MYOSIN VI FUNCTION AND MECHANISM

描述(由申请人提供)：肌球蛋白VI具有许多生化和生物物理特性，使其有别于其他20多种基于肌动蛋白的马达。一个非常独特的特征是它能够朝着肌动蛋白的尖端/负端移动，方向与所有其他家庭成员相反。这表明肌球蛋白VI在体内具有独特的功能。肌球蛋白VI参与了许多不同的细胞过程，包括内吞作用、运动和细胞内定位，这些过程利用其沿肌动蛋白细丝移动和紧密结合肌动蛋白(锚)的双重能力。这些不同的功能被认为需要肌球蛋白VI不同于其他肌球蛋白的结构特征和生物物理性质。然而，几乎没有报道表明在体外测量的耐人寻味和独特的特性在体内是否重要。换句话说，我们知道肌球蛋白VI能做什么，但对它的实际作用知之甚少。提出的研究将以果蝇为模型系统来解决这个问题。到目前为止，已知肌球蛋白VI稳定了参与果蝇精子细胞发育过程中细胞重塑的肌动蛋白结构。在这个过程中，肌球蛋白VI与组织特异性调节分子androcam结合，而不是与其通常的调节因子钙调蛋白结合。这种绑定如何影响它的功能尚不清楚。利用表达肌球蛋白VI突变和截短/缺失形式的转基因果蝇来挽救肌球蛋白VI突变缺陷，体外测定的一些独特性质可能在其参与体内肌动蛋白稳定的能力中起不到关键作用。拟议的研究扩展了这些观察，检查了精子细胞的发育和肌球蛋白VI在其中发挥作用的其他发育过程，以了解在各种细胞环境中体内重要的结构和生化/生物物理特征。将进行GFP标记分子的本地化研究和动力学测量，以了解不同的功能如何介导肌球蛋白VI参与广泛的细胞功能。体外生物化学和生物物理特性的测量，以确定我们的操作和ACAM改变的调节如何影响肌球蛋白VI的特性，这将是对这些体内研究的补充。这些研究可能会揭示肌球蛋白VI在体内的机制，这将对了解肌球蛋白VI在其中发挥作用的基本细胞过程和疾病有价值。例如，肌球蛋白VI在几种癌症中表达上调，可能参与了对转移至关重要的运动。此外，一些人类耳聋综合征和相关的肥厚性心肌病是由肌球蛋白VI突变引起的。对肌球蛋白VI的研究有望阐明这些疾病的潜在缺陷，并可能揭示新的治疗方案。对肌球蛋白VI的研究可能有助于更好地理解一些基本细胞过程中涉及的机制，如内吞作用、蛋白质定位的分类途径以及细胞和细胞内的运动。此外，肌球蛋白VI还与几种疾病有关，如前列腺癌和卵巢癌、人类耳聋综合征和肥厚性心肌病。进一步了解肌球蛋白VI在体内的工作原理可能会增强我们对疾病机制的理解，并提供新的治疗途径。

DESCRIPTION (provided by applicant): Myosin VI has a number of biochemical and biophysical properties that distinguish it from the more than 20 other classes of actin-based motors. One strikingly unique feature is its ability to move towards the pointed/minus end of actin, the direction opposite all other family members. This suggests that myosin VI has unique functions in vivo. Myosin VI has been implicated in a large number of different cellular processes, including endocytosis, motility, and intracellular localization, processes that utilize its dual abilities to move along actin filaments and to bind tightly to actin (anchor). These different functions are thought to require structural features and biophysical properties of myosin VI that differ from other myosins. However, virtually no information has been reported that demonstrates whether the intriguing and unique properties measured in vitro are important in vivo. In other words, we know much about what myosin VI can do, but little about what it actually does. The studies proposed will address this question, using the fruitfly Drosophila as a model system. So far, myosin VI is known to stabilize an actin structure involved in cellular remodeling during Drosophila spermatid development. In this process, myosin VI binds to a tissue-specific regulatory molecule, Androcam, rather than its usual regulator, Calmodulin. How this binding affects it function is unknown. Using transgenic Drosophila lines that express mutant and truncated/deleted forms of myosin VI to rescue myosin VI mutant defects has revealed that some unique properties measured in vitro may not play a key role in its ability to participate in actin stabilization in vivo. The proposed studies extend these observations, examining both spermatid development and other developmental processes in which myosin VI works to understand the structural and biochemical/biophysical features that are important in vivo in a wide variety of cellular contexts. Localization studies and measurements of dynamics of GFP tagged molecules will be performed to understand how different features mediate myosin VI participation in a wide variety of cellular functions. Measurements in vitro of biochemical and biophysical properties to determine how our manipulations, and altered regulation by Acam, affect myosin VI's properties will complement these in vivo studies. These studies will likely reveal insights into myosin VI mechanisms in vivo that will be valuable in understanding basic cellular processes and diseases in which myosin VI plays a role. For example, myosin VI is up regulated in several cancers, likely participating in motility important for metastasis. Additionally, several Human deafness syndromes and associated hypertrophic cardiomyopathy are caused by mutations in myosin VI. Studies of myosin VI promise to illuminate the underlying defects in these diseases and potentially reveal new treatment options. Studies of myosin VI are likely to result in a greater understanding of mechanisms involved in a number of basic cellular processes, such as endocytosis, sorting pathways for protein localization and cellular and intracellular motility. In addition, myosin VI has been implicated in several diseases, such as prostate and ovarian cancers, human deafness syndromes, and hypertrophic cardiomyopathy. Further understanding of how myosin VI works in vivo is likely to enhance our understanding of disease mechanisms and provide for new routes of treatment.