Noguchi Tatsuhiko, Frank Deborah J, Isaji Mamiko, Miller Kathryn G
Center for Developmental Biology, Riken, Kobe, Japan.
Mol Biol Cell. 2009 Jan;20(1):358-67. doi: 10.1091/mbc.e08-07-0776. Epub 2008 Nov 12.
Myosin VI is a pointed-end-directed actin motor that is thought to function as both a transporter of cargoes and an anchor, capable of binding cellular components to actin for long periods. Dimerization via a predicted coiled coil was hypothesized to regulate activity and motor properties. However, the importance of the coiled-coil sequence has not been tested in vivo. We used myosin VI's well-defined role in actin stabilization during Drosophila spermatid individualization to test the importance in vivo of the predicted coiled coil. If myosin VI functions as a dimer, a forced dimer should fully rescue myosin VI loss of function defects, including actin stabilization, actin cone movement, and cytoplasmic exclusion by the cones. Conversely, a molecule lacking the coiled coil should not rescue at all. Surprisingly, neither prediction was correct, because each rescued partially and the molecule lacking the coiled coil functioned better than the forced dimer. In extracts, no cross-linking into higher molecular weight forms indicative of dimerization was observed. In addition, a sequence required for altering nucleotide kinetics to make myosin VI dimers processive is not required for myosin VI's actin stabilization function. We conclude that myosin VI does not need to dimerize via the predicted coiled coil to stabilize actin in vivo.
肌球蛋白VI是一种指向尖端的肌动蛋白马达，被认为既作为货物转运体又作为锚定物发挥作用，能够将细胞成分长时间结合到肌动蛋白上。通过预测的卷曲螺旋进行二聚化被假设用于调节活性和马达特性。然而，卷曲螺旋序列在体内的重要性尚未得到测试。我们利用肌球蛋白VI在果蝇精子个体化过程中对肌动蛋白稳定的明确作用，来测试预测的卷曲螺旋在体内的重要性。如果肌球蛋白VI作为二聚体发挥作用，一个强制二聚体应该能完全挽救肌球蛋白VI功能丧失的缺陷，包括肌动蛋白稳定、肌动蛋白锥运动以及锥对细胞质的排斥。相反，一个缺乏卷曲螺旋的分子应该根本无法挽救。令人惊讶的是，两种预测都不正确，因为每种情况都只是部分挽救，而且缺乏卷曲螺旋的分子比强制二聚体功能更好。在提取物中，未观察到交联形成指示二聚化的更高分子量形式。此外，使肌球蛋白VI二聚体具有持续性而改变核苷酸动力学所需的序列，对于肌球蛋白VI的肌动蛋白稳定功能并非必需。我们得出结论，肌球蛋白VI在体内不需要通过预测的卷曲螺旋进行二聚化来稳定肌动蛋白。
