Unraveling the nanoscale architecture and multifunctional roles of the neuronal membrane skeleton with super

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Ruobo Zhou, PhD (Penn State University)

Host: Kaushik Ra gunathan

Abstract: Super-resolution fluorescence imaging has transformed our ability to visualize the molecular organization of cells at nanometer scales, providing unprecedented insights into nanoscale architecture, molecular interactions, and cellular function. Yet, imaging complex molecular assemblies over extended timescales and across multiple protein species remains challenging. We recently developed FLEXTAG, a compact, self-renewable, anti- photobleaching protein-labeling platf orm that enables multiplexed and sustained super- resolution imaging. We applied these approaches to the neuronal membrane-associated periodic skeleton (MPS), a well-defined nanoscale cytoskeletal architecture that provides a powerful model system for understanding how molecular organization at the nanoscale shapes cellular function. The MPS, composed of actin, spectrin, and associated molecules, forms a lattice-like cortic al structure whose molecular composition and functions have remained incompletely understood. Using co-immunoprecipitation and mass spectrometry, we identified hundreds of candidate MPS-interacting proteins spanning diverse functional categories. Super-resolution imaging of representative proteins, including previously unknown structural components, motor proteins, cell adhesion molecules (CAMs), ion channels, and signalin g proteins, revealed periodic distributions characteristic of the MPS along neurites. Genetic perturbations of the MPS and its interacting proteins indicate roles in axon-axon and axon-dendrite interac tions and axon diameter regulation. Functionally, the MPS serves as a dynamic platform for signal integration. It recruits G protein-coupled receptors (GPCRs), CAMs, and receptor tyrosine kinases (RTKs) in respons e to extracellular cues, promoting colocalization and RTK transactivation that trigger extracellular signal- regulated kinase (ERK) signaling. In addition to signaling, the MPS spatially gates major forms of endocytosis by restricting pit formation to MPS-free "clearing" zo nes across axonal and somatodendritic compartments. Disruption of the MPS enhances both basal and ligand-induced endocytosis, while ligand-triggered endocytosis activates ERK signaling to further remodel the MPS, establishing a self-reinforcing feedback circuit. Notably, MPS integrity limits amyloid precursor protein (APP) endocytosis and suppresses amyloid-β1- 42 production, linking nanoscale cytoskeletal organization to neuronal health and disease susceptibility. Together, these findings establish the neuronal MPS as a model biological system for revealing how nanoscale molecular architecture organizes structural integrity, protein interactions, receptor signaling, and membrane trafficking. More broadly, they demonstrate the power of advanced super-resolution fluorescence imaging to move from resolving nanoscale structures to uncovering how molecular architecture actively governs cellular function and homeostasis.