Spatiotemporally tracking of nano-biofilaments inside the nuclear pore complex core
Mahmoud Shaaban Mohamed, Masaharu Hazawa, Akiko Kobayashi, Laurent Guillaud, Takahiro Watanabe‐Nakayama, Mizuho Nakayama, Hanbo Wang, Noriyuki Kodera, Masanobu Oshima, Toshio Ando, Richard W. Wong
Abstract
Nuclear pore complex (NPC) is a gating nanomachine with a central selective barrier composed mainly of Nups, which contain intrinsically disordered (non-structured) regions (IDRs) with phenylalanine-glycine (FG) motifs (FG-NUPs). The NPC central FG network dynamics is poorly understood, as FG-NUPs liquid-liquid phase separation (LLPS) have evaded structural characterization. Moreover, the working mechanism of single FG-NUP-biofilaments residing at the central lumen is unknown. In general, flexible biofilaments are expected to be tangled and knotted during their motion and interaction. However, filament knotting visualization in real-time and space has yet to be visualized at the nanoscale. Here, we report a spatiotemporally tracking method for FG-NUP organization with nanoscale resolution, unveiling FG-NUP conformation in NPCs of colorectal cells and organoids at timescales of ~150 ms using high-speed atomic force microscopy (HS-AFM). Tracking of FG-NUP single filaments revealed that single filaments have a heterogeneous thickness in normal and cancer models which in turn affected the filament rotation and motion. Notably, FG-NUPs are overexpressed in various cancers. Using the FG-NUP inhibitor, trans-1,2-cyclohexanediol, we found that central plug size was significantly reduced and incompletely reversible back to filamentous structures in aggressive colon cancer cells and organoids. These data showed a model of FG-NUPs reversible self-assembly devolving into the central plug partial biogenesis. Taken together, HS-AFM enabled the tracking and manipulation of single filaments of native FG-NUPs which has remained evasive for decades.