Chemical gradients and chemotropism in yeast

Abstract

Chemical gradients of peptide mating pheromones are necessary for directional growth, which is critical for yeast mating. These gradients are generated by cell-type specific secretion or export and specific degradation in receiving cells. Spatial information is sensed by dedicated seven-transmembrane G-protein coupled receptors and yeast cells are able to detect extremely small differences in ligand concentration across their approximately 5-microm cell surface. Here, I will discuss our current knowledge of how cells detect and respond to such shallow chemical gradients and in particular what is known about the proteins that are involved in directional growth and the establishment of the polarity axis during yeast mating.

Figure 1.

Localization of actin cytoskeleton, regions of growth and proteins required for yeast chemotropism in yeast cells treated with mating pheromone. (A) Wild-type MATa yeast cells treated with α‐factor for 2 h. Differential interference contrast image of shmoo. (B) Wild-type yeast MATa cells treated with α‐factor for 2 h and then the actin cytoskeleton was stained with Alexa-568 phalloidin. Fluorescence images are maximum intensity projections of Z sections. (C) Regions of new cell wall growth (red) and old growth (green) in wild-type MATa shmoos. Alexa-Fluor 488 concanavalin A-labeled cells (green) were incubated in YEP 0.1% glucose in the presence of α-factor for 4 h and then stained with Alexa-Fluor 594 concanavalin A (red). (Reprinted from Nern and Arkowitz 2000a.) (D) Localization of α-factor receptor Ste2-GFP in MATa cells incubated 2 h with α‐factor. The brightly fluorescent structures within the cells are the vacuoles, which accumulate receptor and GFP. (Reprinted from Arkowitz 1999.) (E) Localization of Gα (Gpa1-GFP) in elutriated MATa cells incubated 1 h with α‐factor (kindly provided by D. Stone). (F) Localization of Gβ (Ste4-GFP) in elutriated MATa cells incubated 1 h with α‐factor (kindly provided by D. Stone). (G) Localization of Gγ (Ste18-GFP) in MATa cells incubated 2 h with α‐factor. (Reprinted from Nern and Arkowitz 2000a.) (H) Localization of Far1-GFP in MATa cdc28-13 cells that were arrested at 37ºC and then incubated with α‐factor and shifted to 25ºC for 30 min. (Reprinted, with permission, from Nern and Arkowitz 2000b.) (I) Localization of Cdc24-GFP in MATa cdc28-13 cells that were arrested at 37ºC and then incubated with α‐factor and shifted to 25ºC for 30 min. (Reprinted, with permission, from Nern and Arkowitz 2000b.) (J) Localization of GFP-Cdc42 in MATa cells incubated 2 h with α‐factor. (Reprinted, with permission, from Barale et al. 2006.) (K) Localization of Bni1-GFP in MATa cells incubated 2 h with α‐factor. (Reprinted, with permission, from Matheos et al. 2004 [© Matheos et al. 2004; originally published in J. Cell Biol. doi: 10.1083/jcb.200309089.]) (L) Localization of Spa2-GFP in MATa cells incubated 2 h with α‐factor. (Reprinted, with permission, from Nern and Arkowitz 2000a.)

Figure 2.

Cell morphology changes in response to mating pheromone. Spa2-GFP localization (green) in cell treated with pheromone for indicated times. Rhodamine concanavalin A-labeled cells expressing Spa2-GFP were imaged on SC-ura agar containing 30-µM α-factor. Images are projections (arithmetic average) of 3–5 optical 1-µm z-sections. (Reprinted, with permission, from Nern and Arkowitz 2000a.)

Figure 3.

Cell morphology changes and chemotropism during yeast mating. (A) Time-lapse images of wild-type MATα cells mating with GFP-Bud1 expressing wild-type MATa cells (green) mating partner. Images taken at indicated times (at 30ºC) with visible (black) and fluorescence (green) shown. Note cell pairs indicated by arrows are polarized and grow toward one another (0:50–1:25 and 2:20–2:35 times). These cells pairs fuse to form zygotes at 1:30 and 2:40, respectively, and fluorescence signal in MATa cells can now be observed in MATα cells. (Reprinted, with permission, from Barale et al. 2004.) (B) Time-lapse images of wild-type MATα cells mating with GFP-Bud2 expressing MATa cells (green) mating partner. Images taken at indicated times (at 30ºC) with DIC (red) and fluorescence (green) shown. Images are maximum intensity projections 5 optical 0.5-µm z-sections that were deconvolved with SoftWoRx software. Cell pairs that fuse to form zygote are indicated by arrows. These cells are polarized and grow toward one another (0:20–0:40 and 0:40–1:00 times) before fusing.

Figure 4.

Chemotropic response of yeast cells to artificially generated pheromone gradients. (A) Wild-type cells exposed to a micropipet-generated pheromone gradient. The micropipet contained 67 nM mating pheromone and this image was taken after ∼9 h exposure to the gradient at 30°C. The pipet is the elongated out-of-focus object originating in the upper right corner, which is outlined in black. Image kindly provided by J. Segall (Segall 1993 [©National Academy of Sciences]). (B) Mating pheromone gradient generated in a microfluidics chamber. (Adapted from Moore et al. 2008.) Schematic diagram (upper left) of microfluidics Y-device in which media alone is in the left inlet and media containing α-factor together with and Dextran-3000-TRITC (tracking dye) in the right inlet. This results in a gradient across the width of the chamber via diffusion. Five positions descending the length of the main channel, denoted A to E, are indicated and visualized. The gradient slope varies depending on the position along the length of the central chamber (upper right). Note that the gradient is shallower as one progresses down the chamber (the tracking dye and α-factor have more time to diffuse). In position A of microfluidics chamber in 0–100-nM gradient, after 4 h, a narrow band of bar1Δ cells in the middle of the chamber were almost perfectly aligned to source of the gradient (bottom). The direction of the gradient is from left (low) to right (high), shown by the black arrow and scale bar (50 µm).