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用于植物染色质调控因子体内快速单分子追踪的SlimVar

SlimVar for rapid in vivo single-molecule tracking of chromatin regulators in plants.

作者信息

Payne-Dwyer Alex L, Jang Geng-Jen, Dean Caroline, Leake Mark C

机构信息

School of Physics, Engineering and Technology, University of York, York, UK.

Department of Biology, University of York, York, UK.

出版信息

Nat Commun. 2025 Sep 1;16(1):8156. doi: 10.1038/s41467-025-63108-8.

Abstract

Epigenetic regulation occurs over many rounds of cell division in higher organisms. However, visualisation of the regulators in vivo is limited by imaging dynamic molecules deep in tissue. We report a technology-Variable-angle Slimfield microscopy (SlimVar)-that enables tracking of single fluorescent reporters to 30 µm depth through multiple Arabidopsis thaliana root tip cell layers. SlimVar uses rapid photobleaching to resolve tracked particles to molecular steps in intensity. By modifying widefield microscopy to minimise optical aberrations and robustly post-process few-photon signals, SlimVar mitigates performance losses at depth. We use SlimVar to quantify chromatin-protein assemblies in nuclei, finding that two homologous proteins key to epigenetic switching at FLOWERING LOCUS C (FLC) -cold-induced VERNALISATION INSENSITIVE3 (VIN3) and constitutively expressed VERNALISATION 5 (VRN5)-exhibit dynamic assemblies during FLC silencing. Upon cold exposure, the number of assembly molecules increases up to 100% to a median of ~20 molecules. Larger VRN5 assemblies preferentially colocalise with an FLC lacO transgenic reporter during prolonged cold and persist after return to warmth. 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 offers molecular insights into proteins expressed at physiological levels in tissues.

摘要

表观遗传调控发生在高等生物的多轮细胞分裂过程中。然而,体内调控因子的可视化受到组织深处动态分子成像的限制。我们报告了一种技术——可变角度薄场显微镜(SlimVar),它能够通过多个拟南芥根尖细胞层追踪单个荧光报告分子至30微米深度。SlimVar利用快速光漂白将追踪的粒子解析为强度上的分子步骤。通过改进宽场显微镜以最小化光学像差并稳健地后处理少光子信号,SlimVar减轻了深度处的性能损失。我们使用SlimVar对细胞核中的染色质 - 蛋白质组装体进行定量,发现开花位点C(FLC)处表观遗传转换的两个关键同源蛋白——冷诱导的春化不敏感3(VIN3)和组成型表达的春化5(VRN5)——在FLC沉默期间表现出动态组装。在冷暴露后,组装分子的数量增加高达100%,中位数约为20个分子。在长时间寒冷期间,较大的VRN5组装体优先与FLC lacO转基因报告分子共定位,并在恢复温暖后持续存在。我们的研究结果支持一种表观遗传记忆的混合模型,其中组蛋白三甲基化的成核在延长的持续时间内由动态蛋白质组装体辅助。SlimVar为组织中生理水平表达的蛋白质提供了分子见解。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0f7c/12402101/bf826db6ea7e/41467_2025_63108_Fig1_HTML.jpg

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