Caveolae and Bin1 form ring-shaped platforms for T-tubule initiation

Excitation-contraction coupling requires a highly specialized membrane structure, the triad, composed of a plasma membrane invagination, the T-tubule, surrounded by two sarcoplasmic reticulum terminal cisternae. Although the precise mechanisms governing T-tubule biogenesis and triad formation remain largely unknown, studies have shown that caveolae participate in T-tubule formation and mutations of several of their constituents induce muscle weakness and myopathies. Here, we demonstrate that, at the plasma membrane, caveolae composed of caveolin-3 and Bin1 assemble into ring-like structures from which emerge tubes enriched in the dihydropyridine receptor. Overexpression of Bin1 leads to the formation of both rings and tubes and we show that Bin1 forms scaffolds on which caveolae accumulate to form the initial T-tubule. Cav3 deficiency caused by either gene silencing or pathogenic mutations causes defective ring formation and perturbed Bin1-mediated tubulation that may explain defective T-tubule organization in mature muscles. Our results uncover new pathophysiological mechanisms that may prove relevant to myopathies caused by Cav3 or Bin1 variants.

Methods
These data were generated to investigate the organization and dynamics of caveolae and Bin1 on in vitro and in cellulo systems using sub-diffracted fluorescence microscopy, conventional and platinum replica electron microscopy.

兴奋-收缩耦联需要一种高度特化的膜结构——三联体（triad），其由质膜内陷形成的横小管（T-tubule）构成，周围环绕着两个肌浆网终池（sarcoplasmic reticulum terminal cisternae）。尽管目前对于调控横小管生物发生与三联体形成的精确机制仍知之甚少，但已有研究表明细胞质膜微囊（caveolae）参与横小管的形成，且其多种组分的突变可引发肌肉无力与肌病。

本研究证实，在质膜表面，由窖蛋白3（caveolin-3）与Bin1构成的细胞质膜微囊会组装为环状结构，从中延伸出富含二氢吡啶受体（dihydropyridine receptor）的管状结构。Bin1过表达可同时诱导环状结构与管状结构的形成，且研究发现Bin1可构建支架结构，细胞质膜微囊可在该支架上聚集，进而形成初始横小管。无论是通过基因沉默还是致病性突变引发的窖蛋白3（Cav3）缺失，均会导致环状结构形成缺陷，并扰乱Bin1介导的管形成过程，这或可解释成熟肌肉中横小管组织异常的成因。本研究结果揭示了全新的病理生理机制，该机制或与Cav3及Bin1变异引发的肌病密切相关。

方法
本数据集通过亚衍射荧光显微镜（sub-diffracted fluorescence microscopy）、常规电子显微镜与铂复型电子显微镜（platinum replica electron microscopy），在体外与细胞内体系中探究细胞质膜微囊与Bin1的组织形式与动态变化。