VISUALIZATION OF MYOSIN IN LIVING CELLS
VISUALIZATION OF MYOSIN IN LIVING CELLS
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DOI：
10.1083/jcb.105.4.1753
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发表时间：
1987-10-01
影响因子：
7.8
通讯作者：
SANGER, JW
中科院分区：
文献类型：
--
作者：
MITTAL, B;SANGER, JM;SANGER, JW
当显微注射到活肌肉和非肌肉细胞中时，用罗丹明标记的肌球蛋白轻链被掺入含肌球蛋白的结构中。从鸡骨骼肌中制备肌球蛋白轻链混合物，用荧光染料碘乙酰胺罗丹明标记，并分成单独的标记轻链 LC-1、LC-2 和 LC-3。在分离的兔子和昆虫肌原纤维中，荧光轻链在 A 带中以双峰形式结合，而在 A 带中心的无交叉桥区域中没有结合。当注射到活体胚胎鸡肌管和心肌细胞中时，荧光轻链也沿着 A 带的整个长度掺入，伪 H 区除外。在年轻的肌管（3-4 日龄）中，肌球蛋白定位于非周期性和周期性纤维中。双联 A 带图案首先出现在 5 日龄的肌管中，该肌管也表现出最初的收缩迹象。在 6 维和更老的肌管中，A 带变得更加对齐，边缘更加尖锐，并且它们之间的间隔（I 带）更宽。在非肌肉细胞中，显微注射的荧光轻链以条纹图案结合在应力纤维中，并且在病灶和附着斑块中不存在。当用 DMSO 破坏活注射细胞的应力纤维时，荧光标记的肌球蛋白轻链存在于细胞质中，但不进入细胞核。去除 DMSO 导致带状荧光应力纤维在 45 分钟内重新形成。在分裂细胞中，肌球蛋白轻链集中在卵裂沟中，并在胞质分裂后重新掺入应力纤维中。因此，注射的非肌肉细胞可以使用含有肌肉轻链和非肌肉重链的混合肌球蛋白分子来分解和重新组装收缩纤维。我们的实验表明，来自肌肉的荧光标记的肌球蛋白轻链可以很容易地掺入肌肉和非肌肉肌球蛋白中，然后用于跟踪活细胞中肌球蛋白分布的动态。
Myosin light chains labeled with rhodamine are incorporated into myosin-containing structures when microinjected into live muscle and nonmuscle cells. A mixture of myosin light chains was prepared from chicken skeletal muscle, labeled with the fluorescent dye iodoacetamido rhodamine, and separated into individual labeled light chains, LC-1, LC-2, and LC-3. In isolated rabbit and insect myofibrils, the fluorescent light chains bound in a doublet pattern in the A bands with no binding in the cross-bridge-free region in the center of the A bands. When injected into living embryonic chick myotubes and cardiac myocytes, the fluorescent light chains were also incorporated along the complete length of the A band with the exception of the pseudo-H zone. In young myotubes (3-4 d old), myosin was localized in aperiodic as well as periodic fibers. The doublet A band pattern first appeared in 5-d-old myotubes, which also exhibited the first signs of contractility. In 6-d and older myotubes, A bands became increasing more aligned, their edges sharper, and the separation between them (I bands) wider. In nonmuscle cells, the microinjected fluorescent light chains were incorporated in a striated pattern in stress fibers and were absent from foci and attachment plaques. When the stress fibers of live injected cells were disrupted with DMSO, fluorescently labeled myosin light chains were present in the cytoplasm but did not enter the nucleus. Removal of the DMSO led to the reformation of banded, fluorescent stress fibers within 45 min. In dividing cells, myosin light chains were concentrated in the cleavage furrow and became reincorporated in stress fibers after cytokinesis. Thus, injected nonmuscle cells can disassemble and reassemble contractile fibers using hybrid myosin molecules that contain muscle light chains and nonmuscle heavy chains. Our experiments demonstrate that fluorescently labeled myosin light chains from muscle can be readily incorporated into muscle and nonmuscle myosins and then used to follow the dynamics of myosin distribution in living cells.