Single-molecule visualization of temperature adaptation in sub-cellular dynamics and organization across bacteria

Project Information

TEMPADAPT

Grant agreement ID: 101075984

DOI

10.3030/101075984

EC signature date 6 March 2023

Start date 1 May 2023

End date 30 April 2028

Funded under

• European Research Council (ERC)

Total cost

€ 1 792 125,00

EU contribution

€ 1 792 125,00

Coordinated by

AALTO KORKEAKOULUSAATIO SR
Finland

Periodic Reporting for period 1 - TEMPADAPT (Single-molecule visualization of temperature adaptation in sub-cellular dynamics and organization across bacteria)

Reporting period: 2023-05-01 to 2025-10-31

Summary of the context and overall objectives of the project

One of the fundamental questions in biology is how microorganisms are able to thrive across a wide range of temperature while maintaining precise control of cellular processes. Biochemical reaction rates typically double with every 10°C increase in temperature, resulting in faster cellular activities. However, within the complex intracellular environment, such rapid changes can cause large-scale perturbations which could be disastrous to cellular function. Despite this, many microorganisms, including various bacteria, have evolved to survive and grow across large temperature ranges.

Although the temperature-dependent behaviour of biomolecules has been studied extensively, most existing research relies on reconstituted systems where individual components are examined in isolation. These approaches fail to capture the interactions and contributions from a highly dynamic cellular environment, where they operate.

This project seeks to understand how bacteria maintain cellular homeostasis and sub-cellular organization across different growth temperatures by measuring the dynamics of single proteins inside live cells. Using single-molecule tracking, we measure each protein's localization and mobility patterns to infer molecular interactions. This technique not only reveals intra-cellular heterogeneity in molecular behaviour but also shows differences between cells. We further extend these studies to bacteria adapted to extreme temperatures, including psychrophiles and thermophiles, to better understand the evolutionary adaptations that support life under such conditions. To accomplish this, the project will develop new methodologies, including protocols for single-molecule imaging in extremophiles, advanced analytical tools for trajectory quantification, and deep learning algorithms for high-throughput image analysis. These methods will be beneficial to other studies and can be readily applied to other organisms.

The project aims to address the following questions:
• How does temperature influence the dynamics and activity of single molecules within live bacteria?
• How have cytoplasmic dynamics and molecule diffusivity evolved to sustain life at extreme temperatures?
• What molecular mechanisms enable bacteria to withstand and mitigate detrimental effects of temperature fluctuations?

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

To study temperature-dependent molecular dynamics in Escherichia coli, we have created a library of endogenous HaloTag fusion proteins covering different cellular processes. Using a custom state-of-the-art microscope, we have measured single molecule diffusion of these molecules at different temperatures across optimal and restrictive growth temperatures. To quantify cytoplasmic crowding and establish a reference baseline, we have also employed inert probes of various sizes derived from other organisms.

In parallel, we are developing a novel data analysis pipeline capable of multidimensional analysis, combining spatial and temporal data for a more comprehensive understanding of biomolecular processes. This platform leverages advanced machine learning and neural network algorithms to provide automated, accurate, and high-throughput detection and classification of molecular events in live cells.

We are currently applying these methods to extremophilic bacteria, including Thermus thermophilus, Geobacillus stearothermophilus, Psychrobacter cryohalolentis and Pseudoalteromonas haloplanktis. Adapting the methods to these organisms have proven more challenging, requiring the development of novel expression systems and optimization of labelling protocols across a range of temperatures. These efforts are ongoing.

To uncover genes responsible for temperature adaptation, we are performing genome-wide analyses of gene expression profiles at a function of temperature. The work aims to pinpoint key adaptation pathways, which will then be validated through deletion or over-expression mutant strains to assess their impact on single-molecule diffusion and temperature adaptation. We have already collected samples for a subset of the strains while others await further input based on preliminary results.

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)

The project has made significant progress enabling the measurement of single molecule dynamics during rapid temperature changes by integrating a custom-built single molecule tracking microscope with a fast-responding thin-film heating system. This combined with state-of-the-art labelling strategies and the use of brighter, more photostable fluorescent dyes, has allowed us to overcome the limitations of conventional fluorescent proteins to study single molecule dynamics at elevated temperatures.

Additionally, we are establishing novel single molecule labelling and imaging protocols tailored for extremophiles to expand the applicability of above technologies to a broader range of organisms that were previously inaccessible using these approaches. The expected impacts of this work include improved understanding of microbial temperature adaptation, improved analytical tools for cellular biology, and broader application of advanced imaging technologies.