SlimVar: single-molecule tracking in green-lineage biomolecular condensates

Session

Poster Session

Authors

Alex Payne-Dwyer (1), Mark Leake (1)

Affiliations

1. University of York

Keywords

single particle tracking

biomolecular condensates

stepwise photobleaching

single molecule

photosynthesis

epigenetics

Abstract text

SlimVar (Variable Angle Slimfield) is a pipeline for single particle tracking at 50 nm resolution and millisecond timescales in live cells and tissues.  It extends Slimfield microscopy as used in cell monolayers [1,2] and is particularly suited for studying biomolecular condensates in challenging samples such as plants [3] and algae [4]. It simultaneously captures the dynamic behaviour of the liquid compartments, alongside the stoichiometry, diffusivity and colocalisation of constituent molecular complexes.  A major advantage of SlimVar is that it avoids lengthy labelling protocols and requires only single-copy endogenous fusions of monomeric fluorescent proteins such as GFP.   It also employs a simple HILO-type geometry accessible on commercial widefield microscopes with photon-efficient detection beyond the capabilities of standard lightsheet microscopes.

We demonstrate SlimVar in to track vernalisation factors in Arabidopsis nuclei 40 µm deep in live plants, showing the molecular origins of epigenetic memory [5], and track how the carbon-fixation enzymes in Chlamydomonas algae are turbocharged by phase-separating, intrinsically disordered linkers [6].

References

[1] Plank M, Wadhams GH, Leake MC. Millisecond timescale slimfield imaging and automated quantification of single fluorescent protein molecules for use in probing complex biological processes. Integr Biol (Camb). 2009 Oct;1(10):602-12. doi: 10.1039/b907837a.

[2] Syeda AH, Wollman AJM, Hargreaves AL, Howard JAL, Brüning JG, McGlynn P, Leake MC. Single-molecule live cell imaging of Rep reveals the dynamic interplay between an accessory replicative helicase and the replisome. Nucleic Acids Res. 2019 Jul 9;47(12):6287-6298. doi: 10.1093/nar/gkz298

[3] Payne-Dwyer AL, Leake MC. Single-Molecular Quantification of Flowering Control Proteins Within Nuclear Condensates in Live Whole Arabidopsis Root. Methods Mol Biol. 2022;2476:311-328. doi: 10.1007/978-1-0716-2221-6_21

[4] Adler L, Lau CS, Shaikh KM, van Maldegem KA, Payne-Dwyer AL, Lefoulon C, Girr P, Atkinson N, Barrett J, Emrich-Mills TZ, Dukic E, Blatt MR, Leake MC, Peltier G, Spetea C, Burlacot A, McCormick AJ, Mackinder LCM, Walker CE. The role of BST4 in the pyrenoid of Chlamydomonas reinhardtii. bioRxiv [Preprint]. 2023 Nov 17:2023.06.15.545204. doi: 10.1101/2023.06.15.545204

[5] Schulten A, Jang G, Payne-Dwyer AL, Nielsen ML, Bienz M, Leake MC, Dean C.  Functional specialization of Arabidopsis VEL polymerization domains in the switch to Polycomb silencing. bioRxiv [Preprint] 2024.02.15.580496; doi: 10.1101/2024.02.15.580496

[6] Payne-Dwyer AL, Kumar G, Barrett J, Gherman LK, Hodgkinson M, Plevin M, Mackinder L, Leake MC and Schaefer C. Predicting Rubisco:Linker Condensation from Titration in the Dilute Phase. arXiv [Preprint] 2023. doi: 10.48550/arXiv.2301.05681