RII Track-4: Quantifying Muscle Assembly in Live C. elegans Using Super-Resolution Light Microscopy

现代细胞生物学的一个主要目标是了解生物体如何利用遗传信息发育复杂的功能组织。在肌肉中，由重复的结构单元组成的收缩纤维缩短并产生力量，为各种功能提供动力，如运动、进食和繁殖。为了了解肌肉纤维组装是如何发生的，将使用新开发的基因组编辑技术和先进的光学显微镜来观察完整、发育中的圆虫的纤维成分。南阿拉巴马大学和国立卫生研究院的国家生物医学成像和生物技术研究所(NIBIB)之间的这一合作将使机构之间能够进行密集的知识转移，并为本科生和研究生提供身临其境的研究培训机会。我们的观察和测量不仅将揭示特定的肌肉蛋白质在发育过程中如何组装成收缩纤维，而且还将有助于更好地了解肌肉如何在受伤后愈合，适应变化的机械环境，以及对致病突变和衰老做出反应。这项奖学金将允许PI和一名研究生参与，并有潜力使PI能够制定可持续的研究计划，以提高S在该尖端领域的知名度。技术说明条纹肌肉收缩纤维(肌原纤维)是由细丝和粗丝阵列自组装形成的，这些纤维精确地集成到一系列重复的功能单元(肌节)中以产生力量。肌原纤维组装后，肌节成分保持惊人的动态，使肌肉能够在结构上重组，以响应外部信号，如机械负荷和拉伸。在这个项目中，我们将使用NIBIB提供的最先进的技术来观察完整、发育中的线虫(线虫)的横纹肌组织和动态，具有前所未有的分辨率和精确度。一种新的双顺反子标记和截断(BITTS)基因组编辑工具将被用于生产对基因表达影响最小的荧光记者，并将创建特定的、带有荧光标记的蛋白质片段，从而战略性地扰乱肌原纤维的组装和组织。使用低剂量、高速、超分辨率显微镜，我们将快速收集线虫胚胎发育的3D图像，以量化在各种肌肉类型的肌原纤维组装过程中内源蛋白质如何在特定的肌节结构中积累和变化，同时将光毒性降至最低，并确保有机体的正常发育和行为。这项研究旨在研究复杂而重要的问题，例如：细丝和粗丝是如何连接和组织成功能性肌原纤维的？肌节生长过程中细丝和粗丝是如何伸长的？那么，在肌肉细胞伸长过程中，新的肌节是如何增加的？由于不同动物的横纹肌具有保守的结构、功能和组成相似之处，我们的发现将为了解不同的肌肉类型是如何发展的，肌肉在受伤或生长后如何修复和适应肌节，以及肌肉纤维在疾病或衰老期间如何功能障碍提供重要的见解。

Non-technical DescriptionA major goal of modern cell biology is to understand how organisms develop complex functional tissues using genetic information. In muscle, contractile fibers composed of repeating structural units shorten and produce force to power a wide variety of functions, such as locomotion, feeding, and reproduction. To understand how muscle fiber assembly occurs, newly-developed genome editing techniques and advanced light microscopes will be employed to observe fiber components within an intact, developing round worm. This collaboration between the University of South Alabama and the National Institute of Biomedical Imaging and Biotechnology (NIBIB) of the National Institutes of Health will enable intensive knowledge transfer between the institutions and provide immersive research training opportunities for both undergraduate and graduate students. Our observations and measurements will not only reveal how specific muscle proteins assemble into contractile fibers during development, but will also lead to a better understanding of how muscles heal after injury, adapt to altered mechanical environments, and respond to disease-causing mutations and aging. This fellowship will allow participation of both the PI and a graduate student, and has the potnetial to allow the PI to develop a sustainable research program that would raise the instiution?s visibility in this cutting-edge field.Technical DescriptionStriated muscle contractile fibers (myofibrils) form by the self-assembly of thin and thick filament arrays that are precisely integrated into a series of repeating functional units (sarcomeres) to produce force. Sarcomeric components remain surprisingly dynamic after myofibril assembly, which enable muscles to structurally reorganize in response to external signals such as mechanical load and stretch. In this project, we will use state-of-the-art techniques available at the NIBIB to observe striated muscle organization and dynamics within intact, developing roundworms (Caenorhabditis elegans) with unprecedented resolution and precision. A novel bicistronic tagging and truncations (BiTTs) genome editing tool will be used to produce fluorescent reporters with minimal impact on gene expression, and will create specific, fluorescently-tagged protein fragments that strategically disrupt myofibril assembly and organization. Using low-dose, high-speed, super-resolution microscopy, we will rapidly collect 3D images of developing C. elegans embryos to quantify how endogenous proteins accumulate and change within specific sarcomeric structures during myofibril assembly in a variety of muscle types while minimizing phototoxicity and ensuring normal development and behavior of the organism. This research is designed to examine complex, important questions such as: How are thin and thick filaments connected and organized into functional myofibrils? How do thin and thick filaments elongate during sarcomere growth? And, how are new sarcomeres added during muscle cell elongation? Because of the conserved structural, functional, and compositional similarities among striated muscles within different animals, our findings will provide important insight into how diverse muscle types develop, how muscles repair and adapt sarcomeres after injury or growth, and how muscle fibers malfunction during disease or aging.