Expression Studies of Other Unconventional Myosins

Myosin VIIa是一种非常规的肌球蛋白，广泛表达于从变形虫到哺乳动物的生物体中，已被证明在细胞粘附和吞噬中起重要作用。我们研究了Sf9细胞中表达的果蝇myosin VIIa。我们已经证明，这种肌球蛋白具有与进程马达相似的高占空比动力学，但我们也表明，即使在Sf9细胞中表达全长分子，它也不容易二聚化。我们研究了全长肌球蛋白VIIa和各种c端截短片段的酶活性调控。全长myosin VIIa （FLM7a）的Vmax约为1 / s，但对肌动蛋白具有较低的表观亲和力，需要30-50 μ m的acin才能达到半最大激活。各种c端截短片段具有相似的Vmax，但所需的actin要少得多，才能达到最大激活的一半（0.5-1 uM）。这意味着在5um肌动蛋白时，片段的活性接近最大值，而FLM7a的活性仍然几乎没有被激活。即使去掉最后的99个氨基酸也足以引起这种显著的活性变化。我们利用电子显微镜下的单粒子分析，探讨了这种调控的结构基础。我们发现，在ATP存在的情况下，FLM7a紧密折叠成一个紧凑的结构，使得肌凝蛋白运动结构域无法被识别，而在没有ATP的情况下，FLM7a分子更延伸，并显示出一个清晰可区分的运动结构域。尾部小块的移除也伴随着延伸的构象，这使我们认为紧凑的结构代表了分子的抑制状态。c端尾部一对保守带电基团的点突变也会导致分子失去调控和展开。我们表达了myosin VIIa的第二个FERM结构域，发现它可以以ATP依赖的方式与肌动蛋白结合。我们现在正在寻找结合伙伴或翻译后修饰，将折叠的、不活跃的FLM7a转化为扩展的、活跃的形式。

Myosin VIIa 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. We have studied Drosophila myosin VIIa that was expressed in Sf9 cells. We have shown that this myosin has high duty ratio kinetics similar to that of processive motors, but we also showed that it did not easily dimerize even if the full lenght molecule was expressed in Sf9 cells. We have examined the regulation of enzymatic activity of full length myosin VIIa and various C-terminally truncated fragments. Full length myosin VIIa (FLM7a) has a Vmax of about 1 per sec, but has a low apparent affinity for actin, requiring 30-50 uM acin for half-maximal activation. Various C-terminally truncated fragments have a similar Vmax, but much less actin is required for half maximal activation (0.5-1 uM). This means that at 5 uM actin the activity of the fragments is near maximal whereas that of the FLM7a is still barely activated. Removal of even the last 99 amino acids is sufficient to cause this remarkable change in activity. We explored the structural basis for this regulation by using single particle analysis in the electron microscope. We find that in the presence of ATP FLM7a is tightly folded into a compact structure such that the myosin motor domain cannot be discerned whereas in the absence of ATP the molecule is more extended and shows a clearly distinguishable motor domain. Removable of small bits of the tail also is accompanied by the extended conformation leading us to suggest that the compact structure represents an inhibited state of the molecule. Point mutations of a pair of conserved charged groups in the C-terminal tail also result in loss of regulation and unfolding of the molecule. We have expressed the second FERM domain of myosin VIIa and find that it can bind to actin in an ATP dependent manner. We are now searching for binding partners or post-translation modifications that will convert the foled, inactive FLM7a into an extend, active form. We are currently examining the mechanical ability of myosin VIIa using optical trapping nanometry. Preliminary results reveal that this myosin has a long attachment lifetime. We are also studying the localization of GFP-tagged myosin VII and truncation mutants in S2 cells. We are also attempting to make a null mutation in Drosophila myosin VIIb. We have examined the effect of some deafness-associated mutations in the motor domain of human myosin VIIa on the actin activated MgATPase activity and are attempting to express a full length construct of this myosin in the Sf9 system. Myosin XVIIIa has a large predicted coiled-coil forming sequence, but the structure of the myosin has not been examined on a single molecule level. We expressed full length myosin XVIIIa from mouse and showed that it has a long tail similar to that of myosin II. We are examining whether this tail can form filaments. Little is known about the functions of class III unconventional myosins although, with an N-terminal kinase domain, they are potentially both signaling and motor proteins. Limulus myosin III is particularly interesting because it is a phosphoprotein abundant in photoreceptors that becomes more heavily phosphorylated at night by protein kinase A. This enhanced nighttime phosphorylation occurs in response to signals from an endogenous circadian clock and correlates with dramatic changes in photoreceptor structure and function. We seek to understand the role of Limulus myosin III and its phosphorylation in photoreceptors. Here we determined the sites that become phosphorylated in Limulus myosin III and investigated its kinase, actin binding, and myosin ATPase activities. We show that Limulus myosin III exhibits kinase activity and that a major site for both protein kinase A and autophosphorylation is located within loop 2 of the myosin domain, an important actin binding region. We also identify the phosphorylation of an additional protein kinase A and autophosphorylation site near loop 2, and a predicted phosphorylation site within loop 2. We show that the kinase domain of Limulus myosin III shares some pharmacological properties with protein kinase A, and that it is a potential opsin kinase. Finally, we demonstrate that Limulus myosin III binds actin but lacks ATPase activity. We conclude that Limulus myosin III is an actin-binding and signaling protein and speculate that interactions between actin and Limulus myosin III are regulated by both second messenger mediated phosphorylation and autophosphorylation of its myosin domain within and near loop 2.