Regulation of Myosin Ic Mechanochemistry

描述(申请人提供)：人类基因组包含8种不同的肌球蛋白I亚型，使其成为在人类中表达的最大的非传统肌球蛋白家族。肌球蛋白IC可能是研究最好的肌球蛋白I亚型，因为它被认为在毛细胞的动态适应、胰岛素刺激的GLUT-4运输、代偿性胞外分泌和核转录中发挥作用。尽管它与这些细胞过程和先天性耳聋等疾病有关，但myo1c在细胞中的分子作用尚不清楚。已有研究表明，肌球蛋白IC可能作为一种转运体、一种力生成器或一种应变敏感系绳。肌球蛋白IC在这些角色中发挥作用的能力取决于它产生力量和调节其生物化学以响应负荷的能力，每个角色对肌球蛋白提出了非常不同和特定的机械要求。因此，通过了解肌球蛋白IC的机制，可以推断肌球蛋白IC的分子作用。尽管肌球蛋白的力学机制是其细胞功能的核心，但人们对肌球蛋白IC的力学和负荷依赖性的机械力化学知之甚少。此外，有人提出，钙与肌球蛋白IC调节域上的钙调蛋白结合可能在调节其机制和对负荷的反应中发挥核心作用；然而，这一概念从未得到实验验证。此外，由于肌球蛋白IC对短暂的钙浓度升高的反应从未被研究过，因此目前尚不清楚钙引起的肌球蛋白IC的力学和动力学变化是否与细胞功能有关。这项研究将解决我们知识中的这些差距。利用一组单分子技术，本研究的具体目的是：1.测定肌球蛋白IC工作行程的力学性质2.测定肌球蛋白IC的负荷敏感性3.直接测量瞬时钙对肌球蛋白IC力学和动力学的影响所有这些实验都将在有无钙的情况下进行，以测试钙对肌球蛋白动力学和力学的影响。除了阐明肌球蛋白IC在细胞中的分子作用以及这一作用如何受钙和力的调节外，这项研究还将揭示肌球蛋白I超家族的多样性。 与公共健康相关：人体内的分子马达负责产生和响应这些马达故障导致的各种疾病，包括耳聋、高血压、心力衰竭和发育缺陷。虽然对力做出反应的能力是这些马达功能的核心，但人们对这一点的实现知之甚少。利用尖端的单分子技术，这项研究将检测其中一种马达--肌球蛋白IC的力产生和张力传感，帮助我们了解其细胞功能以及张力传感在分子马达中对健康和疾病所起的作用。

DESCRIPTION (provided by applicant): The human genome contains 8 different isoforms of myosin I, making it the largest family of unconventional myosins expressed in humans. Myosin Ic is perhaps the best studied myosin I isoform due to its proposed roles in dynamic adaptation in hair cells, insulin stimulated GLUT-4 transport, compensatory excocytosis, and nuclear transcription. Despite its association with these cellular processes and disease such as congenital deafness, the molecular role of myo1c in the cell is unknown. It has been proposed that myosin Ic may act as a transporter, a force generator, or a strain-sensing tether. The ability of myosin Ic to function in these roles depends on its abilities to generate force and to modulate its biochemistry in response to load, with each of these roles placing very different and specific mechanical requirements on the myosin. Thus, by understanding the mechanics of myosin Ic, the molecular role of myosin Ic can be deduced. Despite the fact that myosin mechanics are at the heart of its cellular function, very little is known about the mechanics and load dependent mechanochemistry of myosin Ic. Furthermore, it has been proposed that calcium binding to calmodulins on the myosin Ic regulatory domain may play a central role in modulating its mechanics and response to load; however this notion has never been tested experimentally. Also, it is unknown whether calcium induced changes in myosin Ic mechanics and kinetics are relevant to cellular function since the response of myosin Ic to transient increases in calcium has never been studied. This research will address these gaps in our knowledge. Using a battery of single molecule techniques, the specific aims of this research are: 1. Determine the mechanical properties of the myosin Ic working stroke 2. Determine the load sensitivity of myosin Ic 3. Directly measure the effects of transient calcium on myosin Ic mechanics and kinetics All of these experiments will be conducted in the presence and the absence of calcium to test how calcium affects myosin kinetics and mechanics. Besides elucidating the molecular role that myosin Ic plays in the cell and how this role is regulated by calcium and force, this research will also shed light on the diversity within the myosin I superfamily. PUBLIC HEALTH RELEVANCE: Molecular motors within the human body are responsible for generating and responding to forces with malfunction of these motors leading to a wide array of diseases including deafness, hypertension, cardiac failure, and developmental defects. While the ability to respond to forces is central to the function of these motors, little is known about how this is accomplished. Using cutting-edge single molecule techniques, this research will examine force generation and tension sensing in of one of these motors, myosin Ic, helping us to understand both its cellular function and the role that tension sensing plays in molecular motors in both health and disease.