Determining structure and organization of neurofilaments in situ using cryo- electron tomography

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Project Summary As one of the major components of the neuronal cytoskeleton network, neurofilaments are essential for the radial growth and conduction velocity of axons. Neurofilaments are involved in the pathogenesis of multiple neurological disorders and serve as important axonal damage biomarkers in neurodegenerative and traumatic diseases. Despite their critical role in the structure and function of axons, little is known about the molecular assembly of neurofilaments. Currently, it is still unclear how individual neurofilament subunits assemble into mature filaments, or how those filaments interact with each other to form the cytoskeleton network that supports the growth of axons. Cryo-electron tomography (CryoET) allows visualization of protein complexes inside cells at nanometer resolution in three dimensions. Detailed structural information of those proteins can be obtained from the tomogram using specialized computational techniques. In this proposal, we aim to study the structure and organization of neurofilaments within axons using CryoET. To achieve this goal, we will image the axon of primary neurons with CryoET and determine the structures of neurofilaments at subnanometer resolution using subtomogram classification and averaging. Molecular models will be constructed from the averaged structures to reveal the in situ assembly of neurofilaments. The structures and models of neurofilaments will be studied in their cellular context to understand how the neuronal cytoskeleton organization evolves, as the axons grow thicker. To make the proposed work possible, novel computational methods will be developed for CryoET data analysis throughout the process. We believe this research will provide the first structural insight into in situ neurofilament assembly and deepen our understanding of neuronal cytoskeleton organization. The structural information acquired from this research will pave the way for future studies of the axon maturation process, as well as the structural basis of various neurological diseases.