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

University Of South Alabama, Mobile AL

Investigators

Abstract

Non-technical Description A 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 Description Striated 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.