SlimVar: tracking liquid condensates, oligomers and chromatin binding, tens of microns deep in live root tissue
DOI: 10.22443/rms.mmc2025.327
Authors
Alex Payne-Dwyer (1), Geng-Jen Jang (2), Caroline Dean (2), Mark Leake (1)
Affiliations
- York
- John Innes Centre
Liquid-liquid phase-separated condensates are ubiquitous and vital drivers of plant function, yet extracting information at the underlying molecular scale presents a challenge. Standard ‘number and brightness’ methods are commonly used to extract oligomeric distributions of labelled proteins, but for low-mobility conditions inside condensates, this requires traces of many seconds. In sharp contrast, we demonstrate that counting single molecules by intensity steps allows distributions and dynamics of oligomers inside condensates to be extracted within tens of milliseconds, even in live tissue.
Fluorescent intensity is typically used to quantify expression within live cells - the preserve of flow cytometers in single-cell biology, and of confocal microscopes in multicellular and plant biology. However, outstanding stability of the brightness of fluorescent proteins is available in most physiological media above pH 6. This property delivers consistent photobleaching intensity steps for each fluorescent molecule under controlled imaging conditions.
Single-molecule microscopy provides the required sensitivity, which typical confocal microscopes lack, to detect these directly. We previously presented a straightforward method [1] of lending this single molecule precision to live confocal imaging of Arabidopsis root epithelia labelled with fluorescent proteins, as direct imaging was not possible in plants.
We now demonstrate direct single molecule detection in plants at depths of up to 30 microns with the SlimVar (Slimfield Variable Angle) microscopy platform [2]. By mitigating wavefront aberrations using optical adjustments to a HILO-like microscope train, combined with postprocessing thresholds, we identify strict single molecule signals within individual particles in plant nuclei. We use this to perform rapid oligomeric counting within condensates and oligomers of Polycomb-binding regulatory proteins that associate with chromatin to facilitate plant flowering. Live imaging of seedlings with a bespoke SlimVar single-molecule microscope generates precise statistics of the number and distribution of molecules in each nucleus. Dynamics including diffusivity and binding to specific DNA sites are also quantified robustly, and we show examples relevant to epigenetic switching and memory of cold conditions.
This approach can be applied widely to access precise protein quantification and track the molecular stoichiometries and diffusivities of individual assemblies, even in traditionally diverse and challenging samples, such as the interaction of liquid condensates and chromatin loci in plant tissues.
References
[1] 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. PMID: 35635712.
[2] Payne-Dwyer et al. SlimVar: rapid in vivo single-molecule tracking of chromatin regulators in plants, arXiv, https://doi.org/10.1101/2024.05.17.594710
Keywords
Single molecule tracking
Single particle tracking
Live imaging
Quantitative binding
Aberration correction
Epigenetics
Plant biology