Single-molecule and super-resolved imaging deciphers membrane behavior of onco-immunogenic CCR5

Hunter, Patrick; Payne-Dwyer, Alex L.; Shaw, Michael J.; Signoret, Nathalie; Leake, Mark C.

doi:10.1016/j.isci.2022.105675

Cited by 4 publications

(4 citation statements)

References 71 publications

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“…Continuous wave lasers (Coherent OBIS) delivered Gaussian beams (TEM 00 ) at 488 nm, 514 nm and 561 nm that were circularised by quarter waveplate [75], de-expanded, then focused at the objective back aperture to collimate them through the sample. SlimVar featured an oblique beam angle similar to HILO [48], PaTCH [76] and VAEM [48], [49]. To optimise background contrast for Arabidopsis root tips, a four-step calibration was performed: the second convex lens in the expansion telescope was mounted on a lateral translation stage with a high-precision micrometer; this shift (up to 2.3 mm) generated an equivalent lateral displacement of the beam at the 6 mm diameter objective back aperture, thereby tilting the beam away from the optic axis at the sample.…”

Section: Methodsmentioning

confidence: 99%

Section: Slimvar Imagingmentioning

confidence: 99%

“…Peaks in this distribution were detected using the MATLAB findpeaks function, and the intervals between nearest neighbour peaks were calculated. The uncertainty in peak-to-peak interval was estimated as the single molecule uncertainty of 0.6 molecules multiplied by the square root of the mean stoichiometry, divided by the square root of the number of interpolated intervals [76]. To suppress noise and spurious intra-peak sampling, all peak intervals smaller than the interval uncertainty were discarded.…”

Section: Stoichiometry Periodicity Analysismentioning

confidence: 99%

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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: Methodsmentioning

confidence: 99%

Section: Slimvar Imagingmentioning

confidence: 99%

Section: Stoichiometry Periodicity Analysismentioning

confidence: 99%

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

Payne-Dwyer,

Jang,

Dean

et al. 2024

Preprint

Self Cite

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“…Stoichiometry distributions may exhibit peaks which are separated by a characteristic interval. The smallest consistent interval between peaks can be used to infer the size of a physical repeat unit or ‘periodicity’ within assemblies (Hunter et al, 2022; Payne-Dwyer et al, 2022). A kernel width of 0.7 molecules was chosen to generate the stoichiometry distribution (Fig.…”

Section: Methodsmentioning

confidence: 99%

The role of BST4 in the pyrenoid ofChlamydomonas reinhardtii

Adler

Lau

Shaikh

et al. 2023

Preprint

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The eukaryotic algal CO2-concentrating mechanism (CCM) is based on a Rubisco-rich organelle called the pyrenoid, which is typically traversed by a network of thylakoid membranes. BST4 is a bestrophin-like transmembrane protein that has previously been identified in the model alga Chlamydomonas reinhardtii as a putative tether that could link the traversing thylakoid membrane network to the Rubisco matrix. In the present study, we show that BST4 forms a higher order complex assembly that localizes to the thylakoid network within the pyrenoid. However, investigation of a bst4 knock-out mutant in Chlamydomonas showed that the absence of BST4 did not result in a CCM-deficient phenotype and that BST4 is not necessary for the formation of the trans-pyrenoid thylakoids. Furthermore, heterologous expression of BST4 was not sufficient to facilitate the incorporation of thylakoids into a reconstituted Rubisco condensate in the land plant Arabidopsis. Subsequent analyses revealed that bst4 was under oxidative stress and showed enhanced non-photochemical quenching associated with CO2 limitation and over acidification of the thylakoid lumen. We conclude that the primary role of BST4 is not as a tethering protein, but rather as an ion channel involved in pH regulation in pyrenoid-based CCMs.

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Bestrophin-like protein 4 is involved in photosynthetic acclimation to light fluctuations in Chlamydomonas

Adler,

Lau,

Shaikh

et al. 2024

Plant Physiology

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In many eukaryotic algae, CO2 fixation by Rubisco is enhanced by a CO2-concentrating mechanism, which utilizes a Rubisco-rich organelle called the pyrenoid. The pyrenoid is traversed by a network of thylakoid membranes called pyrenoid tubules, which are proposed to deliver CO2. In the model alga Chlamydomonas (Chlamydomonas reinhardtii), the pyrenoid tubules have been proposed to be tethered to the Rubisco matrix by a bestrophin-like transmembrane protein, BST4. Here, we show that BST4 forms a complex that localizes to the pyrenoid tubules. A Chlamydomonas mutant impaired in the accumulation of BST4 (bst4) formed normal pyrenoid tubules, and heterologous expression of BST4 in Arabidopsis (Arabidopsis thaliana) did not lead to the incorporation of thylakoids into a reconstituted Rubisco condensate. Chlamydomonas bst4 mutants did not show impaired growth under continuous light at air level CO2 but were impaired in their growth under fluctuating light. By quantifying the non-photochemical quenching (NPQ) of chlorophyll fluorescence, we propose that bst4 has a transiently lower thylakoid lumenal pH during dark-to-light transition compared to control strains. We conclude that BST4 is not a tethering protein but is most likely a pyrenoid tubule ion channel involved in the ion homeostasis of the lumen with particular importance during light fluctuations.

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Single-molecule and super-resolved imaging deciphers membrane behavior of onco-immunogenic CCR5

Cited by 4 publications

References 71 publications

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

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

The role of BST4 in the pyrenoid ofChlamydomonas reinhardtii

Bestrophin-like protein 4 is involved in photosynthetic acclimation to light fluctuations in Chlamydomonas

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