Mohamed Mahmoud Shaaban, Hazawa Masaharu, Kobayashi Akiko, Guillaud Laurent, Watanabe-Nakayama Takahiro, Nakayama Mizuho, Wang Hanbo, Kodera Noriyuki, Oshima Masanobu, Ando Toshio, Wong Richard W
Cell-Bionomics Research Unit, Innovative Integrated Bio-Research Core, Institute for Frontier Science Initiative, Kanazawa University, Kanazawa, 920-1192, Japan; Zoology Department, Faculty of Science, Assiut University, Assiut, 71516, Egypt.
Cell-Bionomics Research Unit, Innovative Integrated Bio-Research Core, Institute for Frontier Science Initiative, Kanazawa University, Kanazawa, 920-1192, Japan.
Biomaterials. 2020 Oct;256:120198. doi: 10.1016/j.biomaterials.2020.120198. Epub 2020 Jun 23.
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.
核孔复合体（NPC）是一种具有中央选择性屏障的门控纳米机器，该屏障主要由核孔蛋白（Nups）组成，这些核孔蛋白包含具有苯丙氨酸-甘氨酸（FG）基序的内在无序（非结构化）区域（IDRs）（FG-NUPs）。由于FG-NUPs的液-液相分离（LLPS）逃避了结构表征，NPC中央FG网络动力学仍知之甚少。此外，位于中央腔的单个FG-NUP生物丝的工作机制尚不清楚。一般来说，柔性生物丝在其运动和相互作用过程中预计会缠结和打结。然而，丝状结构的缠结在纳米尺度上的实时和空间可视化尚未实现。在这里，我们报告了一种用于FG-NUP组织的具有纳米级分辨率的时空跟踪方法，使用高速原子力显微镜（HS-AFM）在~150毫秒的时间尺度上揭示了结肠直肠细胞和类器官的NPC中的FG-NUP构象。对FG-NUP单丝的跟踪表明，在正常和癌症模型中，单丝具有不均匀的厚度，这反过来又影响了丝的旋转和运动。值得注意的是，FG-NUPs在各种癌症中过度表达。使用FG-NUP抑制剂反式-1,2-环己二醇，我们发现侵袭性结肠癌细胞和类器官中的中央栓大小显著减小，并且不完全可逆地恢复为丝状结构。这些数据显示了一个FG-NUPs可逆自组装演变为中央栓部分生物发生的模型。总之，HS-AFM能够跟踪和操纵天然FG-NUPs的单丝，而这几十年来一直难以实现。
