A combined in vitro and in vivo approach to dissect biochemical network evolution.

Project Information

EvoCellBio

Grant agreement ID: 758132

DOI

10.3030/758132

Project closed

EC signature date 21 November 2017

Start date 1 February 2018

End date 31 January 2023

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 1 500 000,00

EU contribution

€ 1 500 000,00

Coordinated by

TECHNISCHE UNIVERSITEIT DELFT
Netherlands

Periodic Reporting for period 4 - EvoCellBio (A combined in vitro and in vivo approach to dissect biochemical network evolution.)

Reporting period: 2022-08-01 to 2023-01-31

Summary of the context and overall objectives of the project

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 cancer 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 focusing 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 wanted to rebuild a simplified version of parts of the polarity network ourselves from scratch, to observe how evolutionary resilience emerges with increasing complexity. Simultaneously we wanted to look for network properties in living cells that we can build and test in the future. To our surprise we (1) found while rebuilding a simplified version of the polarity network that the polarity network was not as specific and modular as it is described in the literature. Rather we found that there is significant synergy between different players in the polarity network as well as that most players can fulfill several roles in the network, making the network rather fluidic. So rather than looking for network structures we are now redefining the problem so that we look for systems properties that arise from multivalent weak interactions. Interestingly our findings in simplified systems align with our findings in living cells. We found that during evolutionary adaptation ~880 genes (~15 percent of all genes) significantly change their fitness effect during evolution, while these are not mutated. These finding strengthens our current hypothesis that adaptation is a systems level property of a cell that emerges from the (weak) interactions between many proteins in a cell rather than that they can be defined as caused by specific changes in a small number of genes. Based on these findings we are now setting up thermodynamic models to understand how evolutionary robustness emerges from mixtures of many molecules with the long-term goal to understand and predict evolution.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

We have set up experiments in yeast to investigate how cells 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 (Braun et al, in press Nature Comms). Our model, verified by our experiments, suggests how subsequent mutants in a previously established evolutionary trajectory can restore fitness in the polarity network. Based on these findings we realized that it may not be fair to take a modular approach but rather that evolvability may be more of a collective property of the whole cell rather than of specific modules (Glazenburg and Laan, JCS, 2023). We built computer models to test this idea (Nghe et al, Annu Rev 2020, Daalman et al, Phil Trans B 2023, Zwicker et al PNAS, 2022) and performed genome wide experiments, that showed that various redundant pathways indeed collectively respond to buffer change during evolution (Kingma et al Biorxiv 2023).
In parallel we built artificial (synthetic) systems to look for network structures or rather collective behavior by a purified a set or polarity proteins. As a first step we tested their activity (that these proteins work) in bulk assays (Tschripke et al Biorxiv 2022). Subsequently we needed proteins that can interact with lipids, as they do during polarity establishment, and therefore we developed a new method to add a hydrophobic tail to one of the proteins. After we obtained all our building blocks we studies how these proteins regulate Cdc42. To our surprise we found strong synergy between the regulatory proteins showing that even within a network of three proteins, collective behavior occurs (Tschirpke, Daalman and Laan, Biorxiv 2023).

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

I expect the work by Kingma et al biorxiv 2023, where we quantify the genome wide consequences for cellular organization during evolutionary repair of the polarity network,
to have impact on the field of cell biology and evolutionary biology. In both fields people typically assume that it is allowed to compare the direct effect of a specific gene between two species or mutants, implicitly assuming that the genome wide cell architecture is stable. Our work shows that this is not necessarily a valid assumption. The papers by Tschirpke et al are steps beyond the state of the art of the characterization of Cdc42 regulation. Cdc42 is a very important protein that has been studied extensively in vivo and with structural biology, however the study of its kinetics and membrane binding are lacking behind compared to many other small GTPases due to the difficulties purifying it. With our studies we show that the regulation of Cdc42 is less specific than typically described in textbooks and also our work opens up the study of Cdc42 regulation to a community of biophysicists, who do not have the relevant biochemistry expertise, by extensively describing our methods.
The third paper (Zwicker et al) has made impact on the large and growing field of phase separation of multicomponent mixtures. By classical methods it is impossible to find solutions for mixtures that contain many different proteins, like in the cell, but our evolutionary method has made the study of these mixtures possible. Not only that, our results show that there are many different solutions that all look quite different but that can all form a set number of phases. And, rather than having a few specific interactions that dominate, typically a large ensemble of weak not so specific interactions evolve to form these phases. This finding surprised us and was in large contrast with the intuition in the field, but consistent with our experimental findings. I consider the review by Glazenburg and myself a useful contribution to the field because it, in a concrete and readable way synthesizes insight of self-organization and physics with cell biology and evolution by focusing on evolution of cell polarity.

cartoon explaining network differences between two mutants that only have three mutations that disti

our methods to put a hydrophobic tail on Cdc42

evolutionary repair of the polarity network

cartoon showing the complexity in network regulation

our assay to study polarity in an artifcial system