FLEXIBILITY OF ACTIN-FILAMENTS DERIVED FROM THERMAL FLUCTUATIONS - EFFECT OF BOUND NUCLEOTIDE, PHALLOIDIN, AND MUSCLE REGULATORY PROTEINS
FLEXIBILITY OF ACTIN-FILAMENTS DERIVED FROM THERMAL FLUCTUATIONS - EFFECT OF BOUND NUCLEOTIDE, PHALLOIDIN, AND MUSCLE REGULATORY PROTEINS
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DOI：
10.1074/jbc.270.19.11437
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发表时间：
1995-05-12
影响因子：
4.8
通讯作者：
CARLIER, MF
中科院分区：
文献类型：
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作者：
ISAMBERT, H;VENIER, P;CARLIER, MF
在20℃的荧光光学视频显微镜下观察到单个肌动蛋白细丝在二维经历布朗运动，持续长度(L(P))是通过分析罗丹明肌动蛋白组装的一系列记录的细丝形状的余弦相关函数或平均横向涨落得出的。Phalloidin稳定的纤维的持续长度为18+/-1微米，这与最近的观察结果一致，在没有Phalloidin的情况下，一旦在适当临界浓度的游离未标记G-肌动蛋白中稀释，就可以在各种溶液条件下观察到罗丹明标记的纤维。这种未稳定的F-ADP-肌动蛋白长丝具有相同的L(P)=9+/-0.5µm，无论它们是由三磷酸腺苷-G-肌动蛋白还是腺苷二磷酸-肌动蛋白组装而成，并且在不考虑紧密结合的二价金属离子的情况下，在模拟三磷酸腺苷的伽马磷酸盐存在下，F-ADP-BeF_3-肌动蛋白的硬度略高一些，L(P)=13.5µm。因此，新形成的F-ADP-P-I-Actin长丝比‘老的’F-ADP-肌动蛋白长丝更硬。在骨骼肌原肌球蛋白和肌钙蛋白存在的情况下，微丝在关闭状态(-Ca~(2+))时是刚性的(L(P)=20+/-1微米)，而在开启状态(+Ca~(2+))时是柔性的(L(P)=12微米)，与空间位阻模型一致，与X射线衍射数据一致，使用平滑肌原肌球蛋白和钙离子时，关闭状态和开启状态之间没有明显的差异(L(P)20+/-1微米)。总之，这种方法可以准确地测量肌动蛋白细丝与其生物功能相关的微小(小于或等于15%)机械性能的变化。
Single actin filaments undergoing brownian movement in two dimensions were observed at 20 degrees C in fluorescence optical video microscopy, The persistence length (L(p)) was derived from the analysis of either the cosine correlation function or the average transverse fluctuations of a series of recorded shapes of filaments assembled from rhodamine actin. Phalloidin-stabilized filaments had a persistence length of 18 +/- 1 mu m, in agreement with recent observations, In the absence of phalloidin, rhodamine-labeled filaments could be observed under a variety of solution conditions once diluted in free unlabeled G-actin at the appropriate critical concentration. Such nonstabilized F-ADP-actin filaments had the same L(p) of 9 +/- 0.5 mu m, whether they had been assembled from ATP-G-actin or from ADP-G-actin, and independently of the tightly bound divalent metal ion, In the presence of BeF3-, which mimics the gamma-phosphate of ATP, F-ADP-BeF3-actin was appreciably more rigid, with L(p) = 13.5 mu m. Hence, newly formed F-ADP-P-i-actin filaments are more rigid than ''old'' F-ADP-actin filaments, a fact which has implications in actin-based motility processes.In the presence of skeletal tropomyosin and troponin, filaments were rigid (L(p) = 20 +/- 1 mu m) in the off state (-Ca2+), and flexible (L(p) = 12 mu m) in the on state (+Ca2+), consistent with the steric blocking model, In agreement with x-ray diffraction data, no appreciable difference was recorded between the off and on states using smooth muscle tropomyosin and caldesmon (L(p) 20 +/- 1 mu m). In conclusion, this method allows accurate measurement of small (less than or equal to 15%) changes in mechanical properties of actin filaments in correlation with their biological functions.