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展复杂的功能组织。 在肌肉中，由重复结构单元组成的收缩纤维缩短并产生力量来为各种功能提供动力，例如运动，进食和繁殖。 为了了解肌肉纤维组装是如何发生的，新开发的基因组编辑技术和先进的光学显微镜将被用来观察完整的、发育中的圆形蠕虫内的纤维成分。 南亚拉巴马大学与美国国立卫生研究院国家生物医学成像和生物技术研究所（NIBIB）之间的合作将使机构之间的密集知识转移，并为本科生和研究生提供沉浸式研究培训机会。 我们的观察和测量不仅将揭示特定肌肉蛋白在发育过程中如何组装成收缩纤维，而且还将更好地了解肌肉在受伤后如何愈合，适应改变的机械环境，以及对致病突变和衰老的反应。该奖学金将允许PI和研究生的参与，并有潜力让PI开发一个可持续的研究计划，将提高机构？技术描述横纹肌收缩纤维（肌原纤维）是由粗细细丝阵列的自组装形成的，这些细丝阵列精确地整合到一系列重复的功能单元（肌节）中以产生力量。 肌节成分在肌原纤维组装后保持惊人的动态，这使得肌肉能够响应外部信号（如机械负荷和拉伸）进行结构重组。 在这个项目中，我们将使用NIBIB现有的最先进的技术，以前所未有的分辨率和精度观察完整的发育中的蛔虫（秀丽隐杆线虫）内的横纹肌组织和动态。 一种新的双顺反子标记和截短（BiTT）基因组编辑工具将用于产生对基因表达影响最小的荧光报告基因，并将产生特异性的荧光标记蛋白片段，这些片段将在战略上破坏肌原纤维组装和组织。 利用低剂量、高速、超分辨率显微镜，我们将快速收集显影C的三维图像。elegans胚胎中，以量化内源性蛋白质在各种肌肉类型的肌原纤维组装过程中如何在特定的肌节结构内积累和变化，同时最大限度地减少光毒性并确保生物体的正常发育和行为。 这项研究旨在研究复杂的，重要的问题，如：细和粗的细丝是如何连接和组织成功能性肌原纤维？ 在肌节生长过程中，细丝和粗丝是如何伸长的？ 而且，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之间保守的结构，功能和组成相似性，我们的研究结果将提供重要的见解，了解不同的肌肉类型如何发展，肌肉如何修复和适应损伤或生长后的肌节，以及肌肉纤维如何在疾病或衰老期间发生故障。

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.