Periodic Reporting for period 3 - EvoCellBio (A combined in vitro and in vivo approach to dissect biochemical network evolution.)
Okres sprawozdawczy: 2021-02-01 do 2022-07-31
Organisms are remarkably resilient, both in their daily life as well as during evolution. We are fascinated to know what this resilience looks like at the molecular level, both out of curiosity, but also because antibiotic or tumour drug resistance are consequences of this resilience. So how is this evolutionary resilience build in the architecture of an organisms? Because this is a very complex problem, we answer this question by focussing on the single celled organism budding yeast and on one cellular function: polarity establishment, which is essential for cell survival. Our goal is to understand, what it is in this specific network of proteins (but in any network really) that makes it so resilient during evolution. To answer this question we rebuild a simplified version of the polarity network ourselves from scratch, to observe how evolutionary resilience emerges with increasing complexity. Simultaneously we look for network properties in living cells that we can build and test in the future.
We have set up in vivo experiments in yeast to look for network structures that help cells to evolve to recover from mutations in the polarity network. Our assays consists of growth rate experiments as well as live cell microscopy experiments, where we can observe the dynamics of the different polarity proteins involved. With these experiments we have tested a mathematical model that we developed in collaboration with a theory group. Our model, verified by our experiments, explains how subsequent mutants in a previously established evolutionary trajectory restore fitness in the polarity network. Based on these findings we have found first network structures that we will test in artificial systems. To build these artificial (synthetic) systems we have purified the relevant proteins and tested their activity (that these proteins work) in bulk assays. Currently we are testing how these proteins interact with lipids and we are introducing them in a biophysical microscopy assay, where we can observe how these proteins interact and how they generate evolutionary resilience.
So far our in vivo studies have led to ground breaking results in the field of predicting of evolution, because we can predict evolution bottom up, thus from the underlying biophysics rather than from genetic data. To the best of our knowledge we are the first to pioneer this biophysical approach, including spatiotemporal dynamics for an existing network of proteins. For our in vitro studies we have set-up together with a chemist an assay to synthetically create a Cdc42 protein that can bind to membranes. In addition we have for the first time shown that the GAP protein Rga2 can regulate Cdc42 activity in a minimal vitro assay and does that synergistically with the GEF Cdc24.