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Robust spatial sensing of mating pheromone gradients by yeast cells.

Moore TI, Chou CS, Nie Q, Jeon NL, Yi TM - PLoS ONE (2008)

Bottom Line: We tested the ability of S. cerevisiaea-cells to sense and respond to spatial gradients of the mating pheromone alpha-factor produced in a microfluidics chamber; the focus was on bar1Delta strains, which do not degrade the pheromone input.Mathematical modeling revealed insights into the mechanism of this amplification and how the supersensitive mutants can disrupt accurate polarization.Together, these data help to specify and elucidate the abilities of yeast cells to sense and respond to spatial gradients of pheromone.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental and Cell Biology, University of California Irvine, Irvine, CA, USA.

ABSTRACT
Projecting or moving up a chemical gradient is a universal behavior of living organisms. We tested the ability of S. cerevisiaea-cells to sense and respond to spatial gradients of the mating pheromone alpha-factor produced in a microfluidics chamber; the focus was on bar1Delta strains, which do not degrade the pheromone input. The yeast cells exhibited good accuracy with the mating projection typically pointing in the correct direction up the gradient ( approximately 80% under certain conditions), excellent sensitivity to shallow gradients, and broad dynamic range so that gradient-sensing was relatively robust over a 1000-fold range of average alpha-factor concentrations. Optimal directional sensing occurred at lower concentrations (5 nM) close to the K(d) of the receptor and with steeper gradient slopes. Pheromone supersensitive mutations (sst2Delta and ste2(300Delta)) that disrupt the down-regulation of heterotrimeric G-protein signaling caused defects in both sensing and response. Interestingly, yeast cells employed adaptive mechanisms to increase the robustness of the process including filamentous growth (i.e. directional distal budding) up the gradient at low pheromone concentrations, bending of the projection to be more aligned with the gradient, and forming a more accurate second projection when the first projection was in the wrong direction. Finally, the cells were able to amplify a shallow external gradient signal of alpha-factor to produce a dramatic polarization of signaling proteins at the front of the cell. Mathematical modeling revealed insights into the mechanism of this amplification and how the supersensitive mutants can disrupt accurate polarization. Together, these data help to specify and elucidate the abilities of yeast cells to sense and respond to spatial gradients of pheromone.

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Microfluidics device generates α-factor gradients and response of yeast cells to gradient.(A) Schematic diagram of microfluidics Y-device. There were two inlets, a central channel containing the cells, and an outlet. (B) Media alone at the left inlet and media containing α-factor and Dextran-3000-TRITC (tracking dye) at the right inlet were infused resulting in a gradient across the width of the chamber as the chemicals diffused. Five positions down the length of the central channel, denoted A to E, were visualized. (C) The gradient slope varied depending on the position along the length. The gradient became shallower further down the chamber as the tracking dye and α-factor had more time to diffuse. (D) A variety of cell morphologies were observed depending on the amount of α-factor the cells were exposed to. We observed bar1Δ cells in position E in a 0–100 nM gradient.
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pone-0003865-g001: Microfluidics device generates α-factor gradients and response of yeast cells to gradient.(A) Schematic diagram of microfluidics Y-device. There were two inlets, a central channel containing the cells, and an outlet. (B) Media alone at the left inlet and media containing α-factor and Dextran-3000-TRITC (tracking dye) at the right inlet were infused resulting in a gradient across the width of the chamber as the chemicals diffused. Five positions down the length of the central channel, denoted A to E, were visualized. (C) The gradient slope varied depending on the position along the length. The gradient became shallower further down the chamber as the tracking dye and α-factor had more time to diffuse. (D) A variety of cell morphologies were observed depending on the amount of α-factor the cells were exposed to. We observed bar1Δ cells in position E in a 0–100 nM gradient.

Mentions: Microfluidics offer a quantitative and well-controlled method for generating spatial gradients on the micron scale in a reproducible fashion [16], [17]. We used a simple “Y-device”, possessing two inlets converging to a central channel or chamber, to produce an α-factor gradient (Fig. 1A). The device consisted of channels in the polymer poly(dimethylsiloxane) (PDMS) that was attached to a glass slide or coverslip. The cell chamber was 800 µm in width, 15 mm in length, and 100 µm in height. One inlet provided α-factor at a certain concentration and the other provided media without α-factor (Fig. 1B). Laminar flow down the length of the chamber ensured even diffusion across the width of the chamber. The gradient was followed using a 3000 MW fluorescent tracer dye (Dextran-3000-TRITC), which diffused in a similar fashion to α-factor labeled with HiLyte-488 (Supporting Information, Fig. S1). The slope of the gradient varied with the position along the length of the chamber, becoming shallower further down the channel (Fig. 1C).


Robust spatial sensing of mating pheromone gradients by yeast cells.

Moore TI, Chou CS, Nie Q, Jeon NL, Yi TM - PLoS ONE (2008)

Microfluidics device generates α-factor gradients and response of yeast cells to gradient.(A) Schematic diagram of microfluidics Y-device. There were two inlets, a central channel containing the cells, and an outlet. (B) Media alone at the left inlet and media containing α-factor and Dextran-3000-TRITC (tracking dye) at the right inlet were infused resulting in a gradient across the width of the chamber as the chemicals diffused. Five positions down the length of the central channel, denoted A to E, were visualized. (C) The gradient slope varied depending on the position along the length. The gradient became shallower further down the chamber as the tracking dye and α-factor had more time to diffuse. (D) A variety of cell morphologies were observed depending on the amount of α-factor the cells were exposed to. We observed bar1Δ cells in position E in a 0–100 nM gradient.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2586657&req=5

pone-0003865-g001: Microfluidics device generates α-factor gradients and response of yeast cells to gradient.(A) Schematic diagram of microfluidics Y-device. There were two inlets, a central channel containing the cells, and an outlet. (B) Media alone at the left inlet and media containing α-factor and Dextran-3000-TRITC (tracking dye) at the right inlet were infused resulting in a gradient across the width of the chamber as the chemicals diffused. Five positions down the length of the central channel, denoted A to E, were visualized. (C) The gradient slope varied depending on the position along the length. The gradient became shallower further down the chamber as the tracking dye and α-factor had more time to diffuse. (D) A variety of cell morphologies were observed depending on the amount of α-factor the cells were exposed to. We observed bar1Δ cells in position E in a 0–100 nM gradient.
Mentions: Microfluidics offer a quantitative and well-controlled method for generating spatial gradients on the micron scale in a reproducible fashion [16], [17]. We used a simple “Y-device”, possessing two inlets converging to a central channel or chamber, to produce an α-factor gradient (Fig. 1A). The device consisted of channels in the polymer poly(dimethylsiloxane) (PDMS) that was attached to a glass slide or coverslip. The cell chamber was 800 µm in width, 15 mm in length, and 100 µm in height. One inlet provided α-factor at a certain concentration and the other provided media without α-factor (Fig. 1B). Laminar flow down the length of the chamber ensured even diffusion across the width of the chamber. The gradient was followed using a 3000 MW fluorescent tracer dye (Dextran-3000-TRITC), which diffused in a similar fashion to α-factor labeled with HiLyte-488 (Supporting Information, Fig. S1). The slope of the gradient varied with the position along the length of the chamber, becoming shallower further down the channel (Fig. 1C).

Bottom Line: We tested the ability of S. cerevisiaea-cells to sense and respond to spatial gradients of the mating pheromone alpha-factor produced in a microfluidics chamber; the focus was on bar1Delta strains, which do not degrade the pheromone input.Mathematical modeling revealed insights into the mechanism of this amplification and how the supersensitive mutants can disrupt accurate polarization.Together, these data help to specify and elucidate the abilities of yeast cells to sense and respond to spatial gradients of pheromone.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental and Cell Biology, University of California Irvine, Irvine, CA, USA.

ABSTRACT
Projecting or moving up a chemical gradient is a universal behavior of living organisms. We tested the ability of S. cerevisiaea-cells to sense and respond to spatial gradients of the mating pheromone alpha-factor produced in a microfluidics chamber; the focus was on bar1Delta strains, which do not degrade the pheromone input. The yeast cells exhibited good accuracy with the mating projection typically pointing in the correct direction up the gradient ( approximately 80% under certain conditions), excellent sensitivity to shallow gradients, and broad dynamic range so that gradient-sensing was relatively robust over a 1000-fold range of average alpha-factor concentrations. Optimal directional sensing occurred at lower concentrations (5 nM) close to the K(d) of the receptor and with steeper gradient slopes. Pheromone supersensitive mutations (sst2Delta and ste2(300Delta)) that disrupt the down-regulation of heterotrimeric G-protein signaling caused defects in both sensing and response. Interestingly, yeast cells employed adaptive mechanisms to increase the robustness of the process including filamentous growth (i.e. directional distal budding) up the gradient at low pheromone concentrations, bending of the projection to be more aligned with the gradient, and forming a more accurate second projection when the first projection was in the wrong direction. Finally, the cells were able to amplify a shallow external gradient signal of alpha-factor to produce a dramatic polarization of signaling proteins at the front of the cell. Mathematical modeling revealed insights into the mechanism of this amplification and how the supersensitive mutants can disrupt accurate polarization. Together, these data help to specify and elucidate the abilities of yeast cells to sense and respond to spatial gradients of pheromone.

Show MeSH