Tracking live-cell single-molecule dynamics enables measurements of heterochromatinassociated protein-protein interactions.
Tracking live-cell single-molecule dynamics enables measurements of heterochromatinassociated protein-protein interactions.
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跟踪活细胞单分子动力学可以测量异染色质相关的蛋白质-蛋白质相互作用。
DOI：
10.1101/2023.03.08.531771
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Ragunathan,Kaushik
中科院分区：
文献类型：
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作者：
Chen,Ziyuan;Seman,Melissa;Farhat,Ali;Fyodorova,Yekaterina;Biswas,Saikat;Levashkevich,Alexander;Freddolino,PLydia;Biteen,JulieS;Ragunathan,Kaushik
可视化和测量活细胞中分子尺度的相互作用是一项重大挑战，但单分子超分辨率显微镜的最新进展使我们更接近实现这一目标。单分子超分辨率显微镜能够对活细胞中分子的位置和运动进行高分辨率和灵敏的成像。 HP1 蛋白是基因表达的重要调节因子，因为它们选择性结合并识别 H3K9 甲基化 (H3K9me) 组蛋白，形成异染色质相关蛋白复合物，从而沉默基因表达，但该过程的几个重要机制细节尚未探索。在这里，我们扩展了裂殖酵母中的活细胞单分子追踪研究，以确定 HP1 蛋白如何与其在细胞核中的结合伙伴相互作用。我们测量了影响 H3K9me 的遗传扰动如何改变 HP1 蛋白及其结合伴侣的扩散特性，并推断出它们最可能的相互作用位点。我们的结果表明，H3K9 甲基化在空间上限制 HP1 蛋白及其相互作用物，从而促进染色质上三元复合物的形成，同时抑制染色质外结合。与直接 HP1 结合的惰性平台相反，我们的研究提出了 H3K9me 的一种新功能，即通过增强活细胞中 HP1-蛋白质复合物的特异性和刺激其组装来促进三元复合物形成。
Visualizing and measuring molecular-scale interactions in living cells represents a major challenge, but recent advances in single-molecule super-resolution microscopy are bringing us closer to achieving this goal. Single-molecule super-resolution microscopy enables high-resolution and sensitive imaging of the positions and movement of molecules in living cells. HP1 proteins are important regulators of gene expression because they selectively bind and recognize H3K9 methylated (H3K9me) histones to form heterochromatin-associated protein complexes that silence gene expression, but several important mechanistic details of this process remain unexplored. Here, we extended live-cell single-molecule tracking studies in fission yeast to determine how HP1 proteins interact with their binding partners in the nucleus. We measured how genetic perturbations that affect H3K9me alter the diffusive properties of HP1 proteins and their binding partners, and we inferred their most likely interaction sites. Our results demonstrate that H3K9 methylation spatially restricts HP1 proteins and their interactors, thereby promoting ternary complex formation on chromatin while simultaneously suppressing off-chromatin binding. As opposed to being an inert platform to direct HP1 binding, our studies propose a novel function for H3K9me in promoting ternary complex formation by enhancing the specificity and stimulating the assembly of HP1–protein complexes in living cells.