Molecular organization of pathways governing cell-shape formation

National Heart, Lung, And Blood Institute

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Abstract

Bottom-up reconstitution of key signaling complexes for cell shape formation and their validation by top-down cellular observation using cryo-ET: One of the most important pathways that is controlling the cellular and neuronal shape formation is focal adhesion (FA) and FA related signaling pathway. FA is a cellular machinery controlled by its master receptor, integrin, which has wide-ranging roles for cell shape formation. FA contains a few hundred molecular players generating a multi-layered protein-protein network at the plasma membrane, which also connects to the actin cytoskeleton as well as cellular signaling factors. As layers of regulations orchestrate the proper functioning of the system, elucidating FA as a whole on a molecular level is not attainable, hindering our understanding of the FA regulation. To test the hypothesis that the molecular functions of key components are regulated in a hierarchical fashion, we employ a bottom-up reconstitution approach and aim to build up the macromolecular machinery that would mimic FA initiation. Using light microscopy and biophysical methods, we elucidated the mechanisms of regulation of the master controllers of FA, talin, vinculin and actin and their assembly process at the membrane surface. Furthermore, using cryo-EM, we solved the structure of the tissue-dominant integrin a5b1 and its extracellular ligand fibronectin, and elucidated the molecular mechanism of outside-in activation of integrin by the extracellular matrix (ECMs). Currently, we are analyzing the functional relevance learned from the in vitro reconstitution in a cellular context. Furthemore, to facilitate the top-down molecular analysis of intracellular components in a cellular context, we have established a pipeline for the preparation of primary mouse neurons, and their observation in situ by cellular cryo-electron tomography (cryo-ET). The shape formation of neurons and the role of cellular signaling activities at axon branching points is of particular interest. To this end, we aim to obtain visual insights into signaling processes and cytoskeleton remodeling during axon morphogenesis and branching. The first exciting results revealed from the successful pipeline establishment; highlighting that axon branching points act as a hotspot for a cellular dynamic activity which is not found at any other places along axon shaft, fostering a higher level of compartmentalization inside axons.