Multi-angle Total Internal Reflection Structured Illumination Microscopy to Investigate Exo-Endocytosis Coupling in Living Cells

Neuronal communication depends on the precise coordination of synaptic vesicle exocytosis and endocytosis at presynaptic terminals. After neurotransmitter release, efficient retrieval of synaptic vesicle membranes through endocytosis is essential for sustaining neurotransmission. Despite its fundamental importance, the mechanisms linking synaptic vesicle exocytosis and endocytosis remain poorly understood, largely due to the limitations of current imaging technologies. Existing methods lack the spatial and temporal resolution needed to directly visualize these nanoscale processes in living cells. To address this critical gap, this Joint NINDS/NIMH Exploratory Neuroscience Research Grant (PA-25-150) proposal aims to develop a three-dimensional (3D) total internal reflection fluorescence structured illumination microscope (TIRF-SIM). By integrating super-resolution imaging with evanescent field nanometry, this technology will enable unprecedented visualization of exo-endocytic coupling at the nanoscale in living neurons. Our research plan consists of two key objectives: (i) We will iteratively optimize the experimental parameters of 3D TIRF-SIM using well-characterized in vitro and in vivo imaging conditions to maximize spatial and temporal resolution. (ii) Using these optimized parameters, we will evaluate the performance of 3D TIRF-SIM for high-resolution reconstruction of clathrin-coated structures both live cultured cells and tissues of Drosophila melanogaster embryos addressing long-standing questions in endocytosis research. Beyond its technological advancements, this proposal directly addresses a critical need in neuroscience research. By enabling direct visualization of synaptic vesicle dynamics at unprecedented resolution, this innovation will fundamentally advance our understanding of neuronal communication. Moreover, this breakthrough in imaging technology has the potential to catalyze the development of novel therapeutic strategies for neurological disorders associated with synaptic dysfunction. Project Number: 1R21NS143024-01A1 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of Neurological Disorders and Stroke (NINDS) | Principal Investigator: Comert Kural | Institution: OHIO STATE UNIVERSITY, Columbus, OH | Award Amount: $433,125 | Activity Code: R21 | Study Section: Special Emphasis Panel[ZRG1 IBV-P (01)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11304724