Differential effects of Latrunculin-A on myofibrils in cultures of skeletal muscle cells: insights into mechanisms of myofibrillogenesis.
Differential effects of Latrunculin-A on myofibrils in cultures of skeletal muscle cells: insights into mechanisms of myofibrillogenesis.
复制标题
Latrunculin-A 对骨骼肌细胞培养物中肌原纤维的不同影响：深入了解肌原纤维发生机制。
DOI：
10.1002/cm.20083
复制
发表时间：
2005
影响因子：
--
通讯作者：
Sanger,JosephW
中科院分区：
文献类型：
--
作者：
Wang,Jushuo;Sanger,JeanM;Sanger,JosephW
为了测试肌原纤维发生的不同模型，我们跟踪了表达连接到肌动蛋白或α辅肌动蛋白的绿色荧光蛋白的活细胞，并分析了鹌鹑肌管培养物中的应力纤维、前肌原纤维和肌原纤维。通过分析Latrunculin-A（Lat-A）（一种单体肌动蛋白结合大环内酯类药物（M.W. = 422道尔顿），在同一培养物中对成纤维细胞中的应力纤维和骨骼肌管中的肌原纤维。低浓度（0.2 μM）的Lat‐A在几小时内诱导成纤维细胞中的应力纤维丢失，当Lat‐A增加到1.0 μM时，在10分钟内诱导成纤维细胞中的应力纤维丢失。当药物被移除时，随着应力纤维的重建，该作用是可逆的。与Lat-A诱导成纤维细胞中应力纤维的解体相反，骨骼肌细胞中组装的肌原纤维不受1.0 μM浓度的Lat-A的影响。然而，随着Lat‐A浓度的增加（高达5 μM）和孵育时间的增加，药物诱导前肌原纤维（成熟肌原纤维的前体）分解，并停止成熟肌原纤维的积累。去除药物导致前肌原纤维的重组和肌管扩张边缘肌原纤维发生的恢复。相比之下，肌管中心轴中的成熟肌原纤维在高达50 μM的Lat-A剂量下稳定。位于肌管末端和侧面的前肌原纤维附近的新组装的成熟肌原纤维对Lat-A的敏感性中等，暴露于10 μM Lat-A 1小时后解体。为了确定在肌原纤维形成过程中肌动蛋白丝的变化如何可能赋予对Lat-A解聚的更大抵抗力，我们用针对CapZ的抗体对肌管进行染色，CapZ是一种阻止单体肌动蛋白从肌动蛋白丝的倒刺末端释放的蛋白质。CapZ在前肌原纤维中不存在。它沿新生肌原纤维沿着均匀分布，其中F-肌动蛋白是无条纹的，并且在成熟肌原纤维中以清晰的条纹Z带图案定位，其中F-肌动蛋白图案是完全条纹的。这些Lat‐A和CapZ的结果在肌原纤维发生的各种模型的背景下进行了讨论。细胞动力。Cytoskeleton 62：35-47，2005.© 2005 Wiley利斯公司
To test different models of myofibrillogenesis, we followed live cells expressing Green Fluorescent Proteins ligated to either actin or alpha‐actinin and analyzed stress fibers, premyofibrils, and myofibrils in quail myotube cultures. Actin filaments in the three types of fibers were compared by analyzing the effects of Latrunculin‐A (Lat‐A), a monomeric actin binding macrolide drug (M.W. = 422 Daltons), on stress fibers in fibroblasts and on myofibrils in skeletal myotubes in the same culture. Lat‐A, at low concentrations (0.2 μM), induced the loss of stress fibers in fibroblasts within a few hours and within 10 min when Lat‐A was increased to 1.0 μM. The effect was reversible with reformation of the stress fibers when the drug was removed. In contrast to the Lat‐A induced disassembly of stress fibers in fibroblasts, assembling myofibrils in the skeletal muscle cells were not affected by 1.0‐μM concentrations of Lat‐A. With increasing concentrations of Lat‐A (up to 5 μM), and increasing incubation times, however, the drug induced premyofibrils, the precursors of mature myofibrils, to disassemble and the accumulation of mature myofibrils to be halted. Removal of the drug led to the reformation of premyofibrils and the resumption of myofibrillogenesis in the spreading edges of the myotubes. In contrast, the mature myofibrils in the central shaft of the myotubes were stable in doses of Lat‐A as high as 50 μM. The newly assembled mature myofibrils located adjacent to the premyofibrils at the ends and sides of the myotube were intermediate in sensitivity to Lat‐A, disassembling when exposed to 10 μM Lat‐A for one hour. To determine how a change in the actin filaments during myofibrillogenesis might confer greater resistance to depolymerization by Lat‐A, we stained the myotubes with an antibody directed against CapZ, a protein that blocks the release of monomer actin from the barbed ends of actin filaments. CapZ was absent from premyofibrils. It was distributed uniformly along nascent myofibrils where F‐actin was unstriated, and was localized in a clearly striated Z‐band pattern in the mature myofibrils where F‐actin patterns were fully striated. These Lat‐A and CapZ results are discussed in the context of various models of myofibrillogenesis. Cell Motil. Cytoskeleton 62:35–47, 2005. © 2005 Wiley‐Liss, Inc.