True Molecular Scale Visualization of Variable Clustering Properties of Ryanodine Receptors.
True Molecular Scale Visualization of Variable Clustering Properties of Ryanodine Receptors.
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Ryanodine受体的可变聚类特性的真实分子尺度可视化。
DOI：
10.1016/j.celrep.2017.12.045
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发表时间：
2018-01-09
期刊：
影响因子：
8.8
通讯作者：
Soeller C
中科院分区：
文献类型：
--
作者：
Jayasinghe I;Clowsley AH;Lin R;Lutz T;Harrison C;Green E;Baddeley D;Di Michele L;Soeller C
信号传导纳米结构域依赖于蛋白质的空间组织以允许受控的细胞内信号传导。实例包括心肌细胞的钙释放位点，其中兰尼碱受体（RyR）与它们的分子伴侣成簇。定位显微镜对于可视化这些纳米结构域至关重要，但受到标记物亮度的限制，限制了聚集在其中的单个蛋白质的分辨率和定量。利用DNA-PAINT的显著定位精度（<10 nm），我们可视化了这些纳米结构域内的点状标记，确认为单个RyR。RyR在质膜下纳米结构域中的位置揭示了它们是如何随机组织成不规则的聚类模式的，从而留下由辅助蛋白或调节蛋白占据的显著间隙。RyR抑制蛋白junctophilin-2出现高度集中的RyR通道附近。分析这些分子图谱显示，即使在附近的纳米结构域之间，亲连接蛋白-2和RyR之间的共簇化学计量也存在显着变化。这构成了RyR排列和钙信号传导调节的额外复杂性水平，内在地构建在纳米结构域中。随着分辨率的提高，在密集簇中观察到单个RyR RyR似乎随机且不受约束地组装。RyR与JPH 2共簇的比率在纳米结构域之间变化很大。基于DNA的超分辨率使得能够在100 nm分辨率下进行完全定量成像。用DNA-PAINT（一种超分辨率显微镜方法）解析心肌细胞胞内信号结构域中单个兰尼碱受体（RyR）的分布。单个RyR在不规则的簇阵列内分辨。定量成像揭示了RyRs和调节蛋白junctophilin-2之间的共簇化学计量的显着变化。
Signaling nanodomains rely on spatial organization of proteins to allow controlled intracellular signaling. Examples include calcium release sites of cardiomyocytes where ryanodine receptors (RyRs) are clustered with their molecular partners. Localization microscopy has been crucial to visualizing these nanodomains but has been limited by brightness of markers, restricting the resolution and quantification of individual proteins clustered within. Harnessing the remarkable localization precision of DNA-PAINT (<10 nm), we visualized punctate labeling within these nanodomains, confirmed as single RyRs. RyR positions within sub-plasmalemmal nanodomains revealed how they are organized randomly into irregular clustering patterns leaving significant gaps occupied by accessory or regulatory proteins. RyR-inhibiting protein junctophilin-2 appeared highly concentrated adjacent to RyR channels. Analyzing these molecular maps showed significant variations in the co-clustering stoichiometry between junctophilin-2 and RyR, even between nearby nanodomains. This constitutes an additional level of complexity in RyR arrangement and regulation of calcium signaling, intrinsically built into the nanodomains. With enhanced resolution, individual RyRs were observed within dense clusters RyRs appear to assemble randomly and unconstrained The ratio of RyR co-clustering with JPH2 varies considerably between nanodomains DNA-based super-resolution enables fully quantitative imaging at ∼10 nm resolution Jayasinghe et al. resolve the distribution of single ryanodine receptors (RyRs) within intracellular signaling domains in cardiac myocytes with DNA-PAINT, a super-resolution microscopy approach. Individual RyRs are resolved within irregular cluster arrays. Quantitative imaging reveals significant variation in the co-clustering stoichiometry between RyRs and the regulatory protein junctophilin-2.
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