Myosin-XIX and the molecular mechanism of actin-based mitochondrial organization

这项研究项目的长期目标是了解酶特性、马达特性和 肌球蛋白XIX(MYO19)参与转运和定位的分子运动蛋白的细胞功能 线粒体是一种对多种细胞过程起中心作用的细胞器。线粒体动力学已经显示出 部分以肌动蛋白为基础，参与线粒体动力学的其他基因突变导致人类 疾病。此外，基因芯片分析表明，MYO19在某些癌症中的表达增加， 与正常组织相比。先前的研究表明MYO19在线粒体运动中起作用。 培养细胞表达GFP标记的MYO19结构改变肺线粒体动力学 在癌细胞系和培养的神经细胞系中。此外，MYO19的耗尽会导致 线粒体在细胞分裂中的分布。综上所述，MYO19蛋白序列的分析和 生化结合对培养细胞中MYO19表达变化的影响的分析导致了 假设MYO19在线粒体动力学中起中心作用，因为它含有保守的肌球蛋白 运动域，并通过其尾部区域与线粒体相互作用。我们将同时使用Myo19击倒和 在细胞培养模型中过表达功能性和非功能性Myo19结构以确定什么作用 Myo19可能在线粒体发挥核心作用的许多过程中发挥作用。原核表达与纯化 MYO19运动域的研究将允许对速率和平衡常数进行完整的体外分析 与MYO19的马达特性有关，以及特定类别的序列差异对 那些财产。对MYO19的酶和运动特性进行体外检测将使 MYO19运动产生力量的机制的确定和因素的确定 这可能会调节它在体内的功能。通过研究功能角色、酶特性和马达 MYO19的特性建议的研究将进一步加深我们对健康状态下线粒体动力学的理解 和疾病。

The long-term goal of this research project is to understand the enzymatic properties, motor properties, and cellular functions of myosin XIX (MYO19), a molecular motor protein involved in the transport and localization of mitochondria, an organelle central to multiple cellular processes. Mitochondrial dynamics have been shown to be partially actin-based, and mutations in other genes involved in mitochondrial dynamics lead to human diseases. Additionally, microarray analyses indicate that MYO19 expression is increased in certain cancers, compared to normal tissues. Previous studies show that MYO19 plays a role in mitochondrial motility in cultured cells as expression of GFP-tagged MYO19 constructs alters mitochondrial dynamics in a pulmonary cancer cell line and in a cultured neuronal cell line. Additionally, MYO19 depletion leads to defects in mitochondrial distribution during cell division. Taken together, analysis of the MYO19 protein sequence and biochemistry paired with analysis of the effect of altered MYO19 expression in cultured cells leads to the hypothesis that MYO19 plays a central role in mitochondrial dynamics, as it contains a conserved myosin motor domain and interacts with mitochondria via its tail domain. We will use both Myo19 knock-down and overexpression of functional and non-functional Myo19 constructs in cell culture models to determine what role Myo19 may play in a number of processes where mitochondria play a central role. Expression and purification of MYO19 motor domain will allow for a complete in vitro analysis of the rate and equilibrium constants associated with the motor properties of MYO19, and the contributions of class-specific sequence differences to those properties. Examination of both the enzymatic and motor characteristics of MYO19 in vitro will allow the determination of the mechanism by which MYO19 motor activity generates force, and determination of factors that may regulate its function in vivo. By examining the functional roles, enzymatic properties, and motor properties of MYO19 the proposed studies will further our understanding of mitochondrial dynamics in health and disease.