MYOSIN LINKED REGULATION IN MUSCLE AND NON-MUSCLE CELLS

这项建议旨在加深对该机制的了解，并 肌球蛋白连接调节的功能，在肌肉和非肌肉细胞中都有。 在调节性肌球蛋白中，肌动蛋白和肌球蛋白之间的相互作用是 单独由肌球蛋白分子上发生的事件控制，或者 对直接的钙结合或磷酸化作出反应。 我们将利用扇贝独特而多样的特性 横纹内收肌球蛋白，以结合外源轻链，两者 调节的和必要的，进入肌球蛋白分子。这些轻链 将在其主要序列中的不同位置进行修饰。我们的 实验将通过荧光监测头部内部构象的变化 共振能量转移(FRET)。这些事件将表现为更改 在放置在两个上的每个上的探头之间的距离和/或方向上 轻链类型以及轻链上的探针与活性上的探针之间 或肌动蛋白和肌球蛋白上的其他位置。学位的变化 将根据不断变化的条件寻求转移，旨在 模仿“休息”、“僵硬”或“活动状态”的生理状态。 在以下情况下，还将使用蛋白质交联的补充技术 恰如其分。 我们还打算对两种调节肌球蛋白进行比较研究。 它们同时表现出轻链和重链的磷酸化，并 观察这种调节与特定功能的关系 肌球蛋白原位。通过使用软体动物捕捉内收肌球蛋白，我们将进一步 我们对渔获物收缩规律的理解。通过使用肌球蛋白 从神经来源的细胞系分离出来，我们最终将能够 评价肌球蛋白在突触神经递质释放中的作用 变速箱。这种肌球蛋白的抗体将使我们能够探测 肌球蛋白在发育中的中枢神经系统中的定位和功能 系统。 对这些机制的详细了解是至关重要的。

This proposal is aimed at furthering an understanding of the mechanism and function of myosin-linked regulation, both in muscle and non-muscle cells. In regulatory myosins, the interaction between actin and myosin is controlled solely by events occurring on the myosin molecule alone, either in response to direct calcium binding or phosphorylation. We will take advantage of the unique and versatile properties of scallop striated adductor myosin in order to incorporate foreign light-chains, both regulatory and essential, into the myosin molecule. These light-chains will be modified at differing locations in their primary sequence. Our experiments will monitor intra-head conformational changes by fluorescence resonance energy transfer (FRET). These events will be manifest as changes in distance and/or orientation between probes placed on each of the two light-chain types and between probes on either light-chain and the active site, or other locations on both actin and myosin. Changes in the degree of transfer will be sought in response to changing conditions designed to mimic the physiological states of 'rest', 'rigor' or the 'active state.' The complementary technique of protein crosslinking will also be used, when appropriate. We also intend to perform a comparative study of two regulatory myosins which exhibit both light-chain and heavy-chain phosphorylation and to observe the relationship of such regulation to the specific functions of myosin in situ. By using molluscan catch adductor myosin we will further our understanding of the regulation of catch contraction. By using myosin isolated from a neuronally-derived cell line, we will ultimately be able to evaluate the role of myosin in neurotransmitter release during synaptic transmission. An antibody to this myosin will enable us to probe the location and function of myosin within the developing central nervous system. A detailed understanding of these mechanisms is of fundamental importance.