Fission yeast tropomyosin specifies directed transport of myosin-V along actin cables.
Fission yeast tropomyosin specifies directed transport of myosin-V along actin cables.
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DOI：
10.1091/mbc.e13-04-0200
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发表时间：
2014-01
影响因子：
3.3
通讯作者：
Lord M
中科院分区：
文献类型：
--
作者：
Clayton JE;Pollard LW;Sckolnick M;Bookwalter CS;Hodges AR;Trybus KM;Lord M
裂变酵母原肌球蛋白通过促进马达的持续合成能力将肌球蛋白-V靶向肌动蛋白电缆。活细胞成像用于估计体内每个能动颗粒的肌球蛋白-V分子的数量。体外重建证明了基于原肌球蛋白的靶向该马达的生理相关性。V类肌球蛋白的一个标志是它们的加工能力--沿着沿着肌动蛋白丝采取多个步骤而不解离的能力。然而，我们以前的工作表明，裂殖酵母肌球蛋白-V（Myo 52 p）是一种非进行性马达，其活性由原肌球蛋白（Cdc 8 p）增强。在这里，我们调查的分子机制和生理相关性的原肌球蛋白介导的调节Myo 52 p的运输，在体外和体内的方法相结合。单分子Myo 52 p，可视化全内反射荧光显微镜，移动procancerly只有当Cdc 8 p存在于肌动蛋白丝。与量子点结合的Myo 52 p的小集合，模仿与生理货物结合的电机数量，也需要Cdc 8 p来进行连续运动。虽然缺乏货物结合结构域的Myo 52 p的截短形式未能支持体内功能，但它仍然经历肌动蛋白依赖性运动到极化生长位点。该结果表明，缺乏货物的截短Myo 52 p或具有小货物的野生型Myo 52 p的单个分子可以在体内沿着沿着肌动蛋白-Cdc 8 p电缆进行进行性运动。我们的研究结果概述了一种机制，通过这种机制，原肌球蛋白通过开启肌球蛋白的持续合成能力，促进细胞内特定肌动蛋白轨道的运输。
Fission yeast tropomyosin targets myosin-V to actin cables by favoring processivity of the motor. Live-cell imaging is used to estimate the number of myosin-V molecules per motile particle in vivo. In vitro reconstitution demonstrates the physiological relevance of tropomyosin-based targeting of this motor. A hallmark of class-V myosins is their processivity—the ability to take multiple steps along actin filaments without dissociating. Our previous work suggested, however, that the fission yeast myosin-V (Myo52p) is a nonprocessive motor whose activity is enhanced by tropomyosin (Cdc8p). Here we investigate the molecular mechanism and physiological relevance of tropomyosin-mediated regulation of Myo52p transport, using a combination of in vitro and in vivo approaches. Single molecules of Myo52p, visualized by total internal reflection fluorescence microscopy, moved processively only when Cdc8p was present on actin filaments. Small ensembles of Myo52p bound to a quantum dot, mimicking the number of motors bound to physiological cargo, also required Cdc8p for continuous motion. Although a truncated form of Myo52p that lacked a cargo-binding domain failed to support function in vivo, it still underwent actin-dependent movement to polarized growth sites. This result suggests that truncated Myo52p lacking cargo, or single molecules of wild-type Myo52p with small cargoes, can undergo processive movement along actin-Cdc8p cables in vivo. Our findings outline a mechanism by which tropomyosin facilitates sorting of transport to specific actin tracks within the cell by switching on myosin processivity.