Molecular organization of pathways governing cell-shape formation

National Heart, Lung, And Blood Institute

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Abstract

Top-down cellular observation using light microscope combined with cryo-ET To facilitate the molecular analysis of intracellular components, 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. Therefore, we have conducted ultrastructural analyses of branching axons from the central nervous system (CNS). As the first time observation, we have obtained a number of visual insights into signaling processes and cytoskeleton remodeling during axon morphogenesis and branching. Through a systematic survey of >100 axon branches by cryo-ET, we found that axon branching points act as hotspots for cellular dynamic activities, which is not found at any other places along the axon shaft, fostering a higher level of compartmentalization inside axons. When a branch is still nascent and premature, it is reversibly formed via a formation of actin-based filopodia but no microtubules have been established to reinforce the branch. At this stage, we observed an accumulation of mitochondria and short fragments of actin filaments, making an aligned organization towards the tip of filopodia. We discovered the coexistence of ribosomes in cytosol, as well as on ER exclusively at the axon branching point, which supports the hypothesis of local protein synthesis occurring at axon branch points, necessary for the new axon formation. The ER, which normally forms a thin, smooth formation along the axon shaft, expands at the axon branch point into a sheet-like formation. It fosters the binding of ribosomes and acts as a synthesis platform for secretory and membrane proteins. Maturation of the axon branch occurs by the entering of short microtubules into the branch. Here, we observed the co-migration of ER and microtubules, which is necessary for establishing a matured axon branch. ER membranes were tangled around the microtubules so that they tether each other to facilitate the comigration. We obtained a roadmap of events allowing axon branching sites to serve as unique control hubs for axon development and downstream neural network formation. After this showcase study, we aim to elucidate mechanisms underlying the maintenance and regeneration of neurons. We developed a pipeline to mimic axon injury (axotomy) and tested a series of factors that might be key for axon regeneration. We have identified key targets and obtained preliminary results showing the reorganization of cellular components. Bottom-up reconstitution of key signaling complexes for cell shape formation and their validation. One of the most important pathways that is controlling the cellular and neuronal shape formation is focal adhesion (FA) and FA-related signaling. 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 ultimately 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 and 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, cryo-EM and biophysical methods, we have elucidated the mechanisms of regulation of the master controllers of FA, integrin, talin, vinculin and actin and their assembly process at the membrane surface. We analyze the downstream of the FA activation, by obtaining structural insights into actin-talin-vinculin interactions by using cryo-EM approaches in combination with biochemical interaction analyses. Furthermore, we are currently working on the integrin activation mechanisms that rely on Rap1 signaling using cryo-EM. Rap1 mediates the crosstalk between the FA pathway and other cellular/signaling pathways. We are analyzing the functional relevance of Rap1 in the switching process. Lessons learned from the in vitro reconstitution are tested in a cellular context using the top-down approach mentioned above.