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Back to 2005 Research
Erin O'Shea Group
Cells live in a constantly changing environment. The availability of essential nutrients can vary widely, yet cells can respond to these changes to maintain nearly constant nutrient levels inside the cell. We conducted a series of experiments to gain insight into the function of the yeast phosphate-sensing pathway (Pho).
Approximately 22 genes are induced during phosphate starvation. The induction of all of these genes depends on the Pho-regulated transcription factor, Pho4. At least one of the induced genes, Pho84, which codes for a high-affinity phosphate transporter, is expressed in a bimodal, i.e. all or none fashion, in response to phosphate starvation.
Project 1
Bistability in biological systems can arise from a number of mechanisms. One mechanism is the combination of a positive feedback loop with a non-linear system response in the signaling pathway. We proposed a model suggesting that positive feedback in the Pho pathway could arise due to down-regulation of low affinity phosphate transporters upon phosphate starvation (Figure 1).
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Figure 1
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Our first line of experiments utilized flow cytometry to measure expression and bistability of Pho84 in single cells. Yeast cultures were grown in a range of phosphate concentrations and the expression of Pho84 was quantitated by measuring GFP fluorescence in single cells (Figure 2). Additionally, we measured expression and bistability from a yeast strain that was deleted for Spl2, the gene responsible for positive feedback in the Pho pathway (Figure 3). We found that Pho84 expression was bistable. Additionally, we found that bistability was abolished in the Spl2 mutant.
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Figure 2
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Figure 3
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We were also interested in determining the source of non-linearity in the Pho pathway. We proposed that the response of the Pho84 promoter to Pho4 could be a source of non-linearity. To test this, we measured the transcriptional response of the Pho84 promoter in single cells to varying levels of Pho4 (Figure 4). To make measurements in the absence of feedback, Pho4 levels were varied through the use of a doxycycline-inducible promoter. We determined that the Pho84 promoter indeed responded in a non-linear fashion, with a Hill coefficient of approximately 6.
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Figure 4
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In addition to being regulated at the transcriptional level, Pho84 is regulated at the level of endocytosis. Upon being shifted from low to high phosphate conditions, Pho84 is rapidly endocytosed from the plasma membrane. We were interested in determining if the endocytosis rate depends on the concentration of external phosphate. To measure the steady-state endocytosis rate, we constitutively expressed Pho84 and then measured its steady state concentration in cells grown in a range of phosphate concentrations using spinning disk confocal microscopy (Figure 5). Our preliminary results suggest that the steady state rate of endocytosis does not depend on the phosphate concentration in the media.
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Figure 5
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Project 2
We were interested in quantifying the dynamics and steady-state localization of the Pho4 transcription factor at high and low phosphate conditions (see Figure 1). Do so, we utilized yeast strains where the Pho4 transcription factor was tagged with YFP and the nucleus was marked with RFP (Figure 6). Our first set of experiments involved measuring steady-state localization at various concentrations of external phosphate. As expected, nuclear localization increased with decreasing phosphate concentration (Figure 7), but we were unable to resolve whether this transition was abrupt (as we would expect) or graded. Additional experiments need to be performed and finer gradations of external phosphate concentration.
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Figure 6
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Figure 7
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We also measured Pho4 localization in two different genetic backgrounds that eliminated the negative and positive feedback loops. PHO84 overexpression eliminates the negative feedback loop. As expected, PHO4 levels were clearly nuclear even at high phosphate concentrations in these strains. SPL2 overexpression short circuits the positive feedback loop, leaving cells with no low affinity transporters. As expected, in these cells PHO4 migrates to the nucleus, even at high phosphate conditions, to induce PHO84 expression. However, this change in localization was not as extreme as we expected (Figure 8).
For these two sets of experiments, one of the technical challenges was quantifying low levels of PHO4 transcription factor by microscopy. Our ambiguous results are due in part to the noise in quantitation coming from cellular autofluorescence and how one defines the nuclear marker. Both these problems (especially the latter) are surmountable obstacles that suggest a slightly different method of analysis. Specifically, only cells with nuclei completely in focus should be analyzed
Our last experiment was to quantify the dynamics of PHO4-YFP localization upon a step change from high to low phosphate conditions. To do so, we employed a microfluidic device in which cells could be loaded and observed by real time fluorescence microscopy as flow conditions were changed. We observed PHO4-YFP localization in a wild type strain was quicker in smaller cells and slower in larger cells. In contrast, PHO-YFP localization was extremely quick in all cells regardless of size when using a strain defective for vacuolar storage of intracellular phosphate. This strongly suggests that vacuolar stores can buffer intracellular phosphate levels, allowing cells to experience low phosphate conditions for 30-60 minutes without turning on the PHO pathway.
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