Spatiotemporally tracking of nano-biofilaments inside the nuclear pore complex core
Spatiotemporally tracking of nano-biofilaments inside the nuclear pore complex core
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DOI：
10.1016/j.biomaterials.2020.120198
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发表时间：
2020-10-01
期刊：
影响因子：
14
通讯作者：
Wong, Richard W.
中科院分区：
文献类型：
--
作者：
Mohamed, Mahmoud Shaaban;Hazawa, Masaharu;Wong, Richard W.
核孔复合物（NPC）是一种具有中心选择屏障的门控纳米机器，主要由Nups组成，Nups包含具有苯丙氨酸-甘氨酸（FG）基序（FG-NUPs）的内在无序（非结构化）区域（IDR）。NPC中心FG网络动力学知之甚少，因为FG-NUPs液-液相分离（LLPS）回避了结构表征。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，柔性生物丝在运动和相互作用过程中预计会缠结和打结。然而，在实时和空间中的细丝打结可视化尚未在纳米级可视化。在这里，我们报告了一种具有纳米级分辨率的FGNUP组织的时空跟踪方法，使用高速原子力显微镜（HS-AFM）以类似于150 ms的时间尺度揭示了结直肠细胞和类器官NPC中的FG-NUP构象。FG-NUP单丝的跟踪显示，单丝在正常和癌症模型中具有不均匀的厚度，这反过来影响了细丝的旋转和运动。值得注意的是，FG-NUPs在各种癌症中过表达。使用FG-NUP抑制剂，反式-1，2-环己二醇，我们发现在侵袭性结肠癌细胞和类器官中，中心栓的大小显著减小，并且不完全可逆回到丝状结构。这些数据显示了FG-NUPs可逆自组装演变为中央栓部分生物起源的模型。总之，HS-AFM能够跟踪和操纵几十年来一直回避的天然FG-NUPs的单丝。
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-NUPbiofilaments 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 FGNUP organization with nanoscale resolution, unveiling FG-NUP conformation in NPCs of colorectal cells and organoids at timescales of similar to 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.