Molecular organization of pathways governing cell-shape formation

National Heart, Lung, And Blood Institute

Investigators

Linked publications, trials & patents

Abstract

Top-down cellular observation using integrative methods of structural biology We developed a robust comprehensive pipeline that integrates light microscopy with cellular cryo-ET with an ability of manipulation of axon remodeling, facilitating the in situ molecular imaging of neurons at near atomic resolution. This integrative approach facilitated the elucidation of the molecular events of intracellular organization, organelle redistributions and cytoskeletal remodeling under physiological and pathological conditions. Particularly, we developed an axtonomy platform, where we can investigate axon maintenance and mimic injury to observe the axon response through combined biophysical cell biological and in situ cryo-ET approaches. We ultimately seek for the key factors how to induce axon for a pro-regenerative state. Through this, we discovered a drug-induced regeneration mechanism; microtubules-stabilizing agents significantly enhanced axon regeneration, while their absence completely inhibited regeneration. We solved the structure of in situ axonal microtubules specifically at the axon regenerating site, and visualized the engagement of the drug for the regeneration. Our findings provide the first structural understanding of axon injury and its response, and in addition the response of axon to a drug, pave the way for in situ interventions to address axon injuries. Bottom-Up Reconstruction of factors that control cell shape formation: Focal Adhesion Machinery and SSNA1-Driven Remodeling In parallel, we are reconstituting macromolecular complexes comprising focal adhesion machineries such as integrin, talin, vinculin, and actin on synthetic membranes to understand the assembly of focal adhesion (FA) signaling networks in a mechanistic manner: Among these efforts, using cryo-EM, we resolved the force-sensitive talin actin binding domains in complex with F-actin complex at 2.9 Å, providing insights into mechanotransduction at adhesion sites. Building on this bottom-up framework, we also published a paper on a protein SSNA1. SSNA1 is an enigmatic protein that associates with centriole, basal body or microtubule organizing center, therefore it is critical for cell division and morphogenesis. We purified the protein and engineered to be amenable for structural analysis, and solved a structure of SSNA1 at 4.5 Å resolution, showing how SSNA1 assembles into a robust scaffold to maintain centriole structure. We finally demonstrated that SSNA1 localizes to centrioles and functions as a structural regulator, coordinating the nucleation. As centrioles are critical for spatial control of microtubule organization and key for cell duplication, our findings link SSNA1 to higher-order regulation of cell shape formation, providing a crucial element for understanding polarized growth in specialized cells. Integration of top-down and bottom-up approaches We provide a multi-scale view of signaling regulation during cell shape formation by combining top-down cellular cryo-ET with bottom-up in vitro reconstitution. These insights directly connect molecular-scale mechanisms to cellular-level morphogenesis.