Effects of low-shear modeled microgravity on cell function, gene expression, and phenotype in Saccharomyces cerevisiae

Abstract

Only limited information is available concerning the effects of low-shear modeled microgravity (LSMMG) on cell function and morphology. We examined the behavior of Saccharomyces cerevisiae grown in a high-aspect-ratio vessel, which simulates the low-shear and microgravity conditions encountered in spaceflight. With the exception of a shortened lag phase (90 min less than controls; P < 0.05), yeast cells grown under LSMMG conditions did not differ in growth rate, size, shape, or viability from the controls but did differ in the establishment of polarity as exhibited by aberrant (random) budding compared to the usual bipolar pattern of controls. The aberrant budding was accompanied by an increased tendency of cells to clump, as indicated by aggregates containing five or more cells. We also found significant changes (greater than or equal to twofold) in the expression of genes associated with the establishment of polarity (BUD5), bipolar budding (RAX1, RAX2, and BUD25), and cell separation (DSE1, DSE2, and EGT2). Thus, low-shear environments may significantly alter yeast gene expression and phenotype as well as evolutionary conserved cellular functions such as polarization. The results provide a paradigm for understanding polarity-dependent cell responses to microgravity ranging from pathogenesis in fungi to the immune response in mammals.

FIG. 1.

(A) Epifluorescent micrographs of budding cells with normal (1 to 5) and random (6 to 10) budding patterns stained with calcofluor white. Thin white arrows on cell 6 demonstrate chitin-rich bud scarring (bottom), and the relatively darker protruded region indicates chitin-poor birth scarring (top). Thick white arrows indicate random budding patterns as revealed by chitin staining. The scale bar is 15 μm. (B) Percentage of random budding cells at different growth stages in LSMMG (▪) and in 1 × g (□). Each point on the graph is a mean ± SE from at least three samples from three independent experiments. At least 200 cells were scored for each replicate experiment.

FIG. 2.

(A) S. cerevisiae growth kinetics. The percentage of metabolically inactive cells counted in FUN1-stained cells (B) and volume of individual cells (μm³) grown in LSMMG (▪) or 1 × g (□) at different stages of the growth cycle (C). Each data point on panel A is a mean ± SE of three samples from three independent replicate experiments, with at least 500 and 100 individual measurements for each replicate for panels B and C, respectively.

FIG. 3.

Increased frequency of clumped cells was observed when cells were grown in LSMMG compared to when they were grown under the control condition 1 × g. Transmitted images of control (1 × g) cells (A) and LSMMG cells (B) during the early-stationary phase (OD₆₀₀ of 1.5) are shown. A representative cellular clump consisting of ≥5 individual cells observed in LSMMG is indicated by a large white arrow, and two individual clumps consisting of five individual cells are indicated by two smaller arrows (B). Scale bar, 35 μm (A and B). (C) The percentage of cells that are found in the multiple cell arrangement (≥5) in LSMMG (▪) and 1 × g (□) at different growth stages. Each point on the graph is a mean ± SE from at least three independent experiments with at least 300 cell counts for each replicate.

FIG. 4.

(A) Representative images of 3.7% formaldehyde-treated and calcofluor-stained cell clumps that are linked through chitin. Scale bar, 15 μm. (B) The percentage of 3.7% formaldehyde-treated cells that were in arrangements of 3 to 4 cells at different growth stages.