Novel Mechanisms of Myosin-II Mediated Motility

描述（由申请人提供）：肌球蛋白-II细丝的形成是收缩和运动过程的基础。了解细丝组装和拆卸是如何控制的，对于确定肌球蛋白II如何快速响应细胞内变化的条件（例如，在细胞分裂期间或响应趋化信号）至关重要。几种蛋白质已被证明可以稳定丝状肌球蛋白-II组装体;然而，作为Ca 2+调节的S100蛋白质家族的成员，mts 1是第一个被鉴定为促进单体未组装状态的蛋白质。除了参与全身细胞运动外，mts 1还直接参与间质-上皮转化，是一个主要的转移因子。因此，MTS 1是一个很好的目标，研究控制本地组装/拆卸的肌球蛋白-II的运动，发展和转移相关的分子机制。生物化学研究将用于建立mts 1调节肌球蛋白-II的单体-聚合物平衡的分子基础，以及肌球蛋白-II重链磷酸化如何调节mts 1活性。高分辨率的结构研究MTS 1绑定到肌球蛋白-II重链将允许一个详细的原子描述的MTS 1/肌球蛋白-II的相互作用，提供限制的生化机制，MTS 1调节肌球蛋白-II的功能，并提供特定的信息，将有助于生物传感器的建设和诱变。利用对溶剂极性敏感的环境敏感的荧光团，我们将开发新的mts 1生物传感器，用于报告单个活细胞中的钙或靶结合。这些独特的试剂将允许在运动周期中直接检查激活的MTS 1的时间和空间分布，并将允许在定向运动期间MTS 1的局部瞬时激活与特定肌球蛋白-II组装的调节之间的直接相关性。总之，这些信息将被利用来建立一个完整的生化模型，在细胞运动过程中的mts 1介导的肌球蛋白II功能的调节。

DESCRIPTION (provided by applicant): The formation of myosin-lI filaments is fundamental to contractile and motile processes. Understanding how filament assembly and disassembly are controlled is essential for determining how myosin-II rapidly responds to changing conditions within the cell (e.g. during cell division or in response to a chemotactic signal). Several proteins have been shown to stabilize filamentous myosin-II assemblies; however mts1, a member of the Ca2+regulated S100 family of proteins, is the first protein to be identified that promotes the monomeric, unassembled state. In addition to its involvement in generalized cell motility, mts1 is directly implicated in the mesenchymal-epithelial transition and is a major metastatic factor. Thus mts1 is an excellent target for investigating the molecular mechanisms controlling the localized assembly/disassembly of myosin-II that are relevant to motility, development and metastasis. Biochemical studies will be used to establish the molecular basis by which mts1 regulates the monomer-polymer equilibrium of myosin-II and how myosin-II heavy chain phosphorylation regulates mts1 activity. High resolution structural studies of mts1 bound to the myosin-II heavy chain will allow a detailed atomic description of the mts1/myosin-II interaction, provide constraints for the biochemical mechanism by which mts1 regulates myosin-II function and provide specific information that will assist in biosensor construction and mutagenesis. Using environmentally sensitive fluorophores that are sensitive to solvent polarity, we will develop novel mts1 biosensors that report calcium or target binding for use in individual living cells. These unique reagents will allow the temporal and spatial distribution of activated mts1 to be examined directly during the motility cycle, and will permit a direct correlation between localized, transient activation of mts1 and the regulation of specific myosin-II assemblies during directed motility. Altogether, this information will be leveraged to establish a complete biochemical model for mts1-mediated regulation of myosin-II function during cellular motility.