Figure 1.
Model: Confining cell wall degrading enzymes in the fusion zone leads to cell wall destruction.
A. Isotropically growing cells increase the size of their cell walls equally in all directions to grow larger while maintaining an ellipsoidal shape, so cell wall remodeling enzymes are secreted equally in all directions. B. Polarized cells grow anisotropically, so they polarize secretion of cell wall remodeling enzymes to expand their cell walls in the direction of polarization. C. When pheromone stimulated cells are unattached, the cell wall remodeling enzymes secreted from the shmoo tip exit the cell wall along the shortest path by traveling perpendicular to the plasma membrane. These enzymes break cell wall bonds as they diffuse through the wall to allow continual expansion of the shmoo up the pheromone gradient, but the wall is not breached. D. When two pheromone-stimulated cells are attached by mating agglutinins, the cell wall remodeling enzymes secreted into the future fusion zone must now travel further to exit the cell wall, traveling parallel to the plasma membrane until they reach the edge of the agglutinated zone, increasing the local concentration of cell wall remodeling enzymes in this zone. The cell wall remodeling enzymes dissolve the two cell walls at the point of contact while cell wall synthesizing enzymes simultaneously interlock them, allowing the plasma membranes of the two cells to contact one another and fuse without exposing the cell to osmotic lysis. E. We mimicked the attachment of two cells by tightly apposing single cells to impermeable surfaces, forcing cell wall remodeling enzymes to exit the wall by traveling parallel to the plasma membrane until they reached bulk solution and thus increasing the concentration of cell wall remodeling enzymes at the point of attachment to the impermeable surface. This causes a hole to form in the cell wall, exposing the plasma membrane to the extracellular environment and causing the cell to undergo osmotic lysis.
Figure 2.
The role of radial diffusion through the cell wall of apposed cells in increasing the concentration of cell wall degrading enzymes.
The graph shows analytical results for the relative enzyme concentration in two scenarios: red, diffusion through the cell wall, perpendicular to the cell surface, of a cell free in solution and not in contact with other cells or solid surfaces, and blue, diffusion through the cell wall, parallel to the cell surface, of a cell that is apposed to a solid surface, with a circular contact area whose radius is 1 µm.
Figure 3.
Pheromone-induced cell death increases with increasing attachments to an impermeable surface.
A. Cells grown in bulk culture were incubated in test tubes on roller drums in liquid media without any enforced contact with impermeable surfaces. Cells grown in a concanavalin A (ConA) chamber were grown in a chamber whose depth was many times the diameter of a single yeast cell and attached to a single surface of the chamber (the ceiling provided by a glass coverslip) using the lectin, concanavalin A. For confinement, cells were loaded into a microfluidic chamber which traps cells between a ceiling and floor separated by the diameter of a single yeast cell, causing enforced contact with two surfaces. Medium is then constantly perfused through the chamber. B. Percent of MATa bar1Δ cells that died after exposure to 50nM α-factor for five hours in three different physical environments. Error bars represent the standard deviation of at least three independent experiments. C. Time course of MATa bar1Δ cells incubated in 50nM α-factor for the indicated amount of time in the flow chamber. Yellow arrows indicate cells that died since the previous time point. White arrows indicate cells that died earlier. The scale bar indicates 10 µm.
Figure 4.
Pheromone-induced cell death increases with increased polarization.
A. Fraction of MATa bar1Δ cells that died after five hours exposure to various concentrations of α-factor in the flow chamber relative to the fraction of MATa bar1Δ cells that died after five hours exposure to 50nM α-factor. Error bars represent the standard deviation of at least three independent experiments. B. MATa bar1Δ cells incubated in the indicated concentration of α-factor for five hours in the flow chamber. Yellow arrows indicate dead cells. The scale bar indicates 10 µm. C. MATa bar1Δ cells exposed to 0.1mM benomyl for five hours in the flow chamber. The scale bar indicates 10 µm. D. Fraction of MATa bar1Δ cells that died after five hours exposure to either 0.1mM benomyl or 50nM α-factor in the flow chamber relative to the fraction of MATa bar1Δ cells that died after five hours exposure to 50nM α-factor (Student's t-test, p<10−6). Error bars represent the standard deviation of at least three independent experiments.
Figure 5.
Pheromone-induced cell death is due to osmotic lysis.
A. MATa bar1Δ cells were grown in a flow chamber for five hours in medium with 50nM α-factor and 1M sorbitol. After five hours, the sorbitol was washed out, and the cells were incubated in medium with 50nM α-factor and no sorbitol. The fraction of dead cells 10 minutes before and 10 minutes after the 1M sorbitol was washed out relative to the fraction of cells that die when exposed to 50nM α-factor for five hours without the addition of sorbitol was determined (Student's t-test, p = 2×10−4). Error bars represent the standard deviation of at least three independent experiments. B. Cells imaged after 290 minutes in medium with 1M sorbitol and 50nM α-factor (Before sorbitol washout) and 10 minutes after the medium was replaced with medium with 50nM α-factor and no sorbitol (After sorbitol washout). Yellow arrows indicate the cells that died during the 290 minutes of pheromone treatment prior to the sorbitol washout. White arrows in the “After sorbitol washout” picture indicate cells that died during the twenty minute period that spanned the last 10 minutes with sorbitol and the first 10 minutes after the sorbitol washout. The scale bar indicates 10 µm. C. Cells were grown in the flow chamber for 80 minutes in medium with 50nM α-factor. After 80 minutes, 1M sorbitol was added to the medium such that the cells were incubated in medium with 1M sorbitol and 50nM α-factor. The fold change in the number of cells that died during the 80 minutes prior to and 60 minutes after the sorbitol wash-in was determined (Sorbitol wash-in). In control chambers (No sorbitol), no sorbitol was added to the medium, and the fold change in the number of cells that died in the two corresponding periods was determined (Student's t-test, p = 9×10−5). Error bars represent the standard deviation of at least three independent experiments.
Figure 6.
Pheromone-induced cell death is dependent on cell fusion proteins and putative glucanases.
A. Fraction of dead MATa bar1Δ cells deleted for different combinations of FUS1 and FUS2 relative to the fraction of dead MATa bar1Δ cells incubated in 50nM α-factor for five hours in the flow chamber. Error bars represent the standard deviation of at least three independent experiments. B. Fraction of dead MATa bar1Δ cells deleted for different combinations of putative cell wall glucanases relative to the fraction of dead MATa bar1Δ cells incubated in 50nM α-factor for five hours in the flow chamber. Error bars represent the standard deviation of at least three independent experiments.
Table 1.
Strains used in this study.