Understanding feedback loops in membrane dynamics using a tuneable synthetic system in Saccharomyces cerevisiae

The spatio-temporal organisation of molecules in a cell plays an important role in regulating important biological processes such as cell differentiation or cell death. Molecular organisation at cell membranes is an important process through which cells carry out crucial functions. For example, at the plasma membrane, molecules that are involved in signal transduction processes form enriched regions on the membrane to allow the cell to respond to changes in its environment. The enrichment of molecules at membranes is thought to be mainly governed by positive feedback; however, as cellular membranes constitute several types of molecules that interact with one another it is difficult to identify what mechanisms trigger the switch between diffused and enriched states at the membrane. Many competing theoretical models have been proposed to describe what governs the switch between the two states but little experimental data is available to validate these models.

The aim of this thesis is to build a quantifiable synthetic system, which is uncoupled from other feedback in the cell so that it can be used to understand the roles of positive feedback at the plasma membrane. To do this, we have constructed a tuneable synthetic system that exploits the absence of PI(3,4,5)P3 in cellular membranes and the presence of its precursor PI(4,5)P2 at the plasma membrane of Saccharomyces cerevisiae cells. In this thesis, I show that using the catalytic subunit, p110 and PTEN, we can alter phosphatidylinositol metabolism in Saccharomyces cerevisiae cells without observing toxic effects on the cell. I will also introduce the method that was developed to detect and quantify the relative changes in PI(3,4,5)P3 levels at the plasma membrane using a dual reporter system. A machine-learning algorithm was developed to automate quantification of the differences across cells with different PI(3,4,5)P3 conditions which is reflected in the PIP3-Index. Statistical significance of the PIP3-Index across the population distribution was determined using Mann-Whitney test and significance of bimodality was determined using Hartigan's Dip-test.

Using the methods developed in this study, the simplified positive feedback system, was able to show bistability and hysteresis across the cell population. Cells 45 that contain the positive feedback versions of p110 and PTEN (two-loop) and had previously encountered high PI(3,4,5)P3 levels, maintained high levels even after coexpression of p110 and PTEN in the cell. Significant differences (p<0.05) were observed between the response to co-expression of p110α and PTEN in cells that previously experienced high PI(3,4,5)P3 levels, compared to cells that did not experience high PI(3,4,5)P3. The difference between the population means of experienced and naïve cells (or cells that did not experience high levels of PI(3,4,5)P3) was 20%. Bistability in the cell was associated with a higher range of bimodality observed in the two-loop construct compared to the one-loop construct and hysteresis was observed at high levels of PTEN expression in the two-loop construct. The one-loop construct was made up of a the positive feedback version of p110α and a cytosolic version of PTEN and did not show significant bistability across experienced and naïve cell populations. This study confirms that positive feedback at the plasma membrane can result in bistability and memory across the population. These results provide insight for future models of molecular organisation at the membrane that should incorporate elements in addition to a two-loop positive feedback.