Regulation of Myosin Ic Mechanochemistry

描述（由申请人提供）：人类基因组包含8种不同的肌球蛋白I亚型，使其成为人类表达的最大的非常规肌球蛋白家族。肌球蛋白I可能是研究得最好的肌球蛋白I异构体，因为它在毛细胞的动态适应、胰岛素刺激的GLUT-4运输、代偿性胞外吞噬和核转录中起着重要作用。尽管它与这些细胞过程和疾病（如先天性耳聋）有关，但myo1c在细胞中的分子作用尚不清楚。有人提出肌球蛋白i可能作为一种转运体、一种力发生器或一种应变感应系绳。肌凝蛋白Ic发挥这些作用的能力取决于其产生力和根据负载调节其生物化学的能力，每种作用都对肌凝蛋白提出了非常不同和特定的机械要求。因此，通过了解肌凝蛋白Ic的机制，可以推断肌凝蛋白Ic的分子作用。尽管肌凝蛋白的力学是其细胞功能的核心，但对肌凝蛋白Ic的力学和负荷依赖的机械化学知之甚少。此外，有人提出，钙在肌凝蛋白Ic调节区域上与钙调素的结合可能在调节其力学和对负荷的反应中发挥核心作用；然而，这一观点从未经过实验验证。此外，钙诱导的肌球蛋白Ic的力学和动力学变化是否与细胞功能有关尚不清楚，因为肌球蛋白Ic对钙的短暂增加的反应从未被研究过。这项研究将弥补我们知识上的这些空白。利用一系列单分子技术，本研究的具体目标是：1。测定肌球蛋白i工作行程2的机械性能。测定肌球蛋白ic3的负荷敏感性。直接测量瞬态钙对肌凝蛋白力学和动力学的影响所有这些实验都将在有钙和没有钙的情况下进行，以测试钙如何影响肌凝蛋白动力学和力学。除了阐明肌凝蛋白I在细胞中的分子作用以及钙和力如何调节这一作用外，本研究还将揭示肌凝蛋白I超家族的多样性。

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.