Mechanisms of gradient decoding in fission yeast

Project Information

SexYeast

Grant agreement ID: 101019630

DOI

10.3030/101019630

EC signature date 20 June 2021

Start date 1 April 2022

End date 31 March 2027

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 2 191 927,00

EU contribution

€ 2 191 927,00

Coordinated by

UNIVERSITE DE GENEVE
Switzerland

Periodic Reporting for period 3 - SexYeast (Mechanisms of gradient decoding in fission yeast)

Reporting period: 2024-08-01 to 2026-01-31

Summary of the context and overall objectives of the project

How does a cell orient in response to a chemical cue? This basic problem is at the core of how cells interact with their environment. Indeed, the ability to move towards the source of a chemical gradient underlies behaviours as diverse as feeding, identifying a foreign invader or building interconnected cellular networks. Furthermore, chemo-orientation forms the basis of gamete recognition, and is thus critical for genome diversification through sexual reproduction.

This proposal makes use of one of the simplest eukaryotic models, the fission yeast S. pombe, to systematically dissect the mechanisms of gradient detection, namely how cells interpret the spatial information in chemical signals. In fission yeast cells, gradient sensing occurs during sexual reproduction, when cells secrete pheromone to attract partners. This process is akin to speed-dating for mate pairing, where cells exhibit dynamic cortical polarity patches that probe the membrane. These serve to secrete and perceive pheromones and become stabilized in response to those produced by the other mating type. Chemo-detection depends on a minimal kit of conserved eukaryotic proteins, suggesting that a complete understanding of gradient decoding is possible.

The project is divided in four parts, which aim to
1. establish the rules of gradient decoding, using unbiased image analysis and caged pheromones;
2. define the architecture of the communication site, by super-resolution and correlative light-electron microscopy;
3. probe the molecular regulation of cellular speed-dating, which we hypothesize consists of a spatial decoder and dynamic oscillator;
4. reveal natural modifiers of speed-dating for mate selection, by exploiting the diversity of wild strain isolates.

By combining advanced live and correlative imaging with optogenetic, genetic, biochemical and genomic methods, this project will bring a conceptual and molecular understanding to the problem of gradient decoding. Due to the ancient and fundamental nature of gradient orientation, our discoveries will have impact on several fields of research, including those of immunity, wound healing, development, sexual reproduction and 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

In this first period, we have established many tools and made important advance on several of the proposed aims. I briefly describe our progress for each of the four aims below.

Aim 1: We established robust segmentation tools and started dissecting the rules of mate selection. The tools will be made widely available and be broadly useful for the analysis of 4D timelapse data. We have also tested caged pheromones and obtained one form for which proof-of-concept experiments were successful.

Aim 2: We have established a method to record the trajectory of the centroid of fluorescence signals on three-color time lapse acquisitions. Using spatial and temporal references for averaging of trajectories, we have now obtained a super-resolved map of the communication site. This revealed that the communication site at the site of fusion is highly patterned, with individual proteins exhibiting distinct trajectories. It also revealed a novel, unexpected function for type V myosin in organization of the site. This will soon be described in a publication.

Aim 3: We have established robust purification and IP-MS strategies and systematically probed the binding partners of the pheromone signaling cascade proteins. This led to identifying the long sought-after scaffold of the pheromone-MAPK signaling pathway. This will soon be described in a publication.

Aim 4: We have established competitive mating assays quantified through split-GFP technology. This allowed us to characterize the diversity in mating strategy of wild strain isolates, leading to the identification of strains with stronger outbreeding activity.

We have published two studies: a study on the relative dynamics of Cdc42 and the membrane (Rutkowski et al, Nature Communications 2024); and a phosphoproteomic description of the signaling changes happening during sexual reproduction (Bérard et al, PLoS Biology, in press).

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 we will achieve the majority of the stated aims by the end of the project.

Aim 1: The segmentation tools will allow dissection of the rules of mate pairing on large numbers of cells. We are currently expanding on the number of recorded markers. The caged pheromone experiments are proving challenging due to degradation of the caged factor by secreted proteases. We are currently probing improved experimental setups. Work from this aim will be presented in at least two publications.

Aim 2: We are extending our work to probe the novel function of type V myosin in organization of the communication site.

Aim 3: We are focusing on the identified MAPK scaffold, and are probing how this protein contributes positive and negative signaling for signal dynamics. Besides the planned publication, we expect to publish at least a second work.

Aim 4: We are striving to identify the genetic causes of the divergent mating strategies in the strains we identified, and will publish our findings.