The accessibility and interconnectivity of the tubular system network in toad skeletal muscle
The accessibility and interconnectivity of the tubular system network in toad skeletal muscle
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DOI：
10.1113/jphysiol.2008.155127
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发表时间：
2008-11-01
影响因子：
5.5
通讯作者：
Launikonis, Bradley S.
中科院分区：
文献类型：
--
作者：
Edwards, Joshua N.;Launikonis, Bradley S.
管状（t）系统对于骨骼肌纤维的正常功能是必不可少的，充当细胞内分子和离子的管道。然而，t系统的可及性和互连性主要在固定细胞中进行评估，其中t系统不再完全代表活细胞的t系统。在这里，不同直径的荧光染料被允许在蟾蜍的完整纤维的t系统内平衡，机械剥皮以捕获染料，然后使用共聚焦显微镜成像以研究t系统的可及性和互连性。rhod-2和500 kDa荧光素葡聚糖的双重成像确定了整个t系统中不同分子量分子可及性的区域。t系统管腔内的限制发生在纵向和横向小管的交界处，以及横向小管分裂成两个小管以保持其与相邻错位肌节的Z线对齐的地方。因此，三种类型的小管，横向，纵向和Z，可以通过它们的管腔直径在这个网络中识别。我们首次将后者定义为具有狭窄管腔的小管，该管腔负责寄存器的变化。牵张诱导的t系统空泡化显示rhod-2独占进入这些结构，表明它们的起源是纵小管。将密封的t系统暴露于高渗溶液中可逆转纵向小管的空泡化，并显示这些小管不可塌陷。荧光恢复后光漂白（FRAP）测量的t系统捕获的fluo-5 N显示通过t系统沿着轴的纤维的互连性。然而，考虑到连接相邻横管的纵管数量已知，扩散速度比预期慢。这可以通过观察到的从横向小管到纵向小管的狭窄开口来解释，减少了分子可以在t系统内移动的有效横截面积。
The tubular (t) system is essential for normal function of skeletal muscle fibre, acting as a conduit for molecules and ions within the cell. However, t system accessibility and interconnectivity have been mainly assessed in fixed cells where the t system no longer fully represents that of the living cell. Here, fluorescent dyes of different diameter were allowed to equilibrate within the t system of intact fibres from toad, mechanically skinned to trap the dyes, and then imaged using confocal microscopy to investigate t system accessibility and interconnectivity. Dual imaging of rhod-2 and a 500 kDa fluorescein dextran identified regions throughout the t system that differed in the accessibility to molecules of different molecular weight. Restrictions within the t system lumen occurred at the junctions of the longitudinal and transverse tubules and also where a transverse tubule split into two tubules to maintain their alignment with Z-lines of adjacent mis-registered sarcomeres. Thus, three types of tubule, transverse, longitudinal and Z, can be identified by their lumenal diameter in this network. The latter we define for the first time as a tubule with a narrow lumen that is responsible for the change in register. Stretch-induced t system vacuolation showed exclusive access of rhod-2 to these structures indicating their origin was the longitudinal tubules. Exposing the sealed t system to highly hypertonic solution reversed vacuolation of longitudinal tubules and also revealed that these tubules are not collapsible. Fluorescence recovery after photobleaching (FRAP) measurements of t system-trapped fluo-5 N showed interconnectivity through the t system along the axis of the fibre. However, diffusion occurred at a rate slower than expected given the known number of longitudinal tubules linking adjacent transverse tubules. This could be explained by the observed narrow opening to the longitudinal tubules from transverse tubules, reducing the effective cross-sectional area in which molecules could move within the t system.