Cao Tracy T, Chang Wakam, Masters Sarah E, Mooseker Mark S
Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA.
Mol Biol Cell. 2004 Jan;15(1):151-61. doi: 10.1091/mbc.e03-07-0504. Epub 2003 Oct 17.
Myosin-Va was identified as a microtubule binding protein by cosedimentation analysis in the presence of microtubules. Native myosin-Va purified from chick brain, as well as the expressed globular tail domain of this myosin, but not head domain bound to microtubule-associated protein-free microtubules. Binding of myosin-Va to microtubules was saturable and of moderately high affinity (approximately 1:24 Myosin-Va:tubulin; Kd = 70 nM). Myosin-Va may bind to microtubules via its tail domain because microtubule-bound myosin-Va retained the ability to bind actin filaments resulting in the formation of cross-linked gels of microtubules and actin, as assessed by fluorescence and electron microscopy. In low Ca2+, ATP addition induced dissolution of these gels, but not release of myosin-Va from MTs. However, in 10 microM Ca2+, ATP addition resulted in the contraction of the gels into aster-like arrays. These results demonstrate that myosin-Va is a microtubule binding protein that cross-links and mechanochemically couples microtubules to actin filaments.
通过在微管存在下的共沉降分析，肌球蛋白-Va被鉴定为一种微管结合蛋白。从鸡脑中纯化的天然肌球蛋白-Va，以及该肌球蛋白表达的球状尾部结构域，但不是头部结构域，可与不含微管相关蛋白的微管结合。肌球蛋白-Va与微管的结合是可饱和的，且具有中等高度的亲和力（约1:24肌球蛋白-Va:微管蛋白；Kd = 70 nM）。肌球蛋白-Va可能通过其尾部结构域与微管结合，因为通过荧光和电子显微镜评估，结合在微管上的肌球蛋白-Va保留了结合肌动蛋白丝的能力，从而导致微管和肌动蛋白交联凝胶的形成。在低Ca2+浓度下，添加ATP会诱导这些凝胶溶解，但不会使肌球蛋白-Va从微管上释放。然而，在10 microM Ca2+浓度下，添加ATP会导致凝胶收缩成星状阵列。这些结果表明，肌球蛋白-Va是一种微管结合蛋白，它能使微管与肌动蛋白丝交联并进行机械化学偶联。
