Molecular and emergent dynamics of recombinant algal pyrenoids by single-molecule tracking and optical tweezers

Session

Multimodal Microscopy

Authors

Catherine Read (2), Daria Gusew (1), Eleanor Goffee (2), Dr Alex Payne-Dwyer (2)

Affiliations

1. Freie Universitat Berlin
2. University of York

Keywords

Biomolecular condensates

Optical tweezers

Single molecule tracking

Fluorescence recovery after photobleaching (FRAP)

Quantitative phase imaging

Pyrenoid

Abstract text

Most of the oxygen we breathe finds its origins in marine algal photosynthesis.  These algae organise their Rubisco, the enzyme responsible for capturing carbon dioxide and releasing oxygen, into a liquid droplet called the pyrenoid [1]. The algae then concentrate carbon dioxide into the liquid, and the reaction can run around 60% faster compared to the same reaction in plant leaves.  Such a rapidly reversible structure suggests a clear fitness benefit in response to the low CO2 and fluctuating light levels in aquatic surface environments. However, Rubisco requires a counterpart ‘linker’ protein featuring multivalent, transient binding to form this liquid matrix [2].  What then are the minimal properties of a linker and the resulting properties of the droplet that drive internal mixing and this incredible boost in photosynthetic output?  

Our model system uses recombinant Rubisco and the counterpart linker EPYC1 from the green alga Chlamydomonas to nucleate pyrenoid-like droplets. Connecting the molecular and emergent behaviour requires a range of techniques with high specificity and spatiotemporal resolution, as well as the ability to apply perturbative forces.  We correlate widefield fluorescence and optical phase tomography [3] to generate maps of Rubisco and linker concentrations within the droplets.   Combining single-molecule tracking [4] and fluorescence recovery allows us to explore the diffusive dynamics of individual components, while stretching droplets in optical tweezers [5] reveals the emergent rheology of the matrix as a whole. We find that the mechanical stiffness of the pyrenoid is linked to slower rates of fluorescence recovery, with an unexpected differential between the enzyme and linker components.  Since Rubisco is chiefly activated near the pyrenoid surface, this inhibited mixing has implications for the rate of enzymatic carbon fixation.

References

[1] Barrett J. et al. (2021) BBA Mol Cell Res 1868(5):118949,

[2] Mackinder L. et al. (2016) PNAS 113(21):5958-5963

[3] McCall P.M. et al. (2020) bioRxiv 2020.10.25.352823

[4] Payne-Dwyer A.L. et al. (2022) J R Soc Interface 19:20220437

[5] Hargreaves A.L. et al. (2015) J Mol Liq 210:9-19