Revealing the Molecular Basis of Microtubule-Intermediate Filament Interactions. - Project Summary Microtubules (MTs) and intermediate filaments (IFs) are two main components of the cytoskeleton in eukaryotic cells, each with unique structural and regulatory roles. Interactions between MTs and IFs are crucial for maintaining cellular shape, stability, and organization. Despite their wide importance in biology, exactly how MTs and IFs interact at the molecular level remains unclear. A major barrier to elucidating these interactions is the strong context-dependency of cytoskeletal arrangement and function across tissues, cell types, and subcellular locations. MTs and IFs are specialized by subunit composition, bound proteins, and post- translational modifications to create polymers with distinct physical properties, interaction networks, and regulatory mechanisms. Understanding how MTs and IFs are adapted to form morphologically and functionally distinct cytoskeletal arrays will have broad applications in developing therapeutic strategies, improving tissue engineering, and advancing our knowledge of cellular mechanics. The goal of this proposal is to reveal the molecular adaptations of MTs and IFs that regulate cytoskeletal array configuration and function specifically in myelinated axons, which are important for rapid, long distance neuronal communication. All axons contain a staggered, parallel array of highly decorated MTs and neuron-specific IFs called neurofilaments (NFs) that locally regulate cell shape and composition. Myelinated axons regulate their cytoskeletal array through a phosphorylation cascade that directly targets MTs and NFs to change their abundance and rearrange their 3D organization. My research plan builds on the implementation of a novel method for isolating myelinated axons from the brain that preserves the internal configuration of MTs and NFs and will for the first time enable the study of tissue-derived material in its native form. This multifaceted work combines structural determination, biochemical assays, comparative proteomics, and immunofluorescence aiming to reveal how MTs and NFs are adapted and interact inside myelinated axons. Atomic structures of native MTs and NFs determined by cryo-electron microscopy (cryo-EM) will reveal the molecular mechanisms behind their exceptional stability inside the axon, which is important for maintaining cell integrity. Complementary biochemical analyses will investigate how MT and NF turnover is regulated to influence cytoskeletal array composition. To further identify regulators of the MT-NF array, focused ion beam milling (FIB- milling) and cryo-electron tomography (cryo-ET) will reconstruct entire cytoskeletal networks within intact axons to quantify MT-NF network organization and association with cellular cargo. Altogether, support of this research by the R35 MIRA award would establish a framework for future studies on cytoskeletal adaptations in different biological contexts and disease states throughout my career.
