Myosin VI altered at threonine 406 stabilizes actin filaments in vivo
Myosin VI altered at threonine 406 stabilizes actin filaments in vivo
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DOI：
10.1002/cm.20150
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发表时间：
2006-10-01
影响因子：
--
通讯作者：
Hasson, Tama
中科院分区：
文献类型：
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作者：
Naccache, Samia N.;Hasson, Tama
肌凝蛋白VI是一种负端定向的基于肌动蛋白的分子马达，与无包被的内吞囊泡运输有关。最近的动力学研究表明，在不同的负荷条件下，肌凝蛋白VI显示出改变的ADP释放动力学，允许肌凝蛋白VI交替作为转运蛋白或肌动蛋白系链。我们推测，在这些动力学选择之间调节的一个潜在调节事件是肌球蛋白VI运动结构域中保守位点苏氨酸406 （T406）的磷酸化。将模拟磷酸化（T406E）和去磷酸化（T406A）状态的改变引入GFP-myosin VI融合（GFP-M6）。活细胞成像显示GFP-M6（T406E）的表达改变了肌球蛋白VI在未包被的内吞囊泡中的运输路径。与GFP-M6和GFP-M6（T406A）表达细胞中的囊泡向内移动不同，GFP-M6（T406E）将囊泡在不同的周围部位聚集成簇。GFP-M6（T406E）的表达也增加了肌动蛋白细胞骨架的密度。细丝在囊泡团簇部位富集。这不是由于肌动蛋白聚合机制的总体重新分配。相反，丝密度与GFP-M6（T406E）相关的囊泡在f -肌动蛋白上的固定定位相关，导致肌动蛋白解聚受到抑制。我们的研究表明，T406位点的磷酸化改变了体内肌动蛋白VI与肌动蛋白相互作用的性质。
Myosin VI is a minus-end directed actin-based molecular motor implicated in uncoated endocytic vesicle transport. Recent kinetic studies have shown that myosin VI displays altered ADP release kinetics under different load conditions allowing myosin VI to serve alternately as a transporter or as an actin tether. We theorized that one potential regulatory event to modulate between these kinetic choices is phosphorylation at a conserved site, threonine 406 (T406) in the myosin VI motor domain. Alterations mimicking the phosphorylated (T406E) and dephosphorylated state (T406A) were introduced into a GFP-myosin VI fusion (GFP-M6). Live cell imaging revealed that GFP-M6(T406E) expression changed the path myosin VI took in its transport of uncoated endocytic vesicles. Rather than routing vesicles inwards as seen in GFP-M6 and GFP-M6(T406A) expressing cells, GFP-M6(T406E) moved vesicles into clusters at distinct peripheral sites. GFP-M6(T406E) expression also increased the density of the actin cytoskeleton. Filaments were enriched at the vesicle cluster sites. This was not due to a gross redistribution of the actin polymerization machinery. Instead the filament density correlated to the fixed positioning of GFP-M6(T406E)-associated vesicles on F-actin, leading to inhibition of actin depolymerization. Our study suggests that phosphorylation at T406 changes the nature of myosin VI's interaction with actin in vivo.