Formation and destabilization of actin filaments with tetramethylrhodamine-modified actin
Formation and destabilization of actin filaments with tetramethylrhodamine-modified actin
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DOI：
10.1529/biophysj.104.042242
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发表时间：
2004-08-01
影响因子：
3.4
通讯作者：
Reisler, E
中科院分区：
文献类型：
--
作者：
Kudryashov, DS;Phillips, M;Reisler, E
用四甲基罗丹明衍生物（TMR-actin）在Cys（374）处标记肌动蛋白已被广泛用于体外直接观察。遗憾的是，生长，分支，和螺旋铣削，以及在体内可视化的肌动蛋白细胞骨架。TMR-肌动蛋白的优点是它不锁定肌动蛋白。感叹（如罗丹明鬼笔环肽），可能允许其用于研究肌动蛋白聚合物的动态组装行为。虽然已经确定TMR-肌动蛋白单独是不聚合的，但是其与未标记的肌动蛋白的共聚对骨架结构和动力学的影响尚未被测试。在这项研究中，我们表明，TMR-肌动蛋白扰动。感叹结构时，与未标记的肌动蛋白共聚;结果。哀歌比对照组更脆弱和简短。哀叹。因为共聚物的断裂增加。遗憾的是，TMR-肌动蛋白在溶液中也以低于大多数荧光显微镜实验中所用的摩尔比加速未标记的肌动蛋白的聚合。TMR-肌动蛋白的不稳定和切断作用被抵消。lament稳定因子，鬼笔环肽，S1和原肌球蛋白。这些结果表明，TMR-肌动蛋白和切割蛋白质对F-肌动蛋白的影响之间存在类似性，并暗示TMR-肌动蛋白可能不适合用于肌动蛋白的研究。感叹动态。
Actin labeling at Cys(374) with tethramethylrhodamine derivatives (TMR-actin) has been widely used for direct observation of the in vitro. laments growth, branching, and treadmilling, as well as for the in vivo visualization of actin cytoskeleton. The advantage of TMR-actin is that it does not lock actin in. laments (as rhodamine-phalloidin does), possibly allowing for its use in investigating the dynamic assembly behavior of actin polymers. Although it is established that TMR-actin alone is polymerization incompetent, the impact of its copolymerization with unlabeled actin on. lament structure and dynamics has not been tested yet. In this study, we show that TMR-actin perturbs the. laments structure when copolymerized with unlabeled actin; the resulting. laments are more fragile and shorter than the control. laments. Due to the increased severing of copolymer. laments, TMR-actin accelerates the polymerization of unlabeled actin in solution also at mole ratios lower than those used in most fluorescence microscopy experiments. The destabilizing and severing effect of TMR-actin is countered by. lament stabilizing factors, phalloidin, S1, and tropomyosin. These results point to an analogy between the effects of TMR-actin and severing proteins on F-actin, and imply that TMR-actin may be inappropriate for investigations of actin. laments dynamics.