 and five cell wall mutants: chs3Δ, cts1Δ, fks1Δ, knr4Δ and mnn9Δ. The upper array of photos was collected in the Height mode. The height scale above the photos is given in μm. The corresponding lower array of images was obtained in the Peak Force Error mode. The scale bar = 1 μm

Fig. 6

Sensitivity of the BY4741 strain and cell wall mutants with disrupted CHS3, CTS1, FKS1, KNR4, MNN9 genes to Calcofluor white (CW), Congo red (CR), Methyl methanesulfonate (MMS) and sodium chloride (NaCl). Yeast strains were grown in YPD medium (28 °C), spotted onto YPD plates containing the indicated amounts of CW, CR, MMS, NaCl and incubated at 28 °C. Growth on YPD agar plates was treated as a control. Representative results from two independent experiments are shown