Center for Research in Biological Systems, University of California San Diego, La Jolla, CA, USA.
Biophys J. 2013 Jun 4;104(11):L22-4. doi: 10.1016/j.bpj.2013.02.059.
We conducted super-resolution light microscopy (LM) imaging of the distribution of ryanodine receptors (RyRs) and caveolin-3 (CAV3) in mouse ventricular myocytes. Quantitative analysis of data at the surface sarcolemma showed that 4.8% of RyR labeling colocalized with CAV3 whereas 3.5% of CAV3 was in areas with RyR labeling. These values increased to 9.2 and 9.0%, respectively, in the interior of myocytes where CAV3 was widely expressed in the t-system but reduced in regions associated with junctional couplings. Electron microscopic (EM) tomography independently showed only few couplings with caveolae and little evidence for caveolar shapes on the t-system. Unexpectedly, both super-resolution LM and three-dimensional EM data (including serial block-face scanning EM) revealed significant increases in local t-system diameters in many regions associated with junctions. We suggest that this regional specialization helps reduce ionic accumulation and depletion in t-system lumen during excitation-contraction coupling to ensure effective local Ca²⁺ release. Our data demonstrate that super-resolution LM and volume EM techniques complementarily enhance information on subcellular structure at the nanoscale.
我们进行了超分辨率荧光显微镜（LM）成像，以研究 Ryanodine 受体（RyRs）和 caveolin-3（CAV3）在小鼠心室肌细胞中的分布。对表面肌质网数据的定量分析表明，4.8%的 RyR 标记与 CAV3 共定位，而 3.5%的 CAV3 位于具有 RyR 标记的区域。这些值分别增加到 9.2%和 9.0%，在肌细胞内部，CAV3 在 t 系统中广泛表达，但在与连接子偶联相关的区域减少。独立的电子显微镜（EM）断层扫描显示，仅存在少数与 caveolae 的偶联，并且在 t 系统上几乎没有 caveolar 形状的证据。出乎意料的是，超分辨率 LM 和三维 EM 数据（包括连续块面扫描 EM）都显示，在与连接相关的许多区域中，局部 t 系统直径显著增加。我们认为这种区域特化有助于减少兴奋-收缩偶联过程中 t 系统腔中的离子积累和耗竭，以确保有效的局部 Ca²⁺释放。我们的数据表明，超分辨率 LM 和体积 EM 技术互补地增强了纳米尺度亚细胞结构的信息。
