441 Super-resolution imaging for nuclear pore quantification in human keratinocyte differentiation

Nuclear Pore Complexes (NPCs) serve as essential gateways between the nucleus and cytoplasm. Dysregulation of NPC function is associated with aging cancer, yet how implicated in epidermal homeostasis skin disorders remains largely unexplored. Here, we show that 74% genes encoding nucleoporins (NUPs), building blocks constituting NPC, are significantly downregulated (p<0.05) primary human keratinocyte differentiation. NUP93, an NUP for determining protein diffusion size limit across nuclear pores, among most drastically NUPs. To investigate potential changes number composition differentiation, applied Stochastic Optical Reconstruction Microscopy (STORM), which enabled super-resolution optical imaging resolution down to ∼20 nm visualize individual pores quantify pore compositions. Furthermore, developed a “multiplex isolation staining” method, allowed efficient antibody labeling NUPs especially differentiated keratinocytes multiplexed four conditions on single coverslip. Using MAB414 recognizes shared region multiple NUPs, or binds scaffolding NUP133, consistently observe no significant cluster number. These data suggest early differentiation not numbers. Interestingly, NUP93 antibody, identified reproducible reduction density but intensity per cluster, suggesting decreased incorporation into subset Taken together these indicate does involve cell involves altered can impact sizes through pore.