Expression Studies of Other Unconventional Myosins

肌球蛋白VII是一种非传统的肌球蛋白，在从阿米巴到哺乳动物的各种生物中广泛表达，已被证明在细胞黏附和吞噬过程中发挥重要作用。在这里，我们首次研究了果蝇肌球蛋白VIIa的作用机制。我们已经在杆状病毒-Sf9系统中表达了一个截短的单头果蝇肌球蛋白VIIa结构，它结合了钙调蛋白轻链。通过使用稳态和瞬时动力学方法，我们发现肌球蛋白VIIa表现出从肌球蛋白快速释放磷酸盐和缓慢、限速的ADP释放。因此，肌球蛋白VIIa在稳定的ATP水解过程中将主要与肌动蛋白强烈结合(其占空比将至少为80%)。这种动力学模式在许多方面与单分子囊泡转运蛋白myosin V和myosin VI相似。我们表达了果蝇myosin VIIa的两个HMM-like片段，其中一个片段在内源预测的卷曲形成结构域之后添加了亮氨酸拉链基序。我们的研究表明，只有拉链形式的HMM有效地在肌动蛋白细丝上进行移动，这表明非拉链形式本身可能不能有效地二聚。拉链HMM被证明有30纳米的步长，并以手到手的方式在肌动蛋白上移动。这个步长与这个肌球蛋白有一个5IQ图案的脖子是一致的。

Myosin VII is an unconventional myosin widely expressed in organisms ranging from amoebae to mammals that has been shown to play vital roles in cell adhesion and phagocytosis. Here we present the first study of the mechanism of action of a myosin VIIa from Drosophila melanogaster. We have expressed a truncated single-headed Drosophila myosin VIIa construct in the baculovirus-Sf9 system that bound calmodulin light chains. By using steady-state and transient kinetic methods, we showed that myosin VIIa exhibits a fast release of phosphate and a slower, rate-limiting ADP release from actomyosin. As a result, myosin VIIa will be predominantly strongly bound to actin during steady-state ATP hydrolysis (its duty ratio will be at least 80%). This kinetic pattern is, in many respects, similar to that of the single-molecule vesicle transporters myosin V and VI. We expressed two HMM-like fragments of Drosophila myosin VIIa, including one in which a leucine zipper motif was added after the endogenous predicted coiled-coil forming domain. Our studies indicate that only the zippered HMM effectively moved processively on actin filaments, suggesting that the non-zippered form may not dimerize effectively on its own. The zippered HMM was shown to have 30-nm steps and to move in a hand over hand manner on actin. This step size is consistent with this myosin having a neck, which is 5IQ motifs long.