Observation of the molecular organization of calcium release sites in fast- and slow-twitch skeletal muscle with nanoscale imaging
Observation of the molecular organization of calcium release sites in fast- and slow-twitch skeletal muscle with nanoscale imaging
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DOI：
10.1098/rsif.2014.0570
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发表时间：
2014-10-06
影响因子：
3.9
通讯作者：
Soeller, Christian
中科院分区：
文献类型：
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作者：
Jayasinghe, Isuru D.;Munro, Michelle;Soeller, Christian
局部化显微镜是一种最近引入的能够实现纳米级分辨率的超分辨率荧光成像方式。我们已经将这种方法的dSTORM变体应用于骨骼肌纤维中的细胞内分子组装体的成像，骨骼肌纤维是严重依赖于纳米级信号传导域的大细胞，即黑社会。固定的成年大鼠骨骼肌切片中的免疫荧光染色显示，在黑社会中兰尼碱受体（RyRs;主要的钙释放通道）的分子组织中，快肌和慢肌纤维之间存在明显差异。随着dSTORM提供的分辨率的提高，在快纤维的横向视图中观察到邻接的RyR阵列，而慢肌上的碎片化分布则短约1.8倍，由约1.6倍的受体组成。据我们所知，我们第一次使用多色超分辨率量化了三元蛋白质之间的纳米级空间关联，这是一种难以用电子显微镜进行的分析。我们的研究结果证实，junctophilin-1（JPH 1），其中拴系肌浆网（（SR）细胞内钙存储）的管状（t-）系统在三合会，是目前整个RyR阵列，而JPH 2包含在更小的nanodomains。主要SR钙缓冲剂，钙螯合蛋白（CSQ）的类似成像，检测到三重与CSQ在慢收缩肌肉中的重叠较少，支持与快收缩肌肉相比，腔Ca 2+缓冲的空间异质性更大。总的来说，这些纳米级的差异可以解释快肌和慢肌的根本不同生理学。
Localization microscopy is a fairly recently introduced super-resolution fluorescence imaging modality capable of achieving nanometre-scale resolution. We have applied the dSTORM variation of this method to image intracellular molecular assemblies in skeletal muscle fibres which are large cells that critically rely on nanoscale signalling domains, the triads. Immunofluorescence staining in fixed adult rat skeletal muscle sections revealed clear differences between fast-and slow-twitch fibres in the molecular organization of ryanodine receptors (RyRs; the primary calcium release channels) within triads. With the improved resolution offered by dSTORM, abutting arrays of RyRs in transverse view of fast fibres were observed in contrast to the fragmented distribution on slow-twitch muscle that were approximately 1.8 times shorter and consisted of approximately 1.6 times fewer receptors. To the best of our knowledge, for the first time, we have quantified the nanometre-scale spatial association between triadic proteins using multi-colour super-resolution, an analysis difficult to conduct with electron microscopy. Our findings confirmthat junctophilin-1 (JPH1), which tethers the sarcoplasmic reticulum ((SR) intracellular calcium store) to the tubular (t-) system at triads, was present throughout the RyR array, whereas JPH2 was contained within much smaller nanodomains. Similar imaging of the primary SR calcium buffer, calsequestrin (CSQ), detected less overlap of the triad with CSQ in slow-twitch muscle supporting greater spatial heterogeneity in the luminal Ca2+ buffering when compared with fast twitch muscle. Taken together, these nanoscale differences can explain the fundamentally different physiologies of fast-and slow-twitch muscle.