Variable-Angle Epifluorescence Microscopy for Single-Particle Tracking in the Plant ER

Pain, Charlotte; Tynan, Christopher; Botchway, Stanley W.; Kriechbaumer, Verena

doi:10.1007/978-1-0716-3710-4_20

Cited by 2 publications

(2 citation statements)

References 28 publications

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“…In animal tissues, studies using HILO [45] [56], lightsheet [57] [58] or lattice lightsheet [32] have previously demonstrated single-molecule tracking to a standard of 50 ms sampling at ∼300 µm depth, or 10 ms at ∼30 µm depth using dyes, which is comparable to SlimVar with fluorescent proteins. However, in plant tissues, single particle tracking at molecular sensitivity has been demonstrated with TIRF [59], [60], or VAEM [28], [30], [61], [62] only in the vicinity of a surface cell layer. SlimVar therefore advances the ability to track and count single-molecular assemblies in plants, and potentially in a range of tissues, to that of more complex existing microscopy technologies.…”

Section: Discussionmentioning

confidence: 99%

SlimVar: rapidin vivosingle-molecule tracking of chromatin regulators in plants

Payne-Dwyer,

Jang,

Dean

et al. 2024

Preprint

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Epigenetic regulation maintains gene expression patterns over many rounds of cell division in higher organisms. However, visualization of factors regulating epigenetic switches in vivo is limited by the challenge of imaging cells deep in living tissue, with molecular sensitivity and rapid sampling. We report an easy-to-implement method called Variable-angle Slimfield microscopy (SlimVar), which by simple modification of an inverted optical microscope, enables single-molecule tracking of fluorescent reporters in Arabidopsis thaliana. Using SlimVar, we imaged stepwise photobleaching of chromatin-protein assemblies in individual nuclei, 30 microns deep in root tips through multiple cell layers. We find that two homologous proteins key to the epigenetic switch at FLOWERING LOCUS C (FLC) - cold-induced VERNALIZATION INSENSITIVE3 (VIN3) and constitutively expressed VERNALIZATION 5 (VRN5) - exhibit dynamic nuclear assemblies during FLC silencing. Upon cold exposure, these assemblies increase in stoichiometry by up to 100% to a median of ~20 molecules. Larger VRN5 assemblies preferentially co-localize with an FLC lacO transgenic reporter during prolonged cold, persisting after return to warm conditions. Our findings support a hybrid model of epigenetic memory in which nucleation of histone trimethylation is assisted by dynamic protein assemblies over extended durations. SlimVar therefore has potential to offer molecular insights into proteins expressed at physiological levels in a range of tissues.

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Section: Discussionmentioning

confidence: 99%

SlimVar: rapidin vivosingle-molecule tracking of chromatin regulators in plants

Payne-Dwyer,

Jang,

Dean

et al. 2024

Preprint

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“…These technical difficulties have contributed to the fact that dynamic analysis of proteins in plant cells at very high resolution has only recently taken off. The improvement of technical possibilities in microscopy and other methods, such as singlemolecule tracking and cluster analysis, now offers data on dynamic parameters, including diffusion coefficients and nanoscale organization, especially for membrane proteins (Gronnier et al, 2017;Perraki et al, 2018;McKenna et al, 2019;Platre et al, 2019;Smokvarska et al, 2020;Platre et al, 2022;Smokvarska et al, 2023;Pain et al, 2024).…”

Section: Introductionmentioning

confidence: 99%

OneFlowTraX: a user-friendly software for super-resolution analysis of single-molecule dynamics and nanoscale organization

Rohr,

Ehinger,

Rausch

et al. 2024

Front. Plant Sci.

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Super-resolution microscopy (SRM) approaches revolutionize cell biology by providing insights into the nanoscale organization and dynamics of macromolecular assemblies and single molecules in living cells. A major hurdle limiting SRM democratization is post-acquisition data analysis which is often complex and time-consuming. Here, we present OneFlowTraX, a user-friendly and open-source software dedicated to the analysis of single-molecule localization microscopy (SMLM) approaches such as single-particle tracking photoactivated localization microscopy (sptPALM). Through an intuitive graphical user interface, OneFlowTraX provides an automated all-in-one solution for single-molecule localization, tracking, as well as mobility and clustering analyses. OneFlowTraX allows the extraction of diffusion and clustering parameters of millions of molecules in a few minutes. Finally, OneFlowTraX greatly simplifies data management following the FAIR (Findable, Accessible, Interoperable, Reusable) principles. We provide a detailed step-by-step manual and guidelines to assess the quality of single-molecule analyses. Applying different fluorophores including mEos3.2, PA-GFP, and PATagRFP, we exemplarily used OneFlowTraX to analyze the dynamics of plant plasma membrane-localized proteins including an aquaporin, the brassinosteroid receptor Brassinosteroid Insensitive 1 (BRI1) and the Receptor-Like Protein 44 (RLP44).

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Variable-Angle Epifluorescence Microscopy for Single-Particle Tracking in the Plant ER

Cited by 2 publications

References 28 publications

SlimVar: rapidin vivosingle-molecule tracking of chromatin regulators in plants

SlimVar: rapidin vivosingle-molecule tracking of chromatin regulators in plants

OneFlowTraX: a user-friendly software for super-resolution analysis of single-molecule dynamics and nanoscale organization

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